Hindawi Publishing CorporationThe Scientific World JournalVolume 2013 Article ID 958540 10 pageshttpdxdoiorg1011552013958540
Review ArticleEarly Pregnancy Diagnosis in BovinesCurrent Status and Future Directions
Ashok K Balhara1 Meenakshi Gupta2 Surender Singh3
Ashok K Mohanty3 and Inderjeet Singh1
1 Central Institute for Research on Buffaloes Hisar 125001 India2 LLR University of Veterinary and Animal Sciences Hisar 125005 India3 National Dairy Research Institute Karnal 132001 India
Correspondence should be addressed to Ashok K Mohanty ashokmohanty1gmailcom
Received 2 September 2013 Accepted 17 October 2013
Academic Editors P Poltronieri and S Rodtong
Copyright copy 2013 Ashok K Balhara et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited
An early and accurate diagnosis of reproductive dysfunctions or aberrations is crucial to better reproductive management inlivestock High reproductive efficiency is a prerequisite for high life-time production in dairy animals Early pregnancy diagnosisis key to shorten the calving interval through early identification of open animals and their timely treatment and rebreeding so asto maintain a postpartum barren interval close to 60 days A buffalo the most important dairy animal in the Indian subcontinentis known for problems related to high calving interval late puberty and high incidence of anestrus Lack of reliable cow-sideearly pregnancy diagnosis methods further aggravates the situation Several methods of pregnancy diagnosis are being practiced inbovine species yet none qualifies as the ideal pregnancy diagnosis method due to the inherent limitations of sensitivity accuracyspecificity speed and ease of performing the testThe advancement of molecular techniques like proteomics and their applicationsin animal research has given a new hope to look for pregnancy biomarker molecules in these animals This review attemptsto examine common pregnancy diagnosis methods available for dairy animals while assessing the usefulness of the moderntechnologies in detecting novel pregnancy markers and designing future strategies for research in this area
1 Introduction
An early and precise pregnancy diagnosis is an importantcriterion for better reproductivemanagement in livestock likecows and buffaloes High reproductive efficiency is a prereq-uisite to realization of high life-time production from dairyanimals Early pregnancy diagnosis is crucial to shorteningthe calving interval through enabling the farmer to identifyopen animals so as to treat andor rebreed them at the earliestopportunity Ideally a 60-day postparturient barren intervalin dairy animals is recommended for breeding Dairy farmersneed to recognize nonpregnancy at the earliest opportunity soas to rebreed the dam at the very next opportunity The earlyembryonic period in cattle has beendescribed to be lasting forapproximately 42 days postinsemination [1] encompassing aseries of events starting with fertilization and culminating inimplantation (Table 1) After implantation embryonic lossesdue to noninfectious causes are rare and the pregnancy
becomesmore secure [2 3] Studies on levels of progesteronepregnancy associated glycoproteins (PAGs) interferon tauand early pregnancy factor are some of the common clinicallypractised pregnancy detection methods in bovines and eachhas its own benefits and limitations Buffaloes are the mostimportant dairy animal of the Indian subcontinent yet theyexperience problems related to reproduction especially highcalving interval late puberty and high incidence of anestrusLack of reliable early pregnancy diagnosis methods furtheraggravates the problems Many methods of pregnancy diag-nosis both direct and indirect are being practiced in bovinespecies none till date actually qualifies as the ideal pregnancydiagnosis method due to the limitations they inherit Theadvancement of molecular techniques like proteomics andtheir applications in animal research has opened up oppor-tunities for research communities to look for pregnancy bio-marker molecules in these animals
2 The Scientific World Journal
Table 1 Important events during the early embryonic period
Days of pregnancy Event
Days 0-1 Fertilization single-cell embryo (zygote) inoviduct
Day 2 Early cleavages in the oviduct (up to 8 cellstages) activation of embryonic genome
Days 3-4 Embryo enters the uterus
Days 5-6 16ndash32 cell zona-enclosed embryoprogressing into compact morula stage
Days 7-8 Formation of a blastocoele withdifferentiation of embryonic cells
Days 9-10 Blastocyst expansion and hatching from thezona pellucid
Days 11ndash15 Blastocyst elongation from a tubular to afilamentous structure
Days 14ndash19 Maternal recognition of pregnancyDays 19-20 Implantation beginsDay 21 Caruncles-cotyledons appearDays 22ndash41 Implantation progressesDay 42 Implantation completedCompiled from available information (Morris and Diskin 2008 [3] Hafez1993 [4])
In this review we have described common pregnancydiagnosis methods available for dairy animals assessingthe usefulness of the modern technologies in detectingnovel pregnancy markers and designing future strategies forresearch in this area
2 Pregnancy Detection Methods
21 Direct Method
211 Per-Rectal Palpation Cowie [5] first described transrec-tal palpation of the uterus as a method for pregnancy diagno-sis in cattle which makes it the oldest and most widely prac-tisedmethod for early pregnancy diagnosis in large dairy ani-mals even today Traditionally to confirm pregnancy at aboutday 30 of gestation onwards the practitioners have relied onthe palpation of the amniotic vesicle [6] and slipping of thechorioallantoic membranes between the thumb and forefin-ger [7] In buffaloes too palpation per rectum is a simple eco-nomic and the most widely practised method for pregnancydiagnosis however this method is only accurate from day 45of pregnancy [8]Though per-rectal palpation is the cheapestpregnancy diagnosis method several studies have suggestedthat examining pregnant cows early in gestation by tran-srectal palpation increases the risk of iatrogenic embryonicmortality [9]
212 Ultrasonography By per-rectal palpation an expert canaccurately diagnose an animal pregnant only after day 35 ofgestation but the application of ultrasonography has madediagnosis possible as early as day 28 after insemination [9] oreven earlier [10]The first visible changes appearing by day 21after breeding when fetal heartbeat can be visualized also
helped confirm a viable pregnancy [11] though it is not aroutinely assessed parameter for pregnancy diagnosis Tran-srectal ultrasonography has the added advantage of providingadditional information on ovarian structures identificationof twins and determination of fetal viability age and sex[10 12] Transrectal ultrasonographymade a thorough exam-ination of the reproductive health of the animal possible andtherefore it has now become an established research tool tostudy bovine reproductive biology in cattle [12] and buffaloes[10] Ultrasound is a minimally invasive accurate and effi-cient technique for early pregnancy diagnosis [13 14] andmayminimize the rare incidence of palpation-induced abortions
Most studies on the utility of transrectal ultrasonographyfor pregnancy diagnosis have been conducted in cattle butlately it has found utility in buffalo cows as well In buffaloestransrectal ultrasonography is most commonly used to deter-mine pregnancy fetal age and sex as well as ovarian activity[15] In early 1990s various workers started using transrectalultrasonography in buffaloes with visualization of the embry-onic vesicle and embryo proper in pregnant buffalo cowsbetween 19 and 22 days after AI [16] In a field study on 260buffaloes between 30 and 45 days afterAI sensitivity of detec-tion of pregnancy was observed to be 979 [17] Howeverunpublished data from researchers at the Central Institute forResearch on Buffaloes Hisar India suggest the accuracy forselecting pregnant buffaloes at day 21 afterAI to be about 50which increases to almost 100 by day 30 These findingssupport other findings in cattle which claim that transrectalultrasonography for pregnancy diagnosis between days 21and 25 after breeding has sensitivity and specificity of 448and 823 respectively which further increase to 977 and877 respectively when conducted between 26 and 33 daysafter AI [18] Similarly Nation et al [19] documented that thesensitivity and specificity of pregnancy diagnosis in lactatingdairy cows based on ultrasonographic detection of uterinefluid as well as embryonic membranes from 28 to 35 daysafter AI were 96 and 97 respectively Direct observationof a fetus with ultrasonography was found more accuratethan assays for the presence of pregnancy-specific proteins inplasma but resulted in more false negative diagnoses [20]
Per-rectal palpation and transrectal ultrasonography aredirect and accurate methods for pregnancy diagnosis Bothrequire a great deal of skill and experience Veterinary-gradeultrasound machines equipped with a rectal transducer areexpensive in developing countries and therefore the highinitial cost of this technology partly limits its practical imple-mentation [12]
22 Indirect Method
221 Progesterone Shemesh et al [21] proposed that thedifference in peripheral plasma progesterone levels betweenpregnant and nonpregnant cows 19 days after inseminationcan form the basis for a very early pregnancy test Laing andHeap [22] first documented this in milk to diagnose cows inearly pregnancy Measurement of progesterone is an indirectmethod for pregnancy diagnosis in many livestock speciesincluding cattle buffaloes sheep and goats
The Scientific World Journal 3
Table 2 Progesterone levels in different sample types in bovine species
Cow Milk(i) 0 or 1(ii) 9 or 10(iii) 21 or 22(iv) 27 or 28
15111120125
121033068
Zaied et al 1979 [23]
Cow Milk 18 gt8 mdash Simersky et al 2007 [24]
Buffalo Milk plasma 21ndash35 each 1601 and 361 respectively 041ndash267 Kamboj and Prakash 1993[25]
Buffalo Plasma 0 13 01 and 36 respectively 06 (18th day of cycle) Batra et al 1979 [26]Buffalo Plasma 21 or 22 10 lt07 Perera et al 1980 [27]Cow Milk 20 gt11 lt8 Pennington et al 1985 [28]
Buffalo Milk 18ndash22 2483 289 Singh and Puthiyandy 1980[29]
Cow Faeces 18ndash24 gt50 as compared tononpregnant mdash Isobe et al 2005 [30]
Conception extends the life of the corpus luteum (CL) bypreventing the luteolytic mechanism from being triggeredthus prolonging and maintaining its functional characteris-tics ensuring continuedhigh progesterone levels [31] Proges-terone maintains the uterine endometrium in a state whichsupports embryonic development implantation and foe-toplacental development Progesterone concentrations varywith the stage of the estrous cycle which makes it one of themost commonly studied reproductive hormones in bovineruminants for pregnancy detection and ovarian activity [32]
Studies in the bovine estrous cycle indicate that the milkor serum progesterone concentrations reach a maximumvalue 13-14 days after estrus and if the animal is pregnantthese continue to remain elevated up to day 21 after fertil-ization [33] and beyond These high levels of progesteronein serum or milk between days 18 and 24 after inseminationform the basis of establishment of pregnancy in cattle [34 35]Interferon-120591 exerts its antiluteolytic effect by inhibiting theendometrial expression of oxytocin receptors through whichoxytocin stimulates pulsatile PGF
2120572release [36] Although
low progesterone concentrations at 18 to 24 days after breed-ing can accurately predict nonpregnancy high progesteroneconcentrations during this period are not the specific indica-tors of pregnancy due to variations among cows in durationof the estrous cycle as well as the incidence of early or lateembryonic mortality The advantages of progesterone assayfor pregnancy diagnosis include noninvasive collection ofmilk sample and the feasibility to conduct the test on thefarm using commercial cow-side milk progesterone test kits[28 37ndash39] though the sensitivity gets compromised to someextent with these assay kits Table 2 describes the work indifferent labs on the level of progesterone in pregnant andnonpregnant bovines
In buffalo cows it is quite evident that the progesteronelevels inmilk are four to five times higher than those in bloodplasma [25 26] Just like cattle buffaloes too can be accu-rately diagnosed as nonpregnant by determination of plasmaprogesterone concentrations 21 days after insemination [27]
Amajor constraint in using progesterone assay for pregnancydiagnosis is its use only in cases where AI or breeding datesare knownrecorded and not randomly in the herdNeverthe-less progesterone analysis remains themost common clinicaluse of any of the reproductive hormones
222 Estrone Sulphate Estrone sulphate is a conjugatedsteroid product of estrone present predominantly in thebovine placentomes [40] and it is themajor estrone present inthe fetal (allantoic and amniotic) fluids and maternal periph-eral plasma of cows with measurable quantities detectable byday 52 onwards till the end of gestation [41] Its concentra-tions increase from day 60 and plateau around day 150 afterinsemination [42] However reliable pregnancy detection ispossible only after day 100 of gestation and therefore this testcan only detect late pregnancy [43] Concentration of estronesulphate in the maternal body fluids is a useful indicator forthe placental functions especially those related to embryonicgrowth [44] In zebu and crossbred cattle and Murrahbuffaloes Prakash and Madan [45] reported below detectionlevels (lt50 pgmL) of estrone sulphate during the first twomonths followed by sharp increase in the fourth month andvalues stabilized after reaching the highest levels in the sixthmonth of pregnancy Levels of estrone sulphate in differentmaternal body fluids namely milk and blood plasma canbe utilised as the criteria for confirming pregnancy by after110 day insemination in bovine species [46] Estrone sulphateconcentrations have also been frequently correlated to fetalnumbers [44] as these are higher when the number of devel-oping foetuses ismore than one Yet estrone sulphate is not anideal pregnancy biomarker as the plasma and milk profilesare influenced by many other factors such as genetic makeupweight parity status and environment [46]
223 Conceptus and Placenta Secreted Products The veryfact that pregnancy brings about numerous physiologicalchanges in the female body through secretionaltered secre-tion of various biomolecules which often are proteins or their
4 The Scientific World Journal
metabolites supports research endeavours aimed at identify-ing novel proteins as the candidate molecules for pregnancydetection Human chorionic gonadotropin (hCG) discov-ered for the first time by Aschheim and Zondek [47] in theurine of pregnant women in 1927 is perhaps the best exampleof a placental protein hormone used for pregnancy diagnosisWith the advancement of biotechnological tools hCG basedpregnancy diagnosis has become the simplest cheapestand most commonly practised test for humans to diagnosepregnancy as early as 8ndash10 days after conception [48]Homol-ogous to the human protein only higher primates produce achorionic gonadotropin (CG) for maintaining luteal activityduring early pregnancy while ruminants produce type Iinterferon as an antiluteolytic factor during this period [49]
224 Early Conception Factor (ECF) Early pregnancy fac-tor (EPF also known as early conception factormdashECF)mdasha1084 kDa protein [50] is present in the sera of pregnantmammalian females detectable within 6 to 24 hours of fer-tilization [51] and disappearing within 24 to 48 after death orremoval of the embryo [52] EPF is present in the serum up totwo-thirds of the gestation [53] EPF remains the earliestserum benchmark for positive fertilization and hence suc-cessful conceptionThis novel pregnancy-specific protein hashigh immunosuppressive ability which is demonstrated byrosette inhibition test a bioassay first demonstrated in preg-nant mice [54] Laleh et al [55] demonstrated significant dif-ferences in rosette inhibition titres of pregnant and open cowswith values being 8ndash10 and 3ndash5 respectively
EPF is reported to be present in the pregnant sera ofmost mammalian species including humans [56 57] mice[51] sheep [58 59] cows [60ndash62] pigs [63] mares [64]and some wild animals [65] In buffalo pregnancy Chander[66] demonstrated decreased E-rosette formation but failedto demonstrate the presence of a rosette inhibiting factor (RIFwhich probably would have been EPF) in the serum Anti-bodies raised against a cow serum glycoprotein were used todetect EPF [67] leading to development of a lab methodwhich has been commercialized in the USA as Early Con-ception Factor (ECF) test (Concepto Diagnostics KnoxvilleTN) claiming detection within 48 hours Extensive study onthe effectiveness of the commercial ECF test for diagnosingnonpregnancy revealed a high degree of nonreliability of thetest wherein only 444 and 556of the confirmednonpreg-nant heifers were identified correctly by serum ECF analysisat days 1 to 3 and days 7 to 9 after AI respectively [61] Similarconclusions were drawn by [60] and [62] Although EPF issecreted in early pregnancy it is not strictly pregnancy spe-cific because of its secretion from nonplacental sources suchas tumors and transformed cell lines [50] which makes itan erroneous pregnancy detection method EPF belongs to afamily of heat shock proteins though detected extracellularlyand having immunosuppressive and growth factor properties[68]These properties are crucial to avoid rejection of an anti-genically alien embryo and support its development There-fore with the advent ofmodern biosciences there is hope thatthese changes could be identified and used as diagnostics forvery early detection of pregnancy However the practicability
of such an early test may still remain low due to high inci-dence of losses during the first 15 days of conception [2]
225 Interferon-120591 (IFN-120591) Moor and Rowson [69] thepioneers of sheep embryo transfer transferred embryos ondays 12 13 and 14 to unmated ewes and suggested interactionsbetween the embryo and uterus that influence the lutealfunction and result in establishment of pregnancy Godkinet al [70] purified ovine trophoblastic protein-1 (oTP-1) anearly secretory protein of the sheep blastocyst from in vitrocultured days 14ndash16 conceptusesThey revealed that oTP1 actson the maternal endometrium thereby eliciting maternalresponses which contribute to the maintenance of preg-nancy Imakawa et al [49] reported the primary amino acidsequence of oTP-1 to demonstrate that the protein is mostprobably an interferon-alpha Later research proved that thesecretions from the conceptus are in fact responsible for thematernal recognition of pregnancy [71 72]
Interferon-120591 a novel type I interferon [73] is first pro-duced by the conceptus between days 12-13 after inseminationin sheep and days 14ndash16 in cattle [74ndash76] High ovine IFN-120591levels are attained on days 12-13 before luteolysis could actu-ally be triggered [77] In ruminants IFN-120591 a 172 amino acidpolypeptide [73] blocks transcription of estrogen receptoralpha and oxytocin receptors in endometrial cells [78] whiledownregulating the expression of enzymes cyclooxygenase-2 and prostaglandin F synthase [79] thus preventing PGFrelease necessary for luteolysis
IFN-120591 acting within the uterine cavity [80] withextremely low levels in extrauterine tissues and peripheralcirculation prevents direct use of IFN-120591 as an early pregnancydiagnosis molecule [81] Rapid advancement of moleculartechniques in the last two decades has opened new avenuesfor exploring this unique molecule as a pregnancy markerfor ruminants through studies on IFN-120591 stimulated genes(ISG) namely interferon-stimulated protein 15 kDa (Isg15)myxovirus resistance 2 (MX2) and 21015840-51015840 oligoadenylate syn-thetase (OAS1) in peripheral blood leukocytes during earlypregnancy [82 83] Microarray analysis further indicatedthatmany genes including IFN-120591 stimulated are upregulatedduring early pregnancy [84ndash86] Green et al [84] have how-ever shown that the differential expression of such genes isinfluenced by the parity of the animal being more definite inheifers as compared tomultiparous animals All these experi-ments have suggested IFN-120591 stimulated genes to be potentialpregnancy detection biomarkers still there is no field leveltest available based on these markers
226 Pregnancy Associated Glycoproteins (PAGs) Reloca-tion of the extra embryonic trophoblastic cell layers to theendometrium [87] between days 20 to 28 and secretions fromthe conceptus lead to successful implantation and continua-tion of pregnancy in ruminant species [88] The pregnancyassociated glycoproteins (PAGs) are secretory products fromthe mono- and binucleated trophoblastic cells in bovine pla-centomes [89] Among these glycoproteins Butler et al [90]
The Scientific World Journal 5
detected two pregnancy-specific proteins in the sera of preg-nant cows a 65ndash70 kDa and a 47ndash53 kDa protein at pI 46ndash48 and 40ndash44 respectively Of these the former showed animmune reaction similar to that of 120572
1-fetoprotein while the
latter showed no reactivity with known proteins and it wasgiven the name ldquoprotein Brdquo or the ldquopregnancy-specific proteinBrdquo (PSPB) in bovines Further purification and characteriza-tion of several isoforms from bovine foetal cotyledons foundthat protein B is actually a 67 kDa PAG [91] Biochemicaland functional investigations established these proteins to beenzymatically inactive members of the aspartic proteinasesuperfamily having homology to pepsin chymosin cathep-sins D and enzyme renin [92 93] PAGs are a very complexgroup of proteins a fact proven by the already documented 22distinct cDNA libraries [94] The three most studied bovinePAGs PSPB PAG 67 kDa or bPAG-1 [93] and PSP60 [95]are isomers of the same protein having similar N-terminalsequences [96] Transcription of bPAG-2 and -11 mRNA isseen all through the pregnancy -4 -5 and -9mRNAs in earlypregnancy and bPAG-1 mRNA are detectable only after day45 [97] Interestingly bovine PAG-4 and bPAG-1 mRNA arehighly transcribed till day 250 of gestation but become indis-cernible at the end [97]The six N-glycosylation sites [98] areresponsible for the variations in molecular weight and half-life of PAGs [99] and is also the reason for expression ofdifferent PAGs during different stages of gestation [97 100]
Very recently it has been observed that placental defectscommonly seen during somatic nuclear transfers in cattleare complemented by unusually high plasma levels of PAGsprobably due to diminished clearance of these proteinsfollowing changes in the glycosylation patterns [94] PSPB isdetectable in the serumof pregnant cows over a long period ofgestation starting at about the fourthweek [101] of gestation toseveral weeks after parturition [102]High circulating levels ofthese proteins on days 80 to 100 postpartum restrict their useas a pregnancy diagnosis test except in heifers [102 103]
Sasser and coworkers [104] developed double antibodyradioimmunoassay for the serological detection of PSPB forpregnancy detection in cattle and found serum levels increas-ing progressively from 1 ngmL after day 30 to 9 ngmL35 ngmL and 150 ngmL after three six and nine months ofpregnancy respectivelyThe study claimed PSPB detection tobemore accurate than the traditional rectal palpationmethodfor pregnancy detection Green et al [105] developed a sand-wich ELISA using anti-PAG monoclonal antibodies whichwere able to detect PAG in all pregnant animals with concen-trations of 875 ngmL on day 28 the highest at 5889 ngmLduring the week of parturition and very low levels within4 weeks postpartum Silva et al [106] predicted 937 954and 962 accuracies for first second and third postpartumtimed artificial inseminations which were in agreement withother commonly practiced pregnancy detection methodsDifferent homologous (RIA-497) and heterologous radioim-munoassay systems (RIA-706 RIA-780 RIA-809 and RIA-Pool) developed for measurement of ruminant blood PAGconcentrations are highly correlated and can be used forpregnancy detection of 30ndash80 days [107] Radioimmunoassayof pregnant sera of zebu cattle established PAG concen-trations to be 60 ngmL 1960 ngmL 10956 ngmL and
3484 ngmL at 8 weeks at 35 weeks at term and at 2 weekspostpartum respectively a pattern similar to other breeds ofcattle [108] Results of PAG-RIA based pregnancy diagnosisin buffaloes have also been encouraging with a high degree ofaccuracy of diagnosis as early as day 31 with 100 sensitivityand 90ndash100 specificity [109] PAGs are used for develop-ment of bench-top pregnancy detectionmethods [110] whichare now commercially available as BioPRYN (BioTrackingRussia) DG29 (Genex Cooperative Inc USA) and IDEXX(IDEXX Laboratories Inc USA) BioPRYN blood test is themost extensively used PAG based kit for pregnancy detectionin ruminants By May 2010 already there were 2 millioncattle blood tests conducted for pregnancy detection (httpbiotrackingcomsiteprotectnetabouttimeline)
3 Current Research inBiomarkers for Pregnancy
It is presumed that the monitoring of sequential changes inblood proteome profile from the day of estrus to successfulconception and through progression of gestation can lead todiscovery of molecules which will perhaps be novel and spe-cific to the physiological stage of the animal In order to qual-ify as a marker for pregnancy the candidate molecule shouldbe able to accurately determine the pregnancy status as earlyas possible with minimum false positives or false negativesAdditionally the biological marker for pregnancy shouldhave the following desired characteristics
(i) specifically upregulated or downregulated duringpregnancy
(ii) least affected by nonanimal factors like feed environ-ment and drug interactions
(iii) having the ability to reflect age as well as viability ofthe conceptus
(iv) present in easily accessible body fluids like serummilk urine and vaginal discharge
(v) expressed over a considerable period of time to giveample time for diagnosis
(vi) revealing the result immediately
Proteomics [111] is large scale study of protein func-tions protein expression protein-protein interactions orposttranslational modifications in a particular cell tissue ororganism and is intended for identification of all the proteinspresent Proteomics provides an opportunity to simultane-ously analyse thousands of proteins in a single experimentfrom a complex mixture of proteins in various body fluids[112] This will help in identifying specific and sensitive bio-markers fulfilling the characteristics of uniqueness for a preg-nancy diagnosis molecule Main objectives of the proteomicsresearch include documentation of biomarkers altered pro-tein expression patterns indicative of pathophysiologicalchanges and therapeutically important drug targets [113]Easily reachable body fluids like blood serum and milk havea wide range of abundant proteins and these few proteinsmake up about 97 of the total serum and milk proteome
6 The Scientific World Journal
and thereby interfere in the proteomic analysis (reviewed by[113]) Conversely it is the low abundance proteins whichhave the highest prospect of being the novel biomarkersof changes in internal milieu of body To sort the problemof high abundance proteins two approaches are suggestedremoval of abundant proteins (usually by immunoaffinity)and concentration of the low abundancescarce proteins withsimultaneous removal of high abundance proteins techni-cally known as combinatorial peptide ligand libraries CPLL[114] Commercially available ProteoMiner kit fromMs Bio-Rad is CPLL based Both approaches however lead to loss ofa significant portion of the low abundance proteins along thehigh abundance proteins yet the later approach is preferred[114]
There is limited information on the bovine proteomein relation to pregnancy Jin et al [115] performed pro-teomics analysis using blood serum samples of pregnantand nonpregnant Holstein dairy cattle at 21 and 35 daysafter AI and reported composite profiles of key proteinsinvolved in early pregnancy and suggested the potential use ofidentified proteins to detect early pregnancy in bovinesTheseincluded nine pregnancy-specific spots in day 21 and day 35serum samples Pregnancy-specific proteins were identifiedas transferrin albumin IgG2a heavy chain constant regionand immunoglobulin gamma heavy chain variable regionFurther differential proteomic analysis of milk samples frompregnant and nonpregnant cows revealed 16 protein spots 14pregnancy specific and 2 spots downregulated in the pregnantmilk sample [116] Pregnancy-specific proteins were identi-fied as serum albumin precursor IgG1 heavy chain constantregion conglutinin precursor epithelial keratin 10 and kelch-like ECH-associated protein Though some identified spotswere abundant milk or serum proteins their molecularweights and pI values were different from main milk orserum proteinsThismay suggest that these proteins could bepregnancy-specific subunits or fragments of albumin and IgGor carrying differentially expressed small proteins whichmayultimately have potential for pregnancy detection
Encouragingly enough these studies need further inves-tigations for arriving at some sort of pregnancy detectionmethod Preliminary studies in buffalo cows on blood pro-teome too detected significant changes in many proteins in2DE gels [117] Important proteins found on MS analysis ofthese were synaptojanin-1 apolipoprotein A-1 apolipopro-tein B Keratin 10 and Von Willebrand factors which aredocumented to have a role in embryogenesis and earlypregnancy
Data generated out of sequential blood proteome analysisduring pregnancy can have several other applications as wellfor example studying fetal viability genetic disorders and soforth Trisomy 21 pregnancies can be detectedwith high accu-racy by maternal serum proteomic analysis in humans [118]In the absence of a single indicator for a particular life processa combination of the expression patterns of more than onesubstance can be used for a purposeful analysis as in thequadruple test where levels of four blood constituents (alpha-fetoprotein human chorionic gonadotropin unconjugatedoestriol and inhibin-A) have been used to predict theprobability of Downrsquos syndrome in babies [119] A pregnancy
detection test on the same lines as the human quadruple testcan be tried where instead of one we can consider proteinprofiles of more proteins This approach too will require athorough analysis of the bovine proteome before such a testwith high accuracy is available to the livestock owners
4 Conclusion
Early pregnancy diagnosis is an important aspect for optimiz-ing dairy production yet none of the present day methodsqualifies as an ideal diagnostic due to limitations of accuracylater stages of applicability and requirement for elaborateinstrumentation and laboratory setup This warrants furtherresearch on developing novel early pregnancy diagnosticsfor livestock species Currently available state-of-the-artinstrumentation and proteomics techniques instil hope forfinding moleculesmdashexclusively related to intricate maternalmetabolic alterations necessary to align with physiology ofearly embryonic development and its signalling for maternalrecognition of pregnancy and continued survival Thoughthese techniques are still in their infancy in animal scienceresearch they hold great promise to address a long-awaitedbreakthrough in pregnancy diagnosis in livestock
Acknowledgment
The authors acknowledge their thanks to the National Fundfor Basic Strategic and Frontier Application Research inAgriculture (NFBSFARA) Indian Council of AgriculturalResearch India for the funding support
References
[1] ldquoCommittee on Reproductive Nomenclature Recommenda-tions for standardizing bovine reproductive termsrdquoThe CornellVeterinarian vol 62 no 2 pp 216ndash237 1972
[2] N Ayalon ldquoA review of embryonic mortality in cattlerdquo Journalof Reproduction and Fertility vol 54 no 2 pp 483ndash493 1978
[3] D Morris and M Diskin ldquoEffect of progesterone on embryosurvivalrdquo Animal vol 2 no 8 pp 1112ndash1119 2008
[4] E S E Hafez Reproduction in Farm Animalsedition Lea andFebiger Philadelphia Pa USA 6th edition 1993
[5] T A Cowie Pregnancy Diagnosis Tests A Review Common-wealthAgricultural Bureaux Joint Publication no 13 Common-wealth Agricultural Bureaux Oxford UK 1948
[6] WWisnicky and L E Cassida ldquoAmanual method for diagnosisof pregnancy in cattlerdquo Journal of the American VeterinaryMedical Association vol 113 p 451 1948
[7] R Zemjanis Diagnostic and Therapeutic Techniques in AnimalReproduction Williams and Wilkins Baltimore Md USA 2ndedition 1970
[8] G H Arthur D E Noakes H Pearson and T J ParkinsonldquoReproduction in the buffalordquo in Veterinary Reproduction andObstetrics WB Saunders London UK 1966
[9] O J Franco M Drost M-J Thatcher V M Shille and W WThatcher ldquoFetal survival in the cow after pregnancy diagnosisby palpation per rectumrdquoTheriogenology vol 27 no 4 pp 631ndash644 1987
The Scientific World Journal 7
[10] R K Sharma J K Singh S K Phulia S Khanna and I SinghldquoFetal sex determination with ultrasonography in buffaloesrdquoIndian Veterinary Journal vol 88 no 10 pp 105ndash107 2011
[11] S Curran R A Pierson and O J Ginther ldquoUltrasonographicappearance of the bovine conceptus from days 20 through 60rdquoJournal of the American VeterinaryMedical Association vol 189no 10 pp 1295ndash1302 1986
[12] PM Fricke ldquoScanning the futuremdashultrasonography as a repro-ductive management tool for dairy cattlerdquo Journal of Dairy Sci-ence vol 85 no 8 pp 1918ndash1926 2002
[13] L G Paisley W D Mickelsen and O L Frost ldquoA survey of theincidence of prenatal mortality in cattle following pregnancydiagnosis by rectal palpationrdquoTheriogenology vol 9 no 6 pp481ndash491 1978
[14] D Vaillancourt C J Bierschwal D Ogwu et al ldquoCorrelationbetween pregnancy diagnosis bymembrane slip and embryonicmortalityrdquo Journal of the American Veterinary Medical Associa-tion vol 175 no 5 pp 466ndash468 1979
[15] A Ali and S Fahmy ldquoUltrasonographic fetometry and deter-mination of fetal sex in buffaloes (Bubalus bubalis)rdquo AnimalReproduction Science vol 106 no 1-2 pp 90ndash99 2008
[16] C H Pawshe K B C Appa Rao and S M Totey ldquoUltrasono-graphic imaging to monitor early pregnancy and embryonicdevelopment in the buffalo (Bubalus bubalis)rdquo Theriogenologyvol 41 no 3 pp 697ndash709 1994
[17] M R Bhosreker and IMHangarge ldquoUltrasonography for earlypregnancy diagnosis in buffaloesrdquo Indian Journal of AnimalReproduction vol 21 pp 143ndash144 2000
[18] M C Pieterse O Szenci A H Willemse C S A Bajcsy S JDieleman andM AM Taverne ldquoEarly pregnancy diagnosis incattle bymeans of linear-array real-time ultrasound scanning ofthe uterus and a qualitative and quantitative milk progesteronetestrdquoTheriogenology vol 33 no 3 pp 697ndash707 1990
[19] D P Nation J Malmo G M Davis and K L MacmillanldquoAccuracy of bovine pregnancy detection using transrectalultrasonography at 28 to 35 days after inseminationrdquoAustralianVeterinary Journal vol 81 no 1-2 pp 63ndash65 2003
[20] O Szenci J F Beckers P Humblot et al ldquoComparison of ultra-sonography bovine pregnancy-specific protein B and bovinepregnancy-associated glycoprotein 1 tests for pregnancy detec-tion in dairy cowsrdquo Theriogenology vol 50 no 1 pp 77ndash881998
[21] M Shemesh N Ayalon and H R Lindner ldquoEarly pregnancydiagnosis based upon plasma progesterone levels in the cow andewerdquo Journal of Animal Science vol 36 no 4 pp 726ndash729 1973
[22] J A Laing and R B Heap ldquoThe concentration of progesteronein themilk of cows during the reproductive cyclerdquo British Veter-inary Journal vol 127 no 8 pp 19ndash22 1971
[23] A A Zaied C J Bierschwal R G Elmore R S Youngquist AJ Sharp and H A Garverick ldquoConcentrations of progesteroneinmilk as amonitor of early pregnancy diagnosis in dairy cowsrdquoTheriogenology vol 12 no 1 pp 3ndash11 1979
[24] R Simersky J Swaczynova D A Morris M Franek and MStrnad ldquoDevelopment of an ELISA-based kit for the on-farmdetermination of progesterone in milkrdquo Veterinarni Medicinavol 52 no 1 pp 19ndash28 2007
[25] M Kamboj and B S Prakash ldquoRelationship of progesterone inplasma and whole milk of buffaloes during cyclicity and earlypregnancyrdquo Tropical Animal Health and Production vol 25 no3 pp 185ndash192 1993
[26] S K Batra R C Arora N K Bachlaus andR S Pandey ldquoBloodand milk progesterone in pregnant and nonpregnant buffalordquoJournal of Dairy Science vol 62 no 9 pp 1390ndash1393 1979
[27] BM Perera N Pathiraja S A AbeywardenaM XMotha andH Abeygunawardena ldquoEarly pregnancy diagnosis in buffaloesfrom plasma progesterone concentrationrdquo Veterinary Recordvol 106 no 5 pp 104ndash106 1980
[28] J A Pennington L H Schultz and W F Hoffman ldquoCompari-son of pregnancy diagnosis by milk progesterone on day 21 andday 24 postbreeding field study in dairy cattlerdquo Journal of DairyScience vol 68 no 10 pp 2740ndash2745 1985
[29] A Singh and R Puthiyandy ldquoEstimation of progesterone inbuffalomilk and its application to pregnancy diagnosisrdquo Journalof Reproduction and Fertility vol 59 no 1 pp 89ndash93 1980
[30] N Isobe M Akita T Nakao H Yamashiro and H KubotaldquoPregnancy diagnosis based on the fecal progesterone concen-tration in beef and dairy heifers and beef cowsrdquo Animal Repro-duction Science vol 90 no 3-4 pp 211ndash218 2005
[31] T E Spencer N H Ing T L Ott et al ldquoIntrauterine injectionof ovine interferon-120591 alters oestrogen receptor and oxytocinreceptor expression in the endometrium of cyclic ewesrdquo Journalof Molecular Endocrinology vol 15 no 2 pp 203ndash220 1995
[32] J J Kaneko J W Harvey andM L Bruss Clinical Biochemistryof Domestic Animals Academic Press AmsterdamTheNether-lands 2008
[33] T J Parkinson A Turvey and L J Jenner ldquoA morphometricanalysis of the corpus luteumof the cowduring the estrous cycleand early pregnancyrdquo Theriogenology vol 41 no 5 pp 1115ndash1126 1994
[34] R G Sasser and C A Ruder ldquoDetection of early pregnancy indomestic ruminantsrdquo Journal of Reproduction and Fertility Sup-plement vol 34 pp 261ndash271 1987
[35] M Shemesh N Ayalon and H R Lindner ldquoEarly pregnancydiagnosis based upon plasma progesterone levels in the cow andewerdquo Journal of Animal Science vol 36 no 4 pp 726ndash729 1973
[36] E Wolf G J Arnold S Bauersachs et al ldquoEmbryo-maternalcommunication in bovinemdashstrategies for deciphering a com-plex cross-talkrdquo Reproduction in Domestic Animals vol 38 no4 pp 276ndash289 2003
[37] E W Gowan R J Etches C Bryden and G J King ldquoFactorsaffecting accuracy of pregnancy diagnosis in cattlerdquo Journal ofDairy Science vol 65 no 7 pp 1294ndash1302 1982
[38] R L Nebel W D Whittier B G Cassell and J H Britt ldquoCom-parison of on-farm laboratory milk progesterone assays foridentifying errors in detection of estrus and diagnosis of preg-nancyrdquo Journal of Dairy Science vol 70 no 7 pp 1471ndash14761987
[39] T H Wimpy C F Chang V L Estergreen and J K HillersldquoMilk progesterone enzyme immunoassay modifications anda field trial for pregnancy detection in dairy cowsrdquo Journal ofDairy Science vol 69 no 4 pp 1115ndash1121 1986
[40] R M Eley WWThatcher and F W Bazer ldquoLuteolytic effect ofoestrone sulphate on cyclic beef heifersrdquo Journal of Reproductionand Fertility vol 55 no 1 pp 191ndash193 1979
[41] H A Robertson and G J King ldquoConjugated and unconjugatedoestrogens in fetal and maternal fluids of the cow throughoutpregnancyrdquo Journal of Reproduction and Fertility vol 55 no 2pp 463ndash470 1979
[42] R B Heap and M Hamon ldquoOestrone sulphate in milk as anindicator of a viable conceptus in cowsrdquo British Veterinary Jour-nal vol 135 no 4 pp 355ndash363 1979
8 The Scientific World Journal
[43] M Hamon I R Fleet R J Holdsworth and R B Heap ldquoThetime of detection of oestrone sulphate in milk and the diagnosisof pregnancy in cowsrdquo British Veterinary Journal vol 137 no 1pp 71ndash77 1981
[44] M Hirako T Takahashi and I Domeki ldquoPeripheral changes inestrone sulfate concentration during the first trimester of ges-tation in cattle comparison with unconjugated estrogens andrelationship to fetal numberrdquo Theriogenology vol 57 no 7 pp1939ndash1947 2002
[45] B S Prakash and M L Madan ldquoInfluence of gestation onoestrone sulphate concentration in milk of zebu and crossbredcows and murrah buffaloesrdquo Tropical Animal Health and Pro-duction vol 25 no 2 pp 94ndash100 1993
[46] F Lobago M Bekana H Gustafsson et al ldquoSerum profilesof pregnancy-associated glycoprotein oestrone sulphate andprogesterone during gestation and some factors influencing theprofiles in ethiopian borana and crossbred cattlerdquo Reproductionin Domestic Animals vol 44 no 4 pp 685ndash692 2009
[47] S Aschheim and B Zondek ldquoHypophysenvorderlappenhor-mon undOvarialhormon imHarn von SchwangerenrdquoKlinischeWochenschrift vol 6 no 28 p 1322 1927
[48] J R Marshall C B Hammond G T Ross A Jacobson PRayford andW D Odell ldquoPlasma and urinary chorionic gona-dotropin during early human pregnancyrdquo Obstetrics and Gyne-cology vol 32 no 6 pp 760ndash764 1968
[49] K Imakawa R V Anthony M Kazemi K R Marotti H GPolites and R M Roberts ldquoInterferon-like sequence of ovinetrophoblast protein secreted by embryonic trophectodermrdquoNature vol 330 no 6146 pp 377ndash379 1987
[50] A C Cavanagh ldquoIdentification of early pregnancy factor aschaperonin 10 implications for understanding its rolerdquo Reviewsof Reproduction vol 1 no 1 pp 28ndash32 1996
[51] H Morton V Hegh and G J A Clunie ldquoStudies of the rosetteinhibition test in pregnant mice evidence of immunosuppres-sionrdquo Proceedings of the Royal Society B vol 193 no 1113 pp413ndash419 1976
[52] H Morton B E Rolfe and A C Cavanagh ldquoOvum factor andearly pregnancy factorrdquo Current Topics in Developmental Biol-ogy vol 23 pp 73ndash92 1987
[53] H Morton A C Cavanagh S Athanasas-Platsis K A QuinnandB E Rolfe ldquoEarly pregnancy factor has immunosuppressiveand growth factor propertiesrdquo Reproduction Fertility andDevelopment vol 4 no 4 pp 411ndash422 1992
[54] H Morton V Hegh and G J A Clunie ldquoImmunosuppressiondetected in pregnantmice by rosette inhibition testrdquoNature vol249 no 5456 pp 459ndash460 1974
[55] V G Laleh R G Laleh N Pirany and M M Ahrabi ldquoMea-surement of EPF for detection of cow pregnancy using rosetteinhibition testrdquoTheriogenology vol 70 no 1 pp 105ndash107 2008
[56] H Qin and Z Q Zheng ldquoDetection of early pregnancy factorin human serardquo American Journal of Reproductive Immunologyand Microbiology vol 13 no 1 pp 15ndash18 1987
[57] Y C Smart T K Roberts I S Fraser A W Cripps and R LClancy ldquoValidation of the rosette inhibition test for the detec-tion of early pregnancy in womenrdquo Fertility and Sterility vol 37no 6 pp 779ndash785 1982
[58] HMorton G J A Clunie and F D Shaw ldquoA test for early preg-nancy in sheeprdquo Research in Veterinary Science vol 26 no 2pp 261ndash262 1979
[59] S Wilson R McCarthy and F Clarke ldquoIn search of early preg-nancy factor isolation of active polypeptides from pregnant
ewesrsquo serardquo Journal of Reproductive Immunology vol 5 no 5 pp275ndash286 1983
[60] M C Cordoba R Sartori and P M Fricke ldquoAssessment of acommercially available early conception factor (ECF) test fordetermining pregnancy status of dairy cattlerdquo Journal of DairyScience vol 84 no 8 pp 1884ndash1889 2001
[61] B GandyW Tucker P Ryan et al ldquoEvaluation of the early con-ception factor (ECFŮ) test for the detection of nonpregnancyin dairy cattlerdquoTheriogenology vol 56 no 4 pp 637ndash647 2001
[62] C S Whisnant L A Pagels and M G Daves ldquoCase studyeffectiveness of a commercial early conception factor test for usein cattlerdquo Professional Animal Scientist vol 17 pp 51ndash53 2001
[63] A S Grewal A L C Wallace Y S Pan et al ldquoEvaluation of arosette inhibition test for pregnancy diagnosis in pigsrdquo Journalof Reproductive Immunology vol 7 no 2 pp 129ndash138 1985
[64] K Ohnuma I T O Kazuei Y-I Miyake J Takahashi andY Yasuda ldquoDetection of early pregnancy factor (EPF) in mareserardquo Journal of Reproduction and Development vol 42 no 1pp 23ndash28 1996
[65] Y P Cruz L Selwood H Morton and A C CavanaghldquoSignificance of serum early pregnancy factor concentrationsduring pregnancy and embryonic development in Sminthopsismacroura (Spencer) (Marsupialia Dasyuridae)rdquo Reproductionvol 121 no 6 pp 933ndash939 2001
[66] S Chander Studies on certain immunological aspects of preg-nancy diagnosis in buffaloes [PhD thesis] Chaudhary CharanSingh Haryana Agricultural University Hisar India 1983
[67] W R Threlfall ldquoImmunosuppressive early pregnancy factor(ISEPF) determination for pregnancy diagnosis in dairy cowsrdquoTheriogenology vol 41 p 31 1994
[68] H Morton ldquoEarly pregnancy factor an extracellular chaper-onin 10 homologuerdquo Immunology andCell Biology vol 76 no 6pp 483ndash496 1998
[69] R M Moor and L E A Rowson ldquoInfluence of the embryo anduterus on luteal function in the sheeprdquoNature vol 201 no 4918pp 522ndash523 1964
[70] J D Godkin FW Bazer and RM Roberts ldquoOvine trophoblastprotein 1 an early secreted blastocyst protein binds specifi-cally to uterine endometrium and affects protein synthesisrdquoEndocrinology vol 114 no 1 pp 120ndash130 1984
[71] FW Bazer ldquoMediators of maternal recognition of pregnancy inmammalsrdquo Proceedings of the Society for Experimental Biologyand Medicine vol 199 no 4 pp 373ndash384 1992
[72] R M Roberts D W Leaman and J C Cross ldquoRole of interfer-ons in maternal recognition of pregnancy in ruminantsrdquo Pro-ceedings of the Society for Experimental Biology and Medicinevol 200 no 1 pp 7ndash18 1992
[73] R M Roberts ldquoInterferon-tau a type 1 interferon involvedin maternal recognition of pregnancyrdquo Cytokine and GrowthFactor Reviews vol 18 no 5-6 pp 403ndash408 2007
[74] G E Mann G E Lamming R S Robinson and D C WathesldquoThe regulation of interferon-tau production and uterine hor-mone receptors during early pregnancyrdquo Journal of Reproduc-tion and Fertility Supplement vol 54 pp 317ndash328 1999
[75] R M Roberts A D Ealy A P Alexenko C-S Han and TEzashi ldquoTrophoblast interferonsrdquo Placenta vol 20 no 4 pp259ndash264 1999
[76] WWThatcher M DMeyer and G Danet-Desnoyers ldquoMater-nal recognition of pregnancyrdquo Journal of Reproduction andFertility Supplement vol 49 pp 15ndash28 1995
The Scientific World Journal 9
[77] F W Bazer T E Spencer and T L Ott ldquoInterferon tau anovel pregnancy recognition signalrdquoAmerican Journal of Repro-ductive Immunology vol 37 no 6 pp 412ndash420 1997
[78] T E Spencer and F W Bazer ldquoOvine interferon tau suppressestranscription of the estrogen receptor and oxytocin receptorgenes in the ovine endometriumrdquo Endocrinology vol 137 no3 pp 1144ndash1147 1996
[79] C W Xiao B D Murphy J Sirois and A K GoffldquoDown-regulation of oxytocin-induced cyclooxygenase-2 andprostaglandin F synthase expression by interferon-120591 in bovineendometrial cellsrdquo Biology of Reproduction vol 60 no 3 pp656ndash663 1999
[80] T E Spencer N H Ing T L Ott et al ldquoIntrauterine injectionof ovine interferon-120591 alters oestrogen receptor and oxytocinreceptor expression in the endometrium of cyclic ewesrdquo Journalof Molecular Endocrinology vol 15 no 2 pp 203ndash220 1995
[81] J F Oliveira L E Henkes R L Ashley et al ldquoExpression ofinterferon (IFN)-stimulated genes in extrauterine tissues duringearly pregnancy in sheep is the consequence of endocrine IFN-120591 release from the uterine veinrdquo Endocrinology vol 149 no 3pp 1252ndash1259 2008
[82] C A Gifford K Racicot D S Clark et al ldquoRegulation ofinterferon-stimulated genes in peripheral blood leukocytes inpregnant and bred nonpregnant dairy cowsrdquo Journal of DairyScience vol 90 no 1 pp 274ndash280 2007
[83] H Han K J Austin L A Rempel and T R Hansen ldquoLowblood ISG15 mRNA and progesterone levels are predictive ofnon-pregnant dairy cowsrdquo Journal of Endocrinology vol 191 no2 pp 505ndash512 2006
[84] J C Green C S Okamura S E Poock and M C Lucy ldquoMea-surement of interferon-tau (IFN-120591) stimulated gene expressionin blood leukocytes for pregnancy diagnosis within 18-20d afterinsemination in dairy cattlerdquo Animal Reproduction Science vol121 no 1-2 pp 24ndash33 2010
[85] N Forde F Carter T E Spencer et al ldquoConceptus-inducedchanges in the endometrial transcriptome how soon does thecow know she is pregnantrdquo Biology of Reproduction vol 85 no1 pp 144ndash156 2011
[86] K Kizaki A Shichijo-Kizaki T Furusawa T Takahashi MHosoe andKHashizume ldquoDifferential neutrophil gene expres-sion in early bovine pregnancyrdquoReproductive Biology and Endo-crinology vol 11 article 6 2013
[87] F B P Wooding and D C Wathes ldquoBinucleate cell migrationin the bovine placentomerdquo Journal of Reproduction and Fertilityvol 59 no 2 pp 425ndash430 1980
[88] F W Bazer W W Thatcher P J Hansen M A Mirando TL Ott and C Plante ldquoPhysiological mechanisms of pregnancyrecognition in ruminantsrdquo Journal of Reproduction and FertilitySupplement vol 43 pp 39ndash47 1991
[89] A P Zoli P Demez J F Beckers M Reznik and A BeckersldquoLight and electron microscopic immunolocalization of bovinepregnancy-associated glycoprotein in the bovine placentomerdquoBiology of Reproduction vol 46 no 4 pp 623ndash629 1992
[90] J E Butler W C Hamilton R G Sasser C A Ruder G MHass andR JWilliams ldquoDetection and partial characterizationof two bovine pregnancy-specific proteinsrdquoBiology of Reproduc-tion vol 26 no 5 pp 925ndash933 1982
[91] A P Zoli J-F Beckers P Wouters-Ballman J Closset PFalmagne and F Ectors ldquoPurification and characterization of abovine pregnancy-associated glycoproteinrdquo Biology of Repro-duction vol 45 no 1 pp 1ndash10 1991
[92] S Xie B G Low R J Nagel et al ldquoIdentification of the majorpregnancy-specific antigens of cattle and sheep as inactivemembers of the aspartic proteinase familyrdquo Proceedings of theNational Academy of Sciences of theUnited States of America vol88 no 22 pp 10247ndash10251 1991
[93] S Xie B G Low R J Nagel J-F Beckers and R M RobertsldquoA novel glycoprotein of the aspartic proteinase gene familyexpressed in bovine placental trophectodermrdquoBiology of Repro-duction vol 51 no 6 pp 1145ndash1153 1994
[94] F Constanta S Camousa P Chavatte-Palmera et al ldquoAlteredsecretion of pregnancy-associated glycoproteins during gesta-tion in bovine somatic clonesrdquoTheriogenology vol 76 pp 1006ndash1021 2011
[95] M M Mialon S Camous G Renand J Martal and FMenissier ldquoPeripheral concentrations of a 60-kDa pregnancyserum protein during gestation and after calving and in rela-tionship to embryonic mortality in cattlerdquo Reproduction Nutri-tion Development vol 33 no 3 pp 269ndash282 1993
[96] JMartal N Chene S Camous et al ldquoRecent developments andpotentialities for reducing embryo mortality in ruminants therole of IFN-120591 and other cytokines in early pregnancyrdquo Repro-duction Fertility and Development vol 9 no 3 pp 355ndash3801997
[97] J A Green S Xie X Quan et al ldquoPregnancy-associated bovineand ovine glycoproteins exhibit spatially and temporally dis-tinct expression patterns during pregnancyrdquo Biology of Repro-duction vol 62 no 6 pp 1624ndash1631 2000
[98] S Xie J Green J B Bixby et al ldquoThe diversity and evolutionaryrelationships of the pregnancy-associated glycoproteins anaspartic proteinase subfamily consisting of many trophoblast-expressed genesrdquo Proceedings of the National Academy of Sci-ences of the United States of America vol 94 no 24 pp 12809ndash12816 1997
[99] K Klisch A Boos M Friedrich et al ldquoThe glycosylationof pregnancy-associated glycoproteins and prolactin-relatedprotein-I in bovine binucleate trophoblast giant cells changesbefore parturitionrdquo Reproduction vol 132 no 5 pp 791ndash7982006
[100] B P V L Telugu A M Walker and J A Green ldquoCharacter-ization of the bovine pregnancy-associated glycoprotein genefamilymdashanalysis of gene sequences regulatory regions withinthe promoter and expression of selected genesrdquo BMCGenomicsvol 10 article 185 pp 1ndash17 2009
[101] P Humblot S Camous J Martal et al ldquoDiagnosis of pregnancyby radioimmunoassay of a pregnancy-specific protein in theplasma of dairy cowsrdquo Theriogenology vol 30 no 2 pp 257ndash267 1988
[102] G H Kiracofe J M Wright R R Schalles C A Ruder SParish and R G Sasser ldquoPregnancy-specific protein B in serumof postpartum beef cowsrdquo Journal of Animal Science vol 71 no8 pp 2199ndash2205 1993
[103] A P Zoli L A Guilbault P Delahaut W B Ortiz and J-FBeckers ldquoRadioimmunoassay of a bovine pregnancy-associatedglycoprotein in serum its application for pregnancy diagnosisrdquoBiology of Reproduction vol 46 no 1 pp 83ndash92 1992
[104] R G Sasser C A Ruder K A Ivani J E Butler and W CHamilton ldquoDetection of pregnancy by radioimmunoassay of anovel pregnancy-specific protein in serum of cows and a profileof serum concentrations during gestationrdquo Biology of Reproduc-tion vol 35 no 4 pp 936ndash942 1986
10 The Scientific World Journal
[105] J A Green T E Parks M P Avalle et al ldquoThe establishmentof an ELISA for the detection of pregnancy-associated glyco-proteins (PAGs) in the serum of pregnant cows and heifersrdquoTheriogenology vol 63 no 5 pp 1481ndash1503 2005
[106] E Silva R A Sterry D Kolb et al ldquoAccuracy of a pregnancy-associated glycoprotein ELISA to determine pregnancy statusof lactating dairy cows twenty-seven days after timed artificialinseminationrdquo Journal of Dairy Science vol 90 no 10 pp 4612ndash4622 2007
[107] A Ayad N M Sousa J Sulon J L Hornick M Iguer-Ouada and J F Beckers ldquoCorrelation of five radioimmunoas-say systems for measurement of bovine plasma pregnancy-associated glycoprotein concentrations at early pregnancyperiodrdquo Research in Veterinary Science vol 86 no 3 pp 377ndash382 2009
[108] N M de Sousa M Zongo W Pitala et al ldquoPregnancy-associated glycoprotein concentrations during pregnancy andthe postpartum period in Azawak Zebu cattlerdquoTheriogenologyvol 59 no 5-6 pp 1131ndash1142 2003
[109] A Karen S Darwish A Ramoun et al ldquoAccuracy of ultra-sonography and pregnancy-associated glycoprotein test forpregnancy diagnosis in buffaloesrdquoTheriogenology vol 68 no 8pp 1150ndash1155 2007
[110] N M Sousa A Ayad J F Beckers and Z Gajewski ldquoPreg-nancy-associated glycoproteins (PAG) as pregnancy markers inthe ruminantsrdquo Journal of Physiology and Pharmacology vol 57no 8 pp 153ndash171 2006
[111] P R Graves andTA J Haystead ldquoMolecular biologistrsquos guide toproteomicsrdquo Microbiology and Molecular Biology Reviews vol66 no 1 pp 39ndash63 2002
[112] J D Lippolis and T A Reinhardt ldquoCentennial paper pro-teomics in animal sciencerdquo Journal of Animal Science vol 86no 9 pp 2430ndash2441 2008
[113] D A Colantonio and D W Chan ldquoThe clinical application ofproteomicsrdquo Clinica Chimica Acta vol 357 no 2 pp 151ndash1582005
[114] F di Girolamo E Boschetti M C M Chung F Guadagniand P G Righetti ldquolsquoProteomineeringrsquo or not The debate onbiomarker discovery in sera continuesrdquo Journal of Proteomicsvol 74 no 5 pp 589ndash594 2011
[115] D I Jin H R Lee H R KimH J Lee J T Yoon andC S ParkldquoProteomics analysis of pregnancy-specific serum proteins inbovinerdquoReproduction Fertility andDevelopment vol 18 no 1-2p 183 2005
[116] R Han H R Kim K Naruse et al ldquoIdentification of bovinepregnancy-specific milk proteins using proteomicsrdquo Biology ofReproduction vol 78 p 143 2008
[117] A K Balhara Proteomic analysis of pregnancy-specific serumproteins in buffalo [PhD thesis] LLRUVAS Hisar India 2012
[118] A Busch S Michel C Hoppe D Driesch U Claussen and Fvon Eggeling ldquoProteome analysis of maternal serum samplesfor trisomy 21 pregnancies using ProteinChip arrays and bioin-formaticsrdquo Journal of Histochemistry and Cytochemistry vol 53no 3 pp 341ndash343 2005
[119] P A Benn ldquoAdvances in prenatal screening for Down syn-drome I General principles and second trimester testingrdquoClinica Chimica Acta vol 323 no 1-2 pp 1ndash16 2002
Table 1 Important events during the early embryonic period
Days of pregnancy Event
Days 0-1 Fertilization single-cell embryo (zygote) inoviduct
Day 2 Early cleavages in the oviduct (up to 8 cellstages) activation of embryonic genome
Days 3-4 Embryo enters the uterus
Days 5-6 16ndash32 cell zona-enclosed embryoprogressing into compact morula stage
Days 7-8 Formation of a blastocoele withdifferentiation of embryonic cells
Days 9-10 Blastocyst expansion and hatching from thezona pellucid
Days 11ndash15 Blastocyst elongation from a tubular to afilamentous structure
Days 14ndash19 Maternal recognition of pregnancyDays 19-20 Implantation beginsDay 21 Caruncles-cotyledons appearDays 22ndash41 Implantation progressesDay 42 Implantation completedCompiled from available information (Morris and Diskin 2008 [3] Hafez1993 [4])
In this review we have described common pregnancydiagnosis methods available for dairy animals assessingthe usefulness of the modern technologies in detectingnovel pregnancy markers and designing future strategies forresearch in this area
2 Pregnancy Detection Methods
21 Direct Method
211 Per-Rectal Palpation Cowie [5] first described transrec-tal palpation of the uterus as a method for pregnancy diagno-sis in cattle which makes it the oldest and most widely prac-tisedmethod for early pregnancy diagnosis in large dairy ani-mals even today Traditionally to confirm pregnancy at aboutday 30 of gestation onwards the practitioners have relied onthe palpation of the amniotic vesicle [6] and slipping of thechorioallantoic membranes between the thumb and forefin-ger [7] In buffaloes too palpation per rectum is a simple eco-nomic and the most widely practised method for pregnancydiagnosis however this method is only accurate from day 45of pregnancy [8]Though per-rectal palpation is the cheapestpregnancy diagnosis method several studies have suggestedthat examining pregnant cows early in gestation by tran-srectal palpation increases the risk of iatrogenic embryonicmortality [9]
212 Ultrasonography By per-rectal palpation an expert canaccurately diagnose an animal pregnant only after day 35 ofgestation but the application of ultrasonography has madediagnosis possible as early as day 28 after insemination [9] oreven earlier [10]The first visible changes appearing by day 21after breeding when fetal heartbeat can be visualized also
helped confirm a viable pregnancy [11] though it is not aroutinely assessed parameter for pregnancy diagnosis Tran-srectal ultrasonography has the added advantage of providingadditional information on ovarian structures identificationof twins and determination of fetal viability age and sex[10 12] Transrectal ultrasonographymade a thorough exam-ination of the reproductive health of the animal possible andtherefore it has now become an established research tool tostudy bovine reproductive biology in cattle [12] and buffaloes[10] Ultrasound is a minimally invasive accurate and effi-cient technique for early pregnancy diagnosis [13 14] andmayminimize the rare incidence of palpation-induced abortions
Most studies on the utility of transrectal ultrasonographyfor pregnancy diagnosis have been conducted in cattle butlately it has found utility in buffalo cows as well In buffaloestransrectal ultrasonography is most commonly used to deter-mine pregnancy fetal age and sex as well as ovarian activity[15] In early 1990s various workers started using transrectalultrasonography in buffaloes with visualization of the embry-onic vesicle and embryo proper in pregnant buffalo cowsbetween 19 and 22 days after AI [16] In a field study on 260buffaloes between 30 and 45 days afterAI sensitivity of detec-tion of pregnancy was observed to be 979 [17] Howeverunpublished data from researchers at the Central Institute forResearch on Buffaloes Hisar India suggest the accuracy forselecting pregnant buffaloes at day 21 afterAI to be about 50which increases to almost 100 by day 30 These findingssupport other findings in cattle which claim that transrectalultrasonography for pregnancy diagnosis between days 21and 25 after breeding has sensitivity and specificity of 448and 823 respectively which further increase to 977 and877 respectively when conducted between 26 and 33 daysafter AI [18] Similarly Nation et al [19] documented that thesensitivity and specificity of pregnancy diagnosis in lactatingdairy cows based on ultrasonographic detection of uterinefluid as well as embryonic membranes from 28 to 35 daysafter AI were 96 and 97 respectively Direct observationof a fetus with ultrasonography was found more accuratethan assays for the presence of pregnancy-specific proteins inplasma but resulted in more false negative diagnoses [20]
Per-rectal palpation and transrectal ultrasonography aredirect and accurate methods for pregnancy diagnosis Bothrequire a great deal of skill and experience Veterinary-gradeultrasound machines equipped with a rectal transducer areexpensive in developing countries and therefore the highinitial cost of this technology partly limits its practical imple-mentation [12]
22 Indirect Method
221 Progesterone Shemesh et al [21] proposed that thedifference in peripheral plasma progesterone levels betweenpregnant and nonpregnant cows 19 days after inseminationcan form the basis for a very early pregnancy test Laing andHeap [22] first documented this in milk to diagnose cows inearly pregnancy Measurement of progesterone is an indirectmethod for pregnancy diagnosis in many livestock speciesincluding cattle buffaloes sheep and goats
The Scientific World Journal 3
Table 2 Progesterone levels in different sample types in bovine species
Cow Milk(i) 0 or 1(ii) 9 or 10(iii) 21 or 22(iv) 27 or 28
15111120125
121033068
Zaied et al 1979 [23]
Cow Milk 18 gt8 mdash Simersky et al 2007 [24]
Buffalo Milk plasma 21ndash35 each 1601 and 361 respectively 041ndash267 Kamboj and Prakash 1993[25]
Buffalo Plasma 0 13 01 and 36 respectively 06 (18th day of cycle) Batra et al 1979 [26]Buffalo Plasma 21 or 22 10 lt07 Perera et al 1980 [27]Cow Milk 20 gt11 lt8 Pennington et al 1985 [28]
Buffalo Milk 18ndash22 2483 289 Singh and Puthiyandy 1980[29]
Cow Faeces 18ndash24 gt50 as compared tononpregnant mdash Isobe et al 2005 [30]
Conception extends the life of the corpus luteum (CL) bypreventing the luteolytic mechanism from being triggeredthus prolonging and maintaining its functional characteris-tics ensuring continuedhigh progesterone levels [31] Proges-terone maintains the uterine endometrium in a state whichsupports embryonic development implantation and foe-toplacental development Progesterone concentrations varywith the stage of the estrous cycle which makes it one of themost commonly studied reproductive hormones in bovineruminants for pregnancy detection and ovarian activity [32]
Studies in the bovine estrous cycle indicate that the milkor serum progesterone concentrations reach a maximumvalue 13-14 days after estrus and if the animal is pregnantthese continue to remain elevated up to day 21 after fertil-ization [33] and beyond These high levels of progesteronein serum or milk between days 18 and 24 after inseminationform the basis of establishment of pregnancy in cattle [34 35]Interferon-120591 exerts its antiluteolytic effect by inhibiting theendometrial expression of oxytocin receptors through whichoxytocin stimulates pulsatile PGF
2120572release [36] Although
low progesterone concentrations at 18 to 24 days after breed-ing can accurately predict nonpregnancy high progesteroneconcentrations during this period are not the specific indica-tors of pregnancy due to variations among cows in durationof the estrous cycle as well as the incidence of early or lateembryonic mortality The advantages of progesterone assayfor pregnancy diagnosis include noninvasive collection ofmilk sample and the feasibility to conduct the test on thefarm using commercial cow-side milk progesterone test kits[28 37ndash39] though the sensitivity gets compromised to someextent with these assay kits Table 2 describes the work indifferent labs on the level of progesterone in pregnant andnonpregnant bovines
In buffalo cows it is quite evident that the progesteronelevels inmilk are four to five times higher than those in bloodplasma [25 26] Just like cattle buffaloes too can be accu-rately diagnosed as nonpregnant by determination of plasmaprogesterone concentrations 21 days after insemination [27]
Amajor constraint in using progesterone assay for pregnancydiagnosis is its use only in cases where AI or breeding datesare knownrecorded and not randomly in the herdNeverthe-less progesterone analysis remains themost common clinicaluse of any of the reproductive hormones
222 Estrone Sulphate Estrone sulphate is a conjugatedsteroid product of estrone present predominantly in thebovine placentomes [40] and it is themajor estrone present inthe fetal (allantoic and amniotic) fluids and maternal periph-eral plasma of cows with measurable quantities detectable byday 52 onwards till the end of gestation [41] Its concentra-tions increase from day 60 and plateau around day 150 afterinsemination [42] However reliable pregnancy detection ispossible only after day 100 of gestation and therefore this testcan only detect late pregnancy [43] Concentration of estronesulphate in the maternal body fluids is a useful indicator forthe placental functions especially those related to embryonicgrowth [44] In zebu and crossbred cattle and Murrahbuffaloes Prakash and Madan [45] reported below detectionlevels (lt50 pgmL) of estrone sulphate during the first twomonths followed by sharp increase in the fourth month andvalues stabilized after reaching the highest levels in the sixthmonth of pregnancy Levels of estrone sulphate in differentmaternal body fluids namely milk and blood plasma canbe utilised as the criteria for confirming pregnancy by after110 day insemination in bovine species [46] Estrone sulphateconcentrations have also been frequently correlated to fetalnumbers [44] as these are higher when the number of devel-oping foetuses ismore than one Yet estrone sulphate is not anideal pregnancy biomarker as the plasma and milk profilesare influenced by many other factors such as genetic makeupweight parity status and environment [46]
223 Conceptus and Placenta Secreted Products The veryfact that pregnancy brings about numerous physiologicalchanges in the female body through secretionaltered secre-tion of various biomolecules which often are proteins or their
4 The Scientific World Journal
metabolites supports research endeavours aimed at identify-ing novel proteins as the candidate molecules for pregnancydetection Human chorionic gonadotropin (hCG) discov-ered for the first time by Aschheim and Zondek [47] in theurine of pregnant women in 1927 is perhaps the best exampleof a placental protein hormone used for pregnancy diagnosisWith the advancement of biotechnological tools hCG basedpregnancy diagnosis has become the simplest cheapestand most commonly practised test for humans to diagnosepregnancy as early as 8ndash10 days after conception [48]Homol-ogous to the human protein only higher primates produce achorionic gonadotropin (CG) for maintaining luteal activityduring early pregnancy while ruminants produce type Iinterferon as an antiluteolytic factor during this period [49]
224 Early Conception Factor (ECF) Early pregnancy fac-tor (EPF also known as early conception factormdashECF)mdasha1084 kDa protein [50] is present in the sera of pregnantmammalian females detectable within 6 to 24 hours of fer-tilization [51] and disappearing within 24 to 48 after death orremoval of the embryo [52] EPF is present in the serum up totwo-thirds of the gestation [53] EPF remains the earliestserum benchmark for positive fertilization and hence suc-cessful conceptionThis novel pregnancy-specific protein hashigh immunosuppressive ability which is demonstrated byrosette inhibition test a bioassay first demonstrated in preg-nant mice [54] Laleh et al [55] demonstrated significant dif-ferences in rosette inhibition titres of pregnant and open cowswith values being 8ndash10 and 3ndash5 respectively
EPF is reported to be present in the pregnant sera ofmost mammalian species including humans [56 57] mice[51] sheep [58 59] cows [60ndash62] pigs [63] mares [64]and some wild animals [65] In buffalo pregnancy Chander[66] demonstrated decreased E-rosette formation but failedto demonstrate the presence of a rosette inhibiting factor (RIFwhich probably would have been EPF) in the serum Anti-bodies raised against a cow serum glycoprotein were used todetect EPF [67] leading to development of a lab methodwhich has been commercialized in the USA as Early Con-ception Factor (ECF) test (Concepto Diagnostics KnoxvilleTN) claiming detection within 48 hours Extensive study onthe effectiveness of the commercial ECF test for diagnosingnonpregnancy revealed a high degree of nonreliability of thetest wherein only 444 and 556of the confirmednonpreg-nant heifers were identified correctly by serum ECF analysisat days 1 to 3 and days 7 to 9 after AI respectively [61] Similarconclusions were drawn by [60] and [62] Although EPF issecreted in early pregnancy it is not strictly pregnancy spe-cific because of its secretion from nonplacental sources suchas tumors and transformed cell lines [50] which makes itan erroneous pregnancy detection method EPF belongs to afamily of heat shock proteins though detected extracellularlyand having immunosuppressive and growth factor properties[68]These properties are crucial to avoid rejection of an anti-genically alien embryo and support its development There-fore with the advent ofmodern biosciences there is hope thatthese changes could be identified and used as diagnostics forvery early detection of pregnancy However the practicability
of such an early test may still remain low due to high inci-dence of losses during the first 15 days of conception [2]
225 Interferon-120591 (IFN-120591) Moor and Rowson [69] thepioneers of sheep embryo transfer transferred embryos ondays 12 13 and 14 to unmated ewes and suggested interactionsbetween the embryo and uterus that influence the lutealfunction and result in establishment of pregnancy Godkinet al [70] purified ovine trophoblastic protein-1 (oTP-1) anearly secretory protein of the sheep blastocyst from in vitrocultured days 14ndash16 conceptusesThey revealed that oTP1 actson the maternal endometrium thereby eliciting maternalresponses which contribute to the maintenance of preg-nancy Imakawa et al [49] reported the primary amino acidsequence of oTP-1 to demonstrate that the protein is mostprobably an interferon-alpha Later research proved that thesecretions from the conceptus are in fact responsible for thematernal recognition of pregnancy [71 72]
Interferon-120591 a novel type I interferon [73] is first pro-duced by the conceptus between days 12-13 after inseminationin sheep and days 14ndash16 in cattle [74ndash76] High ovine IFN-120591levels are attained on days 12-13 before luteolysis could actu-ally be triggered [77] In ruminants IFN-120591 a 172 amino acidpolypeptide [73] blocks transcription of estrogen receptoralpha and oxytocin receptors in endometrial cells [78] whiledownregulating the expression of enzymes cyclooxygenase-2 and prostaglandin F synthase [79] thus preventing PGFrelease necessary for luteolysis
IFN-120591 acting within the uterine cavity [80] withextremely low levels in extrauterine tissues and peripheralcirculation prevents direct use of IFN-120591 as an early pregnancydiagnosis molecule [81] Rapid advancement of moleculartechniques in the last two decades has opened new avenuesfor exploring this unique molecule as a pregnancy markerfor ruminants through studies on IFN-120591 stimulated genes(ISG) namely interferon-stimulated protein 15 kDa (Isg15)myxovirus resistance 2 (MX2) and 21015840-51015840 oligoadenylate syn-thetase (OAS1) in peripheral blood leukocytes during earlypregnancy [82 83] Microarray analysis further indicatedthatmany genes including IFN-120591 stimulated are upregulatedduring early pregnancy [84ndash86] Green et al [84] have how-ever shown that the differential expression of such genes isinfluenced by the parity of the animal being more definite inheifers as compared tomultiparous animals All these experi-ments have suggested IFN-120591 stimulated genes to be potentialpregnancy detection biomarkers still there is no field leveltest available based on these markers
226 Pregnancy Associated Glycoproteins (PAGs) Reloca-tion of the extra embryonic trophoblastic cell layers to theendometrium [87] between days 20 to 28 and secretions fromthe conceptus lead to successful implantation and continua-tion of pregnancy in ruminant species [88] The pregnancyassociated glycoproteins (PAGs) are secretory products fromthe mono- and binucleated trophoblastic cells in bovine pla-centomes [89] Among these glycoproteins Butler et al [90]
The Scientific World Journal 5
detected two pregnancy-specific proteins in the sera of preg-nant cows a 65ndash70 kDa and a 47ndash53 kDa protein at pI 46ndash48 and 40ndash44 respectively Of these the former showed animmune reaction similar to that of 120572
1-fetoprotein while the
latter showed no reactivity with known proteins and it wasgiven the name ldquoprotein Brdquo or the ldquopregnancy-specific proteinBrdquo (PSPB) in bovines Further purification and characteriza-tion of several isoforms from bovine foetal cotyledons foundthat protein B is actually a 67 kDa PAG [91] Biochemicaland functional investigations established these proteins to beenzymatically inactive members of the aspartic proteinasesuperfamily having homology to pepsin chymosin cathep-sins D and enzyme renin [92 93] PAGs are a very complexgroup of proteins a fact proven by the already documented 22distinct cDNA libraries [94] The three most studied bovinePAGs PSPB PAG 67 kDa or bPAG-1 [93] and PSP60 [95]are isomers of the same protein having similar N-terminalsequences [96] Transcription of bPAG-2 and -11 mRNA isseen all through the pregnancy -4 -5 and -9mRNAs in earlypregnancy and bPAG-1 mRNA are detectable only after day45 [97] Interestingly bovine PAG-4 and bPAG-1 mRNA arehighly transcribed till day 250 of gestation but become indis-cernible at the end [97]The six N-glycosylation sites [98] areresponsible for the variations in molecular weight and half-life of PAGs [99] and is also the reason for expression ofdifferent PAGs during different stages of gestation [97 100]
Very recently it has been observed that placental defectscommonly seen during somatic nuclear transfers in cattleare complemented by unusually high plasma levels of PAGsprobably due to diminished clearance of these proteinsfollowing changes in the glycosylation patterns [94] PSPB isdetectable in the serumof pregnant cows over a long period ofgestation starting at about the fourthweek [101] of gestation toseveral weeks after parturition [102]High circulating levels ofthese proteins on days 80 to 100 postpartum restrict their useas a pregnancy diagnosis test except in heifers [102 103]
Sasser and coworkers [104] developed double antibodyradioimmunoassay for the serological detection of PSPB forpregnancy detection in cattle and found serum levels increas-ing progressively from 1 ngmL after day 30 to 9 ngmL35 ngmL and 150 ngmL after three six and nine months ofpregnancy respectivelyThe study claimed PSPB detection tobemore accurate than the traditional rectal palpationmethodfor pregnancy detection Green et al [105] developed a sand-wich ELISA using anti-PAG monoclonal antibodies whichwere able to detect PAG in all pregnant animals with concen-trations of 875 ngmL on day 28 the highest at 5889 ngmLduring the week of parturition and very low levels within4 weeks postpartum Silva et al [106] predicted 937 954and 962 accuracies for first second and third postpartumtimed artificial inseminations which were in agreement withother commonly practiced pregnancy detection methodsDifferent homologous (RIA-497) and heterologous radioim-munoassay systems (RIA-706 RIA-780 RIA-809 and RIA-Pool) developed for measurement of ruminant blood PAGconcentrations are highly correlated and can be used forpregnancy detection of 30ndash80 days [107] Radioimmunoassayof pregnant sera of zebu cattle established PAG concen-trations to be 60 ngmL 1960 ngmL 10956 ngmL and
3484 ngmL at 8 weeks at 35 weeks at term and at 2 weekspostpartum respectively a pattern similar to other breeds ofcattle [108] Results of PAG-RIA based pregnancy diagnosisin buffaloes have also been encouraging with a high degree ofaccuracy of diagnosis as early as day 31 with 100 sensitivityand 90ndash100 specificity [109] PAGs are used for develop-ment of bench-top pregnancy detectionmethods [110] whichare now commercially available as BioPRYN (BioTrackingRussia) DG29 (Genex Cooperative Inc USA) and IDEXX(IDEXX Laboratories Inc USA) BioPRYN blood test is themost extensively used PAG based kit for pregnancy detectionin ruminants By May 2010 already there were 2 millioncattle blood tests conducted for pregnancy detection (httpbiotrackingcomsiteprotectnetabouttimeline)
3 Current Research inBiomarkers for Pregnancy
It is presumed that the monitoring of sequential changes inblood proteome profile from the day of estrus to successfulconception and through progression of gestation can lead todiscovery of molecules which will perhaps be novel and spe-cific to the physiological stage of the animal In order to qual-ify as a marker for pregnancy the candidate molecule shouldbe able to accurately determine the pregnancy status as earlyas possible with minimum false positives or false negativesAdditionally the biological marker for pregnancy shouldhave the following desired characteristics
(i) specifically upregulated or downregulated duringpregnancy
(ii) least affected by nonanimal factors like feed environ-ment and drug interactions
(iii) having the ability to reflect age as well as viability ofthe conceptus
(iv) present in easily accessible body fluids like serummilk urine and vaginal discharge
(v) expressed over a considerable period of time to giveample time for diagnosis
(vi) revealing the result immediately
Proteomics [111] is large scale study of protein func-tions protein expression protein-protein interactions orposttranslational modifications in a particular cell tissue ororganism and is intended for identification of all the proteinspresent Proteomics provides an opportunity to simultane-ously analyse thousands of proteins in a single experimentfrom a complex mixture of proteins in various body fluids[112] This will help in identifying specific and sensitive bio-markers fulfilling the characteristics of uniqueness for a preg-nancy diagnosis molecule Main objectives of the proteomicsresearch include documentation of biomarkers altered pro-tein expression patterns indicative of pathophysiologicalchanges and therapeutically important drug targets [113]Easily reachable body fluids like blood serum and milk havea wide range of abundant proteins and these few proteinsmake up about 97 of the total serum and milk proteome
6 The Scientific World Journal
and thereby interfere in the proteomic analysis (reviewed by[113]) Conversely it is the low abundance proteins whichhave the highest prospect of being the novel biomarkersof changes in internal milieu of body To sort the problemof high abundance proteins two approaches are suggestedremoval of abundant proteins (usually by immunoaffinity)and concentration of the low abundancescarce proteins withsimultaneous removal of high abundance proteins techni-cally known as combinatorial peptide ligand libraries CPLL[114] Commercially available ProteoMiner kit fromMs Bio-Rad is CPLL based Both approaches however lead to loss ofa significant portion of the low abundance proteins along thehigh abundance proteins yet the later approach is preferred[114]
There is limited information on the bovine proteomein relation to pregnancy Jin et al [115] performed pro-teomics analysis using blood serum samples of pregnantand nonpregnant Holstein dairy cattle at 21 and 35 daysafter AI and reported composite profiles of key proteinsinvolved in early pregnancy and suggested the potential use ofidentified proteins to detect early pregnancy in bovinesTheseincluded nine pregnancy-specific spots in day 21 and day 35serum samples Pregnancy-specific proteins were identifiedas transferrin albumin IgG2a heavy chain constant regionand immunoglobulin gamma heavy chain variable regionFurther differential proteomic analysis of milk samples frompregnant and nonpregnant cows revealed 16 protein spots 14pregnancy specific and 2 spots downregulated in the pregnantmilk sample [116] Pregnancy-specific proteins were identi-fied as serum albumin precursor IgG1 heavy chain constantregion conglutinin precursor epithelial keratin 10 and kelch-like ECH-associated protein Though some identified spotswere abundant milk or serum proteins their molecularweights and pI values were different from main milk orserum proteinsThismay suggest that these proteins could bepregnancy-specific subunits or fragments of albumin and IgGor carrying differentially expressed small proteins whichmayultimately have potential for pregnancy detection
Encouragingly enough these studies need further inves-tigations for arriving at some sort of pregnancy detectionmethod Preliminary studies in buffalo cows on blood pro-teome too detected significant changes in many proteins in2DE gels [117] Important proteins found on MS analysis ofthese were synaptojanin-1 apolipoprotein A-1 apolipopro-tein B Keratin 10 and Von Willebrand factors which aredocumented to have a role in embryogenesis and earlypregnancy
Data generated out of sequential blood proteome analysisduring pregnancy can have several other applications as wellfor example studying fetal viability genetic disorders and soforth Trisomy 21 pregnancies can be detectedwith high accu-racy by maternal serum proteomic analysis in humans [118]In the absence of a single indicator for a particular life processa combination of the expression patterns of more than onesubstance can be used for a purposeful analysis as in thequadruple test where levels of four blood constituents (alpha-fetoprotein human chorionic gonadotropin unconjugatedoestriol and inhibin-A) have been used to predict theprobability of Downrsquos syndrome in babies [119] A pregnancy
detection test on the same lines as the human quadruple testcan be tried where instead of one we can consider proteinprofiles of more proteins This approach too will require athorough analysis of the bovine proteome before such a testwith high accuracy is available to the livestock owners
4 Conclusion
Early pregnancy diagnosis is an important aspect for optimiz-ing dairy production yet none of the present day methodsqualifies as an ideal diagnostic due to limitations of accuracylater stages of applicability and requirement for elaborateinstrumentation and laboratory setup This warrants furtherresearch on developing novel early pregnancy diagnosticsfor livestock species Currently available state-of-the-artinstrumentation and proteomics techniques instil hope forfinding moleculesmdashexclusively related to intricate maternalmetabolic alterations necessary to align with physiology ofearly embryonic development and its signalling for maternalrecognition of pregnancy and continued survival Thoughthese techniques are still in their infancy in animal scienceresearch they hold great promise to address a long-awaitedbreakthrough in pregnancy diagnosis in livestock
Acknowledgment
The authors acknowledge their thanks to the National Fundfor Basic Strategic and Frontier Application Research inAgriculture (NFBSFARA) Indian Council of AgriculturalResearch India for the funding support
References
[1] ldquoCommittee on Reproductive Nomenclature Recommenda-tions for standardizing bovine reproductive termsrdquoThe CornellVeterinarian vol 62 no 2 pp 216ndash237 1972
[2] N Ayalon ldquoA review of embryonic mortality in cattlerdquo Journalof Reproduction and Fertility vol 54 no 2 pp 483ndash493 1978
[3] D Morris and M Diskin ldquoEffect of progesterone on embryosurvivalrdquo Animal vol 2 no 8 pp 1112ndash1119 2008
[4] E S E Hafez Reproduction in Farm Animalsedition Lea andFebiger Philadelphia Pa USA 6th edition 1993
[5] T A Cowie Pregnancy Diagnosis Tests A Review Common-wealthAgricultural Bureaux Joint Publication no 13 Common-wealth Agricultural Bureaux Oxford UK 1948
[6] WWisnicky and L E Cassida ldquoAmanual method for diagnosisof pregnancy in cattlerdquo Journal of the American VeterinaryMedical Association vol 113 p 451 1948
[7] R Zemjanis Diagnostic and Therapeutic Techniques in AnimalReproduction Williams and Wilkins Baltimore Md USA 2ndedition 1970
[8] G H Arthur D E Noakes H Pearson and T J ParkinsonldquoReproduction in the buffalordquo in Veterinary Reproduction andObstetrics WB Saunders London UK 1966
[9] O J Franco M Drost M-J Thatcher V M Shille and W WThatcher ldquoFetal survival in the cow after pregnancy diagnosisby palpation per rectumrdquoTheriogenology vol 27 no 4 pp 631ndash644 1987
The Scientific World Journal 7
[10] R K Sharma J K Singh S K Phulia S Khanna and I SinghldquoFetal sex determination with ultrasonography in buffaloesrdquoIndian Veterinary Journal vol 88 no 10 pp 105ndash107 2011
[11] S Curran R A Pierson and O J Ginther ldquoUltrasonographicappearance of the bovine conceptus from days 20 through 60rdquoJournal of the American VeterinaryMedical Association vol 189no 10 pp 1295ndash1302 1986
[12] PM Fricke ldquoScanning the futuremdashultrasonography as a repro-ductive management tool for dairy cattlerdquo Journal of Dairy Sci-ence vol 85 no 8 pp 1918ndash1926 2002
[13] L G Paisley W D Mickelsen and O L Frost ldquoA survey of theincidence of prenatal mortality in cattle following pregnancydiagnosis by rectal palpationrdquoTheriogenology vol 9 no 6 pp481ndash491 1978
[14] D Vaillancourt C J Bierschwal D Ogwu et al ldquoCorrelationbetween pregnancy diagnosis bymembrane slip and embryonicmortalityrdquo Journal of the American Veterinary Medical Associa-tion vol 175 no 5 pp 466ndash468 1979
[15] A Ali and S Fahmy ldquoUltrasonographic fetometry and deter-mination of fetal sex in buffaloes (Bubalus bubalis)rdquo AnimalReproduction Science vol 106 no 1-2 pp 90ndash99 2008
[16] C H Pawshe K B C Appa Rao and S M Totey ldquoUltrasono-graphic imaging to monitor early pregnancy and embryonicdevelopment in the buffalo (Bubalus bubalis)rdquo Theriogenologyvol 41 no 3 pp 697ndash709 1994
[17] M R Bhosreker and IMHangarge ldquoUltrasonography for earlypregnancy diagnosis in buffaloesrdquo Indian Journal of AnimalReproduction vol 21 pp 143ndash144 2000
[18] M C Pieterse O Szenci A H Willemse C S A Bajcsy S JDieleman andM AM Taverne ldquoEarly pregnancy diagnosis incattle bymeans of linear-array real-time ultrasound scanning ofthe uterus and a qualitative and quantitative milk progesteronetestrdquoTheriogenology vol 33 no 3 pp 697ndash707 1990
[19] D P Nation J Malmo G M Davis and K L MacmillanldquoAccuracy of bovine pregnancy detection using transrectalultrasonography at 28 to 35 days after inseminationrdquoAustralianVeterinary Journal vol 81 no 1-2 pp 63ndash65 2003
[20] O Szenci J F Beckers P Humblot et al ldquoComparison of ultra-sonography bovine pregnancy-specific protein B and bovinepregnancy-associated glycoprotein 1 tests for pregnancy detec-tion in dairy cowsrdquo Theriogenology vol 50 no 1 pp 77ndash881998
[21] M Shemesh N Ayalon and H R Lindner ldquoEarly pregnancydiagnosis based upon plasma progesterone levels in the cow andewerdquo Journal of Animal Science vol 36 no 4 pp 726ndash729 1973
[22] J A Laing and R B Heap ldquoThe concentration of progesteronein themilk of cows during the reproductive cyclerdquo British Veter-inary Journal vol 127 no 8 pp 19ndash22 1971
[23] A A Zaied C J Bierschwal R G Elmore R S Youngquist AJ Sharp and H A Garverick ldquoConcentrations of progesteroneinmilk as amonitor of early pregnancy diagnosis in dairy cowsrdquoTheriogenology vol 12 no 1 pp 3ndash11 1979
[24] R Simersky J Swaczynova D A Morris M Franek and MStrnad ldquoDevelopment of an ELISA-based kit for the on-farmdetermination of progesterone in milkrdquo Veterinarni Medicinavol 52 no 1 pp 19ndash28 2007
[25] M Kamboj and B S Prakash ldquoRelationship of progesterone inplasma and whole milk of buffaloes during cyclicity and earlypregnancyrdquo Tropical Animal Health and Production vol 25 no3 pp 185ndash192 1993
[26] S K Batra R C Arora N K Bachlaus andR S Pandey ldquoBloodand milk progesterone in pregnant and nonpregnant buffalordquoJournal of Dairy Science vol 62 no 9 pp 1390ndash1393 1979
[27] BM Perera N Pathiraja S A AbeywardenaM XMotha andH Abeygunawardena ldquoEarly pregnancy diagnosis in buffaloesfrom plasma progesterone concentrationrdquo Veterinary Recordvol 106 no 5 pp 104ndash106 1980
[28] J A Pennington L H Schultz and W F Hoffman ldquoCompari-son of pregnancy diagnosis by milk progesterone on day 21 andday 24 postbreeding field study in dairy cattlerdquo Journal of DairyScience vol 68 no 10 pp 2740ndash2745 1985
[29] A Singh and R Puthiyandy ldquoEstimation of progesterone inbuffalomilk and its application to pregnancy diagnosisrdquo Journalof Reproduction and Fertility vol 59 no 1 pp 89ndash93 1980
[30] N Isobe M Akita T Nakao H Yamashiro and H KubotaldquoPregnancy diagnosis based on the fecal progesterone concen-tration in beef and dairy heifers and beef cowsrdquo Animal Repro-duction Science vol 90 no 3-4 pp 211ndash218 2005
[31] T E Spencer N H Ing T L Ott et al ldquoIntrauterine injectionof ovine interferon-120591 alters oestrogen receptor and oxytocinreceptor expression in the endometrium of cyclic ewesrdquo Journalof Molecular Endocrinology vol 15 no 2 pp 203ndash220 1995
[32] J J Kaneko J W Harvey andM L Bruss Clinical Biochemistryof Domestic Animals Academic Press AmsterdamTheNether-lands 2008
[33] T J Parkinson A Turvey and L J Jenner ldquoA morphometricanalysis of the corpus luteumof the cowduring the estrous cycleand early pregnancyrdquo Theriogenology vol 41 no 5 pp 1115ndash1126 1994
[34] R G Sasser and C A Ruder ldquoDetection of early pregnancy indomestic ruminantsrdquo Journal of Reproduction and Fertility Sup-plement vol 34 pp 261ndash271 1987
[35] M Shemesh N Ayalon and H R Lindner ldquoEarly pregnancydiagnosis based upon plasma progesterone levels in the cow andewerdquo Journal of Animal Science vol 36 no 4 pp 726ndash729 1973
[36] E Wolf G J Arnold S Bauersachs et al ldquoEmbryo-maternalcommunication in bovinemdashstrategies for deciphering a com-plex cross-talkrdquo Reproduction in Domestic Animals vol 38 no4 pp 276ndash289 2003
[37] E W Gowan R J Etches C Bryden and G J King ldquoFactorsaffecting accuracy of pregnancy diagnosis in cattlerdquo Journal ofDairy Science vol 65 no 7 pp 1294ndash1302 1982
[38] R L Nebel W D Whittier B G Cassell and J H Britt ldquoCom-parison of on-farm laboratory milk progesterone assays foridentifying errors in detection of estrus and diagnosis of preg-nancyrdquo Journal of Dairy Science vol 70 no 7 pp 1471ndash14761987
[39] T H Wimpy C F Chang V L Estergreen and J K HillersldquoMilk progesterone enzyme immunoassay modifications anda field trial for pregnancy detection in dairy cowsrdquo Journal ofDairy Science vol 69 no 4 pp 1115ndash1121 1986
[40] R M Eley WWThatcher and F W Bazer ldquoLuteolytic effect ofoestrone sulphate on cyclic beef heifersrdquo Journal of Reproductionand Fertility vol 55 no 1 pp 191ndash193 1979
[41] H A Robertson and G J King ldquoConjugated and unconjugatedoestrogens in fetal and maternal fluids of the cow throughoutpregnancyrdquo Journal of Reproduction and Fertility vol 55 no 2pp 463ndash470 1979
[42] R B Heap and M Hamon ldquoOestrone sulphate in milk as anindicator of a viable conceptus in cowsrdquo British Veterinary Jour-nal vol 135 no 4 pp 355ndash363 1979
8 The Scientific World Journal
[43] M Hamon I R Fleet R J Holdsworth and R B Heap ldquoThetime of detection of oestrone sulphate in milk and the diagnosisof pregnancy in cowsrdquo British Veterinary Journal vol 137 no 1pp 71ndash77 1981
[44] M Hirako T Takahashi and I Domeki ldquoPeripheral changes inestrone sulfate concentration during the first trimester of ges-tation in cattle comparison with unconjugated estrogens andrelationship to fetal numberrdquo Theriogenology vol 57 no 7 pp1939ndash1947 2002
[45] B S Prakash and M L Madan ldquoInfluence of gestation onoestrone sulphate concentration in milk of zebu and crossbredcows and murrah buffaloesrdquo Tropical Animal Health and Pro-duction vol 25 no 2 pp 94ndash100 1993
[46] F Lobago M Bekana H Gustafsson et al ldquoSerum profilesof pregnancy-associated glycoprotein oestrone sulphate andprogesterone during gestation and some factors influencing theprofiles in ethiopian borana and crossbred cattlerdquo Reproductionin Domestic Animals vol 44 no 4 pp 685ndash692 2009
[47] S Aschheim and B Zondek ldquoHypophysenvorderlappenhor-mon undOvarialhormon imHarn von SchwangerenrdquoKlinischeWochenschrift vol 6 no 28 p 1322 1927
[48] J R Marshall C B Hammond G T Ross A Jacobson PRayford andW D Odell ldquoPlasma and urinary chorionic gona-dotropin during early human pregnancyrdquo Obstetrics and Gyne-cology vol 32 no 6 pp 760ndash764 1968
[49] K Imakawa R V Anthony M Kazemi K R Marotti H GPolites and R M Roberts ldquoInterferon-like sequence of ovinetrophoblast protein secreted by embryonic trophectodermrdquoNature vol 330 no 6146 pp 377ndash379 1987
[50] A C Cavanagh ldquoIdentification of early pregnancy factor aschaperonin 10 implications for understanding its rolerdquo Reviewsof Reproduction vol 1 no 1 pp 28ndash32 1996
[51] H Morton V Hegh and G J A Clunie ldquoStudies of the rosetteinhibition test in pregnant mice evidence of immunosuppres-sionrdquo Proceedings of the Royal Society B vol 193 no 1113 pp413ndash419 1976
[52] H Morton B E Rolfe and A C Cavanagh ldquoOvum factor andearly pregnancy factorrdquo Current Topics in Developmental Biol-ogy vol 23 pp 73ndash92 1987
[53] H Morton A C Cavanagh S Athanasas-Platsis K A QuinnandB E Rolfe ldquoEarly pregnancy factor has immunosuppressiveand growth factor propertiesrdquo Reproduction Fertility andDevelopment vol 4 no 4 pp 411ndash422 1992
[54] H Morton V Hegh and G J A Clunie ldquoImmunosuppressiondetected in pregnantmice by rosette inhibition testrdquoNature vol249 no 5456 pp 459ndash460 1974
[55] V G Laleh R G Laleh N Pirany and M M Ahrabi ldquoMea-surement of EPF for detection of cow pregnancy using rosetteinhibition testrdquoTheriogenology vol 70 no 1 pp 105ndash107 2008
[56] H Qin and Z Q Zheng ldquoDetection of early pregnancy factorin human serardquo American Journal of Reproductive Immunologyand Microbiology vol 13 no 1 pp 15ndash18 1987
[57] Y C Smart T K Roberts I S Fraser A W Cripps and R LClancy ldquoValidation of the rosette inhibition test for the detec-tion of early pregnancy in womenrdquo Fertility and Sterility vol 37no 6 pp 779ndash785 1982
[58] HMorton G J A Clunie and F D Shaw ldquoA test for early preg-nancy in sheeprdquo Research in Veterinary Science vol 26 no 2pp 261ndash262 1979
[59] S Wilson R McCarthy and F Clarke ldquoIn search of early preg-nancy factor isolation of active polypeptides from pregnant
ewesrsquo serardquo Journal of Reproductive Immunology vol 5 no 5 pp275ndash286 1983
[60] M C Cordoba R Sartori and P M Fricke ldquoAssessment of acommercially available early conception factor (ECF) test fordetermining pregnancy status of dairy cattlerdquo Journal of DairyScience vol 84 no 8 pp 1884ndash1889 2001
[61] B GandyW Tucker P Ryan et al ldquoEvaluation of the early con-ception factor (ECFŮ) test for the detection of nonpregnancyin dairy cattlerdquoTheriogenology vol 56 no 4 pp 637ndash647 2001
[62] C S Whisnant L A Pagels and M G Daves ldquoCase studyeffectiveness of a commercial early conception factor test for usein cattlerdquo Professional Animal Scientist vol 17 pp 51ndash53 2001
[63] A S Grewal A L C Wallace Y S Pan et al ldquoEvaluation of arosette inhibition test for pregnancy diagnosis in pigsrdquo Journalof Reproductive Immunology vol 7 no 2 pp 129ndash138 1985
[64] K Ohnuma I T O Kazuei Y-I Miyake J Takahashi andY Yasuda ldquoDetection of early pregnancy factor (EPF) in mareserardquo Journal of Reproduction and Development vol 42 no 1pp 23ndash28 1996
[65] Y P Cruz L Selwood H Morton and A C CavanaghldquoSignificance of serum early pregnancy factor concentrationsduring pregnancy and embryonic development in Sminthopsismacroura (Spencer) (Marsupialia Dasyuridae)rdquo Reproductionvol 121 no 6 pp 933ndash939 2001
[66] S Chander Studies on certain immunological aspects of preg-nancy diagnosis in buffaloes [PhD thesis] Chaudhary CharanSingh Haryana Agricultural University Hisar India 1983
[67] W R Threlfall ldquoImmunosuppressive early pregnancy factor(ISEPF) determination for pregnancy diagnosis in dairy cowsrdquoTheriogenology vol 41 p 31 1994
[68] H Morton ldquoEarly pregnancy factor an extracellular chaper-onin 10 homologuerdquo Immunology andCell Biology vol 76 no 6pp 483ndash496 1998
[69] R M Moor and L E A Rowson ldquoInfluence of the embryo anduterus on luteal function in the sheeprdquoNature vol 201 no 4918pp 522ndash523 1964
[70] J D Godkin FW Bazer and RM Roberts ldquoOvine trophoblastprotein 1 an early secreted blastocyst protein binds specifi-cally to uterine endometrium and affects protein synthesisrdquoEndocrinology vol 114 no 1 pp 120ndash130 1984
[71] FW Bazer ldquoMediators of maternal recognition of pregnancy inmammalsrdquo Proceedings of the Society for Experimental Biologyand Medicine vol 199 no 4 pp 373ndash384 1992
[72] R M Roberts D W Leaman and J C Cross ldquoRole of interfer-ons in maternal recognition of pregnancy in ruminantsrdquo Pro-ceedings of the Society for Experimental Biology and Medicinevol 200 no 1 pp 7ndash18 1992
[73] R M Roberts ldquoInterferon-tau a type 1 interferon involvedin maternal recognition of pregnancyrdquo Cytokine and GrowthFactor Reviews vol 18 no 5-6 pp 403ndash408 2007
[74] G E Mann G E Lamming R S Robinson and D C WathesldquoThe regulation of interferon-tau production and uterine hor-mone receptors during early pregnancyrdquo Journal of Reproduc-tion and Fertility Supplement vol 54 pp 317ndash328 1999
[75] R M Roberts A D Ealy A P Alexenko C-S Han and TEzashi ldquoTrophoblast interferonsrdquo Placenta vol 20 no 4 pp259ndash264 1999
[76] WWThatcher M DMeyer and G Danet-Desnoyers ldquoMater-nal recognition of pregnancyrdquo Journal of Reproduction andFertility Supplement vol 49 pp 15ndash28 1995
The Scientific World Journal 9
[77] F W Bazer T E Spencer and T L Ott ldquoInterferon tau anovel pregnancy recognition signalrdquoAmerican Journal of Repro-ductive Immunology vol 37 no 6 pp 412ndash420 1997
[78] T E Spencer and F W Bazer ldquoOvine interferon tau suppressestranscription of the estrogen receptor and oxytocin receptorgenes in the ovine endometriumrdquo Endocrinology vol 137 no3 pp 1144ndash1147 1996
[79] C W Xiao B D Murphy J Sirois and A K GoffldquoDown-regulation of oxytocin-induced cyclooxygenase-2 andprostaglandin F synthase expression by interferon-120591 in bovineendometrial cellsrdquo Biology of Reproduction vol 60 no 3 pp656ndash663 1999
[80] T E Spencer N H Ing T L Ott et al ldquoIntrauterine injectionof ovine interferon-120591 alters oestrogen receptor and oxytocinreceptor expression in the endometrium of cyclic ewesrdquo Journalof Molecular Endocrinology vol 15 no 2 pp 203ndash220 1995
[81] J F Oliveira L E Henkes R L Ashley et al ldquoExpression ofinterferon (IFN)-stimulated genes in extrauterine tissues duringearly pregnancy in sheep is the consequence of endocrine IFN-120591 release from the uterine veinrdquo Endocrinology vol 149 no 3pp 1252ndash1259 2008
[82] C A Gifford K Racicot D S Clark et al ldquoRegulation ofinterferon-stimulated genes in peripheral blood leukocytes inpregnant and bred nonpregnant dairy cowsrdquo Journal of DairyScience vol 90 no 1 pp 274ndash280 2007
[83] H Han K J Austin L A Rempel and T R Hansen ldquoLowblood ISG15 mRNA and progesterone levels are predictive ofnon-pregnant dairy cowsrdquo Journal of Endocrinology vol 191 no2 pp 505ndash512 2006
[84] J C Green C S Okamura S E Poock and M C Lucy ldquoMea-surement of interferon-tau (IFN-120591) stimulated gene expressionin blood leukocytes for pregnancy diagnosis within 18-20d afterinsemination in dairy cattlerdquo Animal Reproduction Science vol121 no 1-2 pp 24ndash33 2010
[85] N Forde F Carter T E Spencer et al ldquoConceptus-inducedchanges in the endometrial transcriptome how soon does thecow know she is pregnantrdquo Biology of Reproduction vol 85 no1 pp 144ndash156 2011
[86] K Kizaki A Shichijo-Kizaki T Furusawa T Takahashi MHosoe andKHashizume ldquoDifferential neutrophil gene expres-sion in early bovine pregnancyrdquoReproductive Biology and Endo-crinology vol 11 article 6 2013
[87] F B P Wooding and D C Wathes ldquoBinucleate cell migrationin the bovine placentomerdquo Journal of Reproduction and Fertilityvol 59 no 2 pp 425ndash430 1980
[88] F W Bazer W W Thatcher P J Hansen M A Mirando TL Ott and C Plante ldquoPhysiological mechanisms of pregnancyrecognition in ruminantsrdquo Journal of Reproduction and FertilitySupplement vol 43 pp 39ndash47 1991
[89] A P Zoli P Demez J F Beckers M Reznik and A BeckersldquoLight and electron microscopic immunolocalization of bovinepregnancy-associated glycoprotein in the bovine placentomerdquoBiology of Reproduction vol 46 no 4 pp 623ndash629 1992
[90] J E Butler W C Hamilton R G Sasser C A Ruder G MHass andR JWilliams ldquoDetection and partial characterizationof two bovine pregnancy-specific proteinsrdquoBiology of Reproduc-tion vol 26 no 5 pp 925ndash933 1982
[91] A P Zoli J-F Beckers P Wouters-Ballman J Closset PFalmagne and F Ectors ldquoPurification and characterization of abovine pregnancy-associated glycoproteinrdquo Biology of Repro-duction vol 45 no 1 pp 1ndash10 1991
[92] S Xie B G Low R J Nagel et al ldquoIdentification of the majorpregnancy-specific antigens of cattle and sheep as inactivemembers of the aspartic proteinase familyrdquo Proceedings of theNational Academy of Sciences of theUnited States of America vol88 no 22 pp 10247ndash10251 1991
[93] S Xie B G Low R J Nagel J-F Beckers and R M RobertsldquoA novel glycoprotein of the aspartic proteinase gene familyexpressed in bovine placental trophectodermrdquoBiology of Repro-duction vol 51 no 6 pp 1145ndash1153 1994
[94] F Constanta S Camousa P Chavatte-Palmera et al ldquoAlteredsecretion of pregnancy-associated glycoproteins during gesta-tion in bovine somatic clonesrdquoTheriogenology vol 76 pp 1006ndash1021 2011
[95] M M Mialon S Camous G Renand J Martal and FMenissier ldquoPeripheral concentrations of a 60-kDa pregnancyserum protein during gestation and after calving and in rela-tionship to embryonic mortality in cattlerdquo Reproduction Nutri-tion Development vol 33 no 3 pp 269ndash282 1993
[96] JMartal N Chene S Camous et al ldquoRecent developments andpotentialities for reducing embryo mortality in ruminants therole of IFN-120591 and other cytokines in early pregnancyrdquo Repro-duction Fertility and Development vol 9 no 3 pp 355ndash3801997
[97] J A Green S Xie X Quan et al ldquoPregnancy-associated bovineand ovine glycoproteins exhibit spatially and temporally dis-tinct expression patterns during pregnancyrdquo Biology of Repro-duction vol 62 no 6 pp 1624ndash1631 2000
[98] S Xie J Green J B Bixby et al ldquoThe diversity and evolutionaryrelationships of the pregnancy-associated glycoproteins anaspartic proteinase subfamily consisting of many trophoblast-expressed genesrdquo Proceedings of the National Academy of Sci-ences of the United States of America vol 94 no 24 pp 12809ndash12816 1997
[99] K Klisch A Boos M Friedrich et al ldquoThe glycosylationof pregnancy-associated glycoproteins and prolactin-relatedprotein-I in bovine binucleate trophoblast giant cells changesbefore parturitionrdquo Reproduction vol 132 no 5 pp 791ndash7982006
[100] B P V L Telugu A M Walker and J A Green ldquoCharacter-ization of the bovine pregnancy-associated glycoprotein genefamilymdashanalysis of gene sequences regulatory regions withinthe promoter and expression of selected genesrdquo BMCGenomicsvol 10 article 185 pp 1ndash17 2009
[101] P Humblot S Camous J Martal et al ldquoDiagnosis of pregnancyby radioimmunoassay of a pregnancy-specific protein in theplasma of dairy cowsrdquo Theriogenology vol 30 no 2 pp 257ndash267 1988
[102] G H Kiracofe J M Wright R R Schalles C A Ruder SParish and R G Sasser ldquoPregnancy-specific protein B in serumof postpartum beef cowsrdquo Journal of Animal Science vol 71 no8 pp 2199ndash2205 1993
[103] A P Zoli L A Guilbault P Delahaut W B Ortiz and J-FBeckers ldquoRadioimmunoassay of a bovine pregnancy-associatedglycoprotein in serum its application for pregnancy diagnosisrdquoBiology of Reproduction vol 46 no 1 pp 83ndash92 1992
[104] R G Sasser C A Ruder K A Ivani J E Butler and W CHamilton ldquoDetection of pregnancy by radioimmunoassay of anovel pregnancy-specific protein in serum of cows and a profileof serum concentrations during gestationrdquo Biology of Reproduc-tion vol 35 no 4 pp 936ndash942 1986
10 The Scientific World Journal
[105] J A Green T E Parks M P Avalle et al ldquoThe establishmentof an ELISA for the detection of pregnancy-associated glyco-proteins (PAGs) in the serum of pregnant cows and heifersrdquoTheriogenology vol 63 no 5 pp 1481ndash1503 2005
[106] E Silva R A Sterry D Kolb et al ldquoAccuracy of a pregnancy-associated glycoprotein ELISA to determine pregnancy statusof lactating dairy cows twenty-seven days after timed artificialinseminationrdquo Journal of Dairy Science vol 90 no 10 pp 4612ndash4622 2007
[107] A Ayad N M Sousa J Sulon J L Hornick M Iguer-Ouada and J F Beckers ldquoCorrelation of five radioimmunoas-say systems for measurement of bovine plasma pregnancy-associated glycoprotein concentrations at early pregnancyperiodrdquo Research in Veterinary Science vol 86 no 3 pp 377ndash382 2009
[108] N M de Sousa M Zongo W Pitala et al ldquoPregnancy-associated glycoprotein concentrations during pregnancy andthe postpartum period in Azawak Zebu cattlerdquoTheriogenologyvol 59 no 5-6 pp 1131ndash1142 2003
[109] A Karen S Darwish A Ramoun et al ldquoAccuracy of ultra-sonography and pregnancy-associated glycoprotein test forpregnancy diagnosis in buffaloesrdquoTheriogenology vol 68 no 8pp 1150ndash1155 2007
[110] N M Sousa A Ayad J F Beckers and Z Gajewski ldquoPreg-nancy-associated glycoproteins (PAG) as pregnancy markers inthe ruminantsrdquo Journal of Physiology and Pharmacology vol 57no 8 pp 153ndash171 2006
[111] P R Graves andTA J Haystead ldquoMolecular biologistrsquos guide toproteomicsrdquo Microbiology and Molecular Biology Reviews vol66 no 1 pp 39ndash63 2002
[112] J D Lippolis and T A Reinhardt ldquoCentennial paper pro-teomics in animal sciencerdquo Journal of Animal Science vol 86no 9 pp 2430ndash2441 2008
[113] D A Colantonio and D W Chan ldquoThe clinical application ofproteomicsrdquo Clinica Chimica Acta vol 357 no 2 pp 151ndash1582005
[114] F di Girolamo E Boschetti M C M Chung F Guadagniand P G Righetti ldquolsquoProteomineeringrsquo or not The debate onbiomarker discovery in sera continuesrdquo Journal of Proteomicsvol 74 no 5 pp 589ndash594 2011
[115] D I Jin H R Lee H R KimH J Lee J T Yoon andC S ParkldquoProteomics analysis of pregnancy-specific serum proteins inbovinerdquoReproduction Fertility andDevelopment vol 18 no 1-2p 183 2005
[116] R Han H R Kim K Naruse et al ldquoIdentification of bovinepregnancy-specific milk proteins using proteomicsrdquo Biology ofReproduction vol 78 p 143 2008
[117] A K Balhara Proteomic analysis of pregnancy-specific serumproteins in buffalo [PhD thesis] LLRUVAS Hisar India 2012
[118] A Busch S Michel C Hoppe D Driesch U Claussen and Fvon Eggeling ldquoProteome analysis of maternal serum samplesfor trisomy 21 pregnancies using ProteinChip arrays and bioin-formaticsrdquo Journal of Histochemistry and Cytochemistry vol 53no 3 pp 341ndash343 2005
[119] P A Benn ldquoAdvances in prenatal screening for Down syn-drome I General principles and second trimester testingrdquoClinica Chimica Acta vol 323 no 1-2 pp 1ndash16 2002
Cow Milk(i) 0 or 1(ii) 9 or 10(iii) 21 or 22(iv) 27 or 28
15111120125
121033068
Zaied et al 1979 [23]
Cow Milk 18 gt8 mdash Simersky et al 2007 [24]
Buffalo Milk plasma 21ndash35 each 1601 and 361 respectively 041ndash267 Kamboj and Prakash 1993[25]
Buffalo Plasma 0 13 01 and 36 respectively 06 (18th day of cycle) Batra et al 1979 [26]Buffalo Plasma 21 or 22 10 lt07 Perera et al 1980 [27]Cow Milk 20 gt11 lt8 Pennington et al 1985 [28]
Buffalo Milk 18ndash22 2483 289 Singh and Puthiyandy 1980[29]
Cow Faeces 18ndash24 gt50 as compared tononpregnant mdash Isobe et al 2005 [30]
Conception extends the life of the corpus luteum (CL) bypreventing the luteolytic mechanism from being triggeredthus prolonging and maintaining its functional characteris-tics ensuring continuedhigh progesterone levels [31] Proges-terone maintains the uterine endometrium in a state whichsupports embryonic development implantation and foe-toplacental development Progesterone concentrations varywith the stage of the estrous cycle which makes it one of themost commonly studied reproductive hormones in bovineruminants for pregnancy detection and ovarian activity [32]
Studies in the bovine estrous cycle indicate that the milkor serum progesterone concentrations reach a maximumvalue 13-14 days after estrus and if the animal is pregnantthese continue to remain elevated up to day 21 after fertil-ization [33] and beyond These high levels of progesteronein serum or milk between days 18 and 24 after inseminationform the basis of establishment of pregnancy in cattle [34 35]Interferon-120591 exerts its antiluteolytic effect by inhibiting theendometrial expression of oxytocin receptors through whichoxytocin stimulates pulsatile PGF
2120572release [36] Although
low progesterone concentrations at 18 to 24 days after breed-ing can accurately predict nonpregnancy high progesteroneconcentrations during this period are not the specific indica-tors of pregnancy due to variations among cows in durationof the estrous cycle as well as the incidence of early or lateembryonic mortality The advantages of progesterone assayfor pregnancy diagnosis include noninvasive collection ofmilk sample and the feasibility to conduct the test on thefarm using commercial cow-side milk progesterone test kits[28 37ndash39] though the sensitivity gets compromised to someextent with these assay kits Table 2 describes the work indifferent labs on the level of progesterone in pregnant andnonpregnant bovines
In buffalo cows it is quite evident that the progesteronelevels inmilk are four to five times higher than those in bloodplasma [25 26] Just like cattle buffaloes too can be accu-rately diagnosed as nonpregnant by determination of plasmaprogesterone concentrations 21 days after insemination [27]
Amajor constraint in using progesterone assay for pregnancydiagnosis is its use only in cases where AI or breeding datesare knownrecorded and not randomly in the herdNeverthe-less progesterone analysis remains themost common clinicaluse of any of the reproductive hormones
222 Estrone Sulphate Estrone sulphate is a conjugatedsteroid product of estrone present predominantly in thebovine placentomes [40] and it is themajor estrone present inthe fetal (allantoic and amniotic) fluids and maternal periph-eral plasma of cows with measurable quantities detectable byday 52 onwards till the end of gestation [41] Its concentra-tions increase from day 60 and plateau around day 150 afterinsemination [42] However reliable pregnancy detection ispossible only after day 100 of gestation and therefore this testcan only detect late pregnancy [43] Concentration of estronesulphate in the maternal body fluids is a useful indicator forthe placental functions especially those related to embryonicgrowth [44] In zebu and crossbred cattle and Murrahbuffaloes Prakash and Madan [45] reported below detectionlevels (lt50 pgmL) of estrone sulphate during the first twomonths followed by sharp increase in the fourth month andvalues stabilized after reaching the highest levels in the sixthmonth of pregnancy Levels of estrone sulphate in differentmaternal body fluids namely milk and blood plasma canbe utilised as the criteria for confirming pregnancy by after110 day insemination in bovine species [46] Estrone sulphateconcentrations have also been frequently correlated to fetalnumbers [44] as these are higher when the number of devel-oping foetuses ismore than one Yet estrone sulphate is not anideal pregnancy biomarker as the plasma and milk profilesare influenced by many other factors such as genetic makeupweight parity status and environment [46]
223 Conceptus and Placenta Secreted Products The veryfact that pregnancy brings about numerous physiologicalchanges in the female body through secretionaltered secre-tion of various biomolecules which often are proteins or their
4 The Scientific World Journal
metabolites supports research endeavours aimed at identify-ing novel proteins as the candidate molecules for pregnancydetection Human chorionic gonadotropin (hCG) discov-ered for the first time by Aschheim and Zondek [47] in theurine of pregnant women in 1927 is perhaps the best exampleof a placental protein hormone used for pregnancy diagnosisWith the advancement of biotechnological tools hCG basedpregnancy diagnosis has become the simplest cheapestand most commonly practised test for humans to diagnosepregnancy as early as 8ndash10 days after conception [48]Homol-ogous to the human protein only higher primates produce achorionic gonadotropin (CG) for maintaining luteal activityduring early pregnancy while ruminants produce type Iinterferon as an antiluteolytic factor during this period [49]
224 Early Conception Factor (ECF) Early pregnancy fac-tor (EPF also known as early conception factormdashECF)mdasha1084 kDa protein [50] is present in the sera of pregnantmammalian females detectable within 6 to 24 hours of fer-tilization [51] and disappearing within 24 to 48 after death orremoval of the embryo [52] EPF is present in the serum up totwo-thirds of the gestation [53] EPF remains the earliestserum benchmark for positive fertilization and hence suc-cessful conceptionThis novel pregnancy-specific protein hashigh immunosuppressive ability which is demonstrated byrosette inhibition test a bioassay first demonstrated in preg-nant mice [54] Laleh et al [55] demonstrated significant dif-ferences in rosette inhibition titres of pregnant and open cowswith values being 8ndash10 and 3ndash5 respectively
EPF is reported to be present in the pregnant sera ofmost mammalian species including humans [56 57] mice[51] sheep [58 59] cows [60ndash62] pigs [63] mares [64]and some wild animals [65] In buffalo pregnancy Chander[66] demonstrated decreased E-rosette formation but failedto demonstrate the presence of a rosette inhibiting factor (RIFwhich probably would have been EPF) in the serum Anti-bodies raised against a cow serum glycoprotein were used todetect EPF [67] leading to development of a lab methodwhich has been commercialized in the USA as Early Con-ception Factor (ECF) test (Concepto Diagnostics KnoxvilleTN) claiming detection within 48 hours Extensive study onthe effectiveness of the commercial ECF test for diagnosingnonpregnancy revealed a high degree of nonreliability of thetest wherein only 444 and 556of the confirmednonpreg-nant heifers were identified correctly by serum ECF analysisat days 1 to 3 and days 7 to 9 after AI respectively [61] Similarconclusions were drawn by [60] and [62] Although EPF issecreted in early pregnancy it is not strictly pregnancy spe-cific because of its secretion from nonplacental sources suchas tumors and transformed cell lines [50] which makes itan erroneous pregnancy detection method EPF belongs to afamily of heat shock proteins though detected extracellularlyand having immunosuppressive and growth factor properties[68]These properties are crucial to avoid rejection of an anti-genically alien embryo and support its development There-fore with the advent ofmodern biosciences there is hope thatthese changes could be identified and used as diagnostics forvery early detection of pregnancy However the practicability
of such an early test may still remain low due to high inci-dence of losses during the first 15 days of conception [2]
225 Interferon-120591 (IFN-120591) Moor and Rowson [69] thepioneers of sheep embryo transfer transferred embryos ondays 12 13 and 14 to unmated ewes and suggested interactionsbetween the embryo and uterus that influence the lutealfunction and result in establishment of pregnancy Godkinet al [70] purified ovine trophoblastic protein-1 (oTP-1) anearly secretory protein of the sheep blastocyst from in vitrocultured days 14ndash16 conceptusesThey revealed that oTP1 actson the maternal endometrium thereby eliciting maternalresponses which contribute to the maintenance of preg-nancy Imakawa et al [49] reported the primary amino acidsequence of oTP-1 to demonstrate that the protein is mostprobably an interferon-alpha Later research proved that thesecretions from the conceptus are in fact responsible for thematernal recognition of pregnancy [71 72]
Interferon-120591 a novel type I interferon [73] is first pro-duced by the conceptus between days 12-13 after inseminationin sheep and days 14ndash16 in cattle [74ndash76] High ovine IFN-120591levels are attained on days 12-13 before luteolysis could actu-ally be triggered [77] In ruminants IFN-120591 a 172 amino acidpolypeptide [73] blocks transcription of estrogen receptoralpha and oxytocin receptors in endometrial cells [78] whiledownregulating the expression of enzymes cyclooxygenase-2 and prostaglandin F synthase [79] thus preventing PGFrelease necessary for luteolysis
IFN-120591 acting within the uterine cavity [80] withextremely low levels in extrauterine tissues and peripheralcirculation prevents direct use of IFN-120591 as an early pregnancydiagnosis molecule [81] Rapid advancement of moleculartechniques in the last two decades has opened new avenuesfor exploring this unique molecule as a pregnancy markerfor ruminants through studies on IFN-120591 stimulated genes(ISG) namely interferon-stimulated protein 15 kDa (Isg15)myxovirus resistance 2 (MX2) and 21015840-51015840 oligoadenylate syn-thetase (OAS1) in peripheral blood leukocytes during earlypregnancy [82 83] Microarray analysis further indicatedthatmany genes including IFN-120591 stimulated are upregulatedduring early pregnancy [84ndash86] Green et al [84] have how-ever shown that the differential expression of such genes isinfluenced by the parity of the animal being more definite inheifers as compared tomultiparous animals All these experi-ments have suggested IFN-120591 stimulated genes to be potentialpregnancy detection biomarkers still there is no field leveltest available based on these markers
226 Pregnancy Associated Glycoproteins (PAGs) Reloca-tion of the extra embryonic trophoblastic cell layers to theendometrium [87] between days 20 to 28 and secretions fromthe conceptus lead to successful implantation and continua-tion of pregnancy in ruminant species [88] The pregnancyassociated glycoproteins (PAGs) are secretory products fromthe mono- and binucleated trophoblastic cells in bovine pla-centomes [89] Among these glycoproteins Butler et al [90]
The Scientific World Journal 5
detected two pregnancy-specific proteins in the sera of preg-nant cows a 65ndash70 kDa and a 47ndash53 kDa protein at pI 46ndash48 and 40ndash44 respectively Of these the former showed animmune reaction similar to that of 120572
1-fetoprotein while the
latter showed no reactivity with known proteins and it wasgiven the name ldquoprotein Brdquo or the ldquopregnancy-specific proteinBrdquo (PSPB) in bovines Further purification and characteriza-tion of several isoforms from bovine foetal cotyledons foundthat protein B is actually a 67 kDa PAG [91] Biochemicaland functional investigations established these proteins to beenzymatically inactive members of the aspartic proteinasesuperfamily having homology to pepsin chymosin cathep-sins D and enzyme renin [92 93] PAGs are a very complexgroup of proteins a fact proven by the already documented 22distinct cDNA libraries [94] The three most studied bovinePAGs PSPB PAG 67 kDa or bPAG-1 [93] and PSP60 [95]are isomers of the same protein having similar N-terminalsequences [96] Transcription of bPAG-2 and -11 mRNA isseen all through the pregnancy -4 -5 and -9mRNAs in earlypregnancy and bPAG-1 mRNA are detectable only after day45 [97] Interestingly bovine PAG-4 and bPAG-1 mRNA arehighly transcribed till day 250 of gestation but become indis-cernible at the end [97]The six N-glycosylation sites [98] areresponsible for the variations in molecular weight and half-life of PAGs [99] and is also the reason for expression ofdifferent PAGs during different stages of gestation [97 100]
Very recently it has been observed that placental defectscommonly seen during somatic nuclear transfers in cattleare complemented by unusually high plasma levels of PAGsprobably due to diminished clearance of these proteinsfollowing changes in the glycosylation patterns [94] PSPB isdetectable in the serumof pregnant cows over a long period ofgestation starting at about the fourthweek [101] of gestation toseveral weeks after parturition [102]High circulating levels ofthese proteins on days 80 to 100 postpartum restrict their useas a pregnancy diagnosis test except in heifers [102 103]
Sasser and coworkers [104] developed double antibodyradioimmunoassay for the serological detection of PSPB forpregnancy detection in cattle and found serum levels increas-ing progressively from 1 ngmL after day 30 to 9 ngmL35 ngmL and 150 ngmL after three six and nine months ofpregnancy respectivelyThe study claimed PSPB detection tobemore accurate than the traditional rectal palpationmethodfor pregnancy detection Green et al [105] developed a sand-wich ELISA using anti-PAG monoclonal antibodies whichwere able to detect PAG in all pregnant animals with concen-trations of 875 ngmL on day 28 the highest at 5889 ngmLduring the week of parturition and very low levels within4 weeks postpartum Silva et al [106] predicted 937 954and 962 accuracies for first second and third postpartumtimed artificial inseminations which were in agreement withother commonly practiced pregnancy detection methodsDifferent homologous (RIA-497) and heterologous radioim-munoassay systems (RIA-706 RIA-780 RIA-809 and RIA-Pool) developed for measurement of ruminant blood PAGconcentrations are highly correlated and can be used forpregnancy detection of 30ndash80 days [107] Radioimmunoassayof pregnant sera of zebu cattle established PAG concen-trations to be 60 ngmL 1960 ngmL 10956 ngmL and
3484 ngmL at 8 weeks at 35 weeks at term and at 2 weekspostpartum respectively a pattern similar to other breeds ofcattle [108] Results of PAG-RIA based pregnancy diagnosisin buffaloes have also been encouraging with a high degree ofaccuracy of diagnosis as early as day 31 with 100 sensitivityand 90ndash100 specificity [109] PAGs are used for develop-ment of bench-top pregnancy detectionmethods [110] whichare now commercially available as BioPRYN (BioTrackingRussia) DG29 (Genex Cooperative Inc USA) and IDEXX(IDEXX Laboratories Inc USA) BioPRYN blood test is themost extensively used PAG based kit for pregnancy detectionin ruminants By May 2010 already there were 2 millioncattle blood tests conducted for pregnancy detection (httpbiotrackingcomsiteprotectnetabouttimeline)
3 Current Research inBiomarkers for Pregnancy
It is presumed that the monitoring of sequential changes inblood proteome profile from the day of estrus to successfulconception and through progression of gestation can lead todiscovery of molecules which will perhaps be novel and spe-cific to the physiological stage of the animal In order to qual-ify as a marker for pregnancy the candidate molecule shouldbe able to accurately determine the pregnancy status as earlyas possible with minimum false positives or false negativesAdditionally the biological marker for pregnancy shouldhave the following desired characteristics
(i) specifically upregulated or downregulated duringpregnancy
(ii) least affected by nonanimal factors like feed environ-ment and drug interactions
(iii) having the ability to reflect age as well as viability ofthe conceptus
(iv) present in easily accessible body fluids like serummilk urine and vaginal discharge
(v) expressed over a considerable period of time to giveample time for diagnosis
(vi) revealing the result immediately
Proteomics [111] is large scale study of protein func-tions protein expression protein-protein interactions orposttranslational modifications in a particular cell tissue ororganism and is intended for identification of all the proteinspresent Proteomics provides an opportunity to simultane-ously analyse thousands of proteins in a single experimentfrom a complex mixture of proteins in various body fluids[112] This will help in identifying specific and sensitive bio-markers fulfilling the characteristics of uniqueness for a preg-nancy diagnosis molecule Main objectives of the proteomicsresearch include documentation of biomarkers altered pro-tein expression patterns indicative of pathophysiologicalchanges and therapeutically important drug targets [113]Easily reachable body fluids like blood serum and milk havea wide range of abundant proteins and these few proteinsmake up about 97 of the total serum and milk proteome
6 The Scientific World Journal
and thereby interfere in the proteomic analysis (reviewed by[113]) Conversely it is the low abundance proteins whichhave the highest prospect of being the novel biomarkersof changes in internal milieu of body To sort the problemof high abundance proteins two approaches are suggestedremoval of abundant proteins (usually by immunoaffinity)and concentration of the low abundancescarce proteins withsimultaneous removal of high abundance proteins techni-cally known as combinatorial peptide ligand libraries CPLL[114] Commercially available ProteoMiner kit fromMs Bio-Rad is CPLL based Both approaches however lead to loss ofa significant portion of the low abundance proteins along thehigh abundance proteins yet the later approach is preferred[114]
There is limited information on the bovine proteomein relation to pregnancy Jin et al [115] performed pro-teomics analysis using blood serum samples of pregnantand nonpregnant Holstein dairy cattle at 21 and 35 daysafter AI and reported composite profiles of key proteinsinvolved in early pregnancy and suggested the potential use ofidentified proteins to detect early pregnancy in bovinesTheseincluded nine pregnancy-specific spots in day 21 and day 35serum samples Pregnancy-specific proteins were identifiedas transferrin albumin IgG2a heavy chain constant regionand immunoglobulin gamma heavy chain variable regionFurther differential proteomic analysis of milk samples frompregnant and nonpregnant cows revealed 16 protein spots 14pregnancy specific and 2 spots downregulated in the pregnantmilk sample [116] Pregnancy-specific proteins were identi-fied as serum albumin precursor IgG1 heavy chain constantregion conglutinin precursor epithelial keratin 10 and kelch-like ECH-associated protein Though some identified spotswere abundant milk or serum proteins their molecularweights and pI values were different from main milk orserum proteinsThismay suggest that these proteins could bepregnancy-specific subunits or fragments of albumin and IgGor carrying differentially expressed small proteins whichmayultimately have potential for pregnancy detection
Encouragingly enough these studies need further inves-tigations for arriving at some sort of pregnancy detectionmethod Preliminary studies in buffalo cows on blood pro-teome too detected significant changes in many proteins in2DE gels [117] Important proteins found on MS analysis ofthese were synaptojanin-1 apolipoprotein A-1 apolipopro-tein B Keratin 10 and Von Willebrand factors which aredocumented to have a role in embryogenesis and earlypregnancy
Data generated out of sequential blood proteome analysisduring pregnancy can have several other applications as wellfor example studying fetal viability genetic disorders and soforth Trisomy 21 pregnancies can be detectedwith high accu-racy by maternal serum proteomic analysis in humans [118]In the absence of a single indicator for a particular life processa combination of the expression patterns of more than onesubstance can be used for a purposeful analysis as in thequadruple test where levels of four blood constituents (alpha-fetoprotein human chorionic gonadotropin unconjugatedoestriol and inhibin-A) have been used to predict theprobability of Downrsquos syndrome in babies [119] A pregnancy
detection test on the same lines as the human quadruple testcan be tried where instead of one we can consider proteinprofiles of more proteins This approach too will require athorough analysis of the bovine proteome before such a testwith high accuracy is available to the livestock owners
4 Conclusion
Early pregnancy diagnosis is an important aspect for optimiz-ing dairy production yet none of the present day methodsqualifies as an ideal diagnostic due to limitations of accuracylater stages of applicability and requirement for elaborateinstrumentation and laboratory setup This warrants furtherresearch on developing novel early pregnancy diagnosticsfor livestock species Currently available state-of-the-artinstrumentation and proteomics techniques instil hope forfinding moleculesmdashexclusively related to intricate maternalmetabolic alterations necessary to align with physiology ofearly embryonic development and its signalling for maternalrecognition of pregnancy and continued survival Thoughthese techniques are still in their infancy in animal scienceresearch they hold great promise to address a long-awaitedbreakthrough in pregnancy diagnosis in livestock
Acknowledgment
The authors acknowledge their thanks to the National Fundfor Basic Strategic and Frontier Application Research inAgriculture (NFBSFARA) Indian Council of AgriculturalResearch India for the funding support
References
[1] ldquoCommittee on Reproductive Nomenclature Recommenda-tions for standardizing bovine reproductive termsrdquoThe CornellVeterinarian vol 62 no 2 pp 216ndash237 1972
[2] N Ayalon ldquoA review of embryonic mortality in cattlerdquo Journalof Reproduction and Fertility vol 54 no 2 pp 483ndash493 1978
[3] D Morris and M Diskin ldquoEffect of progesterone on embryosurvivalrdquo Animal vol 2 no 8 pp 1112ndash1119 2008
[4] E S E Hafez Reproduction in Farm Animalsedition Lea andFebiger Philadelphia Pa USA 6th edition 1993
[5] T A Cowie Pregnancy Diagnosis Tests A Review Common-wealthAgricultural Bureaux Joint Publication no 13 Common-wealth Agricultural Bureaux Oxford UK 1948
[6] WWisnicky and L E Cassida ldquoAmanual method for diagnosisof pregnancy in cattlerdquo Journal of the American VeterinaryMedical Association vol 113 p 451 1948
[7] R Zemjanis Diagnostic and Therapeutic Techniques in AnimalReproduction Williams and Wilkins Baltimore Md USA 2ndedition 1970
[8] G H Arthur D E Noakes H Pearson and T J ParkinsonldquoReproduction in the buffalordquo in Veterinary Reproduction andObstetrics WB Saunders London UK 1966
[9] O J Franco M Drost M-J Thatcher V M Shille and W WThatcher ldquoFetal survival in the cow after pregnancy diagnosisby palpation per rectumrdquoTheriogenology vol 27 no 4 pp 631ndash644 1987
The Scientific World Journal 7
[10] R K Sharma J K Singh S K Phulia S Khanna and I SinghldquoFetal sex determination with ultrasonography in buffaloesrdquoIndian Veterinary Journal vol 88 no 10 pp 105ndash107 2011
[11] S Curran R A Pierson and O J Ginther ldquoUltrasonographicappearance of the bovine conceptus from days 20 through 60rdquoJournal of the American VeterinaryMedical Association vol 189no 10 pp 1295ndash1302 1986
[12] PM Fricke ldquoScanning the futuremdashultrasonography as a repro-ductive management tool for dairy cattlerdquo Journal of Dairy Sci-ence vol 85 no 8 pp 1918ndash1926 2002
[13] L G Paisley W D Mickelsen and O L Frost ldquoA survey of theincidence of prenatal mortality in cattle following pregnancydiagnosis by rectal palpationrdquoTheriogenology vol 9 no 6 pp481ndash491 1978
[14] D Vaillancourt C J Bierschwal D Ogwu et al ldquoCorrelationbetween pregnancy diagnosis bymembrane slip and embryonicmortalityrdquo Journal of the American Veterinary Medical Associa-tion vol 175 no 5 pp 466ndash468 1979
[15] A Ali and S Fahmy ldquoUltrasonographic fetometry and deter-mination of fetal sex in buffaloes (Bubalus bubalis)rdquo AnimalReproduction Science vol 106 no 1-2 pp 90ndash99 2008
[16] C H Pawshe K B C Appa Rao and S M Totey ldquoUltrasono-graphic imaging to monitor early pregnancy and embryonicdevelopment in the buffalo (Bubalus bubalis)rdquo Theriogenologyvol 41 no 3 pp 697ndash709 1994
[17] M R Bhosreker and IMHangarge ldquoUltrasonography for earlypregnancy diagnosis in buffaloesrdquo Indian Journal of AnimalReproduction vol 21 pp 143ndash144 2000
[18] M C Pieterse O Szenci A H Willemse C S A Bajcsy S JDieleman andM AM Taverne ldquoEarly pregnancy diagnosis incattle bymeans of linear-array real-time ultrasound scanning ofthe uterus and a qualitative and quantitative milk progesteronetestrdquoTheriogenology vol 33 no 3 pp 697ndash707 1990
[19] D P Nation J Malmo G M Davis and K L MacmillanldquoAccuracy of bovine pregnancy detection using transrectalultrasonography at 28 to 35 days after inseminationrdquoAustralianVeterinary Journal vol 81 no 1-2 pp 63ndash65 2003
[20] O Szenci J F Beckers P Humblot et al ldquoComparison of ultra-sonography bovine pregnancy-specific protein B and bovinepregnancy-associated glycoprotein 1 tests for pregnancy detec-tion in dairy cowsrdquo Theriogenology vol 50 no 1 pp 77ndash881998
[21] M Shemesh N Ayalon and H R Lindner ldquoEarly pregnancydiagnosis based upon plasma progesterone levels in the cow andewerdquo Journal of Animal Science vol 36 no 4 pp 726ndash729 1973
[22] J A Laing and R B Heap ldquoThe concentration of progesteronein themilk of cows during the reproductive cyclerdquo British Veter-inary Journal vol 127 no 8 pp 19ndash22 1971
[23] A A Zaied C J Bierschwal R G Elmore R S Youngquist AJ Sharp and H A Garverick ldquoConcentrations of progesteroneinmilk as amonitor of early pregnancy diagnosis in dairy cowsrdquoTheriogenology vol 12 no 1 pp 3ndash11 1979
[24] R Simersky J Swaczynova D A Morris M Franek and MStrnad ldquoDevelopment of an ELISA-based kit for the on-farmdetermination of progesterone in milkrdquo Veterinarni Medicinavol 52 no 1 pp 19ndash28 2007
[25] M Kamboj and B S Prakash ldquoRelationship of progesterone inplasma and whole milk of buffaloes during cyclicity and earlypregnancyrdquo Tropical Animal Health and Production vol 25 no3 pp 185ndash192 1993
[26] S K Batra R C Arora N K Bachlaus andR S Pandey ldquoBloodand milk progesterone in pregnant and nonpregnant buffalordquoJournal of Dairy Science vol 62 no 9 pp 1390ndash1393 1979
[27] BM Perera N Pathiraja S A AbeywardenaM XMotha andH Abeygunawardena ldquoEarly pregnancy diagnosis in buffaloesfrom plasma progesterone concentrationrdquo Veterinary Recordvol 106 no 5 pp 104ndash106 1980
[28] J A Pennington L H Schultz and W F Hoffman ldquoCompari-son of pregnancy diagnosis by milk progesterone on day 21 andday 24 postbreeding field study in dairy cattlerdquo Journal of DairyScience vol 68 no 10 pp 2740ndash2745 1985
[29] A Singh and R Puthiyandy ldquoEstimation of progesterone inbuffalomilk and its application to pregnancy diagnosisrdquo Journalof Reproduction and Fertility vol 59 no 1 pp 89ndash93 1980
[30] N Isobe M Akita T Nakao H Yamashiro and H KubotaldquoPregnancy diagnosis based on the fecal progesterone concen-tration in beef and dairy heifers and beef cowsrdquo Animal Repro-duction Science vol 90 no 3-4 pp 211ndash218 2005
[31] T E Spencer N H Ing T L Ott et al ldquoIntrauterine injectionof ovine interferon-120591 alters oestrogen receptor and oxytocinreceptor expression in the endometrium of cyclic ewesrdquo Journalof Molecular Endocrinology vol 15 no 2 pp 203ndash220 1995
[32] J J Kaneko J W Harvey andM L Bruss Clinical Biochemistryof Domestic Animals Academic Press AmsterdamTheNether-lands 2008
[33] T J Parkinson A Turvey and L J Jenner ldquoA morphometricanalysis of the corpus luteumof the cowduring the estrous cycleand early pregnancyrdquo Theriogenology vol 41 no 5 pp 1115ndash1126 1994
[34] R G Sasser and C A Ruder ldquoDetection of early pregnancy indomestic ruminantsrdquo Journal of Reproduction and Fertility Sup-plement vol 34 pp 261ndash271 1987
[35] M Shemesh N Ayalon and H R Lindner ldquoEarly pregnancydiagnosis based upon plasma progesterone levels in the cow andewerdquo Journal of Animal Science vol 36 no 4 pp 726ndash729 1973
[36] E Wolf G J Arnold S Bauersachs et al ldquoEmbryo-maternalcommunication in bovinemdashstrategies for deciphering a com-plex cross-talkrdquo Reproduction in Domestic Animals vol 38 no4 pp 276ndash289 2003
[37] E W Gowan R J Etches C Bryden and G J King ldquoFactorsaffecting accuracy of pregnancy diagnosis in cattlerdquo Journal ofDairy Science vol 65 no 7 pp 1294ndash1302 1982
[38] R L Nebel W D Whittier B G Cassell and J H Britt ldquoCom-parison of on-farm laboratory milk progesterone assays foridentifying errors in detection of estrus and diagnosis of preg-nancyrdquo Journal of Dairy Science vol 70 no 7 pp 1471ndash14761987
[39] T H Wimpy C F Chang V L Estergreen and J K HillersldquoMilk progesterone enzyme immunoassay modifications anda field trial for pregnancy detection in dairy cowsrdquo Journal ofDairy Science vol 69 no 4 pp 1115ndash1121 1986
[40] R M Eley WWThatcher and F W Bazer ldquoLuteolytic effect ofoestrone sulphate on cyclic beef heifersrdquo Journal of Reproductionand Fertility vol 55 no 1 pp 191ndash193 1979
[41] H A Robertson and G J King ldquoConjugated and unconjugatedoestrogens in fetal and maternal fluids of the cow throughoutpregnancyrdquo Journal of Reproduction and Fertility vol 55 no 2pp 463ndash470 1979
[42] R B Heap and M Hamon ldquoOestrone sulphate in milk as anindicator of a viable conceptus in cowsrdquo British Veterinary Jour-nal vol 135 no 4 pp 355ndash363 1979
8 The Scientific World Journal
[43] M Hamon I R Fleet R J Holdsworth and R B Heap ldquoThetime of detection of oestrone sulphate in milk and the diagnosisof pregnancy in cowsrdquo British Veterinary Journal vol 137 no 1pp 71ndash77 1981
[44] M Hirako T Takahashi and I Domeki ldquoPeripheral changes inestrone sulfate concentration during the first trimester of ges-tation in cattle comparison with unconjugated estrogens andrelationship to fetal numberrdquo Theriogenology vol 57 no 7 pp1939ndash1947 2002
[45] B S Prakash and M L Madan ldquoInfluence of gestation onoestrone sulphate concentration in milk of zebu and crossbredcows and murrah buffaloesrdquo Tropical Animal Health and Pro-duction vol 25 no 2 pp 94ndash100 1993
[46] F Lobago M Bekana H Gustafsson et al ldquoSerum profilesof pregnancy-associated glycoprotein oestrone sulphate andprogesterone during gestation and some factors influencing theprofiles in ethiopian borana and crossbred cattlerdquo Reproductionin Domestic Animals vol 44 no 4 pp 685ndash692 2009
[47] S Aschheim and B Zondek ldquoHypophysenvorderlappenhor-mon undOvarialhormon imHarn von SchwangerenrdquoKlinischeWochenschrift vol 6 no 28 p 1322 1927
[48] J R Marshall C B Hammond G T Ross A Jacobson PRayford andW D Odell ldquoPlasma and urinary chorionic gona-dotropin during early human pregnancyrdquo Obstetrics and Gyne-cology vol 32 no 6 pp 760ndash764 1968
[49] K Imakawa R V Anthony M Kazemi K R Marotti H GPolites and R M Roberts ldquoInterferon-like sequence of ovinetrophoblast protein secreted by embryonic trophectodermrdquoNature vol 330 no 6146 pp 377ndash379 1987
[50] A C Cavanagh ldquoIdentification of early pregnancy factor aschaperonin 10 implications for understanding its rolerdquo Reviewsof Reproduction vol 1 no 1 pp 28ndash32 1996
[51] H Morton V Hegh and G J A Clunie ldquoStudies of the rosetteinhibition test in pregnant mice evidence of immunosuppres-sionrdquo Proceedings of the Royal Society B vol 193 no 1113 pp413ndash419 1976
[52] H Morton B E Rolfe and A C Cavanagh ldquoOvum factor andearly pregnancy factorrdquo Current Topics in Developmental Biol-ogy vol 23 pp 73ndash92 1987
[53] H Morton A C Cavanagh S Athanasas-Platsis K A QuinnandB E Rolfe ldquoEarly pregnancy factor has immunosuppressiveand growth factor propertiesrdquo Reproduction Fertility andDevelopment vol 4 no 4 pp 411ndash422 1992
[54] H Morton V Hegh and G J A Clunie ldquoImmunosuppressiondetected in pregnantmice by rosette inhibition testrdquoNature vol249 no 5456 pp 459ndash460 1974
[55] V G Laleh R G Laleh N Pirany and M M Ahrabi ldquoMea-surement of EPF for detection of cow pregnancy using rosetteinhibition testrdquoTheriogenology vol 70 no 1 pp 105ndash107 2008
[56] H Qin and Z Q Zheng ldquoDetection of early pregnancy factorin human serardquo American Journal of Reproductive Immunologyand Microbiology vol 13 no 1 pp 15ndash18 1987
[57] Y C Smart T K Roberts I S Fraser A W Cripps and R LClancy ldquoValidation of the rosette inhibition test for the detec-tion of early pregnancy in womenrdquo Fertility and Sterility vol 37no 6 pp 779ndash785 1982
[58] HMorton G J A Clunie and F D Shaw ldquoA test for early preg-nancy in sheeprdquo Research in Veterinary Science vol 26 no 2pp 261ndash262 1979
[59] S Wilson R McCarthy and F Clarke ldquoIn search of early preg-nancy factor isolation of active polypeptides from pregnant
ewesrsquo serardquo Journal of Reproductive Immunology vol 5 no 5 pp275ndash286 1983
[60] M C Cordoba R Sartori and P M Fricke ldquoAssessment of acommercially available early conception factor (ECF) test fordetermining pregnancy status of dairy cattlerdquo Journal of DairyScience vol 84 no 8 pp 1884ndash1889 2001
[61] B GandyW Tucker P Ryan et al ldquoEvaluation of the early con-ception factor (ECFŮ) test for the detection of nonpregnancyin dairy cattlerdquoTheriogenology vol 56 no 4 pp 637ndash647 2001
[62] C S Whisnant L A Pagels and M G Daves ldquoCase studyeffectiveness of a commercial early conception factor test for usein cattlerdquo Professional Animal Scientist vol 17 pp 51ndash53 2001
[63] A S Grewal A L C Wallace Y S Pan et al ldquoEvaluation of arosette inhibition test for pregnancy diagnosis in pigsrdquo Journalof Reproductive Immunology vol 7 no 2 pp 129ndash138 1985
[64] K Ohnuma I T O Kazuei Y-I Miyake J Takahashi andY Yasuda ldquoDetection of early pregnancy factor (EPF) in mareserardquo Journal of Reproduction and Development vol 42 no 1pp 23ndash28 1996
[65] Y P Cruz L Selwood H Morton and A C CavanaghldquoSignificance of serum early pregnancy factor concentrationsduring pregnancy and embryonic development in Sminthopsismacroura (Spencer) (Marsupialia Dasyuridae)rdquo Reproductionvol 121 no 6 pp 933ndash939 2001
[66] S Chander Studies on certain immunological aspects of preg-nancy diagnosis in buffaloes [PhD thesis] Chaudhary CharanSingh Haryana Agricultural University Hisar India 1983
[67] W R Threlfall ldquoImmunosuppressive early pregnancy factor(ISEPF) determination for pregnancy diagnosis in dairy cowsrdquoTheriogenology vol 41 p 31 1994
[68] H Morton ldquoEarly pregnancy factor an extracellular chaper-onin 10 homologuerdquo Immunology andCell Biology vol 76 no 6pp 483ndash496 1998
[69] R M Moor and L E A Rowson ldquoInfluence of the embryo anduterus on luteal function in the sheeprdquoNature vol 201 no 4918pp 522ndash523 1964
[70] J D Godkin FW Bazer and RM Roberts ldquoOvine trophoblastprotein 1 an early secreted blastocyst protein binds specifi-cally to uterine endometrium and affects protein synthesisrdquoEndocrinology vol 114 no 1 pp 120ndash130 1984
[71] FW Bazer ldquoMediators of maternal recognition of pregnancy inmammalsrdquo Proceedings of the Society for Experimental Biologyand Medicine vol 199 no 4 pp 373ndash384 1992
[72] R M Roberts D W Leaman and J C Cross ldquoRole of interfer-ons in maternal recognition of pregnancy in ruminantsrdquo Pro-ceedings of the Society for Experimental Biology and Medicinevol 200 no 1 pp 7ndash18 1992
[73] R M Roberts ldquoInterferon-tau a type 1 interferon involvedin maternal recognition of pregnancyrdquo Cytokine and GrowthFactor Reviews vol 18 no 5-6 pp 403ndash408 2007
[74] G E Mann G E Lamming R S Robinson and D C WathesldquoThe regulation of interferon-tau production and uterine hor-mone receptors during early pregnancyrdquo Journal of Reproduc-tion and Fertility Supplement vol 54 pp 317ndash328 1999
[75] R M Roberts A D Ealy A P Alexenko C-S Han and TEzashi ldquoTrophoblast interferonsrdquo Placenta vol 20 no 4 pp259ndash264 1999
[76] WWThatcher M DMeyer and G Danet-Desnoyers ldquoMater-nal recognition of pregnancyrdquo Journal of Reproduction andFertility Supplement vol 49 pp 15ndash28 1995
The Scientific World Journal 9
[77] F W Bazer T E Spencer and T L Ott ldquoInterferon tau anovel pregnancy recognition signalrdquoAmerican Journal of Repro-ductive Immunology vol 37 no 6 pp 412ndash420 1997
[78] T E Spencer and F W Bazer ldquoOvine interferon tau suppressestranscription of the estrogen receptor and oxytocin receptorgenes in the ovine endometriumrdquo Endocrinology vol 137 no3 pp 1144ndash1147 1996
[79] C W Xiao B D Murphy J Sirois and A K GoffldquoDown-regulation of oxytocin-induced cyclooxygenase-2 andprostaglandin F synthase expression by interferon-120591 in bovineendometrial cellsrdquo Biology of Reproduction vol 60 no 3 pp656ndash663 1999
[80] T E Spencer N H Ing T L Ott et al ldquoIntrauterine injectionof ovine interferon-120591 alters oestrogen receptor and oxytocinreceptor expression in the endometrium of cyclic ewesrdquo Journalof Molecular Endocrinology vol 15 no 2 pp 203ndash220 1995
[81] J F Oliveira L E Henkes R L Ashley et al ldquoExpression ofinterferon (IFN)-stimulated genes in extrauterine tissues duringearly pregnancy in sheep is the consequence of endocrine IFN-120591 release from the uterine veinrdquo Endocrinology vol 149 no 3pp 1252ndash1259 2008
[82] C A Gifford K Racicot D S Clark et al ldquoRegulation ofinterferon-stimulated genes in peripheral blood leukocytes inpregnant and bred nonpregnant dairy cowsrdquo Journal of DairyScience vol 90 no 1 pp 274ndash280 2007
[83] H Han K J Austin L A Rempel and T R Hansen ldquoLowblood ISG15 mRNA and progesterone levels are predictive ofnon-pregnant dairy cowsrdquo Journal of Endocrinology vol 191 no2 pp 505ndash512 2006
[84] J C Green C S Okamura S E Poock and M C Lucy ldquoMea-surement of interferon-tau (IFN-120591) stimulated gene expressionin blood leukocytes for pregnancy diagnosis within 18-20d afterinsemination in dairy cattlerdquo Animal Reproduction Science vol121 no 1-2 pp 24ndash33 2010
[85] N Forde F Carter T E Spencer et al ldquoConceptus-inducedchanges in the endometrial transcriptome how soon does thecow know she is pregnantrdquo Biology of Reproduction vol 85 no1 pp 144ndash156 2011
[86] K Kizaki A Shichijo-Kizaki T Furusawa T Takahashi MHosoe andKHashizume ldquoDifferential neutrophil gene expres-sion in early bovine pregnancyrdquoReproductive Biology and Endo-crinology vol 11 article 6 2013
[87] F B P Wooding and D C Wathes ldquoBinucleate cell migrationin the bovine placentomerdquo Journal of Reproduction and Fertilityvol 59 no 2 pp 425ndash430 1980
[88] F W Bazer W W Thatcher P J Hansen M A Mirando TL Ott and C Plante ldquoPhysiological mechanisms of pregnancyrecognition in ruminantsrdquo Journal of Reproduction and FertilitySupplement vol 43 pp 39ndash47 1991
[89] A P Zoli P Demez J F Beckers M Reznik and A BeckersldquoLight and electron microscopic immunolocalization of bovinepregnancy-associated glycoprotein in the bovine placentomerdquoBiology of Reproduction vol 46 no 4 pp 623ndash629 1992
[90] J E Butler W C Hamilton R G Sasser C A Ruder G MHass andR JWilliams ldquoDetection and partial characterizationof two bovine pregnancy-specific proteinsrdquoBiology of Reproduc-tion vol 26 no 5 pp 925ndash933 1982
[91] A P Zoli J-F Beckers P Wouters-Ballman J Closset PFalmagne and F Ectors ldquoPurification and characterization of abovine pregnancy-associated glycoproteinrdquo Biology of Repro-duction vol 45 no 1 pp 1ndash10 1991
[92] S Xie B G Low R J Nagel et al ldquoIdentification of the majorpregnancy-specific antigens of cattle and sheep as inactivemembers of the aspartic proteinase familyrdquo Proceedings of theNational Academy of Sciences of theUnited States of America vol88 no 22 pp 10247ndash10251 1991
[93] S Xie B G Low R J Nagel J-F Beckers and R M RobertsldquoA novel glycoprotein of the aspartic proteinase gene familyexpressed in bovine placental trophectodermrdquoBiology of Repro-duction vol 51 no 6 pp 1145ndash1153 1994
[94] F Constanta S Camousa P Chavatte-Palmera et al ldquoAlteredsecretion of pregnancy-associated glycoproteins during gesta-tion in bovine somatic clonesrdquoTheriogenology vol 76 pp 1006ndash1021 2011
[95] M M Mialon S Camous G Renand J Martal and FMenissier ldquoPeripheral concentrations of a 60-kDa pregnancyserum protein during gestation and after calving and in rela-tionship to embryonic mortality in cattlerdquo Reproduction Nutri-tion Development vol 33 no 3 pp 269ndash282 1993
[96] JMartal N Chene S Camous et al ldquoRecent developments andpotentialities for reducing embryo mortality in ruminants therole of IFN-120591 and other cytokines in early pregnancyrdquo Repro-duction Fertility and Development vol 9 no 3 pp 355ndash3801997
[97] J A Green S Xie X Quan et al ldquoPregnancy-associated bovineand ovine glycoproteins exhibit spatially and temporally dis-tinct expression patterns during pregnancyrdquo Biology of Repro-duction vol 62 no 6 pp 1624ndash1631 2000
[98] S Xie J Green J B Bixby et al ldquoThe diversity and evolutionaryrelationships of the pregnancy-associated glycoproteins anaspartic proteinase subfamily consisting of many trophoblast-expressed genesrdquo Proceedings of the National Academy of Sci-ences of the United States of America vol 94 no 24 pp 12809ndash12816 1997
[99] K Klisch A Boos M Friedrich et al ldquoThe glycosylationof pregnancy-associated glycoproteins and prolactin-relatedprotein-I in bovine binucleate trophoblast giant cells changesbefore parturitionrdquo Reproduction vol 132 no 5 pp 791ndash7982006
[100] B P V L Telugu A M Walker and J A Green ldquoCharacter-ization of the bovine pregnancy-associated glycoprotein genefamilymdashanalysis of gene sequences regulatory regions withinthe promoter and expression of selected genesrdquo BMCGenomicsvol 10 article 185 pp 1ndash17 2009
[101] P Humblot S Camous J Martal et al ldquoDiagnosis of pregnancyby radioimmunoassay of a pregnancy-specific protein in theplasma of dairy cowsrdquo Theriogenology vol 30 no 2 pp 257ndash267 1988
[102] G H Kiracofe J M Wright R R Schalles C A Ruder SParish and R G Sasser ldquoPregnancy-specific protein B in serumof postpartum beef cowsrdquo Journal of Animal Science vol 71 no8 pp 2199ndash2205 1993
[103] A P Zoli L A Guilbault P Delahaut W B Ortiz and J-FBeckers ldquoRadioimmunoassay of a bovine pregnancy-associatedglycoprotein in serum its application for pregnancy diagnosisrdquoBiology of Reproduction vol 46 no 1 pp 83ndash92 1992
[104] R G Sasser C A Ruder K A Ivani J E Butler and W CHamilton ldquoDetection of pregnancy by radioimmunoassay of anovel pregnancy-specific protein in serum of cows and a profileof serum concentrations during gestationrdquo Biology of Reproduc-tion vol 35 no 4 pp 936ndash942 1986
10 The Scientific World Journal
[105] J A Green T E Parks M P Avalle et al ldquoThe establishmentof an ELISA for the detection of pregnancy-associated glyco-proteins (PAGs) in the serum of pregnant cows and heifersrdquoTheriogenology vol 63 no 5 pp 1481ndash1503 2005
[106] E Silva R A Sterry D Kolb et al ldquoAccuracy of a pregnancy-associated glycoprotein ELISA to determine pregnancy statusof lactating dairy cows twenty-seven days after timed artificialinseminationrdquo Journal of Dairy Science vol 90 no 10 pp 4612ndash4622 2007
[107] A Ayad N M Sousa J Sulon J L Hornick M Iguer-Ouada and J F Beckers ldquoCorrelation of five radioimmunoas-say systems for measurement of bovine plasma pregnancy-associated glycoprotein concentrations at early pregnancyperiodrdquo Research in Veterinary Science vol 86 no 3 pp 377ndash382 2009
[108] N M de Sousa M Zongo W Pitala et al ldquoPregnancy-associated glycoprotein concentrations during pregnancy andthe postpartum period in Azawak Zebu cattlerdquoTheriogenologyvol 59 no 5-6 pp 1131ndash1142 2003
[109] A Karen S Darwish A Ramoun et al ldquoAccuracy of ultra-sonography and pregnancy-associated glycoprotein test forpregnancy diagnosis in buffaloesrdquoTheriogenology vol 68 no 8pp 1150ndash1155 2007
[110] N M Sousa A Ayad J F Beckers and Z Gajewski ldquoPreg-nancy-associated glycoproteins (PAG) as pregnancy markers inthe ruminantsrdquo Journal of Physiology and Pharmacology vol 57no 8 pp 153ndash171 2006
[111] P R Graves andTA J Haystead ldquoMolecular biologistrsquos guide toproteomicsrdquo Microbiology and Molecular Biology Reviews vol66 no 1 pp 39ndash63 2002
[112] J D Lippolis and T A Reinhardt ldquoCentennial paper pro-teomics in animal sciencerdquo Journal of Animal Science vol 86no 9 pp 2430ndash2441 2008
[113] D A Colantonio and D W Chan ldquoThe clinical application ofproteomicsrdquo Clinica Chimica Acta vol 357 no 2 pp 151ndash1582005
[114] F di Girolamo E Boschetti M C M Chung F Guadagniand P G Righetti ldquolsquoProteomineeringrsquo or not The debate onbiomarker discovery in sera continuesrdquo Journal of Proteomicsvol 74 no 5 pp 589ndash594 2011
[115] D I Jin H R Lee H R KimH J Lee J T Yoon andC S ParkldquoProteomics analysis of pregnancy-specific serum proteins inbovinerdquoReproduction Fertility andDevelopment vol 18 no 1-2p 183 2005
[116] R Han H R Kim K Naruse et al ldquoIdentification of bovinepregnancy-specific milk proteins using proteomicsrdquo Biology ofReproduction vol 78 p 143 2008
[117] A K Balhara Proteomic analysis of pregnancy-specific serumproteins in buffalo [PhD thesis] LLRUVAS Hisar India 2012
[118] A Busch S Michel C Hoppe D Driesch U Claussen and Fvon Eggeling ldquoProteome analysis of maternal serum samplesfor trisomy 21 pregnancies using ProteinChip arrays and bioin-formaticsrdquo Journal of Histochemistry and Cytochemistry vol 53no 3 pp 341ndash343 2005
[119] P A Benn ldquoAdvances in prenatal screening for Down syn-drome I General principles and second trimester testingrdquoClinica Chimica Acta vol 323 no 1-2 pp 1ndash16 2002
metabolites supports research endeavours aimed at identify-ing novel proteins as the candidate molecules for pregnancydetection Human chorionic gonadotropin (hCG) discov-ered for the first time by Aschheim and Zondek [47] in theurine of pregnant women in 1927 is perhaps the best exampleof a placental protein hormone used for pregnancy diagnosisWith the advancement of biotechnological tools hCG basedpregnancy diagnosis has become the simplest cheapestand most commonly practised test for humans to diagnosepregnancy as early as 8ndash10 days after conception [48]Homol-ogous to the human protein only higher primates produce achorionic gonadotropin (CG) for maintaining luteal activityduring early pregnancy while ruminants produce type Iinterferon as an antiluteolytic factor during this period [49]
224 Early Conception Factor (ECF) Early pregnancy fac-tor (EPF also known as early conception factormdashECF)mdasha1084 kDa protein [50] is present in the sera of pregnantmammalian females detectable within 6 to 24 hours of fer-tilization [51] and disappearing within 24 to 48 after death orremoval of the embryo [52] EPF is present in the serum up totwo-thirds of the gestation [53] EPF remains the earliestserum benchmark for positive fertilization and hence suc-cessful conceptionThis novel pregnancy-specific protein hashigh immunosuppressive ability which is demonstrated byrosette inhibition test a bioassay first demonstrated in preg-nant mice [54] Laleh et al [55] demonstrated significant dif-ferences in rosette inhibition titres of pregnant and open cowswith values being 8ndash10 and 3ndash5 respectively
EPF is reported to be present in the pregnant sera ofmost mammalian species including humans [56 57] mice[51] sheep [58 59] cows [60ndash62] pigs [63] mares [64]and some wild animals [65] In buffalo pregnancy Chander[66] demonstrated decreased E-rosette formation but failedto demonstrate the presence of a rosette inhibiting factor (RIFwhich probably would have been EPF) in the serum Anti-bodies raised against a cow serum glycoprotein were used todetect EPF [67] leading to development of a lab methodwhich has been commercialized in the USA as Early Con-ception Factor (ECF) test (Concepto Diagnostics KnoxvilleTN) claiming detection within 48 hours Extensive study onthe effectiveness of the commercial ECF test for diagnosingnonpregnancy revealed a high degree of nonreliability of thetest wherein only 444 and 556of the confirmednonpreg-nant heifers were identified correctly by serum ECF analysisat days 1 to 3 and days 7 to 9 after AI respectively [61] Similarconclusions were drawn by [60] and [62] Although EPF issecreted in early pregnancy it is not strictly pregnancy spe-cific because of its secretion from nonplacental sources suchas tumors and transformed cell lines [50] which makes itan erroneous pregnancy detection method EPF belongs to afamily of heat shock proteins though detected extracellularlyand having immunosuppressive and growth factor properties[68]These properties are crucial to avoid rejection of an anti-genically alien embryo and support its development There-fore with the advent ofmodern biosciences there is hope thatthese changes could be identified and used as diagnostics forvery early detection of pregnancy However the practicability
of such an early test may still remain low due to high inci-dence of losses during the first 15 days of conception [2]
225 Interferon-120591 (IFN-120591) Moor and Rowson [69] thepioneers of sheep embryo transfer transferred embryos ondays 12 13 and 14 to unmated ewes and suggested interactionsbetween the embryo and uterus that influence the lutealfunction and result in establishment of pregnancy Godkinet al [70] purified ovine trophoblastic protein-1 (oTP-1) anearly secretory protein of the sheep blastocyst from in vitrocultured days 14ndash16 conceptusesThey revealed that oTP1 actson the maternal endometrium thereby eliciting maternalresponses which contribute to the maintenance of preg-nancy Imakawa et al [49] reported the primary amino acidsequence of oTP-1 to demonstrate that the protein is mostprobably an interferon-alpha Later research proved that thesecretions from the conceptus are in fact responsible for thematernal recognition of pregnancy [71 72]
Interferon-120591 a novel type I interferon [73] is first pro-duced by the conceptus between days 12-13 after inseminationin sheep and days 14ndash16 in cattle [74ndash76] High ovine IFN-120591levels are attained on days 12-13 before luteolysis could actu-ally be triggered [77] In ruminants IFN-120591 a 172 amino acidpolypeptide [73] blocks transcription of estrogen receptoralpha and oxytocin receptors in endometrial cells [78] whiledownregulating the expression of enzymes cyclooxygenase-2 and prostaglandin F synthase [79] thus preventing PGFrelease necessary for luteolysis
IFN-120591 acting within the uterine cavity [80] withextremely low levels in extrauterine tissues and peripheralcirculation prevents direct use of IFN-120591 as an early pregnancydiagnosis molecule [81] Rapid advancement of moleculartechniques in the last two decades has opened new avenuesfor exploring this unique molecule as a pregnancy markerfor ruminants through studies on IFN-120591 stimulated genes(ISG) namely interferon-stimulated protein 15 kDa (Isg15)myxovirus resistance 2 (MX2) and 21015840-51015840 oligoadenylate syn-thetase (OAS1) in peripheral blood leukocytes during earlypregnancy [82 83] Microarray analysis further indicatedthatmany genes including IFN-120591 stimulated are upregulatedduring early pregnancy [84ndash86] Green et al [84] have how-ever shown that the differential expression of such genes isinfluenced by the parity of the animal being more definite inheifers as compared tomultiparous animals All these experi-ments have suggested IFN-120591 stimulated genes to be potentialpregnancy detection biomarkers still there is no field leveltest available based on these markers
226 Pregnancy Associated Glycoproteins (PAGs) Reloca-tion of the extra embryonic trophoblastic cell layers to theendometrium [87] between days 20 to 28 and secretions fromthe conceptus lead to successful implantation and continua-tion of pregnancy in ruminant species [88] The pregnancyassociated glycoproteins (PAGs) are secretory products fromthe mono- and binucleated trophoblastic cells in bovine pla-centomes [89] Among these glycoproteins Butler et al [90]
The Scientific World Journal 5
detected two pregnancy-specific proteins in the sera of preg-nant cows a 65ndash70 kDa and a 47ndash53 kDa protein at pI 46ndash48 and 40ndash44 respectively Of these the former showed animmune reaction similar to that of 120572
1-fetoprotein while the
latter showed no reactivity with known proteins and it wasgiven the name ldquoprotein Brdquo or the ldquopregnancy-specific proteinBrdquo (PSPB) in bovines Further purification and characteriza-tion of several isoforms from bovine foetal cotyledons foundthat protein B is actually a 67 kDa PAG [91] Biochemicaland functional investigations established these proteins to beenzymatically inactive members of the aspartic proteinasesuperfamily having homology to pepsin chymosin cathep-sins D and enzyme renin [92 93] PAGs are a very complexgroup of proteins a fact proven by the already documented 22distinct cDNA libraries [94] The three most studied bovinePAGs PSPB PAG 67 kDa or bPAG-1 [93] and PSP60 [95]are isomers of the same protein having similar N-terminalsequences [96] Transcription of bPAG-2 and -11 mRNA isseen all through the pregnancy -4 -5 and -9mRNAs in earlypregnancy and bPAG-1 mRNA are detectable only after day45 [97] Interestingly bovine PAG-4 and bPAG-1 mRNA arehighly transcribed till day 250 of gestation but become indis-cernible at the end [97]The six N-glycosylation sites [98] areresponsible for the variations in molecular weight and half-life of PAGs [99] and is also the reason for expression ofdifferent PAGs during different stages of gestation [97 100]
Very recently it has been observed that placental defectscommonly seen during somatic nuclear transfers in cattleare complemented by unusually high plasma levels of PAGsprobably due to diminished clearance of these proteinsfollowing changes in the glycosylation patterns [94] PSPB isdetectable in the serumof pregnant cows over a long period ofgestation starting at about the fourthweek [101] of gestation toseveral weeks after parturition [102]High circulating levels ofthese proteins on days 80 to 100 postpartum restrict their useas a pregnancy diagnosis test except in heifers [102 103]
Sasser and coworkers [104] developed double antibodyradioimmunoassay for the serological detection of PSPB forpregnancy detection in cattle and found serum levels increas-ing progressively from 1 ngmL after day 30 to 9 ngmL35 ngmL and 150 ngmL after three six and nine months ofpregnancy respectivelyThe study claimed PSPB detection tobemore accurate than the traditional rectal palpationmethodfor pregnancy detection Green et al [105] developed a sand-wich ELISA using anti-PAG monoclonal antibodies whichwere able to detect PAG in all pregnant animals with concen-trations of 875 ngmL on day 28 the highest at 5889 ngmLduring the week of parturition and very low levels within4 weeks postpartum Silva et al [106] predicted 937 954and 962 accuracies for first second and third postpartumtimed artificial inseminations which were in agreement withother commonly practiced pregnancy detection methodsDifferent homologous (RIA-497) and heterologous radioim-munoassay systems (RIA-706 RIA-780 RIA-809 and RIA-Pool) developed for measurement of ruminant blood PAGconcentrations are highly correlated and can be used forpregnancy detection of 30ndash80 days [107] Radioimmunoassayof pregnant sera of zebu cattle established PAG concen-trations to be 60 ngmL 1960 ngmL 10956 ngmL and
3484 ngmL at 8 weeks at 35 weeks at term and at 2 weekspostpartum respectively a pattern similar to other breeds ofcattle [108] Results of PAG-RIA based pregnancy diagnosisin buffaloes have also been encouraging with a high degree ofaccuracy of diagnosis as early as day 31 with 100 sensitivityand 90ndash100 specificity [109] PAGs are used for develop-ment of bench-top pregnancy detectionmethods [110] whichare now commercially available as BioPRYN (BioTrackingRussia) DG29 (Genex Cooperative Inc USA) and IDEXX(IDEXX Laboratories Inc USA) BioPRYN blood test is themost extensively used PAG based kit for pregnancy detectionin ruminants By May 2010 already there were 2 millioncattle blood tests conducted for pregnancy detection (httpbiotrackingcomsiteprotectnetabouttimeline)
3 Current Research inBiomarkers for Pregnancy
It is presumed that the monitoring of sequential changes inblood proteome profile from the day of estrus to successfulconception and through progression of gestation can lead todiscovery of molecules which will perhaps be novel and spe-cific to the physiological stage of the animal In order to qual-ify as a marker for pregnancy the candidate molecule shouldbe able to accurately determine the pregnancy status as earlyas possible with minimum false positives or false negativesAdditionally the biological marker for pregnancy shouldhave the following desired characteristics
(i) specifically upregulated or downregulated duringpregnancy
(ii) least affected by nonanimal factors like feed environ-ment and drug interactions
(iii) having the ability to reflect age as well as viability ofthe conceptus
(iv) present in easily accessible body fluids like serummilk urine and vaginal discharge
(v) expressed over a considerable period of time to giveample time for diagnosis
(vi) revealing the result immediately
Proteomics [111] is large scale study of protein func-tions protein expression protein-protein interactions orposttranslational modifications in a particular cell tissue ororganism and is intended for identification of all the proteinspresent Proteomics provides an opportunity to simultane-ously analyse thousands of proteins in a single experimentfrom a complex mixture of proteins in various body fluids[112] This will help in identifying specific and sensitive bio-markers fulfilling the characteristics of uniqueness for a preg-nancy diagnosis molecule Main objectives of the proteomicsresearch include documentation of biomarkers altered pro-tein expression patterns indicative of pathophysiologicalchanges and therapeutically important drug targets [113]Easily reachable body fluids like blood serum and milk havea wide range of abundant proteins and these few proteinsmake up about 97 of the total serum and milk proteome
6 The Scientific World Journal
and thereby interfere in the proteomic analysis (reviewed by[113]) Conversely it is the low abundance proteins whichhave the highest prospect of being the novel biomarkersof changes in internal milieu of body To sort the problemof high abundance proteins two approaches are suggestedremoval of abundant proteins (usually by immunoaffinity)and concentration of the low abundancescarce proteins withsimultaneous removal of high abundance proteins techni-cally known as combinatorial peptide ligand libraries CPLL[114] Commercially available ProteoMiner kit fromMs Bio-Rad is CPLL based Both approaches however lead to loss ofa significant portion of the low abundance proteins along thehigh abundance proteins yet the later approach is preferred[114]
There is limited information on the bovine proteomein relation to pregnancy Jin et al [115] performed pro-teomics analysis using blood serum samples of pregnantand nonpregnant Holstein dairy cattle at 21 and 35 daysafter AI and reported composite profiles of key proteinsinvolved in early pregnancy and suggested the potential use ofidentified proteins to detect early pregnancy in bovinesTheseincluded nine pregnancy-specific spots in day 21 and day 35serum samples Pregnancy-specific proteins were identifiedas transferrin albumin IgG2a heavy chain constant regionand immunoglobulin gamma heavy chain variable regionFurther differential proteomic analysis of milk samples frompregnant and nonpregnant cows revealed 16 protein spots 14pregnancy specific and 2 spots downregulated in the pregnantmilk sample [116] Pregnancy-specific proteins were identi-fied as serum albumin precursor IgG1 heavy chain constantregion conglutinin precursor epithelial keratin 10 and kelch-like ECH-associated protein Though some identified spotswere abundant milk or serum proteins their molecularweights and pI values were different from main milk orserum proteinsThismay suggest that these proteins could bepregnancy-specific subunits or fragments of albumin and IgGor carrying differentially expressed small proteins whichmayultimately have potential for pregnancy detection
Encouragingly enough these studies need further inves-tigations for arriving at some sort of pregnancy detectionmethod Preliminary studies in buffalo cows on blood pro-teome too detected significant changes in many proteins in2DE gels [117] Important proteins found on MS analysis ofthese were synaptojanin-1 apolipoprotein A-1 apolipopro-tein B Keratin 10 and Von Willebrand factors which aredocumented to have a role in embryogenesis and earlypregnancy
Data generated out of sequential blood proteome analysisduring pregnancy can have several other applications as wellfor example studying fetal viability genetic disorders and soforth Trisomy 21 pregnancies can be detectedwith high accu-racy by maternal serum proteomic analysis in humans [118]In the absence of a single indicator for a particular life processa combination of the expression patterns of more than onesubstance can be used for a purposeful analysis as in thequadruple test where levels of four blood constituents (alpha-fetoprotein human chorionic gonadotropin unconjugatedoestriol and inhibin-A) have been used to predict theprobability of Downrsquos syndrome in babies [119] A pregnancy
detection test on the same lines as the human quadruple testcan be tried where instead of one we can consider proteinprofiles of more proteins This approach too will require athorough analysis of the bovine proteome before such a testwith high accuracy is available to the livestock owners
4 Conclusion
Early pregnancy diagnosis is an important aspect for optimiz-ing dairy production yet none of the present day methodsqualifies as an ideal diagnostic due to limitations of accuracylater stages of applicability and requirement for elaborateinstrumentation and laboratory setup This warrants furtherresearch on developing novel early pregnancy diagnosticsfor livestock species Currently available state-of-the-artinstrumentation and proteomics techniques instil hope forfinding moleculesmdashexclusively related to intricate maternalmetabolic alterations necessary to align with physiology ofearly embryonic development and its signalling for maternalrecognition of pregnancy and continued survival Thoughthese techniques are still in their infancy in animal scienceresearch they hold great promise to address a long-awaitedbreakthrough in pregnancy diagnosis in livestock
Acknowledgment
The authors acknowledge their thanks to the National Fundfor Basic Strategic and Frontier Application Research inAgriculture (NFBSFARA) Indian Council of AgriculturalResearch India for the funding support
References
[1] ldquoCommittee on Reproductive Nomenclature Recommenda-tions for standardizing bovine reproductive termsrdquoThe CornellVeterinarian vol 62 no 2 pp 216ndash237 1972
[2] N Ayalon ldquoA review of embryonic mortality in cattlerdquo Journalof Reproduction and Fertility vol 54 no 2 pp 483ndash493 1978
[3] D Morris and M Diskin ldquoEffect of progesterone on embryosurvivalrdquo Animal vol 2 no 8 pp 1112ndash1119 2008
[4] E S E Hafez Reproduction in Farm Animalsedition Lea andFebiger Philadelphia Pa USA 6th edition 1993
[5] T A Cowie Pregnancy Diagnosis Tests A Review Common-wealthAgricultural Bureaux Joint Publication no 13 Common-wealth Agricultural Bureaux Oxford UK 1948
[6] WWisnicky and L E Cassida ldquoAmanual method for diagnosisof pregnancy in cattlerdquo Journal of the American VeterinaryMedical Association vol 113 p 451 1948
[7] R Zemjanis Diagnostic and Therapeutic Techniques in AnimalReproduction Williams and Wilkins Baltimore Md USA 2ndedition 1970
[8] G H Arthur D E Noakes H Pearson and T J ParkinsonldquoReproduction in the buffalordquo in Veterinary Reproduction andObstetrics WB Saunders London UK 1966
[9] O J Franco M Drost M-J Thatcher V M Shille and W WThatcher ldquoFetal survival in the cow after pregnancy diagnosisby palpation per rectumrdquoTheriogenology vol 27 no 4 pp 631ndash644 1987
The Scientific World Journal 7
[10] R K Sharma J K Singh S K Phulia S Khanna and I SinghldquoFetal sex determination with ultrasonography in buffaloesrdquoIndian Veterinary Journal vol 88 no 10 pp 105ndash107 2011
[11] S Curran R A Pierson and O J Ginther ldquoUltrasonographicappearance of the bovine conceptus from days 20 through 60rdquoJournal of the American VeterinaryMedical Association vol 189no 10 pp 1295ndash1302 1986
[12] PM Fricke ldquoScanning the futuremdashultrasonography as a repro-ductive management tool for dairy cattlerdquo Journal of Dairy Sci-ence vol 85 no 8 pp 1918ndash1926 2002
[13] L G Paisley W D Mickelsen and O L Frost ldquoA survey of theincidence of prenatal mortality in cattle following pregnancydiagnosis by rectal palpationrdquoTheriogenology vol 9 no 6 pp481ndash491 1978
[14] D Vaillancourt C J Bierschwal D Ogwu et al ldquoCorrelationbetween pregnancy diagnosis bymembrane slip and embryonicmortalityrdquo Journal of the American Veterinary Medical Associa-tion vol 175 no 5 pp 466ndash468 1979
[15] A Ali and S Fahmy ldquoUltrasonographic fetometry and deter-mination of fetal sex in buffaloes (Bubalus bubalis)rdquo AnimalReproduction Science vol 106 no 1-2 pp 90ndash99 2008
[16] C H Pawshe K B C Appa Rao and S M Totey ldquoUltrasono-graphic imaging to monitor early pregnancy and embryonicdevelopment in the buffalo (Bubalus bubalis)rdquo Theriogenologyvol 41 no 3 pp 697ndash709 1994
[17] M R Bhosreker and IMHangarge ldquoUltrasonography for earlypregnancy diagnosis in buffaloesrdquo Indian Journal of AnimalReproduction vol 21 pp 143ndash144 2000
[18] M C Pieterse O Szenci A H Willemse C S A Bajcsy S JDieleman andM AM Taverne ldquoEarly pregnancy diagnosis incattle bymeans of linear-array real-time ultrasound scanning ofthe uterus and a qualitative and quantitative milk progesteronetestrdquoTheriogenology vol 33 no 3 pp 697ndash707 1990
[19] D P Nation J Malmo G M Davis and K L MacmillanldquoAccuracy of bovine pregnancy detection using transrectalultrasonography at 28 to 35 days after inseminationrdquoAustralianVeterinary Journal vol 81 no 1-2 pp 63ndash65 2003
[20] O Szenci J F Beckers P Humblot et al ldquoComparison of ultra-sonography bovine pregnancy-specific protein B and bovinepregnancy-associated glycoprotein 1 tests for pregnancy detec-tion in dairy cowsrdquo Theriogenology vol 50 no 1 pp 77ndash881998
[21] M Shemesh N Ayalon and H R Lindner ldquoEarly pregnancydiagnosis based upon plasma progesterone levels in the cow andewerdquo Journal of Animal Science vol 36 no 4 pp 726ndash729 1973
[22] J A Laing and R B Heap ldquoThe concentration of progesteronein themilk of cows during the reproductive cyclerdquo British Veter-inary Journal vol 127 no 8 pp 19ndash22 1971
[23] A A Zaied C J Bierschwal R G Elmore R S Youngquist AJ Sharp and H A Garverick ldquoConcentrations of progesteroneinmilk as amonitor of early pregnancy diagnosis in dairy cowsrdquoTheriogenology vol 12 no 1 pp 3ndash11 1979
[24] R Simersky J Swaczynova D A Morris M Franek and MStrnad ldquoDevelopment of an ELISA-based kit for the on-farmdetermination of progesterone in milkrdquo Veterinarni Medicinavol 52 no 1 pp 19ndash28 2007
[25] M Kamboj and B S Prakash ldquoRelationship of progesterone inplasma and whole milk of buffaloes during cyclicity and earlypregnancyrdquo Tropical Animal Health and Production vol 25 no3 pp 185ndash192 1993
[26] S K Batra R C Arora N K Bachlaus andR S Pandey ldquoBloodand milk progesterone in pregnant and nonpregnant buffalordquoJournal of Dairy Science vol 62 no 9 pp 1390ndash1393 1979
[27] BM Perera N Pathiraja S A AbeywardenaM XMotha andH Abeygunawardena ldquoEarly pregnancy diagnosis in buffaloesfrom plasma progesterone concentrationrdquo Veterinary Recordvol 106 no 5 pp 104ndash106 1980
[28] J A Pennington L H Schultz and W F Hoffman ldquoCompari-son of pregnancy diagnosis by milk progesterone on day 21 andday 24 postbreeding field study in dairy cattlerdquo Journal of DairyScience vol 68 no 10 pp 2740ndash2745 1985
[29] A Singh and R Puthiyandy ldquoEstimation of progesterone inbuffalomilk and its application to pregnancy diagnosisrdquo Journalof Reproduction and Fertility vol 59 no 1 pp 89ndash93 1980
[30] N Isobe M Akita T Nakao H Yamashiro and H KubotaldquoPregnancy diagnosis based on the fecal progesterone concen-tration in beef and dairy heifers and beef cowsrdquo Animal Repro-duction Science vol 90 no 3-4 pp 211ndash218 2005
[31] T E Spencer N H Ing T L Ott et al ldquoIntrauterine injectionof ovine interferon-120591 alters oestrogen receptor and oxytocinreceptor expression in the endometrium of cyclic ewesrdquo Journalof Molecular Endocrinology vol 15 no 2 pp 203ndash220 1995
[32] J J Kaneko J W Harvey andM L Bruss Clinical Biochemistryof Domestic Animals Academic Press AmsterdamTheNether-lands 2008
[33] T J Parkinson A Turvey and L J Jenner ldquoA morphometricanalysis of the corpus luteumof the cowduring the estrous cycleand early pregnancyrdquo Theriogenology vol 41 no 5 pp 1115ndash1126 1994
[34] R G Sasser and C A Ruder ldquoDetection of early pregnancy indomestic ruminantsrdquo Journal of Reproduction and Fertility Sup-plement vol 34 pp 261ndash271 1987
[35] M Shemesh N Ayalon and H R Lindner ldquoEarly pregnancydiagnosis based upon plasma progesterone levels in the cow andewerdquo Journal of Animal Science vol 36 no 4 pp 726ndash729 1973
[36] E Wolf G J Arnold S Bauersachs et al ldquoEmbryo-maternalcommunication in bovinemdashstrategies for deciphering a com-plex cross-talkrdquo Reproduction in Domestic Animals vol 38 no4 pp 276ndash289 2003
[37] E W Gowan R J Etches C Bryden and G J King ldquoFactorsaffecting accuracy of pregnancy diagnosis in cattlerdquo Journal ofDairy Science vol 65 no 7 pp 1294ndash1302 1982
[38] R L Nebel W D Whittier B G Cassell and J H Britt ldquoCom-parison of on-farm laboratory milk progesterone assays foridentifying errors in detection of estrus and diagnosis of preg-nancyrdquo Journal of Dairy Science vol 70 no 7 pp 1471ndash14761987
[39] T H Wimpy C F Chang V L Estergreen and J K HillersldquoMilk progesterone enzyme immunoassay modifications anda field trial for pregnancy detection in dairy cowsrdquo Journal ofDairy Science vol 69 no 4 pp 1115ndash1121 1986
[40] R M Eley WWThatcher and F W Bazer ldquoLuteolytic effect ofoestrone sulphate on cyclic beef heifersrdquo Journal of Reproductionand Fertility vol 55 no 1 pp 191ndash193 1979
[41] H A Robertson and G J King ldquoConjugated and unconjugatedoestrogens in fetal and maternal fluids of the cow throughoutpregnancyrdquo Journal of Reproduction and Fertility vol 55 no 2pp 463ndash470 1979
[42] R B Heap and M Hamon ldquoOestrone sulphate in milk as anindicator of a viable conceptus in cowsrdquo British Veterinary Jour-nal vol 135 no 4 pp 355ndash363 1979
8 The Scientific World Journal
[43] M Hamon I R Fleet R J Holdsworth and R B Heap ldquoThetime of detection of oestrone sulphate in milk and the diagnosisof pregnancy in cowsrdquo British Veterinary Journal vol 137 no 1pp 71ndash77 1981
[44] M Hirako T Takahashi and I Domeki ldquoPeripheral changes inestrone sulfate concentration during the first trimester of ges-tation in cattle comparison with unconjugated estrogens andrelationship to fetal numberrdquo Theriogenology vol 57 no 7 pp1939ndash1947 2002
[45] B S Prakash and M L Madan ldquoInfluence of gestation onoestrone sulphate concentration in milk of zebu and crossbredcows and murrah buffaloesrdquo Tropical Animal Health and Pro-duction vol 25 no 2 pp 94ndash100 1993
[46] F Lobago M Bekana H Gustafsson et al ldquoSerum profilesof pregnancy-associated glycoprotein oestrone sulphate andprogesterone during gestation and some factors influencing theprofiles in ethiopian borana and crossbred cattlerdquo Reproductionin Domestic Animals vol 44 no 4 pp 685ndash692 2009
[47] S Aschheim and B Zondek ldquoHypophysenvorderlappenhor-mon undOvarialhormon imHarn von SchwangerenrdquoKlinischeWochenschrift vol 6 no 28 p 1322 1927
[48] J R Marshall C B Hammond G T Ross A Jacobson PRayford andW D Odell ldquoPlasma and urinary chorionic gona-dotropin during early human pregnancyrdquo Obstetrics and Gyne-cology vol 32 no 6 pp 760ndash764 1968
[49] K Imakawa R V Anthony M Kazemi K R Marotti H GPolites and R M Roberts ldquoInterferon-like sequence of ovinetrophoblast protein secreted by embryonic trophectodermrdquoNature vol 330 no 6146 pp 377ndash379 1987
[50] A C Cavanagh ldquoIdentification of early pregnancy factor aschaperonin 10 implications for understanding its rolerdquo Reviewsof Reproduction vol 1 no 1 pp 28ndash32 1996
[51] H Morton V Hegh and G J A Clunie ldquoStudies of the rosetteinhibition test in pregnant mice evidence of immunosuppres-sionrdquo Proceedings of the Royal Society B vol 193 no 1113 pp413ndash419 1976
[52] H Morton B E Rolfe and A C Cavanagh ldquoOvum factor andearly pregnancy factorrdquo Current Topics in Developmental Biol-ogy vol 23 pp 73ndash92 1987
[53] H Morton A C Cavanagh S Athanasas-Platsis K A QuinnandB E Rolfe ldquoEarly pregnancy factor has immunosuppressiveand growth factor propertiesrdquo Reproduction Fertility andDevelopment vol 4 no 4 pp 411ndash422 1992
[54] H Morton V Hegh and G J A Clunie ldquoImmunosuppressiondetected in pregnantmice by rosette inhibition testrdquoNature vol249 no 5456 pp 459ndash460 1974
[55] V G Laleh R G Laleh N Pirany and M M Ahrabi ldquoMea-surement of EPF for detection of cow pregnancy using rosetteinhibition testrdquoTheriogenology vol 70 no 1 pp 105ndash107 2008
[56] H Qin and Z Q Zheng ldquoDetection of early pregnancy factorin human serardquo American Journal of Reproductive Immunologyand Microbiology vol 13 no 1 pp 15ndash18 1987
[57] Y C Smart T K Roberts I S Fraser A W Cripps and R LClancy ldquoValidation of the rosette inhibition test for the detec-tion of early pregnancy in womenrdquo Fertility and Sterility vol 37no 6 pp 779ndash785 1982
[58] HMorton G J A Clunie and F D Shaw ldquoA test for early preg-nancy in sheeprdquo Research in Veterinary Science vol 26 no 2pp 261ndash262 1979
[59] S Wilson R McCarthy and F Clarke ldquoIn search of early preg-nancy factor isolation of active polypeptides from pregnant
ewesrsquo serardquo Journal of Reproductive Immunology vol 5 no 5 pp275ndash286 1983
[60] M C Cordoba R Sartori and P M Fricke ldquoAssessment of acommercially available early conception factor (ECF) test fordetermining pregnancy status of dairy cattlerdquo Journal of DairyScience vol 84 no 8 pp 1884ndash1889 2001
[61] B GandyW Tucker P Ryan et al ldquoEvaluation of the early con-ception factor (ECFŮ) test for the detection of nonpregnancyin dairy cattlerdquoTheriogenology vol 56 no 4 pp 637ndash647 2001
[62] C S Whisnant L A Pagels and M G Daves ldquoCase studyeffectiveness of a commercial early conception factor test for usein cattlerdquo Professional Animal Scientist vol 17 pp 51ndash53 2001
[63] A S Grewal A L C Wallace Y S Pan et al ldquoEvaluation of arosette inhibition test for pregnancy diagnosis in pigsrdquo Journalof Reproductive Immunology vol 7 no 2 pp 129ndash138 1985
[64] K Ohnuma I T O Kazuei Y-I Miyake J Takahashi andY Yasuda ldquoDetection of early pregnancy factor (EPF) in mareserardquo Journal of Reproduction and Development vol 42 no 1pp 23ndash28 1996
[65] Y P Cruz L Selwood H Morton and A C CavanaghldquoSignificance of serum early pregnancy factor concentrationsduring pregnancy and embryonic development in Sminthopsismacroura (Spencer) (Marsupialia Dasyuridae)rdquo Reproductionvol 121 no 6 pp 933ndash939 2001
[66] S Chander Studies on certain immunological aspects of preg-nancy diagnosis in buffaloes [PhD thesis] Chaudhary CharanSingh Haryana Agricultural University Hisar India 1983
[67] W R Threlfall ldquoImmunosuppressive early pregnancy factor(ISEPF) determination for pregnancy diagnosis in dairy cowsrdquoTheriogenology vol 41 p 31 1994
[68] H Morton ldquoEarly pregnancy factor an extracellular chaper-onin 10 homologuerdquo Immunology andCell Biology vol 76 no 6pp 483ndash496 1998
[69] R M Moor and L E A Rowson ldquoInfluence of the embryo anduterus on luteal function in the sheeprdquoNature vol 201 no 4918pp 522ndash523 1964
[70] J D Godkin FW Bazer and RM Roberts ldquoOvine trophoblastprotein 1 an early secreted blastocyst protein binds specifi-cally to uterine endometrium and affects protein synthesisrdquoEndocrinology vol 114 no 1 pp 120ndash130 1984
[71] FW Bazer ldquoMediators of maternal recognition of pregnancy inmammalsrdquo Proceedings of the Society for Experimental Biologyand Medicine vol 199 no 4 pp 373ndash384 1992
[72] R M Roberts D W Leaman and J C Cross ldquoRole of interfer-ons in maternal recognition of pregnancy in ruminantsrdquo Pro-ceedings of the Society for Experimental Biology and Medicinevol 200 no 1 pp 7ndash18 1992
[73] R M Roberts ldquoInterferon-tau a type 1 interferon involvedin maternal recognition of pregnancyrdquo Cytokine and GrowthFactor Reviews vol 18 no 5-6 pp 403ndash408 2007
[74] G E Mann G E Lamming R S Robinson and D C WathesldquoThe regulation of interferon-tau production and uterine hor-mone receptors during early pregnancyrdquo Journal of Reproduc-tion and Fertility Supplement vol 54 pp 317ndash328 1999
[75] R M Roberts A D Ealy A P Alexenko C-S Han and TEzashi ldquoTrophoblast interferonsrdquo Placenta vol 20 no 4 pp259ndash264 1999
[76] WWThatcher M DMeyer and G Danet-Desnoyers ldquoMater-nal recognition of pregnancyrdquo Journal of Reproduction andFertility Supplement vol 49 pp 15ndash28 1995
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[77] F W Bazer T E Spencer and T L Ott ldquoInterferon tau anovel pregnancy recognition signalrdquoAmerican Journal of Repro-ductive Immunology vol 37 no 6 pp 412ndash420 1997
[78] T E Spencer and F W Bazer ldquoOvine interferon tau suppressestranscription of the estrogen receptor and oxytocin receptorgenes in the ovine endometriumrdquo Endocrinology vol 137 no3 pp 1144ndash1147 1996
[79] C W Xiao B D Murphy J Sirois and A K GoffldquoDown-regulation of oxytocin-induced cyclooxygenase-2 andprostaglandin F synthase expression by interferon-120591 in bovineendometrial cellsrdquo Biology of Reproduction vol 60 no 3 pp656ndash663 1999
[80] T E Spencer N H Ing T L Ott et al ldquoIntrauterine injectionof ovine interferon-120591 alters oestrogen receptor and oxytocinreceptor expression in the endometrium of cyclic ewesrdquo Journalof Molecular Endocrinology vol 15 no 2 pp 203ndash220 1995
[81] J F Oliveira L E Henkes R L Ashley et al ldquoExpression ofinterferon (IFN)-stimulated genes in extrauterine tissues duringearly pregnancy in sheep is the consequence of endocrine IFN-120591 release from the uterine veinrdquo Endocrinology vol 149 no 3pp 1252ndash1259 2008
[82] C A Gifford K Racicot D S Clark et al ldquoRegulation ofinterferon-stimulated genes in peripheral blood leukocytes inpregnant and bred nonpregnant dairy cowsrdquo Journal of DairyScience vol 90 no 1 pp 274ndash280 2007
[83] H Han K J Austin L A Rempel and T R Hansen ldquoLowblood ISG15 mRNA and progesterone levels are predictive ofnon-pregnant dairy cowsrdquo Journal of Endocrinology vol 191 no2 pp 505ndash512 2006
[84] J C Green C S Okamura S E Poock and M C Lucy ldquoMea-surement of interferon-tau (IFN-120591) stimulated gene expressionin blood leukocytes for pregnancy diagnosis within 18-20d afterinsemination in dairy cattlerdquo Animal Reproduction Science vol121 no 1-2 pp 24ndash33 2010
[85] N Forde F Carter T E Spencer et al ldquoConceptus-inducedchanges in the endometrial transcriptome how soon does thecow know she is pregnantrdquo Biology of Reproduction vol 85 no1 pp 144ndash156 2011
[86] K Kizaki A Shichijo-Kizaki T Furusawa T Takahashi MHosoe andKHashizume ldquoDifferential neutrophil gene expres-sion in early bovine pregnancyrdquoReproductive Biology and Endo-crinology vol 11 article 6 2013
[87] F B P Wooding and D C Wathes ldquoBinucleate cell migrationin the bovine placentomerdquo Journal of Reproduction and Fertilityvol 59 no 2 pp 425ndash430 1980
[88] F W Bazer W W Thatcher P J Hansen M A Mirando TL Ott and C Plante ldquoPhysiological mechanisms of pregnancyrecognition in ruminantsrdquo Journal of Reproduction and FertilitySupplement vol 43 pp 39ndash47 1991
[89] A P Zoli P Demez J F Beckers M Reznik and A BeckersldquoLight and electron microscopic immunolocalization of bovinepregnancy-associated glycoprotein in the bovine placentomerdquoBiology of Reproduction vol 46 no 4 pp 623ndash629 1992
[90] J E Butler W C Hamilton R G Sasser C A Ruder G MHass andR JWilliams ldquoDetection and partial characterizationof two bovine pregnancy-specific proteinsrdquoBiology of Reproduc-tion vol 26 no 5 pp 925ndash933 1982
[91] A P Zoli J-F Beckers P Wouters-Ballman J Closset PFalmagne and F Ectors ldquoPurification and characterization of abovine pregnancy-associated glycoproteinrdquo Biology of Repro-duction vol 45 no 1 pp 1ndash10 1991
[92] S Xie B G Low R J Nagel et al ldquoIdentification of the majorpregnancy-specific antigens of cattle and sheep as inactivemembers of the aspartic proteinase familyrdquo Proceedings of theNational Academy of Sciences of theUnited States of America vol88 no 22 pp 10247ndash10251 1991
[93] S Xie B G Low R J Nagel J-F Beckers and R M RobertsldquoA novel glycoprotein of the aspartic proteinase gene familyexpressed in bovine placental trophectodermrdquoBiology of Repro-duction vol 51 no 6 pp 1145ndash1153 1994
[94] F Constanta S Camousa P Chavatte-Palmera et al ldquoAlteredsecretion of pregnancy-associated glycoproteins during gesta-tion in bovine somatic clonesrdquoTheriogenology vol 76 pp 1006ndash1021 2011
[95] M M Mialon S Camous G Renand J Martal and FMenissier ldquoPeripheral concentrations of a 60-kDa pregnancyserum protein during gestation and after calving and in rela-tionship to embryonic mortality in cattlerdquo Reproduction Nutri-tion Development vol 33 no 3 pp 269ndash282 1993
[96] JMartal N Chene S Camous et al ldquoRecent developments andpotentialities for reducing embryo mortality in ruminants therole of IFN-120591 and other cytokines in early pregnancyrdquo Repro-duction Fertility and Development vol 9 no 3 pp 355ndash3801997
[97] J A Green S Xie X Quan et al ldquoPregnancy-associated bovineand ovine glycoproteins exhibit spatially and temporally dis-tinct expression patterns during pregnancyrdquo Biology of Repro-duction vol 62 no 6 pp 1624ndash1631 2000
[98] S Xie J Green J B Bixby et al ldquoThe diversity and evolutionaryrelationships of the pregnancy-associated glycoproteins anaspartic proteinase subfamily consisting of many trophoblast-expressed genesrdquo Proceedings of the National Academy of Sci-ences of the United States of America vol 94 no 24 pp 12809ndash12816 1997
[99] K Klisch A Boos M Friedrich et al ldquoThe glycosylationof pregnancy-associated glycoproteins and prolactin-relatedprotein-I in bovine binucleate trophoblast giant cells changesbefore parturitionrdquo Reproduction vol 132 no 5 pp 791ndash7982006
[100] B P V L Telugu A M Walker and J A Green ldquoCharacter-ization of the bovine pregnancy-associated glycoprotein genefamilymdashanalysis of gene sequences regulatory regions withinthe promoter and expression of selected genesrdquo BMCGenomicsvol 10 article 185 pp 1ndash17 2009
[101] P Humblot S Camous J Martal et al ldquoDiagnosis of pregnancyby radioimmunoassay of a pregnancy-specific protein in theplasma of dairy cowsrdquo Theriogenology vol 30 no 2 pp 257ndash267 1988
[102] G H Kiracofe J M Wright R R Schalles C A Ruder SParish and R G Sasser ldquoPregnancy-specific protein B in serumof postpartum beef cowsrdquo Journal of Animal Science vol 71 no8 pp 2199ndash2205 1993
[103] A P Zoli L A Guilbault P Delahaut W B Ortiz and J-FBeckers ldquoRadioimmunoassay of a bovine pregnancy-associatedglycoprotein in serum its application for pregnancy diagnosisrdquoBiology of Reproduction vol 46 no 1 pp 83ndash92 1992
[104] R G Sasser C A Ruder K A Ivani J E Butler and W CHamilton ldquoDetection of pregnancy by radioimmunoassay of anovel pregnancy-specific protein in serum of cows and a profileof serum concentrations during gestationrdquo Biology of Reproduc-tion vol 35 no 4 pp 936ndash942 1986
10 The Scientific World Journal
[105] J A Green T E Parks M P Avalle et al ldquoThe establishmentof an ELISA for the detection of pregnancy-associated glyco-proteins (PAGs) in the serum of pregnant cows and heifersrdquoTheriogenology vol 63 no 5 pp 1481ndash1503 2005
[106] E Silva R A Sterry D Kolb et al ldquoAccuracy of a pregnancy-associated glycoprotein ELISA to determine pregnancy statusof lactating dairy cows twenty-seven days after timed artificialinseminationrdquo Journal of Dairy Science vol 90 no 10 pp 4612ndash4622 2007
[107] A Ayad N M Sousa J Sulon J L Hornick M Iguer-Ouada and J F Beckers ldquoCorrelation of five radioimmunoas-say systems for measurement of bovine plasma pregnancy-associated glycoprotein concentrations at early pregnancyperiodrdquo Research in Veterinary Science vol 86 no 3 pp 377ndash382 2009
[108] N M de Sousa M Zongo W Pitala et al ldquoPregnancy-associated glycoprotein concentrations during pregnancy andthe postpartum period in Azawak Zebu cattlerdquoTheriogenologyvol 59 no 5-6 pp 1131ndash1142 2003
[109] A Karen S Darwish A Ramoun et al ldquoAccuracy of ultra-sonography and pregnancy-associated glycoprotein test forpregnancy diagnosis in buffaloesrdquoTheriogenology vol 68 no 8pp 1150ndash1155 2007
[110] N M Sousa A Ayad J F Beckers and Z Gajewski ldquoPreg-nancy-associated glycoproteins (PAG) as pregnancy markers inthe ruminantsrdquo Journal of Physiology and Pharmacology vol 57no 8 pp 153ndash171 2006
[111] P R Graves andTA J Haystead ldquoMolecular biologistrsquos guide toproteomicsrdquo Microbiology and Molecular Biology Reviews vol66 no 1 pp 39ndash63 2002
[112] J D Lippolis and T A Reinhardt ldquoCentennial paper pro-teomics in animal sciencerdquo Journal of Animal Science vol 86no 9 pp 2430ndash2441 2008
[113] D A Colantonio and D W Chan ldquoThe clinical application ofproteomicsrdquo Clinica Chimica Acta vol 357 no 2 pp 151ndash1582005
[114] F di Girolamo E Boschetti M C M Chung F Guadagniand P G Righetti ldquolsquoProteomineeringrsquo or not The debate onbiomarker discovery in sera continuesrdquo Journal of Proteomicsvol 74 no 5 pp 589ndash594 2011
[115] D I Jin H R Lee H R KimH J Lee J T Yoon andC S ParkldquoProteomics analysis of pregnancy-specific serum proteins inbovinerdquoReproduction Fertility andDevelopment vol 18 no 1-2p 183 2005
[116] R Han H R Kim K Naruse et al ldquoIdentification of bovinepregnancy-specific milk proteins using proteomicsrdquo Biology ofReproduction vol 78 p 143 2008
[117] A K Balhara Proteomic analysis of pregnancy-specific serumproteins in buffalo [PhD thesis] LLRUVAS Hisar India 2012
[118] A Busch S Michel C Hoppe D Driesch U Claussen and Fvon Eggeling ldquoProteome analysis of maternal serum samplesfor trisomy 21 pregnancies using ProteinChip arrays and bioin-formaticsrdquo Journal of Histochemistry and Cytochemistry vol 53no 3 pp 341ndash343 2005
[119] P A Benn ldquoAdvances in prenatal screening for Down syn-drome I General principles and second trimester testingrdquoClinica Chimica Acta vol 323 no 1-2 pp 1ndash16 2002
detected two pregnancy-specific proteins in the sera of preg-nant cows a 65ndash70 kDa and a 47ndash53 kDa protein at pI 46ndash48 and 40ndash44 respectively Of these the former showed animmune reaction similar to that of 120572
1-fetoprotein while the
latter showed no reactivity with known proteins and it wasgiven the name ldquoprotein Brdquo or the ldquopregnancy-specific proteinBrdquo (PSPB) in bovines Further purification and characteriza-tion of several isoforms from bovine foetal cotyledons foundthat protein B is actually a 67 kDa PAG [91] Biochemicaland functional investigations established these proteins to beenzymatically inactive members of the aspartic proteinasesuperfamily having homology to pepsin chymosin cathep-sins D and enzyme renin [92 93] PAGs are a very complexgroup of proteins a fact proven by the already documented 22distinct cDNA libraries [94] The three most studied bovinePAGs PSPB PAG 67 kDa or bPAG-1 [93] and PSP60 [95]are isomers of the same protein having similar N-terminalsequences [96] Transcription of bPAG-2 and -11 mRNA isseen all through the pregnancy -4 -5 and -9mRNAs in earlypregnancy and bPAG-1 mRNA are detectable only after day45 [97] Interestingly bovine PAG-4 and bPAG-1 mRNA arehighly transcribed till day 250 of gestation but become indis-cernible at the end [97]The six N-glycosylation sites [98] areresponsible for the variations in molecular weight and half-life of PAGs [99] and is also the reason for expression ofdifferent PAGs during different stages of gestation [97 100]
Very recently it has been observed that placental defectscommonly seen during somatic nuclear transfers in cattleare complemented by unusually high plasma levels of PAGsprobably due to diminished clearance of these proteinsfollowing changes in the glycosylation patterns [94] PSPB isdetectable in the serumof pregnant cows over a long period ofgestation starting at about the fourthweek [101] of gestation toseveral weeks after parturition [102]High circulating levels ofthese proteins on days 80 to 100 postpartum restrict their useas a pregnancy diagnosis test except in heifers [102 103]
Sasser and coworkers [104] developed double antibodyradioimmunoassay for the serological detection of PSPB forpregnancy detection in cattle and found serum levels increas-ing progressively from 1 ngmL after day 30 to 9 ngmL35 ngmL and 150 ngmL after three six and nine months ofpregnancy respectivelyThe study claimed PSPB detection tobemore accurate than the traditional rectal palpationmethodfor pregnancy detection Green et al [105] developed a sand-wich ELISA using anti-PAG monoclonal antibodies whichwere able to detect PAG in all pregnant animals with concen-trations of 875 ngmL on day 28 the highest at 5889 ngmLduring the week of parturition and very low levels within4 weeks postpartum Silva et al [106] predicted 937 954and 962 accuracies for first second and third postpartumtimed artificial inseminations which were in agreement withother commonly practiced pregnancy detection methodsDifferent homologous (RIA-497) and heterologous radioim-munoassay systems (RIA-706 RIA-780 RIA-809 and RIA-Pool) developed for measurement of ruminant blood PAGconcentrations are highly correlated and can be used forpregnancy detection of 30ndash80 days [107] Radioimmunoassayof pregnant sera of zebu cattle established PAG concen-trations to be 60 ngmL 1960 ngmL 10956 ngmL and
3484 ngmL at 8 weeks at 35 weeks at term and at 2 weekspostpartum respectively a pattern similar to other breeds ofcattle [108] Results of PAG-RIA based pregnancy diagnosisin buffaloes have also been encouraging with a high degree ofaccuracy of diagnosis as early as day 31 with 100 sensitivityand 90ndash100 specificity [109] PAGs are used for develop-ment of bench-top pregnancy detectionmethods [110] whichare now commercially available as BioPRYN (BioTrackingRussia) DG29 (Genex Cooperative Inc USA) and IDEXX(IDEXX Laboratories Inc USA) BioPRYN blood test is themost extensively used PAG based kit for pregnancy detectionin ruminants By May 2010 already there were 2 millioncattle blood tests conducted for pregnancy detection (httpbiotrackingcomsiteprotectnetabouttimeline)
3 Current Research inBiomarkers for Pregnancy
It is presumed that the monitoring of sequential changes inblood proteome profile from the day of estrus to successfulconception and through progression of gestation can lead todiscovery of molecules which will perhaps be novel and spe-cific to the physiological stage of the animal In order to qual-ify as a marker for pregnancy the candidate molecule shouldbe able to accurately determine the pregnancy status as earlyas possible with minimum false positives or false negativesAdditionally the biological marker for pregnancy shouldhave the following desired characteristics
(i) specifically upregulated or downregulated duringpregnancy
(ii) least affected by nonanimal factors like feed environ-ment and drug interactions
(iii) having the ability to reflect age as well as viability ofthe conceptus
(iv) present in easily accessible body fluids like serummilk urine and vaginal discharge
(v) expressed over a considerable period of time to giveample time for diagnosis
(vi) revealing the result immediately
Proteomics [111] is large scale study of protein func-tions protein expression protein-protein interactions orposttranslational modifications in a particular cell tissue ororganism and is intended for identification of all the proteinspresent Proteomics provides an opportunity to simultane-ously analyse thousands of proteins in a single experimentfrom a complex mixture of proteins in various body fluids[112] This will help in identifying specific and sensitive bio-markers fulfilling the characteristics of uniqueness for a preg-nancy diagnosis molecule Main objectives of the proteomicsresearch include documentation of biomarkers altered pro-tein expression patterns indicative of pathophysiologicalchanges and therapeutically important drug targets [113]Easily reachable body fluids like blood serum and milk havea wide range of abundant proteins and these few proteinsmake up about 97 of the total serum and milk proteome
6 The Scientific World Journal
and thereby interfere in the proteomic analysis (reviewed by[113]) Conversely it is the low abundance proteins whichhave the highest prospect of being the novel biomarkersof changes in internal milieu of body To sort the problemof high abundance proteins two approaches are suggestedremoval of abundant proteins (usually by immunoaffinity)and concentration of the low abundancescarce proteins withsimultaneous removal of high abundance proteins techni-cally known as combinatorial peptide ligand libraries CPLL[114] Commercially available ProteoMiner kit fromMs Bio-Rad is CPLL based Both approaches however lead to loss ofa significant portion of the low abundance proteins along thehigh abundance proteins yet the later approach is preferred[114]
There is limited information on the bovine proteomein relation to pregnancy Jin et al [115] performed pro-teomics analysis using blood serum samples of pregnantand nonpregnant Holstein dairy cattle at 21 and 35 daysafter AI and reported composite profiles of key proteinsinvolved in early pregnancy and suggested the potential use ofidentified proteins to detect early pregnancy in bovinesTheseincluded nine pregnancy-specific spots in day 21 and day 35serum samples Pregnancy-specific proteins were identifiedas transferrin albumin IgG2a heavy chain constant regionand immunoglobulin gamma heavy chain variable regionFurther differential proteomic analysis of milk samples frompregnant and nonpregnant cows revealed 16 protein spots 14pregnancy specific and 2 spots downregulated in the pregnantmilk sample [116] Pregnancy-specific proteins were identi-fied as serum albumin precursor IgG1 heavy chain constantregion conglutinin precursor epithelial keratin 10 and kelch-like ECH-associated protein Though some identified spotswere abundant milk or serum proteins their molecularweights and pI values were different from main milk orserum proteinsThismay suggest that these proteins could bepregnancy-specific subunits or fragments of albumin and IgGor carrying differentially expressed small proteins whichmayultimately have potential for pregnancy detection
Encouragingly enough these studies need further inves-tigations for arriving at some sort of pregnancy detectionmethod Preliminary studies in buffalo cows on blood pro-teome too detected significant changes in many proteins in2DE gels [117] Important proteins found on MS analysis ofthese were synaptojanin-1 apolipoprotein A-1 apolipopro-tein B Keratin 10 and Von Willebrand factors which aredocumented to have a role in embryogenesis and earlypregnancy
Data generated out of sequential blood proteome analysisduring pregnancy can have several other applications as wellfor example studying fetal viability genetic disorders and soforth Trisomy 21 pregnancies can be detectedwith high accu-racy by maternal serum proteomic analysis in humans [118]In the absence of a single indicator for a particular life processa combination of the expression patterns of more than onesubstance can be used for a purposeful analysis as in thequadruple test where levels of four blood constituents (alpha-fetoprotein human chorionic gonadotropin unconjugatedoestriol and inhibin-A) have been used to predict theprobability of Downrsquos syndrome in babies [119] A pregnancy
detection test on the same lines as the human quadruple testcan be tried where instead of one we can consider proteinprofiles of more proteins This approach too will require athorough analysis of the bovine proteome before such a testwith high accuracy is available to the livestock owners
4 Conclusion
Early pregnancy diagnosis is an important aspect for optimiz-ing dairy production yet none of the present day methodsqualifies as an ideal diagnostic due to limitations of accuracylater stages of applicability and requirement for elaborateinstrumentation and laboratory setup This warrants furtherresearch on developing novel early pregnancy diagnosticsfor livestock species Currently available state-of-the-artinstrumentation and proteomics techniques instil hope forfinding moleculesmdashexclusively related to intricate maternalmetabolic alterations necessary to align with physiology ofearly embryonic development and its signalling for maternalrecognition of pregnancy and continued survival Thoughthese techniques are still in their infancy in animal scienceresearch they hold great promise to address a long-awaitedbreakthrough in pregnancy diagnosis in livestock
Acknowledgment
The authors acknowledge their thanks to the National Fundfor Basic Strategic and Frontier Application Research inAgriculture (NFBSFARA) Indian Council of AgriculturalResearch India for the funding support
References
[1] ldquoCommittee on Reproductive Nomenclature Recommenda-tions for standardizing bovine reproductive termsrdquoThe CornellVeterinarian vol 62 no 2 pp 216ndash237 1972
[2] N Ayalon ldquoA review of embryonic mortality in cattlerdquo Journalof Reproduction and Fertility vol 54 no 2 pp 483ndash493 1978
[3] D Morris and M Diskin ldquoEffect of progesterone on embryosurvivalrdquo Animal vol 2 no 8 pp 1112ndash1119 2008
[4] E S E Hafez Reproduction in Farm Animalsedition Lea andFebiger Philadelphia Pa USA 6th edition 1993
[5] T A Cowie Pregnancy Diagnosis Tests A Review Common-wealthAgricultural Bureaux Joint Publication no 13 Common-wealth Agricultural Bureaux Oxford UK 1948
[6] WWisnicky and L E Cassida ldquoAmanual method for diagnosisof pregnancy in cattlerdquo Journal of the American VeterinaryMedical Association vol 113 p 451 1948
[7] R Zemjanis Diagnostic and Therapeutic Techniques in AnimalReproduction Williams and Wilkins Baltimore Md USA 2ndedition 1970
[8] G H Arthur D E Noakes H Pearson and T J ParkinsonldquoReproduction in the buffalordquo in Veterinary Reproduction andObstetrics WB Saunders London UK 1966
[9] O J Franco M Drost M-J Thatcher V M Shille and W WThatcher ldquoFetal survival in the cow after pregnancy diagnosisby palpation per rectumrdquoTheriogenology vol 27 no 4 pp 631ndash644 1987
The Scientific World Journal 7
[10] R K Sharma J K Singh S K Phulia S Khanna and I SinghldquoFetal sex determination with ultrasonography in buffaloesrdquoIndian Veterinary Journal vol 88 no 10 pp 105ndash107 2011
[11] S Curran R A Pierson and O J Ginther ldquoUltrasonographicappearance of the bovine conceptus from days 20 through 60rdquoJournal of the American VeterinaryMedical Association vol 189no 10 pp 1295ndash1302 1986
[12] PM Fricke ldquoScanning the futuremdashultrasonography as a repro-ductive management tool for dairy cattlerdquo Journal of Dairy Sci-ence vol 85 no 8 pp 1918ndash1926 2002
[13] L G Paisley W D Mickelsen and O L Frost ldquoA survey of theincidence of prenatal mortality in cattle following pregnancydiagnosis by rectal palpationrdquoTheriogenology vol 9 no 6 pp481ndash491 1978
[14] D Vaillancourt C J Bierschwal D Ogwu et al ldquoCorrelationbetween pregnancy diagnosis bymembrane slip and embryonicmortalityrdquo Journal of the American Veterinary Medical Associa-tion vol 175 no 5 pp 466ndash468 1979
[15] A Ali and S Fahmy ldquoUltrasonographic fetometry and deter-mination of fetal sex in buffaloes (Bubalus bubalis)rdquo AnimalReproduction Science vol 106 no 1-2 pp 90ndash99 2008
[16] C H Pawshe K B C Appa Rao and S M Totey ldquoUltrasono-graphic imaging to monitor early pregnancy and embryonicdevelopment in the buffalo (Bubalus bubalis)rdquo Theriogenologyvol 41 no 3 pp 697ndash709 1994
[17] M R Bhosreker and IMHangarge ldquoUltrasonography for earlypregnancy diagnosis in buffaloesrdquo Indian Journal of AnimalReproduction vol 21 pp 143ndash144 2000
[18] M C Pieterse O Szenci A H Willemse C S A Bajcsy S JDieleman andM AM Taverne ldquoEarly pregnancy diagnosis incattle bymeans of linear-array real-time ultrasound scanning ofthe uterus and a qualitative and quantitative milk progesteronetestrdquoTheriogenology vol 33 no 3 pp 697ndash707 1990
[19] D P Nation J Malmo G M Davis and K L MacmillanldquoAccuracy of bovine pregnancy detection using transrectalultrasonography at 28 to 35 days after inseminationrdquoAustralianVeterinary Journal vol 81 no 1-2 pp 63ndash65 2003
[20] O Szenci J F Beckers P Humblot et al ldquoComparison of ultra-sonography bovine pregnancy-specific protein B and bovinepregnancy-associated glycoprotein 1 tests for pregnancy detec-tion in dairy cowsrdquo Theriogenology vol 50 no 1 pp 77ndash881998
[21] M Shemesh N Ayalon and H R Lindner ldquoEarly pregnancydiagnosis based upon plasma progesterone levels in the cow andewerdquo Journal of Animal Science vol 36 no 4 pp 726ndash729 1973
[22] J A Laing and R B Heap ldquoThe concentration of progesteronein themilk of cows during the reproductive cyclerdquo British Veter-inary Journal vol 127 no 8 pp 19ndash22 1971
[23] A A Zaied C J Bierschwal R G Elmore R S Youngquist AJ Sharp and H A Garverick ldquoConcentrations of progesteroneinmilk as amonitor of early pregnancy diagnosis in dairy cowsrdquoTheriogenology vol 12 no 1 pp 3ndash11 1979
[24] R Simersky J Swaczynova D A Morris M Franek and MStrnad ldquoDevelopment of an ELISA-based kit for the on-farmdetermination of progesterone in milkrdquo Veterinarni Medicinavol 52 no 1 pp 19ndash28 2007
[25] M Kamboj and B S Prakash ldquoRelationship of progesterone inplasma and whole milk of buffaloes during cyclicity and earlypregnancyrdquo Tropical Animal Health and Production vol 25 no3 pp 185ndash192 1993
[26] S K Batra R C Arora N K Bachlaus andR S Pandey ldquoBloodand milk progesterone in pregnant and nonpregnant buffalordquoJournal of Dairy Science vol 62 no 9 pp 1390ndash1393 1979
[27] BM Perera N Pathiraja S A AbeywardenaM XMotha andH Abeygunawardena ldquoEarly pregnancy diagnosis in buffaloesfrom plasma progesterone concentrationrdquo Veterinary Recordvol 106 no 5 pp 104ndash106 1980
[28] J A Pennington L H Schultz and W F Hoffman ldquoCompari-son of pregnancy diagnosis by milk progesterone on day 21 andday 24 postbreeding field study in dairy cattlerdquo Journal of DairyScience vol 68 no 10 pp 2740ndash2745 1985
[29] A Singh and R Puthiyandy ldquoEstimation of progesterone inbuffalomilk and its application to pregnancy diagnosisrdquo Journalof Reproduction and Fertility vol 59 no 1 pp 89ndash93 1980
[30] N Isobe M Akita T Nakao H Yamashiro and H KubotaldquoPregnancy diagnosis based on the fecal progesterone concen-tration in beef and dairy heifers and beef cowsrdquo Animal Repro-duction Science vol 90 no 3-4 pp 211ndash218 2005
[31] T E Spencer N H Ing T L Ott et al ldquoIntrauterine injectionof ovine interferon-120591 alters oestrogen receptor and oxytocinreceptor expression in the endometrium of cyclic ewesrdquo Journalof Molecular Endocrinology vol 15 no 2 pp 203ndash220 1995
[32] J J Kaneko J W Harvey andM L Bruss Clinical Biochemistryof Domestic Animals Academic Press AmsterdamTheNether-lands 2008
[33] T J Parkinson A Turvey and L J Jenner ldquoA morphometricanalysis of the corpus luteumof the cowduring the estrous cycleand early pregnancyrdquo Theriogenology vol 41 no 5 pp 1115ndash1126 1994
[34] R G Sasser and C A Ruder ldquoDetection of early pregnancy indomestic ruminantsrdquo Journal of Reproduction and Fertility Sup-plement vol 34 pp 261ndash271 1987
[35] M Shemesh N Ayalon and H R Lindner ldquoEarly pregnancydiagnosis based upon plasma progesterone levels in the cow andewerdquo Journal of Animal Science vol 36 no 4 pp 726ndash729 1973
[36] E Wolf G J Arnold S Bauersachs et al ldquoEmbryo-maternalcommunication in bovinemdashstrategies for deciphering a com-plex cross-talkrdquo Reproduction in Domestic Animals vol 38 no4 pp 276ndash289 2003
[37] E W Gowan R J Etches C Bryden and G J King ldquoFactorsaffecting accuracy of pregnancy diagnosis in cattlerdquo Journal ofDairy Science vol 65 no 7 pp 1294ndash1302 1982
[38] R L Nebel W D Whittier B G Cassell and J H Britt ldquoCom-parison of on-farm laboratory milk progesterone assays foridentifying errors in detection of estrus and diagnosis of preg-nancyrdquo Journal of Dairy Science vol 70 no 7 pp 1471ndash14761987
[39] T H Wimpy C F Chang V L Estergreen and J K HillersldquoMilk progesterone enzyme immunoassay modifications anda field trial for pregnancy detection in dairy cowsrdquo Journal ofDairy Science vol 69 no 4 pp 1115ndash1121 1986
[40] R M Eley WWThatcher and F W Bazer ldquoLuteolytic effect ofoestrone sulphate on cyclic beef heifersrdquo Journal of Reproductionand Fertility vol 55 no 1 pp 191ndash193 1979
[41] H A Robertson and G J King ldquoConjugated and unconjugatedoestrogens in fetal and maternal fluids of the cow throughoutpregnancyrdquo Journal of Reproduction and Fertility vol 55 no 2pp 463ndash470 1979
[42] R B Heap and M Hamon ldquoOestrone sulphate in milk as anindicator of a viable conceptus in cowsrdquo British Veterinary Jour-nal vol 135 no 4 pp 355ndash363 1979
8 The Scientific World Journal
[43] M Hamon I R Fleet R J Holdsworth and R B Heap ldquoThetime of detection of oestrone sulphate in milk and the diagnosisof pregnancy in cowsrdquo British Veterinary Journal vol 137 no 1pp 71ndash77 1981
[44] M Hirako T Takahashi and I Domeki ldquoPeripheral changes inestrone sulfate concentration during the first trimester of ges-tation in cattle comparison with unconjugated estrogens andrelationship to fetal numberrdquo Theriogenology vol 57 no 7 pp1939ndash1947 2002
[45] B S Prakash and M L Madan ldquoInfluence of gestation onoestrone sulphate concentration in milk of zebu and crossbredcows and murrah buffaloesrdquo Tropical Animal Health and Pro-duction vol 25 no 2 pp 94ndash100 1993
[46] F Lobago M Bekana H Gustafsson et al ldquoSerum profilesof pregnancy-associated glycoprotein oestrone sulphate andprogesterone during gestation and some factors influencing theprofiles in ethiopian borana and crossbred cattlerdquo Reproductionin Domestic Animals vol 44 no 4 pp 685ndash692 2009
[47] S Aschheim and B Zondek ldquoHypophysenvorderlappenhor-mon undOvarialhormon imHarn von SchwangerenrdquoKlinischeWochenschrift vol 6 no 28 p 1322 1927
[48] J R Marshall C B Hammond G T Ross A Jacobson PRayford andW D Odell ldquoPlasma and urinary chorionic gona-dotropin during early human pregnancyrdquo Obstetrics and Gyne-cology vol 32 no 6 pp 760ndash764 1968
[49] K Imakawa R V Anthony M Kazemi K R Marotti H GPolites and R M Roberts ldquoInterferon-like sequence of ovinetrophoblast protein secreted by embryonic trophectodermrdquoNature vol 330 no 6146 pp 377ndash379 1987
[50] A C Cavanagh ldquoIdentification of early pregnancy factor aschaperonin 10 implications for understanding its rolerdquo Reviewsof Reproduction vol 1 no 1 pp 28ndash32 1996
[51] H Morton V Hegh and G J A Clunie ldquoStudies of the rosetteinhibition test in pregnant mice evidence of immunosuppres-sionrdquo Proceedings of the Royal Society B vol 193 no 1113 pp413ndash419 1976
[52] H Morton B E Rolfe and A C Cavanagh ldquoOvum factor andearly pregnancy factorrdquo Current Topics in Developmental Biol-ogy vol 23 pp 73ndash92 1987
[53] H Morton A C Cavanagh S Athanasas-Platsis K A QuinnandB E Rolfe ldquoEarly pregnancy factor has immunosuppressiveand growth factor propertiesrdquo Reproduction Fertility andDevelopment vol 4 no 4 pp 411ndash422 1992
[54] H Morton V Hegh and G J A Clunie ldquoImmunosuppressiondetected in pregnantmice by rosette inhibition testrdquoNature vol249 no 5456 pp 459ndash460 1974
[55] V G Laleh R G Laleh N Pirany and M M Ahrabi ldquoMea-surement of EPF for detection of cow pregnancy using rosetteinhibition testrdquoTheriogenology vol 70 no 1 pp 105ndash107 2008
[56] H Qin and Z Q Zheng ldquoDetection of early pregnancy factorin human serardquo American Journal of Reproductive Immunologyand Microbiology vol 13 no 1 pp 15ndash18 1987
[57] Y C Smart T K Roberts I S Fraser A W Cripps and R LClancy ldquoValidation of the rosette inhibition test for the detec-tion of early pregnancy in womenrdquo Fertility and Sterility vol 37no 6 pp 779ndash785 1982
[58] HMorton G J A Clunie and F D Shaw ldquoA test for early preg-nancy in sheeprdquo Research in Veterinary Science vol 26 no 2pp 261ndash262 1979
[59] S Wilson R McCarthy and F Clarke ldquoIn search of early preg-nancy factor isolation of active polypeptides from pregnant
ewesrsquo serardquo Journal of Reproductive Immunology vol 5 no 5 pp275ndash286 1983
[60] M C Cordoba R Sartori and P M Fricke ldquoAssessment of acommercially available early conception factor (ECF) test fordetermining pregnancy status of dairy cattlerdquo Journal of DairyScience vol 84 no 8 pp 1884ndash1889 2001
[61] B GandyW Tucker P Ryan et al ldquoEvaluation of the early con-ception factor (ECFŮ) test for the detection of nonpregnancyin dairy cattlerdquoTheriogenology vol 56 no 4 pp 637ndash647 2001
[62] C S Whisnant L A Pagels and M G Daves ldquoCase studyeffectiveness of a commercial early conception factor test for usein cattlerdquo Professional Animal Scientist vol 17 pp 51ndash53 2001
[63] A S Grewal A L C Wallace Y S Pan et al ldquoEvaluation of arosette inhibition test for pregnancy diagnosis in pigsrdquo Journalof Reproductive Immunology vol 7 no 2 pp 129ndash138 1985
[64] K Ohnuma I T O Kazuei Y-I Miyake J Takahashi andY Yasuda ldquoDetection of early pregnancy factor (EPF) in mareserardquo Journal of Reproduction and Development vol 42 no 1pp 23ndash28 1996
[65] Y P Cruz L Selwood H Morton and A C CavanaghldquoSignificance of serum early pregnancy factor concentrationsduring pregnancy and embryonic development in Sminthopsismacroura (Spencer) (Marsupialia Dasyuridae)rdquo Reproductionvol 121 no 6 pp 933ndash939 2001
[66] S Chander Studies on certain immunological aspects of preg-nancy diagnosis in buffaloes [PhD thesis] Chaudhary CharanSingh Haryana Agricultural University Hisar India 1983
[67] W R Threlfall ldquoImmunosuppressive early pregnancy factor(ISEPF) determination for pregnancy diagnosis in dairy cowsrdquoTheriogenology vol 41 p 31 1994
[68] H Morton ldquoEarly pregnancy factor an extracellular chaper-onin 10 homologuerdquo Immunology andCell Biology vol 76 no 6pp 483ndash496 1998
[69] R M Moor and L E A Rowson ldquoInfluence of the embryo anduterus on luteal function in the sheeprdquoNature vol 201 no 4918pp 522ndash523 1964
[70] J D Godkin FW Bazer and RM Roberts ldquoOvine trophoblastprotein 1 an early secreted blastocyst protein binds specifi-cally to uterine endometrium and affects protein synthesisrdquoEndocrinology vol 114 no 1 pp 120ndash130 1984
[71] FW Bazer ldquoMediators of maternal recognition of pregnancy inmammalsrdquo Proceedings of the Society for Experimental Biologyand Medicine vol 199 no 4 pp 373ndash384 1992
[72] R M Roberts D W Leaman and J C Cross ldquoRole of interfer-ons in maternal recognition of pregnancy in ruminantsrdquo Pro-ceedings of the Society for Experimental Biology and Medicinevol 200 no 1 pp 7ndash18 1992
[73] R M Roberts ldquoInterferon-tau a type 1 interferon involvedin maternal recognition of pregnancyrdquo Cytokine and GrowthFactor Reviews vol 18 no 5-6 pp 403ndash408 2007
[74] G E Mann G E Lamming R S Robinson and D C WathesldquoThe regulation of interferon-tau production and uterine hor-mone receptors during early pregnancyrdquo Journal of Reproduc-tion and Fertility Supplement vol 54 pp 317ndash328 1999
[75] R M Roberts A D Ealy A P Alexenko C-S Han and TEzashi ldquoTrophoblast interferonsrdquo Placenta vol 20 no 4 pp259ndash264 1999
[76] WWThatcher M DMeyer and G Danet-Desnoyers ldquoMater-nal recognition of pregnancyrdquo Journal of Reproduction andFertility Supplement vol 49 pp 15ndash28 1995
The Scientific World Journal 9
[77] F W Bazer T E Spencer and T L Ott ldquoInterferon tau anovel pregnancy recognition signalrdquoAmerican Journal of Repro-ductive Immunology vol 37 no 6 pp 412ndash420 1997
[78] T E Spencer and F W Bazer ldquoOvine interferon tau suppressestranscription of the estrogen receptor and oxytocin receptorgenes in the ovine endometriumrdquo Endocrinology vol 137 no3 pp 1144ndash1147 1996
[79] C W Xiao B D Murphy J Sirois and A K GoffldquoDown-regulation of oxytocin-induced cyclooxygenase-2 andprostaglandin F synthase expression by interferon-120591 in bovineendometrial cellsrdquo Biology of Reproduction vol 60 no 3 pp656ndash663 1999
[80] T E Spencer N H Ing T L Ott et al ldquoIntrauterine injectionof ovine interferon-120591 alters oestrogen receptor and oxytocinreceptor expression in the endometrium of cyclic ewesrdquo Journalof Molecular Endocrinology vol 15 no 2 pp 203ndash220 1995
[81] J F Oliveira L E Henkes R L Ashley et al ldquoExpression ofinterferon (IFN)-stimulated genes in extrauterine tissues duringearly pregnancy in sheep is the consequence of endocrine IFN-120591 release from the uterine veinrdquo Endocrinology vol 149 no 3pp 1252ndash1259 2008
[82] C A Gifford K Racicot D S Clark et al ldquoRegulation ofinterferon-stimulated genes in peripheral blood leukocytes inpregnant and bred nonpregnant dairy cowsrdquo Journal of DairyScience vol 90 no 1 pp 274ndash280 2007
[83] H Han K J Austin L A Rempel and T R Hansen ldquoLowblood ISG15 mRNA and progesterone levels are predictive ofnon-pregnant dairy cowsrdquo Journal of Endocrinology vol 191 no2 pp 505ndash512 2006
[84] J C Green C S Okamura S E Poock and M C Lucy ldquoMea-surement of interferon-tau (IFN-120591) stimulated gene expressionin blood leukocytes for pregnancy diagnosis within 18-20d afterinsemination in dairy cattlerdquo Animal Reproduction Science vol121 no 1-2 pp 24ndash33 2010
[85] N Forde F Carter T E Spencer et al ldquoConceptus-inducedchanges in the endometrial transcriptome how soon does thecow know she is pregnantrdquo Biology of Reproduction vol 85 no1 pp 144ndash156 2011
[86] K Kizaki A Shichijo-Kizaki T Furusawa T Takahashi MHosoe andKHashizume ldquoDifferential neutrophil gene expres-sion in early bovine pregnancyrdquoReproductive Biology and Endo-crinology vol 11 article 6 2013
[87] F B P Wooding and D C Wathes ldquoBinucleate cell migrationin the bovine placentomerdquo Journal of Reproduction and Fertilityvol 59 no 2 pp 425ndash430 1980
[88] F W Bazer W W Thatcher P J Hansen M A Mirando TL Ott and C Plante ldquoPhysiological mechanisms of pregnancyrecognition in ruminantsrdquo Journal of Reproduction and FertilitySupplement vol 43 pp 39ndash47 1991
[89] A P Zoli P Demez J F Beckers M Reznik and A BeckersldquoLight and electron microscopic immunolocalization of bovinepregnancy-associated glycoprotein in the bovine placentomerdquoBiology of Reproduction vol 46 no 4 pp 623ndash629 1992
[90] J E Butler W C Hamilton R G Sasser C A Ruder G MHass andR JWilliams ldquoDetection and partial characterizationof two bovine pregnancy-specific proteinsrdquoBiology of Reproduc-tion vol 26 no 5 pp 925ndash933 1982
[91] A P Zoli J-F Beckers P Wouters-Ballman J Closset PFalmagne and F Ectors ldquoPurification and characterization of abovine pregnancy-associated glycoproteinrdquo Biology of Repro-duction vol 45 no 1 pp 1ndash10 1991
[92] S Xie B G Low R J Nagel et al ldquoIdentification of the majorpregnancy-specific antigens of cattle and sheep as inactivemembers of the aspartic proteinase familyrdquo Proceedings of theNational Academy of Sciences of theUnited States of America vol88 no 22 pp 10247ndash10251 1991
[93] S Xie B G Low R J Nagel J-F Beckers and R M RobertsldquoA novel glycoprotein of the aspartic proteinase gene familyexpressed in bovine placental trophectodermrdquoBiology of Repro-duction vol 51 no 6 pp 1145ndash1153 1994
[94] F Constanta S Camousa P Chavatte-Palmera et al ldquoAlteredsecretion of pregnancy-associated glycoproteins during gesta-tion in bovine somatic clonesrdquoTheriogenology vol 76 pp 1006ndash1021 2011
[95] M M Mialon S Camous G Renand J Martal and FMenissier ldquoPeripheral concentrations of a 60-kDa pregnancyserum protein during gestation and after calving and in rela-tionship to embryonic mortality in cattlerdquo Reproduction Nutri-tion Development vol 33 no 3 pp 269ndash282 1993
[96] JMartal N Chene S Camous et al ldquoRecent developments andpotentialities for reducing embryo mortality in ruminants therole of IFN-120591 and other cytokines in early pregnancyrdquo Repro-duction Fertility and Development vol 9 no 3 pp 355ndash3801997
[97] J A Green S Xie X Quan et al ldquoPregnancy-associated bovineand ovine glycoproteins exhibit spatially and temporally dis-tinct expression patterns during pregnancyrdquo Biology of Repro-duction vol 62 no 6 pp 1624ndash1631 2000
[98] S Xie J Green J B Bixby et al ldquoThe diversity and evolutionaryrelationships of the pregnancy-associated glycoproteins anaspartic proteinase subfamily consisting of many trophoblast-expressed genesrdquo Proceedings of the National Academy of Sci-ences of the United States of America vol 94 no 24 pp 12809ndash12816 1997
[99] K Klisch A Boos M Friedrich et al ldquoThe glycosylationof pregnancy-associated glycoproteins and prolactin-relatedprotein-I in bovine binucleate trophoblast giant cells changesbefore parturitionrdquo Reproduction vol 132 no 5 pp 791ndash7982006
[100] B P V L Telugu A M Walker and J A Green ldquoCharacter-ization of the bovine pregnancy-associated glycoprotein genefamilymdashanalysis of gene sequences regulatory regions withinthe promoter and expression of selected genesrdquo BMCGenomicsvol 10 article 185 pp 1ndash17 2009
[101] P Humblot S Camous J Martal et al ldquoDiagnosis of pregnancyby radioimmunoassay of a pregnancy-specific protein in theplasma of dairy cowsrdquo Theriogenology vol 30 no 2 pp 257ndash267 1988
[102] G H Kiracofe J M Wright R R Schalles C A Ruder SParish and R G Sasser ldquoPregnancy-specific protein B in serumof postpartum beef cowsrdquo Journal of Animal Science vol 71 no8 pp 2199ndash2205 1993
[103] A P Zoli L A Guilbault P Delahaut W B Ortiz and J-FBeckers ldquoRadioimmunoassay of a bovine pregnancy-associatedglycoprotein in serum its application for pregnancy diagnosisrdquoBiology of Reproduction vol 46 no 1 pp 83ndash92 1992
[104] R G Sasser C A Ruder K A Ivani J E Butler and W CHamilton ldquoDetection of pregnancy by radioimmunoassay of anovel pregnancy-specific protein in serum of cows and a profileof serum concentrations during gestationrdquo Biology of Reproduc-tion vol 35 no 4 pp 936ndash942 1986
10 The Scientific World Journal
[105] J A Green T E Parks M P Avalle et al ldquoThe establishmentof an ELISA for the detection of pregnancy-associated glyco-proteins (PAGs) in the serum of pregnant cows and heifersrdquoTheriogenology vol 63 no 5 pp 1481ndash1503 2005
[106] E Silva R A Sterry D Kolb et al ldquoAccuracy of a pregnancy-associated glycoprotein ELISA to determine pregnancy statusof lactating dairy cows twenty-seven days after timed artificialinseminationrdquo Journal of Dairy Science vol 90 no 10 pp 4612ndash4622 2007
[107] A Ayad N M Sousa J Sulon J L Hornick M Iguer-Ouada and J F Beckers ldquoCorrelation of five radioimmunoas-say systems for measurement of bovine plasma pregnancy-associated glycoprotein concentrations at early pregnancyperiodrdquo Research in Veterinary Science vol 86 no 3 pp 377ndash382 2009
[108] N M de Sousa M Zongo W Pitala et al ldquoPregnancy-associated glycoprotein concentrations during pregnancy andthe postpartum period in Azawak Zebu cattlerdquoTheriogenologyvol 59 no 5-6 pp 1131ndash1142 2003
[109] A Karen S Darwish A Ramoun et al ldquoAccuracy of ultra-sonography and pregnancy-associated glycoprotein test forpregnancy diagnosis in buffaloesrdquoTheriogenology vol 68 no 8pp 1150ndash1155 2007
[110] N M Sousa A Ayad J F Beckers and Z Gajewski ldquoPreg-nancy-associated glycoproteins (PAG) as pregnancy markers inthe ruminantsrdquo Journal of Physiology and Pharmacology vol 57no 8 pp 153ndash171 2006
[111] P R Graves andTA J Haystead ldquoMolecular biologistrsquos guide toproteomicsrdquo Microbiology and Molecular Biology Reviews vol66 no 1 pp 39ndash63 2002
[112] J D Lippolis and T A Reinhardt ldquoCentennial paper pro-teomics in animal sciencerdquo Journal of Animal Science vol 86no 9 pp 2430ndash2441 2008
[113] D A Colantonio and D W Chan ldquoThe clinical application ofproteomicsrdquo Clinica Chimica Acta vol 357 no 2 pp 151ndash1582005
[114] F di Girolamo E Boschetti M C M Chung F Guadagniand P G Righetti ldquolsquoProteomineeringrsquo or not The debate onbiomarker discovery in sera continuesrdquo Journal of Proteomicsvol 74 no 5 pp 589ndash594 2011
[115] D I Jin H R Lee H R KimH J Lee J T Yoon andC S ParkldquoProteomics analysis of pregnancy-specific serum proteins inbovinerdquoReproduction Fertility andDevelopment vol 18 no 1-2p 183 2005
[116] R Han H R Kim K Naruse et al ldquoIdentification of bovinepregnancy-specific milk proteins using proteomicsrdquo Biology ofReproduction vol 78 p 143 2008
[117] A K Balhara Proteomic analysis of pregnancy-specific serumproteins in buffalo [PhD thesis] LLRUVAS Hisar India 2012
[118] A Busch S Michel C Hoppe D Driesch U Claussen and Fvon Eggeling ldquoProteome analysis of maternal serum samplesfor trisomy 21 pregnancies using ProteinChip arrays and bioin-formaticsrdquo Journal of Histochemistry and Cytochemistry vol 53no 3 pp 341ndash343 2005
[119] P A Benn ldquoAdvances in prenatal screening for Down syn-drome I General principles and second trimester testingrdquoClinica Chimica Acta vol 323 no 1-2 pp 1ndash16 2002
and thereby interfere in the proteomic analysis (reviewed by[113]) Conversely it is the low abundance proteins whichhave the highest prospect of being the novel biomarkersof changes in internal milieu of body To sort the problemof high abundance proteins two approaches are suggestedremoval of abundant proteins (usually by immunoaffinity)and concentration of the low abundancescarce proteins withsimultaneous removal of high abundance proteins techni-cally known as combinatorial peptide ligand libraries CPLL[114] Commercially available ProteoMiner kit fromMs Bio-Rad is CPLL based Both approaches however lead to loss ofa significant portion of the low abundance proteins along thehigh abundance proteins yet the later approach is preferred[114]
There is limited information on the bovine proteomein relation to pregnancy Jin et al [115] performed pro-teomics analysis using blood serum samples of pregnantand nonpregnant Holstein dairy cattle at 21 and 35 daysafter AI and reported composite profiles of key proteinsinvolved in early pregnancy and suggested the potential use ofidentified proteins to detect early pregnancy in bovinesTheseincluded nine pregnancy-specific spots in day 21 and day 35serum samples Pregnancy-specific proteins were identifiedas transferrin albumin IgG2a heavy chain constant regionand immunoglobulin gamma heavy chain variable regionFurther differential proteomic analysis of milk samples frompregnant and nonpregnant cows revealed 16 protein spots 14pregnancy specific and 2 spots downregulated in the pregnantmilk sample [116] Pregnancy-specific proteins were identi-fied as serum albumin precursor IgG1 heavy chain constantregion conglutinin precursor epithelial keratin 10 and kelch-like ECH-associated protein Though some identified spotswere abundant milk or serum proteins their molecularweights and pI values were different from main milk orserum proteinsThismay suggest that these proteins could bepregnancy-specific subunits or fragments of albumin and IgGor carrying differentially expressed small proteins whichmayultimately have potential for pregnancy detection
Encouragingly enough these studies need further inves-tigations for arriving at some sort of pregnancy detectionmethod Preliminary studies in buffalo cows on blood pro-teome too detected significant changes in many proteins in2DE gels [117] Important proteins found on MS analysis ofthese were synaptojanin-1 apolipoprotein A-1 apolipopro-tein B Keratin 10 and Von Willebrand factors which aredocumented to have a role in embryogenesis and earlypregnancy
Data generated out of sequential blood proteome analysisduring pregnancy can have several other applications as wellfor example studying fetal viability genetic disorders and soforth Trisomy 21 pregnancies can be detectedwith high accu-racy by maternal serum proteomic analysis in humans [118]In the absence of a single indicator for a particular life processa combination of the expression patterns of more than onesubstance can be used for a purposeful analysis as in thequadruple test where levels of four blood constituents (alpha-fetoprotein human chorionic gonadotropin unconjugatedoestriol and inhibin-A) have been used to predict theprobability of Downrsquos syndrome in babies [119] A pregnancy
detection test on the same lines as the human quadruple testcan be tried where instead of one we can consider proteinprofiles of more proteins This approach too will require athorough analysis of the bovine proteome before such a testwith high accuracy is available to the livestock owners
4 Conclusion
Early pregnancy diagnosis is an important aspect for optimiz-ing dairy production yet none of the present day methodsqualifies as an ideal diagnostic due to limitations of accuracylater stages of applicability and requirement for elaborateinstrumentation and laboratory setup This warrants furtherresearch on developing novel early pregnancy diagnosticsfor livestock species Currently available state-of-the-artinstrumentation and proteomics techniques instil hope forfinding moleculesmdashexclusively related to intricate maternalmetabolic alterations necessary to align with physiology ofearly embryonic development and its signalling for maternalrecognition of pregnancy and continued survival Thoughthese techniques are still in their infancy in animal scienceresearch they hold great promise to address a long-awaitedbreakthrough in pregnancy diagnosis in livestock
Acknowledgment
The authors acknowledge their thanks to the National Fundfor Basic Strategic and Frontier Application Research inAgriculture (NFBSFARA) Indian Council of AgriculturalResearch India for the funding support
References
[1] ldquoCommittee on Reproductive Nomenclature Recommenda-tions for standardizing bovine reproductive termsrdquoThe CornellVeterinarian vol 62 no 2 pp 216ndash237 1972
[2] N Ayalon ldquoA review of embryonic mortality in cattlerdquo Journalof Reproduction and Fertility vol 54 no 2 pp 483ndash493 1978
[3] D Morris and M Diskin ldquoEffect of progesterone on embryosurvivalrdquo Animal vol 2 no 8 pp 1112ndash1119 2008
[4] E S E Hafez Reproduction in Farm Animalsedition Lea andFebiger Philadelphia Pa USA 6th edition 1993
[5] T A Cowie Pregnancy Diagnosis Tests A Review Common-wealthAgricultural Bureaux Joint Publication no 13 Common-wealth Agricultural Bureaux Oxford UK 1948
[6] WWisnicky and L E Cassida ldquoAmanual method for diagnosisof pregnancy in cattlerdquo Journal of the American VeterinaryMedical Association vol 113 p 451 1948
[7] R Zemjanis Diagnostic and Therapeutic Techniques in AnimalReproduction Williams and Wilkins Baltimore Md USA 2ndedition 1970
[8] G H Arthur D E Noakes H Pearson and T J ParkinsonldquoReproduction in the buffalordquo in Veterinary Reproduction andObstetrics WB Saunders London UK 1966
[9] O J Franco M Drost M-J Thatcher V M Shille and W WThatcher ldquoFetal survival in the cow after pregnancy diagnosisby palpation per rectumrdquoTheriogenology vol 27 no 4 pp 631ndash644 1987
The Scientific World Journal 7
[10] R K Sharma J K Singh S K Phulia S Khanna and I SinghldquoFetal sex determination with ultrasonography in buffaloesrdquoIndian Veterinary Journal vol 88 no 10 pp 105ndash107 2011
[11] S Curran R A Pierson and O J Ginther ldquoUltrasonographicappearance of the bovine conceptus from days 20 through 60rdquoJournal of the American VeterinaryMedical Association vol 189no 10 pp 1295ndash1302 1986
[12] PM Fricke ldquoScanning the futuremdashultrasonography as a repro-ductive management tool for dairy cattlerdquo Journal of Dairy Sci-ence vol 85 no 8 pp 1918ndash1926 2002
[13] L G Paisley W D Mickelsen and O L Frost ldquoA survey of theincidence of prenatal mortality in cattle following pregnancydiagnosis by rectal palpationrdquoTheriogenology vol 9 no 6 pp481ndash491 1978
[14] D Vaillancourt C J Bierschwal D Ogwu et al ldquoCorrelationbetween pregnancy diagnosis bymembrane slip and embryonicmortalityrdquo Journal of the American Veterinary Medical Associa-tion vol 175 no 5 pp 466ndash468 1979
[15] A Ali and S Fahmy ldquoUltrasonographic fetometry and deter-mination of fetal sex in buffaloes (Bubalus bubalis)rdquo AnimalReproduction Science vol 106 no 1-2 pp 90ndash99 2008
[16] C H Pawshe K B C Appa Rao and S M Totey ldquoUltrasono-graphic imaging to monitor early pregnancy and embryonicdevelopment in the buffalo (Bubalus bubalis)rdquo Theriogenologyvol 41 no 3 pp 697ndash709 1994
[17] M R Bhosreker and IMHangarge ldquoUltrasonography for earlypregnancy diagnosis in buffaloesrdquo Indian Journal of AnimalReproduction vol 21 pp 143ndash144 2000
[18] M C Pieterse O Szenci A H Willemse C S A Bajcsy S JDieleman andM AM Taverne ldquoEarly pregnancy diagnosis incattle bymeans of linear-array real-time ultrasound scanning ofthe uterus and a qualitative and quantitative milk progesteronetestrdquoTheriogenology vol 33 no 3 pp 697ndash707 1990
[19] D P Nation J Malmo G M Davis and K L MacmillanldquoAccuracy of bovine pregnancy detection using transrectalultrasonography at 28 to 35 days after inseminationrdquoAustralianVeterinary Journal vol 81 no 1-2 pp 63ndash65 2003
[20] O Szenci J F Beckers P Humblot et al ldquoComparison of ultra-sonography bovine pregnancy-specific protein B and bovinepregnancy-associated glycoprotein 1 tests for pregnancy detec-tion in dairy cowsrdquo Theriogenology vol 50 no 1 pp 77ndash881998
[21] M Shemesh N Ayalon and H R Lindner ldquoEarly pregnancydiagnosis based upon plasma progesterone levels in the cow andewerdquo Journal of Animal Science vol 36 no 4 pp 726ndash729 1973
[22] J A Laing and R B Heap ldquoThe concentration of progesteronein themilk of cows during the reproductive cyclerdquo British Veter-inary Journal vol 127 no 8 pp 19ndash22 1971
[23] A A Zaied C J Bierschwal R G Elmore R S Youngquist AJ Sharp and H A Garverick ldquoConcentrations of progesteroneinmilk as amonitor of early pregnancy diagnosis in dairy cowsrdquoTheriogenology vol 12 no 1 pp 3ndash11 1979
[24] R Simersky J Swaczynova D A Morris M Franek and MStrnad ldquoDevelopment of an ELISA-based kit for the on-farmdetermination of progesterone in milkrdquo Veterinarni Medicinavol 52 no 1 pp 19ndash28 2007
[25] M Kamboj and B S Prakash ldquoRelationship of progesterone inplasma and whole milk of buffaloes during cyclicity and earlypregnancyrdquo Tropical Animal Health and Production vol 25 no3 pp 185ndash192 1993
[26] S K Batra R C Arora N K Bachlaus andR S Pandey ldquoBloodand milk progesterone in pregnant and nonpregnant buffalordquoJournal of Dairy Science vol 62 no 9 pp 1390ndash1393 1979
[27] BM Perera N Pathiraja S A AbeywardenaM XMotha andH Abeygunawardena ldquoEarly pregnancy diagnosis in buffaloesfrom plasma progesterone concentrationrdquo Veterinary Recordvol 106 no 5 pp 104ndash106 1980
[28] J A Pennington L H Schultz and W F Hoffman ldquoCompari-son of pregnancy diagnosis by milk progesterone on day 21 andday 24 postbreeding field study in dairy cattlerdquo Journal of DairyScience vol 68 no 10 pp 2740ndash2745 1985
[29] A Singh and R Puthiyandy ldquoEstimation of progesterone inbuffalomilk and its application to pregnancy diagnosisrdquo Journalof Reproduction and Fertility vol 59 no 1 pp 89ndash93 1980
[30] N Isobe M Akita T Nakao H Yamashiro and H KubotaldquoPregnancy diagnosis based on the fecal progesterone concen-tration in beef and dairy heifers and beef cowsrdquo Animal Repro-duction Science vol 90 no 3-4 pp 211ndash218 2005
[31] T E Spencer N H Ing T L Ott et al ldquoIntrauterine injectionof ovine interferon-120591 alters oestrogen receptor and oxytocinreceptor expression in the endometrium of cyclic ewesrdquo Journalof Molecular Endocrinology vol 15 no 2 pp 203ndash220 1995
[32] J J Kaneko J W Harvey andM L Bruss Clinical Biochemistryof Domestic Animals Academic Press AmsterdamTheNether-lands 2008
[33] T J Parkinson A Turvey and L J Jenner ldquoA morphometricanalysis of the corpus luteumof the cowduring the estrous cycleand early pregnancyrdquo Theriogenology vol 41 no 5 pp 1115ndash1126 1994
[34] R G Sasser and C A Ruder ldquoDetection of early pregnancy indomestic ruminantsrdquo Journal of Reproduction and Fertility Sup-plement vol 34 pp 261ndash271 1987
[35] M Shemesh N Ayalon and H R Lindner ldquoEarly pregnancydiagnosis based upon plasma progesterone levels in the cow andewerdquo Journal of Animal Science vol 36 no 4 pp 726ndash729 1973
[36] E Wolf G J Arnold S Bauersachs et al ldquoEmbryo-maternalcommunication in bovinemdashstrategies for deciphering a com-plex cross-talkrdquo Reproduction in Domestic Animals vol 38 no4 pp 276ndash289 2003
[37] E W Gowan R J Etches C Bryden and G J King ldquoFactorsaffecting accuracy of pregnancy diagnosis in cattlerdquo Journal ofDairy Science vol 65 no 7 pp 1294ndash1302 1982
[38] R L Nebel W D Whittier B G Cassell and J H Britt ldquoCom-parison of on-farm laboratory milk progesterone assays foridentifying errors in detection of estrus and diagnosis of preg-nancyrdquo Journal of Dairy Science vol 70 no 7 pp 1471ndash14761987
[39] T H Wimpy C F Chang V L Estergreen and J K HillersldquoMilk progesterone enzyme immunoassay modifications anda field trial for pregnancy detection in dairy cowsrdquo Journal ofDairy Science vol 69 no 4 pp 1115ndash1121 1986
[40] R M Eley WWThatcher and F W Bazer ldquoLuteolytic effect ofoestrone sulphate on cyclic beef heifersrdquo Journal of Reproductionand Fertility vol 55 no 1 pp 191ndash193 1979
[41] H A Robertson and G J King ldquoConjugated and unconjugatedoestrogens in fetal and maternal fluids of the cow throughoutpregnancyrdquo Journal of Reproduction and Fertility vol 55 no 2pp 463ndash470 1979
[42] R B Heap and M Hamon ldquoOestrone sulphate in milk as anindicator of a viable conceptus in cowsrdquo British Veterinary Jour-nal vol 135 no 4 pp 355ndash363 1979
8 The Scientific World Journal
[43] M Hamon I R Fleet R J Holdsworth and R B Heap ldquoThetime of detection of oestrone sulphate in milk and the diagnosisof pregnancy in cowsrdquo British Veterinary Journal vol 137 no 1pp 71ndash77 1981
[44] M Hirako T Takahashi and I Domeki ldquoPeripheral changes inestrone sulfate concentration during the first trimester of ges-tation in cattle comparison with unconjugated estrogens andrelationship to fetal numberrdquo Theriogenology vol 57 no 7 pp1939ndash1947 2002
[45] B S Prakash and M L Madan ldquoInfluence of gestation onoestrone sulphate concentration in milk of zebu and crossbredcows and murrah buffaloesrdquo Tropical Animal Health and Pro-duction vol 25 no 2 pp 94ndash100 1993
[46] F Lobago M Bekana H Gustafsson et al ldquoSerum profilesof pregnancy-associated glycoprotein oestrone sulphate andprogesterone during gestation and some factors influencing theprofiles in ethiopian borana and crossbred cattlerdquo Reproductionin Domestic Animals vol 44 no 4 pp 685ndash692 2009
[47] S Aschheim and B Zondek ldquoHypophysenvorderlappenhor-mon undOvarialhormon imHarn von SchwangerenrdquoKlinischeWochenschrift vol 6 no 28 p 1322 1927
[48] J R Marshall C B Hammond G T Ross A Jacobson PRayford andW D Odell ldquoPlasma and urinary chorionic gona-dotropin during early human pregnancyrdquo Obstetrics and Gyne-cology vol 32 no 6 pp 760ndash764 1968
[49] K Imakawa R V Anthony M Kazemi K R Marotti H GPolites and R M Roberts ldquoInterferon-like sequence of ovinetrophoblast protein secreted by embryonic trophectodermrdquoNature vol 330 no 6146 pp 377ndash379 1987
[50] A C Cavanagh ldquoIdentification of early pregnancy factor aschaperonin 10 implications for understanding its rolerdquo Reviewsof Reproduction vol 1 no 1 pp 28ndash32 1996
[51] H Morton V Hegh and G J A Clunie ldquoStudies of the rosetteinhibition test in pregnant mice evidence of immunosuppres-sionrdquo Proceedings of the Royal Society B vol 193 no 1113 pp413ndash419 1976
[52] H Morton B E Rolfe and A C Cavanagh ldquoOvum factor andearly pregnancy factorrdquo Current Topics in Developmental Biol-ogy vol 23 pp 73ndash92 1987
[53] H Morton A C Cavanagh S Athanasas-Platsis K A QuinnandB E Rolfe ldquoEarly pregnancy factor has immunosuppressiveand growth factor propertiesrdquo Reproduction Fertility andDevelopment vol 4 no 4 pp 411ndash422 1992
[54] H Morton V Hegh and G J A Clunie ldquoImmunosuppressiondetected in pregnantmice by rosette inhibition testrdquoNature vol249 no 5456 pp 459ndash460 1974
[55] V G Laleh R G Laleh N Pirany and M M Ahrabi ldquoMea-surement of EPF for detection of cow pregnancy using rosetteinhibition testrdquoTheriogenology vol 70 no 1 pp 105ndash107 2008
[56] H Qin and Z Q Zheng ldquoDetection of early pregnancy factorin human serardquo American Journal of Reproductive Immunologyand Microbiology vol 13 no 1 pp 15ndash18 1987
[57] Y C Smart T K Roberts I S Fraser A W Cripps and R LClancy ldquoValidation of the rosette inhibition test for the detec-tion of early pregnancy in womenrdquo Fertility and Sterility vol 37no 6 pp 779ndash785 1982
[58] HMorton G J A Clunie and F D Shaw ldquoA test for early preg-nancy in sheeprdquo Research in Veterinary Science vol 26 no 2pp 261ndash262 1979
[59] S Wilson R McCarthy and F Clarke ldquoIn search of early preg-nancy factor isolation of active polypeptides from pregnant
ewesrsquo serardquo Journal of Reproductive Immunology vol 5 no 5 pp275ndash286 1983
[60] M C Cordoba R Sartori and P M Fricke ldquoAssessment of acommercially available early conception factor (ECF) test fordetermining pregnancy status of dairy cattlerdquo Journal of DairyScience vol 84 no 8 pp 1884ndash1889 2001
[61] B GandyW Tucker P Ryan et al ldquoEvaluation of the early con-ception factor (ECFŮ) test for the detection of nonpregnancyin dairy cattlerdquoTheriogenology vol 56 no 4 pp 637ndash647 2001
[62] C S Whisnant L A Pagels and M G Daves ldquoCase studyeffectiveness of a commercial early conception factor test for usein cattlerdquo Professional Animal Scientist vol 17 pp 51ndash53 2001
[63] A S Grewal A L C Wallace Y S Pan et al ldquoEvaluation of arosette inhibition test for pregnancy diagnosis in pigsrdquo Journalof Reproductive Immunology vol 7 no 2 pp 129ndash138 1985
[64] K Ohnuma I T O Kazuei Y-I Miyake J Takahashi andY Yasuda ldquoDetection of early pregnancy factor (EPF) in mareserardquo Journal of Reproduction and Development vol 42 no 1pp 23ndash28 1996
[65] Y P Cruz L Selwood H Morton and A C CavanaghldquoSignificance of serum early pregnancy factor concentrationsduring pregnancy and embryonic development in Sminthopsismacroura (Spencer) (Marsupialia Dasyuridae)rdquo Reproductionvol 121 no 6 pp 933ndash939 2001
[66] S Chander Studies on certain immunological aspects of preg-nancy diagnosis in buffaloes [PhD thesis] Chaudhary CharanSingh Haryana Agricultural University Hisar India 1983
[67] W R Threlfall ldquoImmunosuppressive early pregnancy factor(ISEPF) determination for pregnancy diagnosis in dairy cowsrdquoTheriogenology vol 41 p 31 1994
[68] H Morton ldquoEarly pregnancy factor an extracellular chaper-onin 10 homologuerdquo Immunology andCell Biology vol 76 no 6pp 483ndash496 1998
[69] R M Moor and L E A Rowson ldquoInfluence of the embryo anduterus on luteal function in the sheeprdquoNature vol 201 no 4918pp 522ndash523 1964
[70] J D Godkin FW Bazer and RM Roberts ldquoOvine trophoblastprotein 1 an early secreted blastocyst protein binds specifi-cally to uterine endometrium and affects protein synthesisrdquoEndocrinology vol 114 no 1 pp 120ndash130 1984
[71] FW Bazer ldquoMediators of maternal recognition of pregnancy inmammalsrdquo Proceedings of the Society for Experimental Biologyand Medicine vol 199 no 4 pp 373ndash384 1992
[72] R M Roberts D W Leaman and J C Cross ldquoRole of interfer-ons in maternal recognition of pregnancy in ruminantsrdquo Pro-ceedings of the Society for Experimental Biology and Medicinevol 200 no 1 pp 7ndash18 1992
[73] R M Roberts ldquoInterferon-tau a type 1 interferon involvedin maternal recognition of pregnancyrdquo Cytokine and GrowthFactor Reviews vol 18 no 5-6 pp 403ndash408 2007
[74] G E Mann G E Lamming R S Robinson and D C WathesldquoThe regulation of interferon-tau production and uterine hor-mone receptors during early pregnancyrdquo Journal of Reproduc-tion and Fertility Supplement vol 54 pp 317ndash328 1999
[75] R M Roberts A D Ealy A P Alexenko C-S Han and TEzashi ldquoTrophoblast interferonsrdquo Placenta vol 20 no 4 pp259ndash264 1999
[76] WWThatcher M DMeyer and G Danet-Desnoyers ldquoMater-nal recognition of pregnancyrdquo Journal of Reproduction andFertility Supplement vol 49 pp 15ndash28 1995
The Scientific World Journal 9
[77] F W Bazer T E Spencer and T L Ott ldquoInterferon tau anovel pregnancy recognition signalrdquoAmerican Journal of Repro-ductive Immunology vol 37 no 6 pp 412ndash420 1997
[78] T E Spencer and F W Bazer ldquoOvine interferon tau suppressestranscription of the estrogen receptor and oxytocin receptorgenes in the ovine endometriumrdquo Endocrinology vol 137 no3 pp 1144ndash1147 1996
[79] C W Xiao B D Murphy J Sirois and A K GoffldquoDown-regulation of oxytocin-induced cyclooxygenase-2 andprostaglandin F synthase expression by interferon-120591 in bovineendometrial cellsrdquo Biology of Reproduction vol 60 no 3 pp656ndash663 1999
[80] T E Spencer N H Ing T L Ott et al ldquoIntrauterine injectionof ovine interferon-120591 alters oestrogen receptor and oxytocinreceptor expression in the endometrium of cyclic ewesrdquo Journalof Molecular Endocrinology vol 15 no 2 pp 203ndash220 1995
[81] J F Oliveira L E Henkes R L Ashley et al ldquoExpression ofinterferon (IFN)-stimulated genes in extrauterine tissues duringearly pregnancy in sheep is the consequence of endocrine IFN-120591 release from the uterine veinrdquo Endocrinology vol 149 no 3pp 1252ndash1259 2008
[82] C A Gifford K Racicot D S Clark et al ldquoRegulation ofinterferon-stimulated genes in peripheral blood leukocytes inpregnant and bred nonpregnant dairy cowsrdquo Journal of DairyScience vol 90 no 1 pp 274ndash280 2007
[83] H Han K J Austin L A Rempel and T R Hansen ldquoLowblood ISG15 mRNA and progesterone levels are predictive ofnon-pregnant dairy cowsrdquo Journal of Endocrinology vol 191 no2 pp 505ndash512 2006
[84] J C Green C S Okamura S E Poock and M C Lucy ldquoMea-surement of interferon-tau (IFN-120591) stimulated gene expressionin blood leukocytes for pregnancy diagnosis within 18-20d afterinsemination in dairy cattlerdquo Animal Reproduction Science vol121 no 1-2 pp 24ndash33 2010
[85] N Forde F Carter T E Spencer et al ldquoConceptus-inducedchanges in the endometrial transcriptome how soon does thecow know she is pregnantrdquo Biology of Reproduction vol 85 no1 pp 144ndash156 2011
[86] K Kizaki A Shichijo-Kizaki T Furusawa T Takahashi MHosoe andKHashizume ldquoDifferential neutrophil gene expres-sion in early bovine pregnancyrdquoReproductive Biology and Endo-crinology vol 11 article 6 2013
[87] F B P Wooding and D C Wathes ldquoBinucleate cell migrationin the bovine placentomerdquo Journal of Reproduction and Fertilityvol 59 no 2 pp 425ndash430 1980
[88] F W Bazer W W Thatcher P J Hansen M A Mirando TL Ott and C Plante ldquoPhysiological mechanisms of pregnancyrecognition in ruminantsrdquo Journal of Reproduction and FertilitySupplement vol 43 pp 39ndash47 1991
[89] A P Zoli P Demez J F Beckers M Reznik and A BeckersldquoLight and electron microscopic immunolocalization of bovinepregnancy-associated glycoprotein in the bovine placentomerdquoBiology of Reproduction vol 46 no 4 pp 623ndash629 1992
[90] J E Butler W C Hamilton R G Sasser C A Ruder G MHass andR JWilliams ldquoDetection and partial characterizationof two bovine pregnancy-specific proteinsrdquoBiology of Reproduc-tion vol 26 no 5 pp 925ndash933 1982
[91] A P Zoli J-F Beckers P Wouters-Ballman J Closset PFalmagne and F Ectors ldquoPurification and characterization of abovine pregnancy-associated glycoproteinrdquo Biology of Repro-duction vol 45 no 1 pp 1ndash10 1991
[92] S Xie B G Low R J Nagel et al ldquoIdentification of the majorpregnancy-specific antigens of cattle and sheep as inactivemembers of the aspartic proteinase familyrdquo Proceedings of theNational Academy of Sciences of theUnited States of America vol88 no 22 pp 10247ndash10251 1991
[93] S Xie B G Low R J Nagel J-F Beckers and R M RobertsldquoA novel glycoprotein of the aspartic proteinase gene familyexpressed in bovine placental trophectodermrdquoBiology of Repro-duction vol 51 no 6 pp 1145ndash1153 1994
[94] F Constanta S Camousa P Chavatte-Palmera et al ldquoAlteredsecretion of pregnancy-associated glycoproteins during gesta-tion in bovine somatic clonesrdquoTheriogenology vol 76 pp 1006ndash1021 2011
[95] M M Mialon S Camous G Renand J Martal and FMenissier ldquoPeripheral concentrations of a 60-kDa pregnancyserum protein during gestation and after calving and in rela-tionship to embryonic mortality in cattlerdquo Reproduction Nutri-tion Development vol 33 no 3 pp 269ndash282 1993
[96] JMartal N Chene S Camous et al ldquoRecent developments andpotentialities for reducing embryo mortality in ruminants therole of IFN-120591 and other cytokines in early pregnancyrdquo Repro-duction Fertility and Development vol 9 no 3 pp 355ndash3801997
[97] J A Green S Xie X Quan et al ldquoPregnancy-associated bovineand ovine glycoproteins exhibit spatially and temporally dis-tinct expression patterns during pregnancyrdquo Biology of Repro-duction vol 62 no 6 pp 1624ndash1631 2000
[98] S Xie J Green J B Bixby et al ldquoThe diversity and evolutionaryrelationships of the pregnancy-associated glycoproteins anaspartic proteinase subfamily consisting of many trophoblast-expressed genesrdquo Proceedings of the National Academy of Sci-ences of the United States of America vol 94 no 24 pp 12809ndash12816 1997
[99] K Klisch A Boos M Friedrich et al ldquoThe glycosylationof pregnancy-associated glycoproteins and prolactin-relatedprotein-I in bovine binucleate trophoblast giant cells changesbefore parturitionrdquo Reproduction vol 132 no 5 pp 791ndash7982006
[100] B P V L Telugu A M Walker and J A Green ldquoCharacter-ization of the bovine pregnancy-associated glycoprotein genefamilymdashanalysis of gene sequences regulatory regions withinthe promoter and expression of selected genesrdquo BMCGenomicsvol 10 article 185 pp 1ndash17 2009
[101] P Humblot S Camous J Martal et al ldquoDiagnosis of pregnancyby radioimmunoassay of a pregnancy-specific protein in theplasma of dairy cowsrdquo Theriogenology vol 30 no 2 pp 257ndash267 1988
[102] G H Kiracofe J M Wright R R Schalles C A Ruder SParish and R G Sasser ldquoPregnancy-specific protein B in serumof postpartum beef cowsrdquo Journal of Animal Science vol 71 no8 pp 2199ndash2205 1993
[103] A P Zoli L A Guilbault P Delahaut W B Ortiz and J-FBeckers ldquoRadioimmunoassay of a bovine pregnancy-associatedglycoprotein in serum its application for pregnancy diagnosisrdquoBiology of Reproduction vol 46 no 1 pp 83ndash92 1992
[104] R G Sasser C A Ruder K A Ivani J E Butler and W CHamilton ldquoDetection of pregnancy by radioimmunoassay of anovel pregnancy-specific protein in serum of cows and a profileof serum concentrations during gestationrdquo Biology of Reproduc-tion vol 35 no 4 pp 936ndash942 1986
10 The Scientific World Journal
[105] J A Green T E Parks M P Avalle et al ldquoThe establishmentof an ELISA for the detection of pregnancy-associated glyco-proteins (PAGs) in the serum of pregnant cows and heifersrdquoTheriogenology vol 63 no 5 pp 1481ndash1503 2005
[106] E Silva R A Sterry D Kolb et al ldquoAccuracy of a pregnancy-associated glycoprotein ELISA to determine pregnancy statusof lactating dairy cows twenty-seven days after timed artificialinseminationrdquo Journal of Dairy Science vol 90 no 10 pp 4612ndash4622 2007
[107] A Ayad N M Sousa J Sulon J L Hornick M Iguer-Ouada and J F Beckers ldquoCorrelation of five radioimmunoas-say systems for measurement of bovine plasma pregnancy-associated glycoprotein concentrations at early pregnancyperiodrdquo Research in Veterinary Science vol 86 no 3 pp 377ndash382 2009
[108] N M de Sousa M Zongo W Pitala et al ldquoPregnancy-associated glycoprotein concentrations during pregnancy andthe postpartum period in Azawak Zebu cattlerdquoTheriogenologyvol 59 no 5-6 pp 1131ndash1142 2003
[109] A Karen S Darwish A Ramoun et al ldquoAccuracy of ultra-sonography and pregnancy-associated glycoprotein test forpregnancy diagnosis in buffaloesrdquoTheriogenology vol 68 no 8pp 1150ndash1155 2007
[110] N M Sousa A Ayad J F Beckers and Z Gajewski ldquoPreg-nancy-associated glycoproteins (PAG) as pregnancy markers inthe ruminantsrdquo Journal of Physiology and Pharmacology vol 57no 8 pp 153ndash171 2006
[111] P R Graves andTA J Haystead ldquoMolecular biologistrsquos guide toproteomicsrdquo Microbiology and Molecular Biology Reviews vol66 no 1 pp 39ndash63 2002
[112] J D Lippolis and T A Reinhardt ldquoCentennial paper pro-teomics in animal sciencerdquo Journal of Animal Science vol 86no 9 pp 2430ndash2441 2008
[113] D A Colantonio and D W Chan ldquoThe clinical application ofproteomicsrdquo Clinica Chimica Acta vol 357 no 2 pp 151ndash1582005
[114] F di Girolamo E Boschetti M C M Chung F Guadagniand P G Righetti ldquolsquoProteomineeringrsquo or not The debate onbiomarker discovery in sera continuesrdquo Journal of Proteomicsvol 74 no 5 pp 589ndash594 2011
[115] D I Jin H R Lee H R KimH J Lee J T Yoon andC S ParkldquoProteomics analysis of pregnancy-specific serum proteins inbovinerdquoReproduction Fertility andDevelopment vol 18 no 1-2p 183 2005
[116] R Han H R Kim K Naruse et al ldquoIdentification of bovinepregnancy-specific milk proteins using proteomicsrdquo Biology ofReproduction vol 78 p 143 2008
[117] A K Balhara Proteomic analysis of pregnancy-specific serumproteins in buffalo [PhD thesis] LLRUVAS Hisar India 2012
[118] A Busch S Michel C Hoppe D Driesch U Claussen and Fvon Eggeling ldquoProteome analysis of maternal serum samplesfor trisomy 21 pregnancies using ProteinChip arrays and bioin-formaticsrdquo Journal of Histochemistry and Cytochemistry vol 53no 3 pp 341ndash343 2005
[119] P A Benn ldquoAdvances in prenatal screening for Down syn-drome I General principles and second trimester testingrdquoClinica Chimica Acta vol 323 no 1-2 pp 1ndash16 2002
[10] R K Sharma J K Singh S K Phulia S Khanna and I SinghldquoFetal sex determination with ultrasonography in buffaloesrdquoIndian Veterinary Journal vol 88 no 10 pp 105ndash107 2011
[11] S Curran R A Pierson and O J Ginther ldquoUltrasonographicappearance of the bovine conceptus from days 20 through 60rdquoJournal of the American VeterinaryMedical Association vol 189no 10 pp 1295ndash1302 1986
[12] PM Fricke ldquoScanning the futuremdashultrasonography as a repro-ductive management tool for dairy cattlerdquo Journal of Dairy Sci-ence vol 85 no 8 pp 1918ndash1926 2002
[13] L G Paisley W D Mickelsen and O L Frost ldquoA survey of theincidence of prenatal mortality in cattle following pregnancydiagnosis by rectal palpationrdquoTheriogenology vol 9 no 6 pp481ndash491 1978
[14] D Vaillancourt C J Bierschwal D Ogwu et al ldquoCorrelationbetween pregnancy diagnosis bymembrane slip and embryonicmortalityrdquo Journal of the American Veterinary Medical Associa-tion vol 175 no 5 pp 466ndash468 1979
[15] A Ali and S Fahmy ldquoUltrasonographic fetometry and deter-mination of fetal sex in buffaloes (Bubalus bubalis)rdquo AnimalReproduction Science vol 106 no 1-2 pp 90ndash99 2008
[16] C H Pawshe K B C Appa Rao and S M Totey ldquoUltrasono-graphic imaging to monitor early pregnancy and embryonicdevelopment in the buffalo (Bubalus bubalis)rdquo Theriogenologyvol 41 no 3 pp 697ndash709 1994
[17] M R Bhosreker and IMHangarge ldquoUltrasonography for earlypregnancy diagnosis in buffaloesrdquo Indian Journal of AnimalReproduction vol 21 pp 143ndash144 2000
[18] M C Pieterse O Szenci A H Willemse C S A Bajcsy S JDieleman andM AM Taverne ldquoEarly pregnancy diagnosis incattle bymeans of linear-array real-time ultrasound scanning ofthe uterus and a qualitative and quantitative milk progesteronetestrdquoTheriogenology vol 33 no 3 pp 697ndash707 1990
[19] D P Nation J Malmo G M Davis and K L MacmillanldquoAccuracy of bovine pregnancy detection using transrectalultrasonography at 28 to 35 days after inseminationrdquoAustralianVeterinary Journal vol 81 no 1-2 pp 63ndash65 2003
[20] O Szenci J F Beckers P Humblot et al ldquoComparison of ultra-sonography bovine pregnancy-specific protein B and bovinepregnancy-associated glycoprotein 1 tests for pregnancy detec-tion in dairy cowsrdquo Theriogenology vol 50 no 1 pp 77ndash881998
[21] M Shemesh N Ayalon and H R Lindner ldquoEarly pregnancydiagnosis based upon plasma progesterone levels in the cow andewerdquo Journal of Animal Science vol 36 no 4 pp 726ndash729 1973
[22] J A Laing and R B Heap ldquoThe concentration of progesteronein themilk of cows during the reproductive cyclerdquo British Veter-inary Journal vol 127 no 8 pp 19ndash22 1971
[23] A A Zaied C J Bierschwal R G Elmore R S Youngquist AJ Sharp and H A Garverick ldquoConcentrations of progesteroneinmilk as amonitor of early pregnancy diagnosis in dairy cowsrdquoTheriogenology vol 12 no 1 pp 3ndash11 1979
[24] R Simersky J Swaczynova D A Morris M Franek and MStrnad ldquoDevelopment of an ELISA-based kit for the on-farmdetermination of progesterone in milkrdquo Veterinarni Medicinavol 52 no 1 pp 19ndash28 2007
[25] M Kamboj and B S Prakash ldquoRelationship of progesterone inplasma and whole milk of buffaloes during cyclicity and earlypregnancyrdquo Tropical Animal Health and Production vol 25 no3 pp 185ndash192 1993
[26] S K Batra R C Arora N K Bachlaus andR S Pandey ldquoBloodand milk progesterone in pregnant and nonpregnant buffalordquoJournal of Dairy Science vol 62 no 9 pp 1390ndash1393 1979
[27] BM Perera N Pathiraja S A AbeywardenaM XMotha andH Abeygunawardena ldquoEarly pregnancy diagnosis in buffaloesfrom plasma progesterone concentrationrdquo Veterinary Recordvol 106 no 5 pp 104ndash106 1980
[28] J A Pennington L H Schultz and W F Hoffman ldquoCompari-son of pregnancy diagnosis by milk progesterone on day 21 andday 24 postbreeding field study in dairy cattlerdquo Journal of DairyScience vol 68 no 10 pp 2740ndash2745 1985
[29] A Singh and R Puthiyandy ldquoEstimation of progesterone inbuffalomilk and its application to pregnancy diagnosisrdquo Journalof Reproduction and Fertility vol 59 no 1 pp 89ndash93 1980
[30] N Isobe M Akita T Nakao H Yamashiro and H KubotaldquoPregnancy diagnosis based on the fecal progesterone concen-tration in beef and dairy heifers and beef cowsrdquo Animal Repro-duction Science vol 90 no 3-4 pp 211ndash218 2005
[31] T E Spencer N H Ing T L Ott et al ldquoIntrauterine injectionof ovine interferon-120591 alters oestrogen receptor and oxytocinreceptor expression in the endometrium of cyclic ewesrdquo Journalof Molecular Endocrinology vol 15 no 2 pp 203ndash220 1995
[32] J J Kaneko J W Harvey andM L Bruss Clinical Biochemistryof Domestic Animals Academic Press AmsterdamTheNether-lands 2008
[33] T J Parkinson A Turvey and L J Jenner ldquoA morphometricanalysis of the corpus luteumof the cowduring the estrous cycleand early pregnancyrdquo Theriogenology vol 41 no 5 pp 1115ndash1126 1994
[34] R G Sasser and C A Ruder ldquoDetection of early pregnancy indomestic ruminantsrdquo Journal of Reproduction and Fertility Sup-plement vol 34 pp 261ndash271 1987
[35] M Shemesh N Ayalon and H R Lindner ldquoEarly pregnancydiagnosis based upon plasma progesterone levels in the cow andewerdquo Journal of Animal Science vol 36 no 4 pp 726ndash729 1973
[36] E Wolf G J Arnold S Bauersachs et al ldquoEmbryo-maternalcommunication in bovinemdashstrategies for deciphering a com-plex cross-talkrdquo Reproduction in Domestic Animals vol 38 no4 pp 276ndash289 2003
[37] E W Gowan R J Etches C Bryden and G J King ldquoFactorsaffecting accuracy of pregnancy diagnosis in cattlerdquo Journal ofDairy Science vol 65 no 7 pp 1294ndash1302 1982
[38] R L Nebel W D Whittier B G Cassell and J H Britt ldquoCom-parison of on-farm laboratory milk progesterone assays foridentifying errors in detection of estrus and diagnosis of preg-nancyrdquo Journal of Dairy Science vol 70 no 7 pp 1471ndash14761987
[39] T H Wimpy C F Chang V L Estergreen and J K HillersldquoMilk progesterone enzyme immunoassay modifications anda field trial for pregnancy detection in dairy cowsrdquo Journal ofDairy Science vol 69 no 4 pp 1115ndash1121 1986
[40] R M Eley WWThatcher and F W Bazer ldquoLuteolytic effect ofoestrone sulphate on cyclic beef heifersrdquo Journal of Reproductionand Fertility vol 55 no 1 pp 191ndash193 1979
[41] H A Robertson and G J King ldquoConjugated and unconjugatedoestrogens in fetal and maternal fluids of the cow throughoutpregnancyrdquo Journal of Reproduction and Fertility vol 55 no 2pp 463ndash470 1979
[42] R B Heap and M Hamon ldquoOestrone sulphate in milk as anindicator of a viable conceptus in cowsrdquo British Veterinary Jour-nal vol 135 no 4 pp 355ndash363 1979
8 The Scientific World Journal
[43] M Hamon I R Fleet R J Holdsworth and R B Heap ldquoThetime of detection of oestrone sulphate in milk and the diagnosisof pregnancy in cowsrdquo British Veterinary Journal vol 137 no 1pp 71ndash77 1981
[44] M Hirako T Takahashi and I Domeki ldquoPeripheral changes inestrone sulfate concentration during the first trimester of ges-tation in cattle comparison with unconjugated estrogens andrelationship to fetal numberrdquo Theriogenology vol 57 no 7 pp1939ndash1947 2002
[45] B S Prakash and M L Madan ldquoInfluence of gestation onoestrone sulphate concentration in milk of zebu and crossbredcows and murrah buffaloesrdquo Tropical Animal Health and Pro-duction vol 25 no 2 pp 94ndash100 1993
[46] F Lobago M Bekana H Gustafsson et al ldquoSerum profilesof pregnancy-associated glycoprotein oestrone sulphate andprogesterone during gestation and some factors influencing theprofiles in ethiopian borana and crossbred cattlerdquo Reproductionin Domestic Animals vol 44 no 4 pp 685ndash692 2009
[47] S Aschheim and B Zondek ldquoHypophysenvorderlappenhor-mon undOvarialhormon imHarn von SchwangerenrdquoKlinischeWochenschrift vol 6 no 28 p 1322 1927
[48] J R Marshall C B Hammond G T Ross A Jacobson PRayford andW D Odell ldquoPlasma and urinary chorionic gona-dotropin during early human pregnancyrdquo Obstetrics and Gyne-cology vol 32 no 6 pp 760ndash764 1968
[49] K Imakawa R V Anthony M Kazemi K R Marotti H GPolites and R M Roberts ldquoInterferon-like sequence of ovinetrophoblast protein secreted by embryonic trophectodermrdquoNature vol 330 no 6146 pp 377ndash379 1987
[50] A C Cavanagh ldquoIdentification of early pregnancy factor aschaperonin 10 implications for understanding its rolerdquo Reviewsof Reproduction vol 1 no 1 pp 28ndash32 1996
[51] H Morton V Hegh and G J A Clunie ldquoStudies of the rosetteinhibition test in pregnant mice evidence of immunosuppres-sionrdquo Proceedings of the Royal Society B vol 193 no 1113 pp413ndash419 1976
[52] H Morton B E Rolfe and A C Cavanagh ldquoOvum factor andearly pregnancy factorrdquo Current Topics in Developmental Biol-ogy vol 23 pp 73ndash92 1987
[53] H Morton A C Cavanagh S Athanasas-Platsis K A QuinnandB E Rolfe ldquoEarly pregnancy factor has immunosuppressiveand growth factor propertiesrdquo Reproduction Fertility andDevelopment vol 4 no 4 pp 411ndash422 1992
[54] H Morton V Hegh and G J A Clunie ldquoImmunosuppressiondetected in pregnantmice by rosette inhibition testrdquoNature vol249 no 5456 pp 459ndash460 1974
[55] V G Laleh R G Laleh N Pirany and M M Ahrabi ldquoMea-surement of EPF for detection of cow pregnancy using rosetteinhibition testrdquoTheriogenology vol 70 no 1 pp 105ndash107 2008
[56] H Qin and Z Q Zheng ldquoDetection of early pregnancy factorin human serardquo American Journal of Reproductive Immunologyand Microbiology vol 13 no 1 pp 15ndash18 1987
[57] Y C Smart T K Roberts I S Fraser A W Cripps and R LClancy ldquoValidation of the rosette inhibition test for the detec-tion of early pregnancy in womenrdquo Fertility and Sterility vol 37no 6 pp 779ndash785 1982
[58] HMorton G J A Clunie and F D Shaw ldquoA test for early preg-nancy in sheeprdquo Research in Veterinary Science vol 26 no 2pp 261ndash262 1979
[59] S Wilson R McCarthy and F Clarke ldquoIn search of early preg-nancy factor isolation of active polypeptides from pregnant
ewesrsquo serardquo Journal of Reproductive Immunology vol 5 no 5 pp275ndash286 1983
[60] M C Cordoba R Sartori and P M Fricke ldquoAssessment of acommercially available early conception factor (ECF) test fordetermining pregnancy status of dairy cattlerdquo Journal of DairyScience vol 84 no 8 pp 1884ndash1889 2001
[61] B GandyW Tucker P Ryan et al ldquoEvaluation of the early con-ception factor (ECFŮ) test for the detection of nonpregnancyin dairy cattlerdquoTheriogenology vol 56 no 4 pp 637ndash647 2001
[62] C S Whisnant L A Pagels and M G Daves ldquoCase studyeffectiveness of a commercial early conception factor test for usein cattlerdquo Professional Animal Scientist vol 17 pp 51ndash53 2001
[63] A S Grewal A L C Wallace Y S Pan et al ldquoEvaluation of arosette inhibition test for pregnancy diagnosis in pigsrdquo Journalof Reproductive Immunology vol 7 no 2 pp 129ndash138 1985
[64] K Ohnuma I T O Kazuei Y-I Miyake J Takahashi andY Yasuda ldquoDetection of early pregnancy factor (EPF) in mareserardquo Journal of Reproduction and Development vol 42 no 1pp 23ndash28 1996
[65] Y P Cruz L Selwood H Morton and A C CavanaghldquoSignificance of serum early pregnancy factor concentrationsduring pregnancy and embryonic development in Sminthopsismacroura (Spencer) (Marsupialia Dasyuridae)rdquo Reproductionvol 121 no 6 pp 933ndash939 2001
[66] S Chander Studies on certain immunological aspects of preg-nancy diagnosis in buffaloes [PhD thesis] Chaudhary CharanSingh Haryana Agricultural University Hisar India 1983
[67] W R Threlfall ldquoImmunosuppressive early pregnancy factor(ISEPF) determination for pregnancy diagnosis in dairy cowsrdquoTheriogenology vol 41 p 31 1994
[68] H Morton ldquoEarly pregnancy factor an extracellular chaper-onin 10 homologuerdquo Immunology andCell Biology vol 76 no 6pp 483ndash496 1998
[69] R M Moor and L E A Rowson ldquoInfluence of the embryo anduterus on luteal function in the sheeprdquoNature vol 201 no 4918pp 522ndash523 1964
[70] J D Godkin FW Bazer and RM Roberts ldquoOvine trophoblastprotein 1 an early secreted blastocyst protein binds specifi-cally to uterine endometrium and affects protein synthesisrdquoEndocrinology vol 114 no 1 pp 120ndash130 1984
[71] FW Bazer ldquoMediators of maternal recognition of pregnancy inmammalsrdquo Proceedings of the Society for Experimental Biologyand Medicine vol 199 no 4 pp 373ndash384 1992
[72] R M Roberts D W Leaman and J C Cross ldquoRole of interfer-ons in maternal recognition of pregnancy in ruminantsrdquo Pro-ceedings of the Society for Experimental Biology and Medicinevol 200 no 1 pp 7ndash18 1992
[73] R M Roberts ldquoInterferon-tau a type 1 interferon involvedin maternal recognition of pregnancyrdquo Cytokine and GrowthFactor Reviews vol 18 no 5-6 pp 403ndash408 2007
[74] G E Mann G E Lamming R S Robinson and D C WathesldquoThe regulation of interferon-tau production and uterine hor-mone receptors during early pregnancyrdquo Journal of Reproduc-tion and Fertility Supplement vol 54 pp 317ndash328 1999
[75] R M Roberts A D Ealy A P Alexenko C-S Han and TEzashi ldquoTrophoblast interferonsrdquo Placenta vol 20 no 4 pp259ndash264 1999
[76] WWThatcher M DMeyer and G Danet-Desnoyers ldquoMater-nal recognition of pregnancyrdquo Journal of Reproduction andFertility Supplement vol 49 pp 15ndash28 1995
The Scientific World Journal 9
[77] F W Bazer T E Spencer and T L Ott ldquoInterferon tau anovel pregnancy recognition signalrdquoAmerican Journal of Repro-ductive Immunology vol 37 no 6 pp 412ndash420 1997
[78] T E Spencer and F W Bazer ldquoOvine interferon tau suppressestranscription of the estrogen receptor and oxytocin receptorgenes in the ovine endometriumrdquo Endocrinology vol 137 no3 pp 1144ndash1147 1996
[79] C W Xiao B D Murphy J Sirois and A K GoffldquoDown-regulation of oxytocin-induced cyclooxygenase-2 andprostaglandin F synthase expression by interferon-120591 in bovineendometrial cellsrdquo Biology of Reproduction vol 60 no 3 pp656ndash663 1999
[80] T E Spencer N H Ing T L Ott et al ldquoIntrauterine injectionof ovine interferon-120591 alters oestrogen receptor and oxytocinreceptor expression in the endometrium of cyclic ewesrdquo Journalof Molecular Endocrinology vol 15 no 2 pp 203ndash220 1995
[81] J F Oliveira L E Henkes R L Ashley et al ldquoExpression ofinterferon (IFN)-stimulated genes in extrauterine tissues duringearly pregnancy in sheep is the consequence of endocrine IFN-120591 release from the uterine veinrdquo Endocrinology vol 149 no 3pp 1252ndash1259 2008
[82] C A Gifford K Racicot D S Clark et al ldquoRegulation ofinterferon-stimulated genes in peripheral blood leukocytes inpregnant and bred nonpregnant dairy cowsrdquo Journal of DairyScience vol 90 no 1 pp 274ndash280 2007
[83] H Han K J Austin L A Rempel and T R Hansen ldquoLowblood ISG15 mRNA and progesterone levels are predictive ofnon-pregnant dairy cowsrdquo Journal of Endocrinology vol 191 no2 pp 505ndash512 2006
[84] J C Green C S Okamura S E Poock and M C Lucy ldquoMea-surement of interferon-tau (IFN-120591) stimulated gene expressionin blood leukocytes for pregnancy diagnosis within 18-20d afterinsemination in dairy cattlerdquo Animal Reproduction Science vol121 no 1-2 pp 24ndash33 2010
[85] N Forde F Carter T E Spencer et al ldquoConceptus-inducedchanges in the endometrial transcriptome how soon does thecow know she is pregnantrdquo Biology of Reproduction vol 85 no1 pp 144ndash156 2011
[86] K Kizaki A Shichijo-Kizaki T Furusawa T Takahashi MHosoe andKHashizume ldquoDifferential neutrophil gene expres-sion in early bovine pregnancyrdquoReproductive Biology and Endo-crinology vol 11 article 6 2013
[87] F B P Wooding and D C Wathes ldquoBinucleate cell migrationin the bovine placentomerdquo Journal of Reproduction and Fertilityvol 59 no 2 pp 425ndash430 1980
[88] F W Bazer W W Thatcher P J Hansen M A Mirando TL Ott and C Plante ldquoPhysiological mechanisms of pregnancyrecognition in ruminantsrdquo Journal of Reproduction and FertilitySupplement vol 43 pp 39ndash47 1991
[89] A P Zoli P Demez J F Beckers M Reznik and A BeckersldquoLight and electron microscopic immunolocalization of bovinepregnancy-associated glycoprotein in the bovine placentomerdquoBiology of Reproduction vol 46 no 4 pp 623ndash629 1992
[90] J E Butler W C Hamilton R G Sasser C A Ruder G MHass andR JWilliams ldquoDetection and partial characterizationof two bovine pregnancy-specific proteinsrdquoBiology of Reproduc-tion vol 26 no 5 pp 925ndash933 1982
[91] A P Zoli J-F Beckers P Wouters-Ballman J Closset PFalmagne and F Ectors ldquoPurification and characterization of abovine pregnancy-associated glycoproteinrdquo Biology of Repro-duction vol 45 no 1 pp 1ndash10 1991
[92] S Xie B G Low R J Nagel et al ldquoIdentification of the majorpregnancy-specific antigens of cattle and sheep as inactivemembers of the aspartic proteinase familyrdquo Proceedings of theNational Academy of Sciences of theUnited States of America vol88 no 22 pp 10247ndash10251 1991
[93] S Xie B G Low R J Nagel J-F Beckers and R M RobertsldquoA novel glycoprotein of the aspartic proteinase gene familyexpressed in bovine placental trophectodermrdquoBiology of Repro-duction vol 51 no 6 pp 1145ndash1153 1994
[94] F Constanta S Camousa P Chavatte-Palmera et al ldquoAlteredsecretion of pregnancy-associated glycoproteins during gesta-tion in bovine somatic clonesrdquoTheriogenology vol 76 pp 1006ndash1021 2011
[95] M M Mialon S Camous G Renand J Martal and FMenissier ldquoPeripheral concentrations of a 60-kDa pregnancyserum protein during gestation and after calving and in rela-tionship to embryonic mortality in cattlerdquo Reproduction Nutri-tion Development vol 33 no 3 pp 269ndash282 1993
[96] JMartal N Chene S Camous et al ldquoRecent developments andpotentialities for reducing embryo mortality in ruminants therole of IFN-120591 and other cytokines in early pregnancyrdquo Repro-duction Fertility and Development vol 9 no 3 pp 355ndash3801997
[97] J A Green S Xie X Quan et al ldquoPregnancy-associated bovineand ovine glycoproteins exhibit spatially and temporally dis-tinct expression patterns during pregnancyrdquo Biology of Repro-duction vol 62 no 6 pp 1624ndash1631 2000
[98] S Xie J Green J B Bixby et al ldquoThe diversity and evolutionaryrelationships of the pregnancy-associated glycoproteins anaspartic proteinase subfamily consisting of many trophoblast-expressed genesrdquo Proceedings of the National Academy of Sci-ences of the United States of America vol 94 no 24 pp 12809ndash12816 1997
[99] K Klisch A Boos M Friedrich et al ldquoThe glycosylationof pregnancy-associated glycoproteins and prolactin-relatedprotein-I in bovine binucleate trophoblast giant cells changesbefore parturitionrdquo Reproduction vol 132 no 5 pp 791ndash7982006
[100] B P V L Telugu A M Walker and J A Green ldquoCharacter-ization of the bovine pregnancy-associated glycoprotein genefamilymdashanalysis of gene sequences regulatory regions withinthe promoter and expression of selected genesrdquo BMCGenomicsvol 10 article 185 pp 1ndash17 2009
[101] P Humblot S Camous J Martal et al ldquoDiagnosis of pregnancyby radioimmunoassay of a pregnancy-specific protein in theplasma of dairy cowsrdquo Theriogenology vol 30 no 2 pp 257ndash267 1988
[102] G H Kiracofe J M Wright R R Schalles C A Ruder SParish and R G Sasser ldquoPregnancy-specific protein B in serumof postpartum beef cowsrdquo Journal of Animal Science vol 71 no8 pp 2199ndash2205 1993
[103] A P Zoli L A Guilbault P Delahaut W B Ortiz and J-FBeckers ldquoRadioimmunoassay of a bovine pregnancy-associatedglycoprotein in serum its application for pregnancy diagnosisrdquoBiology of Reproduction vol 46 no 1 pp 83ndash92 1992
[104] R G Sasser C A Ruder K A Ivani J E Butler and W CHamilton ldquoDetection of pregnancy by radioimmunoassay of anovel pregnancy-specific protein in serum of cows and a profileof serum concentrations during gestationrdquo Biology of Reproduc-tion vol 35 no 4 pp 936ndash942 1986
10 The Scientific World Journal
[105] J A Green T E Parks M P Avalle et al ldquoThe establishmentof an ELISA for the detection of pregnancy-associated glyco-proteins (PAGs) in the serum of pregnant cows and heifersrdquoTheriogenology vol 63 no 5 pp 1481ndash1503 2005
[106] E Silva R A Sterry D Kolb et al ldquoAccuracy of a pregnancy-associated glycoprotein ELISA to determine pregnancy statusof lactating dairy cows twenty-seven days after timed artificialinseminationrdquo Journal of Dairy Science vol 90 no 10 pp 4612ndash4622 2007
[107] A Ayad N M Sousa J Sulon J L Hornick M Iguer-Ouada and J F Beckers ldquoCorrelation of five radioimmunoas-say systems for measurement of bovine plasma pregnancy-associated glycoprotein concentrations at early pregnancyperiodrdquo Research in Veterinary Science vol 86 no 3 pp 377ndash382 2009
[108] N M de Sousa M Zongo W Pitala et al ldquoPregnancy-associated glycoprotein concentrations during pregnancy andthe postpartum period in Azawak Zebu cattlerdquoTheriogenologyvol 59 no 5-6 pp 1131ndash1142 2003
[109] A Karen S Darwish A Ramoun et al ldquoAccuracy of ultra-sonography and pregnancy-associated glycoprotein test forpregnancy diagnosis in buffaloesrdquoTheriogenology vol 68 no 8pp 1150ndash1155 2007
[110] N M Sousa A Ayad J F Beckers and Z Gajewski ldquoPreg-nancy-associated glycoproteins (PAG) as pregnancy markers inthe ruminantsrdquo Journal of Physiology and Pharmacology vol 57no 8 pp 153ndash171 2006
[111] P R Graves andTA J Haystead ldquoMolecular biologistrsquos guide toproteomicsrdquo Microbiology and Molecular Biology Reviews vol66 no 1 pp 39ndash63 2002
[112] J D Lippolis and T A Reinhardt ldquoCentennial paper pro-teomics in animal sciencerdquo Journal of Animal Science vol 86no 9 pp 2430ndash2441 2008
[113] D A Colantonio and D W Chan ldquoThe clinical application ofproteomicsrdquo Clinica Chimica Acta vol 357 no 2 pp 151ndash1582005
[114] F di Girolamo E Boschetti M C M Chung F Guadagniand P G Righetti ldquolsquoProteomineeringrsquo or not The debate onbiomarker discovery in sera continuesrdquo Journal of Proteomicsvol 74 no 5 pp 589ndash594 2011
[115] D I Jin H R Lee H R KimH J Lee J T Yoon andC S ParkldquoProteomics analysis of pregnancy-specific serum proteins inbovinerdquoReproduction Fertility andDevelopment vol 18 no 1-2p 183 2005
[116] R Han H R Kim K Naruse et al ldquoIdentification of bovinepregnancy-specific milk proteins using proteomicsrdquo Biology ofReproduction vol 78 p 143 2008
[117] A K Balhara Proteomic analysis of pregnancy-specific serumproteins in buffalo [PhD thesis] LLRUVAS Hisar India 2012
[118] A Busch S Michel C Hoppe D Driesch U Claussen and Fvon Eggeling ldquoProteome analysis of maternal serum samplesfor trisomy 21 pregnancies using ProteinChip arrays and bioin-formaticsrdquo Journal of Histochemistry and Cytochemistry vol 53no 3 pp 341ndash343 2005
[119] P A Benn ldquoAdvances in prenatal screening for Down syn-drome I General principles and second trimester testingrdquoClinica Chimica Acta vol 323 no 1-2 pp 1ndash16 2002
[43] M Hamon I R Fleet R J Holdsworth and R B Heap ldquoThetime of detection of oestrone sulphate in milk and the diagnosisof pregnancy in cowsrdquo British Veterinary Journal vol 137 no 1pp 71ndash77 1981
[44] M Hirako T Takahashi and I Domeki ldquoPeripheral changes inestrone sulfate concentration during the first trimester of ges-tation in cattle comparison with unconjugated estrogens andrelationship to fetal numberrdquo Theriogenology vol 57 no 7 pp1939ndash1947 2002
[45] B S Prakash and M L Madan ldquoInfluence of gestation onoestrone sulphate concentration in milk of zebu and crossbredcows and murrah buffaloesrdquo Tropical Animal Health and Pro-duction vol 25 no 2 pp 94ndash100 1993
[46] F Lobago M Bekana H Gustafsson et al ldquoSerum profilesof pregnancy-associated glycoprotein oestrone sulphate andprogesterone during gestation and some factors influencing theprofiles in ethiopian borana and crossbred cattlerdquo Reproductionin Domestic Animals vol 44 no 4 pp 685ndash692 2009
[47] S Aschheim and B Zondek ldquoHypophysenvorderlappenhor-mon undOvarialhormon imHarn von SchwangerenrdquoKlinischeWochenschrift vol 6 no 28 p 1322 1927
[48] J R Marshall C B Hammond G T Ross A Jacobson PRayford andW D Odell ldquoPlasma and urinary chorionic gona-dotropin during early human pregnancyrdquo Obstetrics and Gyne-cology vol 32 no 6 pp 760ndash764 1968
[49] K Imakawa R V Anthony M Kazemi K R Marotti H GPolites and R M Roberts ldquoInterferon-like sequence of ovinetrophoblast protein secreted by embryonic trophectodermrdquoNature vol 330 no 6146 pp 377ndash379 1987
[50] A C Cavanagh ldquoIdentification of early pregnancy factor aschaperonin 10 implications for understanding its rolerdquo Reviewsof Reproduction vol 1 no 1 pp 28ndash32 1996
[51] H Morton V Hegh and G J A Clunie ldquoStudies of the rosetteinhibition test in pregnant mice evidence of immunosuppres-sionrdquo Proceedings of the Royal Society B vol 193 no 1113 pp413ndash419 1976
[52] H Morton B E Rolfe and A C Cavanagh ldquoOvum factor andearly pregnancy factorrdquo Current Topics in Developmental Biol-ogy vol 23 pp 73ndash92 1987
[53] H Morton A C Cavanagh S Athanasas-Platsis K A QuinnandB E Rolfe ldquoEarly pregnancy factor has immunosuppressiveand growth factor propertiesrdquo Reproduction Fertility andDevelopment vol 4 no 4 pp 411ndash422 1992
[54] H Morton V Hegh and G J A Clunie ldquoImmunosuppressiondetected in pregnantmice by rosette inhibition testrdquoNature vol249 no 5456 pp 459ndash460 1974
[55] V G Laleh R G Laleh N Pirany and M M Ahrabi ldquoMea-surement of EPF for detection of cow pregnancy using rosetteinhibition testrdquoTheriogenology vol 70 no 1 pp 105ndash107 2008
[56] H Qin and Z Q Zheng ldquoDetection of early pregnancy factorin human serardquo American Journal of Reproductive Immunologyand Microbiology vol 13 no 1 pp 15ndash18 1987
[57] Y C Smart T K Roberts I S Fraser A W Cripps and R LClancy ldquoValidation of the rosette inhibition test for the detec-tion of early pregnancy in womenrdquo Fertility and Sterility vol 37no 6 pp 779ndash785 1982
[58] HMorton G J A Clunie and F D Shaw ldquoA test for early preg-nancy in sheeprdquo Research in Veterinary Science vol 26 no 2pp 261ndash262 1979
[59] S Wilson R McCarthy and F Clarke ldquoIn search of early preg-nancy factor isolation of active polypeptides from pregnant
ewesrsquo serardquo Journal of Reproductive Immunology vol 5 no 5 pp275ndash286 1983
[60] M C Cordoba R Sartori and P M Fricke ldquoAssessment of acommercially available early conception factor (ECF) test fordetermining pregnancy status of dairy cattlerdquo Journal of DairyScience vol 84 no 8 pp 1884ndash1889 2001
[61] B GandyW Tucker P Ryan et al ldquoEvaluation of the early con-ception factor (ECFŮ) test for the detection of nonpregnancyin dairy cattlerdquoTheriogenology vol 56 no 4 pp 637ndash647 2001
[62] C S Whisnant L A Pagels and M G Daves ldquoCase studyeffectiveness of a commercial early conception factor test for usein cattlerdquo Professional Animal Scientist vol 17 pp 51ndash53 2001
[63] A S Grewal A L C Wallace Y S Pan et al ldquoEvaluation of arosette inhibition test for pregnancy diagnosis in pigsrdquo Journalof Reproductive Immunology vol 7 no 2 pp 129ndash138 1985
[64] K Ohnuma I T O Kazuei Y-I Miyake J Takahashi andY Yasuda ldquoDetection of early pregnancy factor (EPF) in mareserardquo Journal of Reproduction and Development vol 42 no 1pp 23ndash28 1996
[65] Y P Cruz L Selwood H Morton and A C CavanaghldquoSignificance of serum early pregnancy factor concentrationsduring pregnancy and embryonic development in Sminthopsismacroura (Spencer) (Marsupialia Dasyuridae)rdquo Reproductionvol 121 no 6 pp 933ndash939 2001
[66] S Chander Studies on certain immunological aspects of preg-nancy diagnosis in buffaloes [PhD thesis] Chaudhary CharanSingh Haryana Agricultural University Hisar India 1983
[67] W R Threlfall ldquoImmunosuppressive early pregnancy factor(ISEPF) determination for pregnancy diagnosis in dairy cowsrdquoTheriogenology vol 41 p 31 1994
[68] H Morton ldquoEarly pregnancy factor an extracellular chaper-onin 10 homologuerdquo Immunology andCell Biology vol 76 no 6pp 483ndash496 1998
[69] R M Moor and L E A Rowson ldquoInfluence of the embryo anduterus on luteal function in the sheeprdquoNature vol 201 no 4918pp 522ndash523 1964
[70] J D Godkin FW Bazer and RM Roberts ldquoOvine trophoblastprotein 1 an early secreted blastocyst protein binds specifi-cally to uterine endometrium and affects protein synthesisrdquoEndocrinology vol 114 no 1 pp 120ndash130 1984
[71] FW Bazer ldquoMediators of maternal recognition of pregnancy inmammalsrdquo Proceedings of the Society for Experimental Biologyand Medicine vol 199 no 4 pp 373ndash384 1992
[72] R M Roberts D W Leaman and J C Cross ldquoRole of interfer-ons in maternal recognition of pregnancy in ruminantsrdquo Pro-ceedings of the Society for Experimental Biology and Medicinevol 200 no 1 pp 7ndash18 1992
[73] R M Roberts ldquoInterferon-tau a type 1 interferon involvedin maternal recognition of pregnancyrdquo Cytokine and GrowthFactor Reviews vol 18 no 5-6 pp 403ndash408 2007
[74] G E Mann G E Lamming R S Robinson and D C WathesldquoThe regulation of interferon-tau production and uterine hor-mone receptors during early pregnancyrdquo Journal of Reproduc-tion and Fertility Supplement vol 54 pp 317ndash328 1999
[75] R M Roberts A D Ealy A P Alexenko C-S Han and TEzashi ldquoTrophoblast interferonsrdquo Placenta vol 20 no 4 pp259ndash264 1999
[76] WWThatcher M DMeyer and G Danet-Desnoyers ldquoMater-nal recognition of pregnancyrdquo Journal of Reproduction andFertility Supplement vol 49 pp 15ndash28 1995
The Scientific World Journal 9
[77] F W Bazer T E Spencer and T L Ott ldquoInterferon tau anovel pregnancy recognition signalrdquoAmerican Journal of Repro-ductive Immunology vol 37 no 6 pp 412ndash420 1997
[78] T E Spencer and F W Bazer ldquoOvine interferon tau suppressestranscription of the estrogen receptor and oxytocin receptorgenes in the ovine endometriumrdquo Endocrinology vol 137 no3 pp 1144ndash1147 1996
[79] C W Xiao B D Murphy J Sirois and A K GoffldquoDown-regulation of oxytocin-induced cyclooxygenase-2 andprostaglandin F synthase expression by interferon-120591 in bovineendometrial cellsrdquo Biology of Reproduction vol 60 no 3 pp656ndash663 1999
[80] T E Spencer N H Ing T L Ott et al ldquoIntrauterine injectionof ovine interferon-120591 alters oestrogen receptor and oxytocinreceptor expression in the endometrium of cyclic ewesrdquo Journalof Molecular Endocrinology vol 15 no 2 pp 203ndash220 1995
[81] J F Oliveira L E Henkes R L Ashley et al ldquoExpression ofinterferon (IFN)-stimulated genes in extrauterine tissues duringearly pregnancy in sheep is the consequence of endocrine IFN-120591 release from the uterine veinrdquo Endocrinology vol 149 no 3pp 1252ndash1259 2008
[82] C A Gifford K Racicot D S Clark et al ldquoRegulation ofinterferon-stimulated genes in peripheral blood leukocytes inpregnant and bred nonpregnant dairy cowsrdquo Journal of DairyScience vol 90 no 1 pp 274ndash280 2007
[83] H Han K J Austin L A Rempel and T R Hansen ldquoLowblood ISG15 mRNA and progesterone levels are predictive ofnon-pregnant dairy cowsrdquo Journal of Endocrinology vol 191 no2 pp 505ndash512 2006
[84] J C Green C S Okamura S E Poock and M C Lucy ldquoMea-surement of interferon-tau (IFN-120591) stimulated gene expressionin blood leukocytes for pregnancy diagnosis within 18-20d afterinsemination in dairy cattlerdquo Animal Reproduction Science vol121 no 1-2 pp 24ndash33 2010
[85] N Forde F Carter T E Spencer et al ldquoConceptus-inducedchanges in the endometrial transcriptome how soon does thecow know she is pregnantrdquo Biology of Reproduction vol 85 no1 pp 144ndash156 2011
[86] K Kizaki A Shichijo-Kizaki T Furusawa T Takahashi MHosoe andKHashizume ldquoDifferential neutrophil gene expres-sion in early bovine pregnancyrdquoReproductive Biology and Endo-crinology vol 11 article 6 2013
[87] F B P Wooding and D C Wathes ldquoBinucleate cell migrationin the bovine placentomerdquo Journal of Reproduction and Fertilityvol 59 no 2 pp 425ndash430 1980
[88] F W Bazer W W Thatcher P J Hansen M A Mirando TL Ott and C Plante ldquoPhysiological mechanisms of pregnancyrecognition in ruminantsrdquo Journal of Reproduction and FertilitySupplement vol 43 pp 39ndash47 1991
[89] A P Zoli P Demez J F Beckers M Reznik and A BeckersldquoLight and electron microscopic immunolocalization of bovinepregnancy-associated glycoprotein in the bovine placentomerdquoBiology of Reproduction vol 46 no 4 pp 623ndash629 1992
[90] J E Butler W C Hamilton R G Sasser C A Ruder G MHass andR JWilliams ldquoDetection and partial characterizationof two bovine pregnancy-specific proteinsrdquoBiology of Reproduc-tion vol 26 no 5 pp 925ndash933 1982
[91] A P Zoli J-F Beckers P Wouters-Ballman J Closset PFalmagne and F Ectors ldquoPurification and characterization of abovine pregnancy-associated glycoproteinrdquo Biology of Repro-duction vol 45 no 1 pp 1ndash10 1991
[92] S Xie B G Low R J Nagel et al ldquoIdentification of the majorpregnancy-specific antigens of cattle and sheep as inactivemembers of the aspartic proteinase familyrdquo Proceedings of theNational Academy of Sciences of theUnited States of America vol88 no 22 pp 10247ndash10251 1991
[93] S Xie B G Low R J Nagel J-F Beckers and R M RobertsldquoA novel glycoprotein of the aspartic proteinase gene familyexpressed in bovine placental trophectodermrdquoBiology of Repro-duction vol 51 no 6 pp 1145ndash1153 1994
[94] F Constanta S Camousa P Chavatte-Palmera et al ldquoAlteredsecretion of pregnancy-associated glycoproteins during gesta-tion in bovine somatic clonesrdquoTheriogenology vol 76 pp 1006ndash1021 2011
[95] M M Mialon S Camous G Renand J Martal and FMenissier ldquoPeripheral concentrations of a 60-kDa pregnancyserum protein during gestation and after calving and in rela-tionship to embryonic mortality in cattlerdquo Reproduction Nutri-tion Development vol 33 no 3 pp 269ndash282 1993
[96] JMartal N Chene S Camous et al ldquoRecent developments andpotentialities for reducing embryo mortality in ruminants therole of IFN-120591 and other cytokines in early pregnancyrdquo Repro-duction Fertility and Development vol 9 no 3 pp 355ndash3801997
[97] J A Green S Xie X Quan et al ldquoPregnancy-associated bovineand ovine glycoproteins exhibit spatially and temporally dis-tinct expression patterns during pregnancyrdquo Biology of Repro-duction vol 62 no 6 pp 1624ndash1631 2000
[98] S Xie J Green J B Bixby et al ldquoThe diversity and evolutionaryrelationships of the pregnancy-associated glycoproteins anaspartic proteinase subfamily consisting of many trophoblast-expressed genesrdquo Proceedings of the National Academy of Sci-ences of the United States of America vol 94 no 24 pp 12809ndash12816 1997
[99] K Klisch A Boos M Friedrich et al ldquoThe glycosylationof pregnancy-associated glycoproteins and prolactin-relatedprotein-I in bovine binucleate trophoblast giant cells changesbefore parturitionrdquo Reproduction vol 132 no 5 pp 791ndash7982006
[100] B P V L Telugu A M Walker and J A Green ldquoCharacter-ization of the bovine pregnancy-associated glycoprotein genefamilymdashanalysis of gene sequences regulatory regions withinthe promoter and expression of selected genesrdquo BMCGenomicsvol 10 article 185 pp 1ndash17 2009
[101] P Humblot S Camous J Martal et al ldquoDiagnosis of pregnancyby radioimmunoassay of a pregnancy-specific protein in theplasma of dairy cowsrdquo Theriogenology vol 30 no 2 pp 257ndash267 1988
[102] G H Kiracofe J M Wright R R Schalles C A Ruder SParish and R G Sasser ldquoPregnancy-specific protein B in serumof postpartum beef cowsrdquo Journal of Animal Science vol 71 no8 pp 2199ndash2205 1993
[103] A P Zoli L A Guilbault P Delahaut W B Ortiz and J-FBeckers ldquoRadioimmunoassay of a bovine pregnancy-associatedglycoprotein in serum its application for pregnancy diagnosisrdquoBiology of Reproduction vol 46 no 1 pp 83ndash92 1992
[104] R G Sasser C A Ruder K A Ivani J E Butler and W CHamilton ldquoDetection of pregnancy by radioimmunoassay of anovel pregnancy-specific protein in serum of cows and a profileof serum concentrations during gestationrdquo Biology of Reproduc-tion vol 35 no 4 pp 936ndash942 1986
10 The Scientific World Journal
[105] J A Green T E Parks M P Avalle et al ldquoThe establishmentof an ELISA for the detection of pregnancy-associated glyco-proteins (PAGs) in the serum of pregnant cows and heifersrdquoTheriogenology vol 63 no 5 pp 1481ndash1503 2005
[106] E Silva R A Sterry D Kolb et al ldquoAccuracy of a pregnancy-associated glycoprotein ELISA to determine pregnancy statusof lactating dairy cows twenty-seven days after timed artificialinseminationrdquo Journal of Dairy Science vol 90 no 10 pp 4612ndash4622 2007
[107] A Ayad N M Sousa J Sulon J L Hornick M Iguer-Ouada and J F Beckers ldquoCorrelation of five radioimmunoas-say systems for measurement of bovine plasma pregnancy-associated glycoprotein concentrations at early pregnancyperiodrdquo Research in Veterinary Science vol 86 no 3 pp 377ndash382 2009
[108] N M de Sousa M Zongo W Pitala et al ldquoPregnancy-associated glycoprotein concentrations during pregnancy andthe postpartum period in Azawak Zebu cattlerdquoTheriogenologyvol 59 no 5-6 pp 1131ndash1142 2003
[109] A Karen S Darwish A Ramoun et al ldquoAccuracy of ultra-sonography and pregnancy-associated glycoprotein test forpregnancy diagnosis in buffaloesrdquoTheriogenology vol 68 no 8pp 1150ndash1155 2007
[110] N M Sousa A Ayad J F Beckers and Z Gajewski ldquoPreg-nancy-associated glycoproteins (PAG) as pregnancy markers inthe ruminantsrdquo Journal of Physiology and Pharmacology vol 57no 8 pp 153ndash171 2006
[111] P R Graves andTA J Haystead ldquoMolecular biologistrsquos guide toproteomicsrdquo Microbiology and Molecular Biology Reviews vol66 no 1 pp 39ndash63 2002
[112] J D Lippolis and T A Reinhardt ldquoCentennial paper pro-teomics in animal sciencerdquo Journal of Animal Science vol 86no 9 pp 2430ndash2441 2008
[113] D A Colantonio and D W Chan ldquoThe clinical application ofproteomicsrdquo Clinica Chimica Acta vol 357 no 2 pp 151ndash1582005
[114] F di Girolamo E Boschetti M C M Chung F Guadagniand P G Righetti ldquolsquoProteomineeringrsquo or not The debate onbiomarker discovery in sera continuesrdquo Journal of Proteomicsvol 74 no 5 pp 589ndash594 2011
[115] D I Jin H R Lee H R KimH J Lee J T Yoon andC S ParkldquoProteomics analysis of pregnancy-specific serum proteins inbovinerdquoReproduction Fertility andDevelopment vol 18 no 1-2p 183 2005
[116] R Han H R Kim K Naruse et al ldquoIdentification of bovinepregnancy-specific milk proteins using proteomicsrdquo Biology ofReproduction vol 78 p 143 2008
[117] A K Balhara Proteomic analysis of pregnancy-specific serumproteins in buffalo [PhD thesis] LLRUVAS Hisar India 2012
[118] A Busch S Michel C Hoppe D Driesch U Claussen and Fvon Eggeling ldquoProteome analysis of maternal serum samplesfor trisomy 21 pregnancies using ProteinChip arrays and bioin-formaticsrdquo Journal of Histochemistry and Cytochemistry vol 53no 3 pp 341ndash343 2005
[119] P A Benn ldquoAdvances in prenatal screening for Down syn-drome I General principles and second trimester testingrdquoClinica Chimica Acta vol 323 no 1-2 pp 1ndash16 2002
[77] F W Bazer T E Spencer and T L Ott ldquoInterferon tau anovel pregnancy recognition signalrdquoAmerican Journal of Repro-ductive Immunology vol 37 no 6 pp 412ndash420 1997
[78] T E Spencer and F W Bazer ldquoOvine interferon tau suppressestranscription of the estrogen receptor and oxytocin receptorgenes in the ovine endometriumrdquo Endocrinology vol 137 no3 pp 1144ndash1147 1996
[79] C W Xiao B D Murphy J Sirois and A K GoffldquoDown-regulation of oxytocin-induced cyclooxygenase-2 andprostaglandin F synthase expression by interferon-120591 in bovineendometrial cellsrdquo Biology of Reproduction vol 60 no 3 pp656ndash663 1999
[80] T E Spencer N H Ing T L Ott et al ldquoIntrauterine injectionof ovine interferon-120591 alters oestrogen receptor and oxytocinreceptor expression in the endometrium of cyclic ewesrdquo Journalof Molecular Endocrinology vol 15 no 2 pp 203ndash220 1995
[81] J F Oliveira L E Henkes R L Ashley et al ldquoExpression ofinterferon (IFN)-stimulated genes in extrauterine tissues duringearly pregnancy in sheep is the consequence of endocrine IFN-120591 release from the uterine veinrdquo Endocrinology vol 149 no 3pp 1252ndash1259 2008
[82] C A Gifford K Racicot D S Clark et al ldquoRegulation ofinterferon-stimulated genes in peripheral blood leukocytes inpregnant and bred nonpregnant dairy cowsrdquo Journal of DairyScience vol 90 no 1 pp 274ndash280 2007
[83] H Han K J Austin L A Rempel and T R Hansen ldquoLowblood ISG15 mRNA and progesterone levels are predictive ofnon-pregnant dairy cowsrdquo Journal of Endocrinology vol 191 no2 pp 505ndash512 2006
[84] J C Green C S Okamura S E Poock and M C Lucy ldquoMea-surement of interferon-tau (IFN-120591) stimulated gene expressionin blood leukocytes for pregnancy diagnosis within 18-20d afterinsemination in dairy cattlerdquo Animal Reproduction Science vol121 no 1-2 pp 24ndash33 2010
[85] N Forde F Carter T E Spencer et al ldquoConceptus-inducedchanges in the endometrial transcriptome how soon does thecow know she is pregnantrdquo Biology of Reproduction vol 85 no1 pp 144ndash156 2011
[86] K Kizaki A Shichijo-Kizaki T Furusawa T Takahashi MHosoe andKHashizume ldquoDifferential neutrophil gene expres-sion in early bovine pregnancyrdquoReproductive Biology and Endo-crinology vol 11 article 6 2013
[87] F B P Wooding and D C Wathes ldquoBinucleate cell migrationin the bovine placentomerdquo Journal of Reproduction and Fertilityvol 59 no 2 pp 425ndash430 1980
[88] F W Bazer W W Thatcher P J Hansen M A Mirando TL Ott and C Plante ldquoPhysiological mechanisms of pregnancyrecognition in ruminantsrdquo Journal of Reproduction and FertilitySupplement vol 43 pp 39ndash47 1991
[89] A P Zoli P Demez J F Beckers M Reznik and A BeckersldquoLight and electron microscopic immunolocalization of bovinepregnancy-associated glycoprotein in the bovine placentomerdquoBiology of Reproduction vol 46 no 4 pp 623ndash629 1992
[90] J E Butler W C Hamilton R G Sasser C A Ruder G MHass andR JWilliams ldquoDetection and partial characterizationof two bovine pregnancy-specific proteinsrdquoBiology of Reproduc-tion vol 26 no 5 pp 925ndash933 1982
[91] A P Zoli J-F Beckers P Wouters-Ballman J Closset PFalmagne and F Ectors ldquoPurification and characterization of abovine pregnancy-associated glycoproteinrdquo Biology of Repro-duction vol 45 no 1 pp 1ndash10 1991
[92] S Xie B G Low R J Nagel et al ldquoIdentification of the majorpregnancy-specific antigens of cattle and sheep as inactivemembers of the aspartic proteinase familyrdquo Proceedings of theNational Academy of Sciences of theUnited States of America vol88 no 22 pp 10247ndash10251 1991
[93] S Xie B G Low R J Nagel J-F Beckers and R M RobertsldquoA novel glycoprotein of the aspartic proteinase gene familyexpressed in bovine placental trophectodermrdquoBiology of Repro-duction vol 51 no 6 pp 1145ndash1153 1994
[94] F Constanta S Camousa P Chavatte-Palmera et al ldquoAlteredsecretion of pregnancy-associated glycoproteins during gesta-tion in bovine somatic clonesrdquoTheriogenology vol 76 pp 1006ndash1021 2011
[95] M M Mialon S Camous G Renand J Martal and FMenissier ldquoPeripheral concentrations of a 60-kDa pregnancyserum protein during gestation and after calving and in rela-tionship to embryonic mortality in cattlerdquo Reproduction Nutri-tion Development vol 33 no 3 pp 269ndash282 1993
[96] JMartal N Chene S Camous et al ldquoRecent developments andpotentialities for reducing embryo mortality in ruminants therole of IFN-120591 and other cytokines in early pregnancyrdquo Repro-duction Fertility and Development vol 9 no 3 pp 355ndash3801997
[97] J A Green S Xie X Quan et al ldquoPregnancy-associated bovineand ovine glycoproteins exhibit spatially and temporally dis-tinct expression patterns during pregnancyrdquo Biology of Repro-duction vol 62 no 6 pp 1624ndash1631 2000
[98] S Xie J Green J B Bixby et al ldquoThe diversity and evolutionaryrelationships of the pregnancy-associated glycoproteins anaspartic proteinase subfamily consisting of many trophoblast-expressed genesrdquo Proceedings of the National Academy of Sci-ences of the United States of America vol 94 no 24 pp 12809ndash12816 1997
[99] K Klisch A Boos M Friedrich et al ldquoThe glycosylationof pregnancy-associated glycoproteins and prolactin-relatedprotein-I in bovine binucleate trophoblast giant cells changesbefore parturitionrdquo Reproduction vol 132 no 5 pp 791ndash7982006
[100] B P V L Telugu A M Walker and J A Green ldquoCharacter-ization of the bovine pregnancy-associated glycoprotein genefamilymdashanalysis of gene sequences regulatory regions withinthe promoter and expression of selected genesrdquo BMCGenomicsvol 10 article 185 pp 1ndash17 2009
[101] P Humblot S Camous J Martal et al ldquoDiagnosis of pregnancyby radioimmunoassay of a pregnancy-specific protein in theplasma of dairy cowsrdquo Theriogenology vol 30 no 2 pp 257ndash267 1988
[102] G H Kiracofe J M Wright R R Schalles C A Ruder SParish and R G Sasser ldquoPregnancy-specific protein B in serumof postpartum beef cowsrdquo Journal of Animal Science vol 71 no8 pp 2199ndash2205 1993
[103] A P Zoli L A Guilbault P Delahaut W B Ortiz and J-FBeckers ldquoRadioimmunoassay of a bovine pregnancy-associatedglycoprotein in serum its application for pregnancy diagnosisrdquoBiology of Reproduction vol 46 no 1 pp 83ndash92 1992
[104] R G Sasser C A Ruder K A Ivani J E Butler and W CHamilton ldquoDetection of pregnancy by radioimmunoassay of anovel pregnancy-specific protein in serum of cows and a profileof serum concentrations during gestationrdquo Biology of Reproduc-tion vol 35 no 4 pp 936ndash942 1986
10 The Scientific World Journal
[105] J A Green T E Parks M P Avalle et al ldquoThe establishmentof an ELISA for the detection of pregnancy-associated glyco-proteins (PAGs) in the serum of pregnant cows and heifersrdquoTheriogenology vol 63 no 5 pp 1481ndash1503 2005
[106] E Silva R A Sterry D Kolb et al ldquoAccuracy of a pregnancy-associated glycoprotein ELISA to determine pregnancy statusof lactating dairy cows twenty-seven days after timed artificialinseminationrdquo Journal of Dairy Science vol 90 no 10 pp 4612ndash4622 2007
[107] A Ayad N M Sousa J Sulon J L Hornick M Iguer-Ouada and J F Beckers ldquoCorrelation of five radioimmunoas-say systems for measurement of bovine plasma pregnancy-associated glycoprotein concentrations at early pregnancyperiodrdquo Research in Veterinary Science vol 86 no 3 pp 377ndash382 2009
[108] N M de Sousa M Zongo W Pitala et al ldquoPregnancy-associated glycoprotein concentrations during pregnancy andthe postpartum period in Azawak Zebu cattlerdquoTheriogenologyvol 59 no 5-6 pp 1131ndash1142 2003
[109] A Karen S Darwish A Ramoun et al ldquoAccuracy of ultra-sonography and pregnancy-associated glycoprotein test forpregnancy diagnosis in buffaloesrdquoTheriogenology vol 68 no 8pp 1150ndash1155 2007
[110] N M Sousa A Ayad J F Beckers and Z Gajewski ldquoPreg-nancy-associated glycoproteins (PAG) as pregnancy markers inthe ruminantsrdquo Journal of Physiology and Pharmacology vol 57no 8 pp 153ndash171 2006
[111] P R Graves andTA J Haystead ldquoMolecular biologistrsquos guide toproteomicsrdquo Microbiology and Molecular Biology Reviews vol66 no 1 pp 39ndash63 2002
[112] J D Lippolis and T A Reinhardt ldquoCentennial paper pro-teomics in animal sciencerdquo Journal of Animal Science vol 86no 9 pp 2430ndash2441 2008
[113] D A Colantonio and D W Chan ldquoThe clinical application ofproteomicsrdquo Clinica Chimica Acta vol 357 no 2 pp 151ndash1582005
[114] F di Girolamo E Boschetti M C M Chung F Guadagniand P G Righetti ldquolsquoProteomineeringrsquo or not The debate onbiomarker discovery in sera continuesrdquo Journal of Proteomicsvol 74 no 5 pp 589ndash594 2011
[115] D I Jin H R Lee H R KimH J Lee J T Yoon andC S ParkldquoProteomics analysis of pregnancy-specific serum proteins inbovinerdquoReproduction Fertility andDevelopment vol 18 no 1-2p 183 2005
[116] R Han H R Kim K Naruse et al ldquoIdentification of bovinepregnancy-specific milk proteins using proteomicsrdquo Biology ofReproduction vol 78 p 143 2008
[117] A K Balhara Proteomic analysis of pregnancy-specific serumproteins in buffalo [PhD thesis] LLRUVAS Hisar India 2012
[118] A Busch S Michel C Hoppe D Driesch U Claussen and Fvon Eggeling ldquoProteome analysis of maternal serum samplesfor trisomy 21 pregnancies using ProteinChip arrays and bioin-formaticsrdquo Journal of Histochemistry and Cytochemistry vol 53no 3 pp 341ndash343 2005
[119] P A Benn ldquoAdvances in prenatal screening for Down syn-drome I General principles and second trimester testingrdquoClinica Chimica Acta vol 323 no 1-2 pp 1ndash16 2002
[105] J A Green T E Parks M P Avalle et al ldquoThe establishmentof an ELISA for the detection of pregnancy-associated glyco-proteins (PAGs) in the serum of pregnant cows and heifersrdquoTheriogenology vol 63 no 5 pp 1481ndash1503 2005
[106] E Silva R A Sterry D Kolb et al ldquoAccuracy of a pregnancy-associated glycoprotein ELISA to determine pregnancy statusof lactating dairy cows twenty-seven days after timed artificialinseminationrdquo Journal of Dairy Science vol 90 no 10 pp 4612ndash4622 2007
[107] A Ayad N M Sousa J Sulon J L Hornick M Iguer-Ouada and J F Beckers ldquoCorrelation of five radioimmunoas-say systems for measurement of bovine plasma pregnancy-associated glycoprotein concentrations at early pregnancyperiodrdquo Research in Veterinary Science vol 86 no 3 pp 377ndash382 2009
[108] N M de Sousa M Zongo W Pitala et al ldquoPregnancy-associated glycoprotein concentrations during pregnancy andthe postpartum period in Azawak Zebu cattlerdquoTheriogenologyvol 59 no 5-6 pp 1131ndash1142 2003
[109] A Karen S Darwish A Ramoun et al ldquoAccuracy of ultra-sonography and pregnancy-associated glycoprotein test forpregnancy diagnosis in buffaloesrdquoTheriogenology vol 68 no 8pp 1150ndash1155 2007
[110] N M Sousa A Ayad J F Beckers and Z Gajewski ldquoPreg-nancy-associated glycoproteins (PAG) as pregnancy markers inthe ruminantsrdquo Journal of Physiology and Pharmacology vol 57no 8 pp 153ndash171 2006
[111] P R Graves andTA J Haystead ldquoMolecular biologistrsquos guide toproteomicsrdquo Microbiology and Molecular Biology Reviews vol66 no 1 pp 39ndash63 2002
[112] J D Lippolis and T A Reinhardt ldquoCentennial paper pro-teomics in animal sciencerdquo Journal of Animal Science vol 86no 9 pp 2430ndash2441 2008
[113] D A Colantonio and D W Chan ldquoThe clinical application ofproteomicsrdquo Clinica Chimica Acta vol 357 no 2 pp 151ndash1582005
[114] F di Girolamo E Boschetti M C M Chung F Guadagniand P G Righetti ldquolsquoProteomineeringrsquo or not The debate onbiomarker discovery in sera continuesrdquo Journal of Proteomicsvol 74 no 5 pp 589ndash594 2011
[115] D I Jin H R Lee H R KimH J Lee J T Yoon andC S ParkldquoProteomics analysis of pregnancy-specific serum proteins inbovinerdquoReproduction Fertility andDevelopment vol 18 no 1-2p 183 2005
[116] R Han H R Kim K Naruse et al ldquoIdentification of bovinepregnancy-specific milk proteins using proteomicsrdquo Biology ofReproduction vol 78 p 143 2008
[117] A K Balhara Proteomic analysis of pregnancy-specific serumproteins in buffalo [PhD thesis] LLRUVAS Hisar India 2012
[118] A Busch S Michel C Hoppe D Driesch U Claussen and Fvon Eggeling ldquoProteome analysis of maternal serum samplesfor trisomy 21 pregnancies using ProteinChip arrays and bioin-formaticsrdquo Journal of Histochemistry and Cytochemistry vol 53no 3 pp 341ndash343 2005
[119] P A Benn ldquoAdvances in prenatal screening for Down syn-drome I General principles and second trimester testingrdquoClinica Chimica Acta vol 323 no 1-2 pp 1ndash16 2002
