Ž . Animal Reproduction Science 62 2000 77–111 www.elsevier.comrlocateranireprosci Storage of ram semen S. Salamon, W.M.C. Maxwell ) Department of Animal Science, UniÕersity of Sydney, Sydney, NSW 2006, Australia Abstract Storage of ram semen in liquid and frozen state, the diluents used for both methods, processing, cooling, freezing and thawing of semen are reviewed. Factors influencing the fertility of stored semen and methods used for improvement are discussed, and fertility results of long-term frozen stored ram semen are also given. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Spermatozoa; Liquid and frozen storage; Diluents; Protective agent; Freezing; Thawing; Cervical and intrauterine insemination 1. Introduction Ž . Investigations on artificial insemination AI of sheep were initiated at the beginning Ž . of the twentieth century by Ivanov 1907, 1912 , whose studies on diluting media and reproduction led to the development and the practical application of AI with farm animals. After the First World War, further intensive studies, under the leadership of Milovanov, were undertaken in the former Soviet Union, and by the early 1930s AI with fresh and diluted semen was used on a large scale in sheep breeding programmes. The need to fertilise large numbers of ewes with semen of outstanding rams required transport of semen from the points or centres of collection to the sites of insemination at more distant farms. The necessity to use the rams over extended periods, or at different times of the year, stimulated research on storage of spermatozoa under artificial conditions. This could be achieved by methods that reduced or arrested the metabolism of spermatozoa and thereby prolonged their fertile life. Accordingly, investigators exam- Ž. Ž . ined storage of semen in i a liquid unfrozen state, using reduced temperatures or ) Corresponding author. Tel.: q 61-29-351-4864; fax: q 61-29-351-3957. Ž . E-mail address: [email protected]W.M.C. Maxwell . 0378-4320r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. Ž . PII: S0378-4320 00 00155-X
Department of Animal Science, UniÕersity of Sydney, Sydney, NSW 2006, Australia
Abstract
Storage of ram semen in liquid and frozen state, the diluents used for both methods,processing, cooling, freezing and thawing of semen are reviewed. Factors influencing the fertilityof stored semen and methods used for improvement are discussed, and fertility results oflong-term frozen stored ram semen are also given. q 2000 Elsevier Science B.V. All rightsreserved.
Ž .Investigations on artificial insemination AI of sheep were initiated at the beginningŽ .of the twentieth century by Ivanov 1907, 1912 , whose studies on diluting media and
reproduction led to the development and the practical application of AI with farmanimals. After the First World War, further intensive studies, under the leadership ofMilovanov, were undertaken in the former Soviet Union, and by the early 1930s AI withfresh and diluted semen was used on a large scale in sheep breeding programmes.
The need to fertilise large numbers of ewes with semen of outstanding rams requiredtransport of semen from the points or centres of collection to the sites of insemination atmore distant farms. The necessity to use the rams over extended periods, or at differenttimes of the year, stimulated research on storage of spermatozoa under artificialconditions.
This could be achieved by methods that reduced or arrested the metabolism ofspermatozoa and thereby prolonged their fertile life. Accordingly, investigators exam-
Ž . Ž .ined storage of semen in i a liquid unfrozen state, using reduced temperatures or
Ž .other means to depress sperm metabolism; and ii in a frozen state which involvedpreservation at sub-zero temperatures. Comprehensive reviews have been published on
Ž . Žliquid Maxwell and Salamon, 1993 and frozen storage of ram semen Salamon and.Maxwell, 1995a,b .
2. Liquid storage of semen
ŽThe main methods of storage of semen in a liquid state are storage at reduced 0–5 or.10–158C and at ambient temperatures by reversible inactivation of spermatozoa.
2.1. Storage at reduced temperatures
Ž .Milovanov 1940, 1951 described the early work and discussed the problems ofŽstorage of ram spermatozoa in a chilled state. Whereas British investigators Chang and
.Walton, 1940 and some Soviet workers claimed that 10–158C was the ‘‘optimum’’Ž .temperature for liquid storage, most Soviet investigators reported that survival of ram
and bull spermatozoa was better after storage at 0–58C.When semen was stored at a low temperature, care had to be taken not to subject the
spermatozoa to cold shock. This caused irreversible changes in sperm cells cooled totemperatures close to 08C. The detrimental effects of cold shock, discovered in 1931Ž .Milovanov, 1951 , were overcome either by gradual cooling of semen from roomtemperature, or by addition of lipid to the diluent. The value of lipids from various
Ž .sources egg yolk, testicles, corpus luteum, brain and soy beans in the protection ofspermatozoa against cold shock was initially reported by Milovanov and Selivanova in
Ž . Ž .1932 cited by Milovanov, 1962 . Lardy and Phillips 1939 and Phillips and LardyŽ .1940 applied these observations in the development of the egg yolk–phosphate diluentfor the preservation of bull semen. Another generally accepted storage medium, the egg
Ž .yolk–citrate diluent, was elaborated later by Salisbury et al. 1941 and Willett andŽ .Salisbury 1942 .
Before 1940, the main diluent used in the former Soviet Union for liquid storage ofŽ . Ž .ram semen was glucose–phosphate–phospholipid soy extract medium GFO-5 . This
was subsequently replaced by glucose–citrate–egg yolk as the generally acceptedŽ . Ždiluent for ram semen Milovanov, 1962 . The egg yolk component and more likely its
.high molecular weight low density lipoprotein fraction , apart from providing protectionagainst cold shock, has been shown to reduce the loss of acrosomal enzymes and preventdegenerative changes in the acrosome during liquid storage. The composition of thecitrate diluent as used nowadays is:
Ž .2.37 g sodium citrate 2H O2
0.50 g glucose15 ml egg yolk100,000 i.u. penicillin100 mg streptomycinglass distilled water to 100 ml
Although fructose is the only simple carbohydrate present in ram semen, spermatozoacan also metabolise glucose and mannose when these sugars are included in the storagediluent. No other sugars act as energy sources, but a range of sugars have beenexamined for their ability to preserve the motility of spermatozoa. Most investigators
Žused glucose and fructose as sugar components of the diluent. Some other sugars e.g..sucrose and lactose may only act extracellularly to maintain the osmotic pressure of the
diluent and membrane integrity of the sperm cells during storage.Attempts to replace the traditional citrate buffer with glycine or addition of glycine to
the citrate buffer gave contradictory results regarding the survival of spermatozoa, andwere abandoned. Other diluents investigated, such as citrate–glycol–egg yolk–mucinaseŽ . Ž .Sokolovskaja et al., 1956 and citrate–boric acid–egg yolk Yoshioka et al., 1951 ,were variants of the above citrate diluent and remained only in experimental use.
During the early and mid-1970s, several studies concentrated on the use of organicbuffers, on the basis that these have better buffering capacity than phosphate or citrate,are relatively non-toxic to living cells, and may penetrate the sperm cell thereby actingas intracellular buffers against pH shifts, or may increase the tolerance of the cell to anintracellular increase in monovalent cations. Organic buffers often increase the overalltonicity of the diluent, which is important when the semen is stored.
Ž . Ž .Diluents containing tris hydroxymethyl aminomethane Tris as the main componentŽhave been examined for storage of semen from the bull, boar and ram reviewed by
.Visser, 1975 . Tris concentrations ranging from 10 to 50 mM were reported to have littleor no effect on motility and metabolism of ram spermatozoa. Subsequently, higherconcentrations of tris were reported to be advantageous in diluents for chilled storage.More research is required, particularly on those buffers with efficient hydrogen ionbuffering capacity in the ‘‘sperm tolerant’’ range of pH 6.5–7.5, for example Tesw Ž . x wN-Tris hydroxymethyl -methylaminoethane-sulfonic acid , Hepes N-2-hydroxyethyl-
X x w Ž . xpiperazine-N -2-ethanesulfonic acid , Mops 3- N-morpholino propane sulfonic acid ,w Ž . x Ž . ŽMes 2 N-morpholino ethane sulfonic acid and Pipes piperazine-N, N-bis 2-ethane
.sulphonic acid .ŽNowadays, the tris-based medium is a recommended extender for ram semen Evans
.and Maxwell, 1987 , and its composition is as follows:
3.63 g tris0.50 g fructose1.99 g citric acid14 ml egg yolk100,000 i.u. penicillin100 mg streptomycinglass-distilled water to 100 ml
Whole, skim or reconstituted milk have also been used for many years as extendersfor ram semen. The success of this diluent has been attributed to its protein fraction,which may act as a buffer against changes in pH and as a chelating agent against anyheavy metals present. It may also partially protect spermatozoa during reduction oftemperature for storage. Cows’ milk has been preferable to the milk of other species.
Before dilution of semen, the whole, skim or reconstituted milk should be heated at92–958C for 8–10 min to inactivate the lactenin in the protein fraction which is toxic tospermatozoa. The skim milk powder must be carefully selected because some samplesmay not be satisfactory for preparation of diluent. The reconstituted milk diluent isprepared by dissolving 9 g of skim milk powder in 100 ml of glass-distilled water. To
Žcontrol microbial growth, antibiotics e.g. 1000 i.u. sodium penicillin and 1 mg.streptomycin sulphate should be added to each milliliter of whole, skim and reconsti-
tuted milk diluents.Some investigators found that skim milk was better than whole milk for storage of
semen at 2–58C. When combined with antibiotics, it was as effective as egg yolk–glu-cose–citrate diluent for chilled storage of ram semen. Addition of 5% egg yolk and 1%glucose to the skim milk was claimed to improve the viability of spermatozoa during
Ž .chilled storage Feredean et al., 1967 . Regarding reconstituted skim milk, Frenchworkers found that viability and fertility of ram spermatozoa was better after storage for
Ž .8–16 h at 158C than at 58C Colas et al., 1968, 1974 .Ž .In past years, commercially available ultra-heat-treated UHT ‘‘long-life’’ milk has
proved a satisfactory diluent for fresh semen and also for maintaining the viability ofspermatozoa during liquid storage. The UHT milk is sterile, does not require heating andmay be used directly as a diluent without further treatment; a freshly opened carton ofUHT milk is required for use each day.
Ž .Recent studies on the suitability of a ‘‘chemically defined’’ diluent RSD-1 , withand without combination with antioxidants, for liquid storage of ram semen have yet to
Ž .improve fertility over other diluents Upreti et al., 1997 .
2.2. Storage at ambient temperatures by reÕersible inactiÕation of spermatozoa
The first experiments on the inhibitory effect of carbon dioxide on the motility ofspermatozoa of different species were performed in 1924 by Krshyshkovsky and PavlovŽ .cited by Milovanov, 1962 . More than three decades later, this method was re-examined
Ž .and a new extender for bull semen, called the Illini Variable Temperature IVT diluent,Ž .was elaborated Salisbury and VanDemark, 1961 . The diluent required gassing with
carbon dioxide before use.Another diluent, also developed for storage of bull semen at ambient temperature, the
Ž .Cornell University Extender CUE , is a self-gassing medium in which the carbonŽdioxide is derived from the reaction of citric acid and sodium bicarbonate Foote et al.,
.1958 . Both diluents have been modified for use with ram semen; they were saturatedwith carbon dioxide by gassing to pH 6.3 for about 10 min before use, or were‘‘self-carbonating’’ as a result of their carbonate or bicarbonate and acid content.
Ž .As an alternative to gassing, diluents containing volatile organic oxalic and aceticŽ .acids were also examined Habibulin, 1963 .
2.3. Fertility of semen after liquid storage and factors influencing it
There are many reports on the fertility of liquid stored semen after cervical insemina-tion, mainly from the former Soviet Union. A critical assessment of most reports is
difficult, as they do not provide sufficient information on essential experimentalprocedures. Details such as type or composition of diluent, temperature of storage,dilution rate, dose of inseminate, type of teaser rams used for the detection of oestrous
Ž .ewes vasectomised or aproned entire rams , number of inseminations within one oestrusŽ .and the method of determining the fertility lambing data were often not stated. Several
papers were in the form of reports of selected data from large-scale field inseminationprogrammes, rather than from proper experiments.
Ž .In critical studies reviewed by Maxwell and Salamon, 1993 , fertility declinedrapidly when semen stored for more than 24 h was used for cervical insemination. Thedecrease in fertility was at a rate of 10–35% per day of storage. Thus, while 68–75% ofewes lambed from insemination with fresh semen in a single cycle, the lambing rates forsemen stored for 24, 48 and 72 h were 45–50%, 25–30% and 15–20%, respectively.
Irrespective of the diluent, dilution rate, temperature or conditions of storage, thespermatozoa deteriorated as the duration of storage increased. The main changes thatoccurred during storage included reduction in motility and morphological integrity ofspermatozoa. These changes may be contributed to by the accumulation of the toxic
Ž .products of metabolism, mainly of reactive oxygen species ROS formed through lipidperoxidation of the membranes of spermatozoa. The above events are accompanied by adecline in transport and survival of spermatozoa in the female reproductive tract andreduction in fertility.
The cervix constitutes the initial barrier to the ascent of spermatozoa. By comparisonto fresh spermatozoa, a relatively small proportion of the stored cells deposited at theexternal os cervix during insemination penetrate the cervical canal and migrate throughthe tract of the ewe to the site of fertilisation. Establishment of an adequate cervicalsperm population is important with the insemination of sheep, as spermatozoa maycontinuously ascend to the oviducts from this reservoir. However, spermatozoa function-ally affected during liquid storage may not migrate, or may migrate slowly, and theirsurvival in the female tract is also reduced to about half that of fresh spermatozoaŽ .Lopyrin, 1971 . Attempts to improve the transport of spermatozoa from the posterior
Ž .cervix to the oviducts of oestrous ewes by prostaglandins PGF a or PGE added to2 2Žthe stored semen have given conflicting results reviewed by Maxwell and Salamon,
.1993 .Ž .It is also possible that the process of liquid storage like that of frozen storage
advances the maturation of sperm membranes, thus increasing the proportion of capaci-tated and acrosome reacted cells. Capacitated spermatozoa have reduced viability andlimited fertile life; or they may be rendered incapable of fertilisation if they are further
Žaged in the female reproductive tract after cervical insemination Maxwell and Watson,.1996 .
Early embryonic mortality is considered to be a further, or even one of the maincauses of low fertility. The condition of gametes at the time of fertilisation is known toaffect embryonic survival. An increase in abnormalities of embryonic developmentassociated with ageing of spermatozoa has been observed for several species, andconsiderable attention has been focused on possible changes in the haploid genome of
Ž .the sperm cell due to ageing Salisbury et al., 1976 . There is some evidence thatembryonic loss may be increased when stored spermatozoa are further aged in the
female tract resulting in asynchrony between the age of the spermatozoa and ovaŽ .Salamon et al., 1979 . Thus, there was a steeper decline with age of semen inembryonic survival after single than after double insemination, pointing to the impor-tance of time of semen deposition into the cervix to avoid additional ageing ofspermatozoa.
The problem of low fertility after cervical insemination with liquid stored ram semenstill remains unresolved. Increased depth of cervical insemination may improve thefertility, but it has so far been problematic in achieving sufficient penetration andobtaining acceptably high conception rates. A number of attempts have also been made
Žto penetrate the cervical canal and to deposit the semen in the uterus see Sections 3 and.3.6 . Improvement of the techniques proposed for transcervical insemination and further
research in this area seem warranted.The fertility of liquid stored semen can be improved by carefully timed intrauterine
insemination by laparoscopy, and when semen containing antioxidants is used. Surgicaluterine insemination after laparotomy resulted in acceptable fertility with chilled semenstored for 8 days, and some spermatozoa retained their fertilising capacity for up to 10
Ž . Ž .days Salamon et al., 1979 . Several antioxidants, such as superoxide dismutase SOD ,Ž .catalase CAT , cytochrome c and glutathione peroxidase had beneficial effects on
maintenance of motility and acrosome integrity of spermatozoa during chilled storagewhen added to the storage diluent. In fertility tests, using laparoscopic insemination,
Ž . Ž .SOD 800 Urml and CAT 200 Urml added to tris–glucose–egg yolk diluentimproved the fertility and extended the ‘‘useful’’ storage time to 14 days, when egg
Žfertilisation and pregnancy rates of 50% and 33% have been obtained Maxwell and.Stojanov, 1996 .
Storage of semen packaged in encapsulated form, as proposed for bovine semen byŽ . Ž .Nebel et al. 1993 , has been examined in the ram by Maxwell et al. 1996b . After 8
days storage at 58C, the viability and acrosome integrity of ram spermatozoa was lowerŽ . Žfor encapsulated in D-lysine and poly D-lysine microcapsules than for control unen-
.capsulated semen. In a fertility test, using semen stored at 58C for 20 h for intrauterineinsemination, there was no difference between encapsulated and control semen regarding
Ž .fertilisation rates 67% of 33 vs. 79% of 44 . Improvement of the viability ofencapsulated spermatozoa and attempts to use micro-encapsulated ram semen forcervical insemination remain future tasks.
3. Frozen storage of semen
Ž .The first records on freezing ram semen are by Bernstein and Petropavlovsky 1937Ž . Žwho successfully used glycerol solution 1 M, 9.2% for storage of mammalian rabbit,
. Ž .guinea pig, ram, bull, boar, stallion and avian fowl and duck spermatozoa at y218C;Ž .glycerol at 18% concentration had a toxic effect on spermatozoa. Smirnov 1949, 1950
Ž . Žduring 1947–1950 investigated vitrification without glycerol of small volumes 0.05–.0.10 ml of ram and bull semen; in a fertility trial in 1949, of 19 ewes receiving three to
Ž .five inseminations with frozen semen held for 15 min at y788C , seven ewes produced11 lambs. In 1951, insemination of 11 cows resulted in the birth of five calves. The
Ž .cryoprotective property of glycerol was brought into prominence by Polge et al. 1949whose finding gave a big impetus to research on cryopreservation of mammalian semen.
Considerable experimentation was conducted on frozen storage of ram semen duringthe 1950s. Initially the method and diluent for freezing bull semen was applied to ram
Žsemen with poor fertility results 5% lambing: Blackshaw and Emmens, 1953; Dauzier,.1956 . It became obvious that the ‘‘bull method’’ had limitations when adopted for ram
semen. Over the years of research, a wealth of information has been accumulated onaspects of frozen storage of ram semen such as various diluents, cryoprotective agents,processing, freezing and thawing methods, cryogenic damage and fertility of ramspermatozoa. These aspects will be discussed in the sections that follow.
3.1. Diluents
The diluents used for preservation of ram semen, as for other species, generallyshould have adequate pH and buffering capacity, suitable osmolality, and should protectspermatozoa from cryogenic injury. The diluents discussed here have been grouped onthe basis of their chronological use or development, with observations given on theirefficiency with regard to fertility.
3.1.1. Citrate–sugar-based diluentsOpinions of investigators differed on the ‘‘suitable’’ type of sugar to be included in
citrate medium. Using the post-thawing survival of spermatozoa as the criterion, someinvestigators have chosen arabinose, others fructose or glucose, as components of thecitrate diluent. Due to the decrease in osmotic pressure caused by the glycerol in theextender, hypertonic citrate–glucose–yolk or citrate–fructose–yolk diluents with anosmotic pressure of 8–12 atm were generally used. Ram semen, with a mean osmoticpressure of 7.7 atm, tolerated well the above range in diluent tonicity. Also, ramspermatozoa can tolerate a glucose or fructose concentration twice that of isotonicity dueto the capacity of monosaccharides to permeate the sperm cells, and thus equalise theosmotic gradient.
Ž . ŽThe lambing results for semen frozen in ampoules with citrate–glucose or.fructose –yolk diluents in experiments conducted under controlled conditions varied
Žbetween 17% and 40% after one to three inseminations reviewed by Salamon and.Maxwell, 1995a . The relatively high lambing rates claimed by some workers in the
former Soviet Union could not be confirmed and were severely questioned on the basisŽ .of experimental artefacts Lopyrin, 1971 .
The citrate–sugar-based diluents were seldom used for freezing ram semen after thelate 1960s.
3.1.2. Milk diluentsMilk has been adapted for freezing ram semen, mostly in reconstituted form,
combined with arabinose, fructose or egg yolk. In comparative laboratory studies, someworkers found that pasteurised whole or reconstituted skim milk, citrate–yolk, milk–glucose–yolk and citrate–glucose–yolk were of equal value. Other investigators re-
ported better post-thaw sperm survival after freezing with reconstituted skim milk thancitrate–yolk, fructose- or lactose-based diluents. Addition of egg yolk to heated ho-mogenised milk did not increase post-thaw sperm survival. Freezing in homogenisedmilk resulted in better recovery of spermatozoa than when frozen in Norman–JohnsonŽ . Ž .N-J-1 and N-J-2 media Patt and Nath, 1969 , but not when frozen in test-yolk, whichalso prevented the leakage of glutamic oxaloacetic transaminase from spermatozoaŽ .Hinshelwood et al., 1980 . In some laboratory studies, milk preparations were includedin synthetic diluents containing sugars and electrolytes.
Ž . Ž .The INRA-patented milk–citrate medium was used by several mainly Frenchworkers as a glycerolated portion of the two-step dilution method, and was added to the
Ž .cooled and partially diluted with lactose–yolk semen. Skim milk has been used as bothportions of the two-step dilution, and freezing of semen for practical use in SwedenŽ .Soderquist et al., 1996 .¨
The fertility results after cervical insemination with milk-frozen semen have beenŽ . Ž .variable reviewed by Salamon and Maxwell, 1995a . Some were poor 0–23% , othersŽ . Ž .moderate 30–45% . Reports on lambing rates of 50–75% or higher should be
regarded with caution.
3.1.3. Lactose-based diluentsSuccessful use of lactose as the main component of diluents for freezing bull semen
stimulated its application for ram semen. The initial in vitro studies with lactose-baseddiluents gave contradictory results, and the first fertility tests were also not convincing.In subsequent tests, the lambing results for lactose–yolk frozen semen were poorŽ . Ž .12–29% or moderate 40–50% . Lactose–yolk was used for both the non-glycerolatedand glycerolated diluent portions, or only for the non-glycerolated portion followed byglycerolated INRA medium.
Ž .Disaccharides lactose and saccharose were found more effective in lowering thecrystallisation temperature during freezing than monosaccharides, and this was increased
Ž .when they were combined with EDTA–Na and diethylamine Platov, 1988 . Inclusion2
of gum arabic, a complex polysaccharide, in a lactose-based diluent has also beenŽ . Ž .examined Platov, 1977 . Gum arabic is a high molecular weight )37,000 , water-
soluble substance secreted by acacia plants, and at 1.5–15.0% concentration in thediluent, is well tolerated by spermatozoa and has extracellular cryoprotective properties.
Ž .In the studies of Platov 1977 , the semen was diluted in two steps after collection, firstwith non-glycerolated lactose–yolk and then, after mixing, with lactose–gum arabic–yolk–glycerol or with lactose–yolk–EDTA–Na -diethylamine–gum arabic–glycerol.2
In further studies, for practical application, Platov et al. elaborated and recommendedŽ .the so-called VNII-plem system Russian Animal Research Institute, Lesnie Poljany for
Ž .processing and freezing of ram semen Platov, 1988 . According to this system, thesemen is diluted in two steps: first, 1.5- to 2-fold with non-glycerolated extender at288C; second, 2-fold at 248C with glycerolated medium. For both steps of dilution, either
Ž . Ž . Ž . Ž . Ž .lactose 9% –dextran 5% –EDTA–Na 0.135% –tris 0.105% –yolk 20% q2Ž . Ž .antibiotics q14% glycerol for the second step is used Variant 1 , or lactose
Ž . Ž . Ž .12% –yolk 20% qantibiotics for the first step and glycerolated 17% lactoseŽ . Ž . Ž . Ž . Ž .14.5% –gum arabic 6% –tris 0.6% –citrate 0.27% –yolk 20% qantibiotics for the
Ž . Ž .second step Variant 2 . The semen is cooled to 28C in 2 h, then pelleted 0.2 ml on dryice or a polymer plate at y808C to y908C.
In fertility tests comparing the diluent variants of Platov, the claimed lambing resultsvaried from 26% to 60%.
Ž . ŽArabonic acid extracted from tree gums substituted for gum arabic Sadykov et al.,. Ž1985 and inclusion of 5–6% polyglukine or 10% reopolyglukine glucose polymers
. Ž .with high molecular weight: 30–60,000 in lactose–yolk Zheltobjuk et al., 1981 didnot give better fertility than Platov’s diluents. However, insemination with semen frozenin lactose–dextran-based diluents resulted in more acceptable lambing rates.
3.1.4. Saccharose-based diluentsSaccharose was used as the main component of synthetic diluents because it was
found to protect the acrosome integrity of spermatozoa better than glucose, fructose orŽ .lactose Milovanov and Sokolovskaja, 1980b . Synthetic antioxidants have generally
been added to saccharose diluents to inhibit the peroxidation of sperm phospholipids,particularly of unsaturated fatty acids. Peroxidation may be prevented by processing in
Ž .anaerobic conditions, and inclusion of antioxidants and chelating agents e.g. EDTA inŽ .the diluent. According to Nauk 1991 , lipid peroxidation, as measured by the malon-
Ž .dialdehyde MDA; a secondary peroxide product content of thawed semen, is notŽ .prevented by the freeze–thawing procedure. However, Erochin and Derjazencev 1991
reported that MDA content was similar in fresh-diluted and thawed ram semen, and itincreased only during post-thawing incubation at 378C.
A substantial amount of work on the inhibition of peroxidation in saccharose-baseddiluents has been done by workers in the former Soviet Union. Inhibition of peroxida-
Ž .tion by providing anaerobic conditions hydrogen or nitrogen atmosphere duringprocessing of semen for freezing proved effective, but technically difficult. Therefore,attention was focused on the use of antioxidants, first of natural or synthetic tocopherolŽ .vitamin E . Tocopherol was most effective when included in saccharide-based diluentscontaining glucose, EDTA–Na and tris, and when the semen was processed in2
anaerobic conditions. Subsequently, the diluent was replaced by a saccharose–EDTA–ŽCaNa complex medium EDTA–Na saturated with Ca ions; Milovanov and2 2
.Sokolovskaja, 1980a , and several synthetic antioxidants were examined such as buty-Ž . Ž . Ž .lated hydroxytoluene BHT , 6-ditrat-butyl-1,4-kresol ‘‘ionol’’ or ‘‘topanol’’ DTBK ,
Ž . Žmonoethanolamine ‘‘kolamine’’ , phosphoethanolamine, and echinochrome A an ex-.tract from sea urchins with activity 10 times higher than DTBK .
Most workers claimed that processing and freezing of semen in diluents containingthe above antioxidants improved lambing rates after cervical insemination. Otherinvestigators, however, reported that BHT and vitamin E gave little protection to ramspermatozoa, although it was found that BHT could stabilise the membrane phospho-
Ž .lipids and decrease the permeability of the sperm membrane Hammerstedt et al., 1978 ,Ž .and that DTBK has a cryoprotective effect Milovanov et al., 1985 .
Several variants of the saccharose-based diluents served as a basis of the so-calledŽ .VIZ system All-Union Animal Research Institute, Moscow; Milovanov et al., 1985 .
The features of this system, recommended by the authors for practical use were: 3- toŽ .4-fold dilution of semen at room temperature with saccharose 9.8% –EDTA–CaNa2
Ž . Ž . Ž . Ž .0.84% –DTBK antioxidant 0.5% –egg yolk 10% –glycerol 5% qantibiotics, cool-Ž . Ž .ing to 2–48C in 3 h, and pellet freezing 0.2 ml on a polymer plate or dry ice . The
claimed lambing rates for semen frozen in the saccharose-based diluents with anti-oxidants varied from 19% to 55%.
ŽSaccharose-based diluents were used also in the KazNITIO system Kasakh Research.and Technological Institute for Sheep Breeding, Alma-Ata; Kasymov, 1984 and the
ŽKirgNPOZ system Kirghiz Association for Animal Research and Production; Sadykov.et al., 1985 . The composition of diluents used in the above two systems are described
Ž .by Salamon and Maxwell 1995a .
3.1.5. Raffinose-based diluentsRaffinose as a diluent component was brought into prominence by the observation of
Ž .Nagase and Graham 1964 with bull semen, that sugars of high molecular weightprovide better protection to spermatozoa during fast freezing than do those of lowmolecular weight. The better cryoprotective effect of trisaccharides in comparison to
Ž .mono- and disaccharides has also been reported when freezing ram semen. Nauk 1991found the order of efficiency in stabilising the protein–lipid complex of the cellmembrane to be: raffinose)saccharose) lactose) fructose)glucose, and the protec-tive effect of sugars in the higher rank was better when they were combined in certainproportions with other sugars, than when used alone.
Comprehensive studies on combinations of sodium citrate with various concentrationsŽ .of sugars arabinose, glucose, lactose, raffinose led to the elaboration of the raffinose
Ž . Ž . Ž . Ž .9.9% –citrate 2H O; 2% –egg yolk 15% –glycerol 5% medium for pellet freezing2Ž .of ram semen Salamon and Lightfoot, 1969; Lightfoot and Salamon, 1969a . These
workers found that the optimum osmotic pressure varied with the type of diluent, andthat hypertonic media were required for successful freezing of ram semen by the pelletmethod. The optimum osmotic pressure for the raffinose–citrate–yolk diluent was375–485 mosM. The high osmotic pressures due to increased raffinose concentrationswere less harmful to the survival of spermatozoa than high citrate concentrations. Thelambing results were 40–50% after cervical inseminations with semen frozen in this
Ždiluent using 150–180 million motile sperm for single and double insemination;.Lightfoot and Salamon, 1970a,b; Salamon and Lightfoot, 1970 .
Inclusion of glutamic acid and methionine in raffinose–citrate–yolk resulted inŽmodest lambing after both single and double insemination 31.3% and 39.8%; Smirnov
.et al., 1978 , and lambing was not improved when tris buffer was added to aŽ .raffinose–glucose–saccharose diluent Ivakhnenko and Aibasov, 1982 . However, it was
Ž .claimed Ostashko and Kantsedal, 1976 that, as with bull semen, addition of theŽ .lysomal ferment b-glucuronidase 150 Urml to raffinose–citrate–yolk improved post-
Ž .thawing survival and fertility of pellet-frozen ram semen 54.3% vs. 40.8% control . TheŽ .lambing was also improved in comparison to the control 37% when peroxidation was
Ž .inhibited by processing the semen in a hydrogen atmosphere 44% and also in theŽ .presence of an antioxidant 48% in the combined raffinose–lactose–fructose–mag-
Ž .nesium–yolk medium Nauk, 1991 .ŽRaffinose formed the basis of the so-called VNIIOK freezing system All-Union
.Sheep and Goat Research Institute, Stavropol elaborated and recommended for practical
Ž .use by Zheltobrjuk et al. 1987 . The system consisted of 2.5- to 3-fold dilution ofŽ . Ž .semen at 25–308C with a diluent containing raffinose 15% –saccharose 3% –glucose
Ž . Ž . Ž . Ž .0.5% –PVP 3%, molecular weight 8000–20,000 –tris 0.07% –yolk 20% –glycerolŽ . Ž .4.2% qantibiotics, cooling to 2–48C in 3–4 h before pellet freezing 0.15–0.20 ml on
Ž .a polymer plate y858C to y958C .
3.1.6. Tris-based diluentsTris in diluents for freezing ram semen was reported by several workers in the early
1970s. Systematic examination of tris as a basic diluent component for pellet freezingŽ .was undertaken by Salamon and Visser 1972 . In their studies, ram spermatozoa
tolerated a range of tris concentrations from 250 to 400 mM, and glucose was a moresuitable sugar component in the tris medium than fructose, lactose or raffinose. In
Ž . Ž . Ž .fertility tests, semen diluted 5-fold with tris 300 mM –glucose 27.75 mM –citricŽ . Ž . Ž .acid 94.7 mM –egg yolk 15% –glycerol 5% qantibiotics and pellet-frozen on dry
Žice, yielded lambing rates from 30–57% after cervical insemination Visser and Sala-.mon, 1973, 1974a .
Ž .Fischer 1990 found that tris with an osmolality of 375 mosMrkg containing 2%egg yolk was the best in preserving acrosomal integrity and motility after thawing.
Ž .Combination of 30–290 mM sugars mono- and disaccharides with 100–300 mM trisŽ .maintaining 325 mosmol osmolality did not influence post-thawing motility and
Ž .fertility of spermatozoa Molinia et al., 1994 . However, there are also reports that trisperformed better in vitro when combined with lactose rather than glucose, saccharose or
Ž .milk Zamfirescu et al., 1980 , although trisqskim milkqorvus-es-paste offered someŽ .protection to acrosome integrity Tekin and Gunzel, 1986 .¨
w Ž .In studies in vitro, Triladil a tris-based diluent proved to be of equal value or abetter freezing medium than lactose–yolk and saccharose–lactose–yolk, and addition of2% bovine serum albumin to Triladilw improved its protective effect on acrosomeintegrity. However, after insemination the conception rate was better for semen frozen in
w Ž .saccharose-based medium than in Triladil Marinov et al., 1983 . Subsequently,w ŽTriladil -frozen semen yielded promising fertility after transcervical insemination Hal-
.bert et al., 1990 .Tris–fructose was used as the first diluent part in the two-step method of Fiser et al.
Ž .1987 , and tris has also been included as a component in some lactose- and saccharose-Ž .based diluents. The tris–glucose diluent elaborated by Salamon and Visser 1972 is
Žwidely used and recommended for the frozen storage of ram semen Salamon, 1976;.Evans and Maxwell, 1987 .
3.1.7. Other diluentsZwitterion buffers such as tes, hepes and pipes have been used with varying success
as the basis of diluents for freezing ram semen. Fertility of semen frozen in test mediumŽ . Ž .tes titrated with tris varied from poor to satisfactory 13–67% lambing . Test combinedwith skim milk and the zwitterion buffers tes, hepes and pipes were better thantris–glucose–yolk diluents in terms of post-thawing motility, but inferior in preservingacrosomal integrity of spermatozoa. Fertility was lower for semen frozen in zwitterionbuffers than in tris–glucose–yolk.
Noteworthy among other substances examined are dextran and hydroxyethyl starch.ŽWhen these were added to a combined medium tes–citrate–glycine–lactose–
.raffinose–fructose–citric acid and used as the non-glycerolated diluent portion, fol-lowed by glycerolated milk–citrate, more effective cryoprotection was provided tospermatozoa than by using lactose–yolk as the first and INRA medium as the second
Ž .diluent portion Fiser, 1975 .
3.2. ProtectiÕe agents and methods of addition to semen
3.2.1. GlycerolGlycerol is the most commonly used protective substance in diluents for freezing ram
semen. For semen frozen by the slow ‘‘conventional’’ method, and using mainlyhypertonic diluents, most investigators found that the optimal glycerol concentration waswithin the range of 6–8%; spermatozoa frozen rapidly by the pellet method survived
Ž .best with 3–4% glycerol in the diluent. Graham et al. 1978 reported that glycerollevels above 6% were detrimental to post-thawing survival of spermatozoa. The actionof glycerol on ram and bull spermatozoa seems to be different, as it more easily
Ž .penetrates ram spermatozoa Nauk et al., 1970 . It was suggested that the optimalglycerol concentration in diluted semen is also related to its final concentration relative
Ž .to the spermatozoa Colas, 1975 .The level of glycerol included in diluents for frozen storage of ram semen is
Ž .ultimately limited by its toxicity Fahy, 1986 , which in turn depends on cooling andfreezing rate, diluent composition, and method of addition of glycerol. Examination of
Žthe combined effects of glycerol concentrations and cooling rates 0–8% and pelletingon dry ice and into liquid nitrogen, Visser and Salamon, 1974c; 0–16% and cooling at
.1–1008Crmin, Fiser and Fairfull, 1984 showed that the higher the cooling rate, thelower the optimum glycerol concentration, and the best post-thaw sperm survival ratesobserved by the latter investigators were for 4–6% glycerol and a freezing rate of10–1008Crmin.
Apart from the cooling rate, the optimum glycerol concentration also depends on theŽcomposition of the diluent and in particular on its osmotic pressure Salamon, 1968;
.Lightfoot and Salamon, 1969a . Furthermore, the glycerol concentration may be influ-enced by the egg yolk level in the diluent, as increased concentrations of egg yolk may
Ž .reduce the required concentration of glycerol Watson and Martin, 1974 .There are reports indicating that inclusion of antioxidants with a cryoprotective effect
in the diluent may also allow the use of a relatively low glycerol concentration of 3.5%,Ž .2.5% or 2.0% Varnavskij and Varnavskaja, 1976; Milovanov et al., 1985 .
Ž .Glycerol can be added to the semen in a separate diluent fraction two-step dilution ,Ž .or by a single addition of the diluent containing glycerol one-step method . Initially, it
was found better to add the glycerolated diluent portion at 298C than at 58C, but insubsequent tests addition at 58C was more suitable. Addition of glycerolated diluent at4–58C was preferred by most workers, but some found no difference between addition ateither 328C or 38C or at 228C or 58C.
The glycerol-containing diluent generally was of the same type as that used for initialŽ .extension at 308C , but media of differing composition have also been used. French
workers found that post-thawing recovery and fertility of spermatozoa were better whenthe partially diluted semen was glycerolated with INRA medium than with the initiallactose–yolk extender, and lambing was also better when glycerolisation occurred at 48C
Ž .rather than at 308C Colas, 1975; Colas and Brice, 1975 .Ž .Colas 1975 suggested that glycerol may be slightly toxic to spermatozoa even at a
concentration of 4% and its harmful effect is less when added at a temperature close to08C. Early workers using two-step dilution recommended a gradual addition of theglycerol-containing diluent at 2–58C. However, it has been found that single addition of
Ž .the glycerolated portion before freezing at 58C was as effective, or better than, thegradual addition in divided portions.
Ž .Contrary to the work recommending the use of two-step dilution, Salamon 1968 andŽ .Lightfoot and Salamon 1969a reported that a single dilution of semen with glycerol-
Ž .containing diluent at 308C ‘‘one-step’’ method was as good as two-step dilution, butthe efficiency of the former method depended on the sugar component of the diluent.One-step addition of glycerol at 308C is a practical and widely used method for dilution
Ž .of ram semen for frozen storage Salamon, 1976; Evans and Maxwell, 1987 .While glycerol offers cryoprotection to spermatozoa, it may also cause structural
damage during pre-freeze processing. Consequently, it was suggested that glycerolshould be added no more than 20–30 min before freezing. Effective cryoprotection after
Ž .short 5–10 s contact with glycerol has been demonstrated for bull and boar, and alsoŽ .for ram semen 0–5 min , which supports the earlier view that penetration of glycerol
into the cell is not essential for protection. Removal of glycerol from thawed semen bycentrifugation or by dialysis had no effect on lambing.
Successes in obtaining fertility with ram semen frozen without glycerol have beenŽ . Ž .reported by Lopatko 1976 and Abdelhakeam et al. 1991a who claimed 26% and 52%
lambing rates, respectively. For freezing ram semen without glycerol, Abdelhakeam etŽ .al. 1991b diluted the semen at 58C, 3 h after collection, with hypertonic test buffer
containing 25–30% egg yolk and 10% maltose monohydrate. The efficacy of theprocedure remains to be confirmed.
Several agents other than glycerol have also been examined for their cryoprotectiveŽ .action on ram spermatozoa. Such agents were dimethylsulfoxide DMSO , ethylene
glycol, albumin, low molecular weight polyols, polymeric compounds, surfactants, highŽconcentrations of sugars of various types, compatible solutes proline, glycine betaine,
. Žtaurine and antifreeze proteins from polar fish reviewed by Salamon and Maxwell,.1995a . None of the agents examined proved better than glycerol.
3.2.2. Egg yolkEgg yolk is a common constituent of semen diluents, protects the spermatozoa
against cold shock and confers protection during freezing and thawing. It is believed toact at the level of the cell membrane, and to have a bigger effect for bull than ramspermatozoa.
A wide range of egg yolk concentrations have been examined in diluents for freezingof ram semen. Early workers used 30–50%, but most subsequent investigators includedlower concentrations in the diluent. For freezing ram semen in ampoules, egg yolkconcentrations of 3–6% seemed sufficient, but for pellet freezing 15% was the optimum
Žconcentration, although the effect depended on the composition of the diluent Salamon.and Lightfoot, 1969 . Some investigators examined egg yolk concentrations as low as
Ž .1.5–3.75%, but others Graham et al., 1978 found that low levels were unsatisfactory.Ž .The saccharose–EDTA–CaNa complex diluent allowed the use of low 4–5% egg2
Ž .yolk concentrations and even its elimination was suggested Milovanov et al., 1985 . OnŽ .the other hand, in the attempt of Abdelhakeam et al. 1991b to freeze ram semen
without glycerol, it was necessary to increase the egg yolk to 25–30% in the test diluentfor successful freezing.
Ž .Egg yolk, although sometimes partially replaced by substance s with similar activity,is still likely to remain an important component of diluents for freezing ram semen,particularly due to its protective effect on the plasma membrane.
3.3. Processing and freezing of semen
The success of deep freezing depends to a notable degree on the rate of dilution ofsemen. Originally, semen was diluted to protect spermatozoa during cooling, freezingand thawing, but the rate of dilution was often changed for technical reasons, such as toincrease the number of females which could be inseminated with each ejaculate, or tostandardise the number of spermatozoa in each dose of frozen–thawed semen.
The pre-freezing dilution rate used by early workers varied from 11- to 26-fold, butŽ .the majority of subsequent investigators used 2- to 6-fold vrv dilution rates. As the
diluent composition was not adjusted for the different rates of dilution, it could not bedetermined whether any effect on spermatozoa was due to dilution per se, or to variationof the diluent components in the diluted semen. For freezing ram semen nowadays,
Ž .generally 2- to 5-fold vrv pre-freezing dilution rates are used, with the diluentŽcomposition adjusted for the rate of dilution Salamon, 1976; Evans and Maxwell,
.1987 .After dilution, the semen is cooled to a temperature close to 08C. Cooling is a period
of adaptation of spermatozoa to reduced metabolism. Traditionally, equilibration hasbeen regarded as the total time spermatozoa remain in contact with glycerol beforefreezing, during which it penetrates into the sperm cell to establish a balanced intra-cellular and extracellular concentration. It should not be overlooked that the term‘‘equilibration’’ includes the concentration balance not only of glycerol, but also of theother osmotically active diluent components. Glycerol has been implicated in someundesirable phenomena occurring during equilibration such as changes in the structureand biochemical integrity of spermatozoa and acceleration of the acrosome reaction.Although these may be overlooked in the light of the other favourable cryogenicproperties of glycerol, it should also be noted that this medium may, to a certain extent,adversely affect the fertility.
With the two-step method of dilution the contact with glycerol begins at 2–58C.However, when one-step dilution is adopted, glycerolisation begins at 308C. In bothmethods, a period of cooling to 2–58C is required, the duration of which generally variesfrom 1 to 3 h. Rapid cooling of extended semen from 308C to about 158C may have noeffect on survival of spermatozoa, but fast cooling from 308C to 108C, 58C or 08C
Ž .decreases the post-thaw motility of spermatozoa Fiser and Fairfull, 1986 .
In the case of one-step dilution to the final rate at 308C with the glycerolatedŽ .medium, a combined cooling–equilibration storage is involved. In the studies on pellet
Ž .freezing of ram semen Lightfoot and Salamon, 1969a , the optimum period of storageŽ .equilibration after one-step dilution and cooling to 58C depended on the sugarcomposition of the diluent and the glycerol concentration. The presence of raffinose inthe diluent allowed a decrease in equilibration from 2.5 to 1 h. Nowadays the one-stepmethod of dilution with glycerolated medium is generally used and it is recommended to
Žfreeze the semen after 1.5 to 2 h cooling to 58C Salamon, 1976, Evans and Maxwell,.1987 . There seems to be no convincing evidence supporting either a longer
coolingrequilibration period, or the need for the more complicated two-step dilutionprocedure.
The cooling rate of diluted semen from temperatures just above 08C can significantlyinfluence the survival of spermatozoa after freezing. Ram semen frozen in ampoules hasgenerally been cooled at the slow ‘‘conventional’’ rate applied for bull semen. Thismethod is seldom used today, and freezing is done in PVC straws and minitubes or inpellet form.
Freezing of semen in straws is performed by suspension in liquid nitrogen vapour,and the velocity of cooling is regulated by the distance of the straws from the level ofthe liquid nitrogen. Ram spermatozoa tolerate a range of freezing velocities in straws.Thus, suspension of straws in liquid nitrogen vapour between y758C and y1258C had
Ž .no effect, but vapour at y558C reduced the survival of spermatozoa Colas, 1975 .Although cooling rate was found to interact with glycerol concentration, the coolingrates from 10 to 1008Crmin had a smaller effect on the survival of spermatozoa than
Ž .glycerol concentration Visser and Salamon, 1974c; Fiser and Fairfull, 1984 .The shape of the freezing curve may also be important for straw freezing. Instead of a
linear decrease in temperature, it is better to cool the semen according to a parabola-shaped curve, which can be achieved by suspending the straws 4–6 cm above the liquidnitrogen surface. Post-thaw recovery of motility of spermatozoa frozen in straws andminitubes on the surface of dry ice was poorer than of pelleted spermatozoa, but this
Ž .was not reflected in the fertility rate Maxwell et al., 1995a .Pellet-freezing is generally done by placing semen droplets into depressions on dry
Ž .ice or on a polymer plate cooled to y808C to y958C in the former Soviet Union . Thevelocity of cooling can be regulated by the volume of the semen pellet and thetemperature of the freezing agent. Nevertheless, pellet volumes from 0.03 to 0.86 ml,with different surface to volume ratios, have been frozen on dry ice without a reduction
Ž .in post-thaw motility, although the cooling rates varied 95 to 308Crmin for pellet sizesŽ .and also within the pellet Lightfoot and Salamon, 1969b; Visser and Salamon, 1974b .
Increased cooling rates obtained by pelleting on a stainless steel plate or on dry iceŽ .cooled in liquid nitrogen y1008C to y1608C had no effect on recovery of sperm
Ž .motility Salamon, 1970 and lambing for semen pellet-frozen at y798C or y1408CŽ .was not different Salamon, 1971 . Dropping the semen directly into liquid nitrogen
resulted in poor recovery of motility, or all spermatozoa died. The reason for this maybe that liquid nitrogen is not a good medium for very fast freezing. Since it is a liquid atits boiling point, it has a calefactory effect which delays the thermal contact with the
Ž . Ž .semen Rey, 1957 . However, when semen droplets 0.03 ml were pipetted into liquid
Ž .nitrogen, or vials with semen 1.5–2.5 ml were immersed into liquid nitrogen for 5–7 s,then elevated and held in liquid nitrogen vapour for a few minutes before re-immersioninto liquid nitrogen, post-thaw motility of pelleted spermatozoa was nearly similar to
Ž .that of sperm pelleted onto dry ice Visser and Salamon, 1974c , and after inseminationŽ .with the semen thawed in vials satisfactory pregnancy rates 51–69% were claimed
Ž .Kasymov and Ashimov, 1975 .
3.4. Thawing of semen
In the freeze–thaw procedure, the warming phase is just as important to the survivalof spermatozoa as the cooling phase. Spermatozoa that have survived cooling toy1968C still face the challenge of warming and thawing, and thus must traverse twice
Ž .the critical temperature zone y158C to y608C .Both rate of cooling and thawing exert an effect on the survival of spermatozoa
Ž .Mazur, 1985 . The effect depends on whether the rate of cooling has been sufficientlyhigh to induce intracellular freezing, or low enough to produce cell dehydration. In theformer case, fast thawing is required to prevent recrystallisation of any intracellular icepresent in the spermatozoa. Spermatozoa thawed at a fast rate may also be exposed for a
Ž .shorter time to the concentrated solute and cryoprotectant glycerol , and the restorationof the intracellular and extracellular equilibrium is more rapid than for slow thawing.
There was no agreement among early investigators on the optimum temperature forthawing of semen frozen in ampoules. Some found that ram spermatozoa cooled slowlyin ampoules by ‘‘the bull method’’ revived better when thawed at 40–438C, than slowly‘‘by reversing the cooling procedure’’. Other workers thawed the ampoules in ice water,
Ž .in water at 2–58C, 6–88C or at room temperature 18–208C with conflicting resultsregarding recovery of spermatozoa. In the majority of reports, ampoule–frozen ramsemen was thawed in a water bath at 378C.
Ram semen frozen in straws has been thawed by most investigators at 38–428C.Ž .Some workers reported that thawing at high temperatures 60–758C was comparable to
that at 38–428C in terms of post-thaw motility, acrosome integrity and fertility ofspermatozoa.
Ž .Pellet-frozen ram semen may be thawed either in a solution wet thawing or in dryŽ .tubes dry thawing . In the former case, not only the presence, but also the composition
of the thawing solution influenced the recovery of sperm motility, and the effectŽdepended on the composition of the sugar–citrate–yolk freezing diluent Lightfoot and
.Salamon, 1969b . Inositol–citrate, glucose–citrate and fructose solutions were the bestŽ .and of equal value for thawing at 378C semen pellets frozen in raffinose–citrate–yolk
diluent. A relationship between composition of freezing diluent and of thawing solutionŽwas also found when the tris-based freezing media were used Salamon and Visser,
.1972 . Further, there were interactions between the composition of freezing diluents andŽ .method of dry or wet thawing.
Thawing temperatures between 378C and 758C have been examined for both wet anddry thawing, and generally improvements in post-thaw motility rates were observed withincreasing temperature. In order to maintain the benefit of a high rate of warming,
without the risk of overheating the semen, interrupted thawing or the use of specialthawing devices have been recommended. In the former case the semen pellet, placed in
Ž .warm thawing solution 60–808C , was transferred to a water bath at 35–408C whenŽ .about two-thirds of the pellet has melted Smirnov et al., 1978 . The thawing devices
assure a continuous and fast separation of the liquid portion from the remaining solidpellet, and thus prevent further heating of the thawed semen portion. The different types
Ž .of thawing device have been described by Salamon and Maxwell 1995a .
3.5. Causes of low fertility after cerÕical insemination
Satisfactory sperm survival rates were obtained with a number of diluents and thecomplex technology of freezing ram semen appears to be satisfactorily developed.Nevertheless, final judgement on the technologies elaborated should be tempered,because lambing results similar to those for fresh semen have only rarely been obtainedafter cervical insemination. The low fertility after cervical insemination with frozen–thawed ram semen could be due to a variety of factors.
3.5.1. Damage to spermatozoa during freeze–thawingŽ .Although a relatively high proportion 40–60% of ram spermatozoa preserve their
motility after freeze–thawing, only about 20–30% remain biologically undamaged. Aspermatozoon may be motile, but damaged, in which case it is doubtful if such a cellwill fertilise the egg. Motility and structure of the spermatozoa are affected to differentextents, and it is not known whether the changes occur simultaneously or are caused atdifferent stages of the freeze–thaw procedure.
Ž .The basic cryogenic damage to spermatozoa may be ultrastructural physical ,biochemical or functional. Ultrastructural damage occurs to the plasma and acrosomemembranes, the acrosome, the mitochondrial sheath and the axoneme. Ultrastructuraldamage generally is more severe for ram than bull spermatozoa. After both slow and fastfreezing of ram semen, motility is better preserved than the morphological integrity ofspermatozoa. The plasma and acrosome membranes are more sensitive than the nucleus
Ž .and locomotor mid-piece part of the sperm cell. The outer membrane of the acrosomeis more vulnerable than its inner part and internal membrane. It has been observed thatthe mitochondrial architecture is altered by freeze–thawing, but the tail filament andfibrils showed no detectable change after freeze–thawing.
Ultrastructural damage during freeze–thawing is accompanied by biochemical changesor loss of their vital contents. Some of the measured losses and changes include the
Ž .release of glutamic oxaloacetic transaminase GOT , losses of lipoproteins and aminoacids, decrease in phosphatase activity, decrease in loosely bound cholesterol protein,increase in sodium and decrease in potassium content, inactivation of hyaluronidase andacrosin enzymes, loss of prostaglandins, reduction of ATP and ADP synthesis, decrease
Ž .in acrosomal proteolytic activity reviewed by Salamon and Maxwell, 1995b . Denatura-tion of DNA is also possible, as modifications in the chromatin structure of bull, boar,
Žcat, human and recently of ram spermatozoa have been observed Gillan and Maxwell,.1999 .
These ultrastructural and biochemical cryogenic changes to spermatozoa could beresponsible for a decrease in their functional integrity, survival in vivo, and fertilisingcapacity.
3.5.2. Transport, Õiability and fertilising ability of spermatozoa in the female genitaltract
ŽA number of investigators have examined these aspects reviewed by Salamon and.Maxwell, 1995b and found that there is inadequate transport and reduced viability of
frozen–thawed spermatozoa in the genital tract of the ewe, which are the major causesof low fertility after cervical insemination. After cervical insemination, frozen–thawedspermatozoa were expelled faster than fresh spermatozoa from the female reproductive
Ž .tract Gillan and Maxwell, 1999 . However, when the thawed semen was deposited intoŽ .the uterus or oviducts, high 85–95% egg fertilisation rates have been obtained,
showing that the presumably biologically intact spermatozoa maintained their fertilisingcapacity after freeze–thawing.
3.5.3. CapacitationŽ .It has been advocated Watson, 1995; Maxwell and Watson, 1996 that some aspects
Ž .of the cooling–freezing–thawing cycle similar to liquid storage may advance thematuration of sperm membranes, and increase the proportion of capacitated and acro-some reacted spermatozoa. Changes to the membranes of spermatozoa may not affectmotility, but shorten the lifespan of the cells and render them less fertile or incapable offertilisation. Ageing of capacitated spermatozoa in the reproductive tract after cervicalinsemination may further aggravate the situation. If such spermatozoa are not exposed tooocytes within a short time after insemination, fertilisation will not occur, as the cells
Žmay prematurely die in the lower part of the reproductive tract. In vitro studies Gillan.and Maxwell, 1999 showed that frozen–thawed spermatozoa are released earlier than
fresh spermatozoa after binding to oviduct cells, confirming their physiological readinessŽto participate in fertilisation. The membrane status of spermatozoa intact, capacitated
. Žand acrosome reacted can be assessed by functional assay zona-free hamster eggw x .penetration: Garde et al., 1993; and chlortetracycline CTC staining: Gillan et al., 1997 .
Ž .Where hamsters are not available like Australia, due to quarantine restrictions , in vitroŽ .matured zona-intact sheep oocytes may be used for penetrationrfertilization assays
Ž .Gillan and Maxwell, 1999 .
3.5.4. Embryonic mortalitySpermatozoa weakened or not mortally injured during freeze–thawing, and those
which after thawing and insemination are aged in the female tract may initiate embryosŽ .that are not viable and perish at an early stage. Maxwell et al. 1996a found that after in
vitro fertilisation of in vitro matured sheep oocytes, the stage of fertilisation may bemore advanced for oocytes inseminated with frozen–thawed than fresh spermatozoa.The fast cleavage observed also after cervical insemination with liquid stored semenŽ .Lopyrin and Rabocev, 1968 , can be considered to presage embryonic mortality. There
Ž .is some evidence Gillan et al., 1997 that ewes inseminated with frozen–thawedŽ .capacitated spermatozoa into the uterine horns, and at a time close to ovulation aremore likely to produce viable embryos.
3.5.5. Other causes of low fertilityLate oestrus is not favourable for transport and survival of spermatozoa and fertilisa-
tion is generally lower for short than long oestrus. Fertility may be reduced if eCG is notused in combination with a progestagen sponge for synchronisation and induction of
Ž . Ž .oestrus, and fertility is also low when oestrus occurs early 36 h rather than late 48 hafter progestagen sponge removal. The optimum time of insemination is between 48 and58 h after sponge removal.
3.6. Methods and attempts to improÕe fertility after cerÕical insemination
Since the low fertility obtained after cervical insemination with frozen–thawed semenis primarily due to failure of establishment of an adequate cervical population offunctionally intact spermatozoa, which is associated with impaired sperm transport in theewe’s genital tract, several methods and substances have been used in attempts toimprove the situation.
3.6.1. Increased concentration of spermatozoaAn obvious method was the reconcentration of the thawed semen by centrifugation in
order to deposit high numbers of spermatozoa at insemination. This resulted in anincreased number of sperm cells throughout the tract up to the oviduct, improved egg
Žfertilisation and lambing Lightfoot and Salamon, 1970b; Salamon and Lightfoot, 1970;.Colas and Guerin, 1981 . Reconcentration of thawed semen by filtration through a´
Ž .milliporous membrane Volkov, 1974 and insemination of high numbers of spermato-Žzoa in increased volumes have also been used to increase fertility Colas and Brice,
.1976; Colas, 1975 . However, the above methods were of low efficiency, as only a fewewes could be inseminated with one frozen–thawed ejaculate. Dilution of semen at a
Ž .low rate 1.5-fold before freezing in order to obtain inseminates with a high concentra-tion of thawed spermatozoa yielded poor lambing.
3.6.2. Use of relaxin, oxytocin, prostaglandins, other substances and methodsInjection of ewes with relaxin, oxytocin and prostaglandins had two possible aims: to
relax the cervix, which would permit deep cervical or uterine deposition of frozen–thawed semen, or to increase the contractility of the genital tract and thus improve thetransport of spermatozoa.
Relaxin injected 12 h before insemination had no relaxation effect on the cervix andŽ .did not affect the mean depth of insemination Salamon and Lightfoot, 1970 . Oxytocin
stimulated the contractile activity of the cervix and uterus in vivo and in vitro, dilatedthe cervix and improved sperm transport after mating, but not after insemination withfrozen–thawed semen in which case lambing was also depressed.
Introduction of the muscle relaxant cocaine into the cervix, or electrical stimulationŽ . Ž .3.7 V of the cervix before insemination to induce uterine contraction , had no
Ž . w Žbeneficial effect on lambing Varnavskij and Turbin, 1974 . Injection of Partusisten a.tocolytic substance 20 min before insemination improved the penetrability of the cervixŽ .but not the lambing Nehring et al., 1989 .
Ž .Prostaglandin PG E , PGE and PGF a stimulated the contractile activity of the1 2 2
cervix and uterus in vitro and in vivo, and supplementation of semen with PGE and1
PGF a before freezing also improved the transport of spermatozoa in the genital tract.2
PG improved the sperm motility and lambing when added to thawed semen, but notwhen used before freezing. Injection of ewes with PGF a before insemination with2
thawed semen also improved the lambing results, and hylase and buscolysin injectedŽintramuscularly increased the penetrability of the cervix reviewed by Salamon and
.Maxwell, 1995b .Treatment of frozen–thawed semen with metabolic stimulants such as methylxan-
thines caffeine and pentoxifylline, proteinase, kallikrein calcium, cyclic adenosine-3X,X Ž .5 -monophosphate cAMP , and 2-deoxyadenosine, which in a number of other species
stimulated sperm motility, had no such effect on ram spermatozoa, except for pentoxi-fylline. However, as the increase in motility by the latter compound was not reflected in
Ž .improved fertility after intrauterine insemination Maxwell et al., 1995b , it is doubtfulwhether pentoxifylline would improve the fertility in the case of cervical insemination.
3.6.3. Double inseminationDouble insemination is a generally accepted method of increasing fertility. The
magnitude of the response may depend on the sperm concentration in the inseminate andthe timing of insemination in relation to the stage of oestrus. Little advantage was gainedfrom the second insemination when the first had been performed at about the middle of
Ž .oestrus Salamon, 1972 . It should also be noted that in studies on double insemination,the number of motile frozen–thawed spermatozoa deposited by two inseminations wastwice that deposited by single insemination, and in such a case the benefit of double
Žinsemination can be accounted for by the increased number of spermatozoa Visser and.Salamon, 1974a . When equal total numbers of motile spermatozoa were deposited by
Ž .single and double inseminations within the optimum time range 12–28 h afterdetection of oestrus, the lambing results were similar, and the number of motilespermatozoa inseminated was more important than whether single or double insemina-
Ž .tions were performed Salamon, 1977 . Regarding the time interval between insemina-tions, a better lambing result can be expected when the first, performed on the morningof detection of oestrus, is followed by the second 8–10 h rather than 24 h later.
3.6.4. Depth of inseminationPenetration of the ewe’s cervix is difficult due to its peculiar structure, and for
deposition of thawed semen deep into the cervical canal different techniques and devicesŽhave been adopted. The cervical traction method which consisted of pulling the
.entrance of the cervix into the vagina by forceps and the cervical traction combinedŽ .with digital manipulation of the cervix through the rectum Andersen et al., 1973
allowed semen deposition to a depth of 2–5 cm, and the latter method even uterinedeposition of semen. Both methods improved the fertility, but were stressful to theanimal. Deep cervical semen deposition was easier to achieve by special inseminating
Ž .pipettes and devices described by Salamon and Maxwell, 1995b . The lambing resultsŽ .improved with increased depth of insemination into the cervix Fig. 1 , and were
Fig. 1. Effect of depth of insemination on fertility of frozen–thawed ram semen introduced through the cervixŽ .weighted means from 26 publications; 9716 ewes .
relatively consistent between workers inseminating to approximately the same depth.Deep cervical insemination allowed a decrease in the number of motile spermatozoa to adose of 20 or 40 million, which after single insemination gave 50% and 53% lambingrespectively, compared with 51% for control insemination with 80–100 million motile
Ž .spermatozoa Stojanov, 1980 .
3.6.5. Intrauterine inseminationThe problem of the cervical barrier in the ewe can be overcome by deposition of
Ž .frozen–thawed semen into the uterus via the cervix transcervical insemination ordirectly into the uterus by laparotomy or laparoscopy.
Success of penetration transcervically into the uterus varied between investigatorsfrom 39% to 62%. By using the ‘‘Guelph system’’ which involved traction of the cervix,
Ž .87% successful penetration has been claimed Buckrell et al., 1992 . The transcervicalinsemination methods, although offering success for intrauterine insemination, varyregarding the repeatability of penetration, are time consuming and rather stressful to theanimal. In an experiment conducted in Australia, the pregnancy rate at day 70 for ewes
Ž .inseminated by laparoscopy 48% was higher than for ewes inseminated by transcervi-Ž . Ž . Ž .cal intrauterine 32% or cervical 9% methods Windsor et al., 1994 .
Surgical insemination by laparotomy was used by early investigators to examine thefertilising capacity of frozen–thawed spermatozoa. Since the introduction of laparo-
Ž .scopic semen deposition Killeen and Caffery, 1982 , this method has been generallyŽadopted for intrauterine insemination in most sheep producing countries Evans, 1991;
.Haresign, 1992 , as it offers reliable and predictable lambing results. Noteworthy reportsare summarised in Table 1.
Laparoscopic insemination has been mainly performed at a synchronised oestrus andto a lesser extent in natural oestrus. In the case of synchronised oestrus, the mainvariables examined have been type of synchronising device, season of synchronisation,time of insemination, dose of inseminate and site of uterine deposition.
3.6.5.1. Type of synchronising deÕice. Laparoscopic insemination yielded better lambingŽ .in ewes synchronised with fluorogestone acetate FGA; 30 mg Chronogest than with
Ž .medroxy-progesterone acetate MAP; 60 mg Repromap sponges, and the time of
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.M.C
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Table 1Summary of noteworthy results on fertility of frozen–thawed semen after intrauterine insemination by laparoscopy
Reference Diluent Inseminated Time of No. of ewes Fertility Comments:Ždose total no. insemination inseminated % method of
6 Žsperm=10 in hours synchronisation:two uterine after sponge sponge, or
a b c. .horns removal other fertility data , or
Ž .Maxwell 1984 Tris–glucose– 40 motile 60–66 837 50.7 Mean from 17 flocks2yolk–glycerol FGA sponge
Fukui et al. Tris–glucose– 100–160 36–60 75 54.7 MAP spongea
Ž .1993a yolk–glycerolw a69 49.3 CIDR
Fukui et al. Tris–glucose– 36 44–52 55 56.4 0.05 ml inseminate dose Data pooled for progesteronew aŽ .1993b yolk–glycerol 71 59 62.7 0.1 ml inseminate dose sponge and CIDR
a Percentage of ewes lambing per inseminated.b Percentage of ewes pregnant per inseminated, determined by ultrasound 45–110 days after insemination.c Percentage of ewes pregnant at slaughter 50–100 days after insemination.dSemen frozen in pellets.eSemen frozen in 0.25 ml straws.gAI 12 h after oestrus detection.
Ž .insemination after sponge removal 48, 60, 72 h was more critical in MAP- than inŽ .FGA-treated ewes Eppleston and Roberts, 1986 . Also, lambing depended on the
wintravaginal device used for synchronisation of oestrus in the autumn 54.7 vs. 67.4 forŽ . xCIDR controlled internal drug release vs. Chronogest sponge , but not in the spring
Ž . Ž .59.2 vs. 56.2 respectively Wilson and Maxwell, 1989 .
3.6.5.2. Time of insemination. The appropriate time of insemination after spongeŽ .removal was found by different workers to be between 48 and 65 h. Early 36 h or late
Ž . Ž72–78 h insemination after sponge removal generally reduced the fertility see Table.1 . Differences in the median time of ovulation in different breeds of ewe at different
Ž .locations may explain the variations in fertility. Maxwell 1986a reported median timesof 55.8 and 59.7 h after FGA sponge removal for teased and unteased Merino ewesrespectively, compared with a mean time of 65.8 in the reports of Findlater et al.Ž .1988a,b for Bluefaced Leicester=Swaledale ewes. In some reports where the time ofovulation was known, inseminations before ovulation were most effective, whereas inothers later inseminations after the estimated time of ovulation were as efficient asinseminations before ovulation. Intrauterine insemination performed close to or coinci-dent with the time of ovulation resulted in low fertility.
In superovulated ewes, the effective time of intrauterine insemination was observedto vary within the range of 44 to 64 h after removal of progestagen sponges. Thedifferences may be attributed to variation in the precision and timing of ovulation due to
Ž .different superovulation regimes. The use of gonadotrophin releasing hormone GnRHin superovulated ewes improved the synchrony of ovulation, and thus increased thefertility after intrauterine insemination. Most workers have not been able to demonstratea beneficial effect of GnRH on the fertility of non-superovulated ewes.
Superovulation was found to impair both transport and viability of spermatozoa aftervaginal or cervical insemination, and also after late or early intrauterine insemination.Late insemination has been associated with abnormal egg fertilisation or failure offertilised eggs to cleave. It has also been suggested that the uterotubal junction may actas a barrier to the transport of frozen–thawed spermatozoa in superovulated ewes afterovulation, and that laparoscopy may interfere with normal ova capture and transport by
Žthe oviduct if performed close to ovulation in superovulated ewes reviewed by Salamon.and Maxwell, 1995b .
3.6.5.3. Dose of spermatozoa for insemination. Intrauterine insemination allows adecrease in the number of spermatozoa deposited in comparison with cervical insemina-tion. Nevertheless, account should be taken of the possible impairment of spermtransport through the uterotubal junction if the number of spermatozoa deposited in the
Ž .uterus is excessively low Hunter et al., 1982 .Ž .Fertility data for ewes inseminated with a range 10 to 200 million total of
frozen–thawed sperm doses are presented in Table 1. There are also reports onlaparoscopic insemination with lower numbers of spermatozoa. Fertility was not differ-
Ž .ent after deposition of 1 and 10 million total motile spermatozoa Walker et al., 1984 .The proportion of superovulated ewes with fertilised eggs after insemination of low
Ž .numbers of spermatozoa 0.1, 0.5, 1.0 million total spermatozoa was better for tubal
aAt day 50.b Pooled data for 10, 20, 40 and 80=106 motile sperm doses.
Ž . Ž56%, 70% and 100%, respectively than uterine insemination 0%, 11% and 40%,. Ž .respectively Maxwell et al., 1993 . It seems that the minimum number of frozen–thawed
motile spermatozoa which still yield satisfactory fertility after intrauterine inseminationof ewes in synchronised oestrus is about 1 million.
3.6.5.4. Site of intrauterine insemination by laparoscopy. The site of intrauterineinsemination may be a factor influencing fertility. Deposition of frozen–thawed semenin the middle and bottom of the uterine horns tended to be better than into the tip of the
Ž .horns 53% and 53% vs. 44% lambing, respectively and there was also an advantageŽ .from insemination into both rather than only one horn Maxwell, 1986b . However,
others have reported no difference between insemination in the tip or middle of the leftŽ .or right uterine horn or at the junction of the horns Reinhold et al., 1990 , and no effect
Ž .of site one or both horns of intrauterine insemination on fertility in synchronisedŽ .superovulated Evans et al., 1984 or non-superovulated ewes comparing two horns vs.Ž .body of uterus Eppleston, 1993 .
At present, intrauterine insemination by laparoscopy offers not only satisfactory andrepeatable lambing results with frozen–thawed ram semen, but also allows a substantialreduction in the number of motile spermatozoa deposited. It should be noted, however,that a number of studies encouraged the belief that the technique of transcervicalinsemination has the potential to replace the laparoscopic method in some situations.Further research on its application and a better understanding of the requirements for
Ž .cervical penetration is required. A recent study Maxwell et al., 1999 indicates thatfrozen–thawed spermatozoa supplemented with ram seminal plasma may yield accept-able fertility after cervical insemination. The efficiency and applicability of the methodsneeds confirmation.
4. Long-term frozen storage of semen
One of the important questions when storing frozen semen for a prolonged period iswhether or not there will be any decrease in fertility during storage. To answer thisquestion, a semen bank was laid down in 1968 and fertility tests were conducted after 3,
Ž .5, 7, 11, 16 and 27 years of storage Salamon et al., 1996 . The fertility results arepresented in Table 2. In tests 1 to 4, cervical inseminations were performed with a high
Ž .concentration of motile spermatozoa in the inseminate Lightfoot and Salamon, 1970b .The fertility results for semen stored for 3, 5, 7 and 11 years were not different.Intrauterine insemination of 10 to 80 million motile spermatozoa in test 5 gaveindistinguishable lambing results. A period of 27 years of storage had no effect onfertility, which shows that long-term frozen storage of ram semen is feasible and makespossible the banking of genetic resources in sheep breeding.
5. Concluding remarks
Several diluting–protecting media and different methods have been used for storageof ram semen in liquid and frozen state. Irrespective of the factors examined, thespermatozoa deteriorate during liquid storage and freeze–thawing of semen. The mainchanges that occur are ultrastructural, biochemical and functional. These are accompa-nied by impaired transport, decline in survival of spermatozoa in the female reproductivetract and reduced fertility.
Of the several methods used to improve the fertility after insemination, the mosteffective was laparoscopic instrauterine insemination. The method requires expertise andis relatively costly, but allows substantial reduction in the number of spermatozoainseminated and gives satisfactory fertility results. It is hoped that further research mayoffer cost-effective, yet efficient methods of use of stored ram semen.
References
Abdelhakeam, A.A., Graham, E.F., Vazquez, I.A., 1991a. Studies on the presence and absence of glycerol inunfrozen and frozen semen: fertility trials and the effect of dilution methods on freezing ram semen in theabsence of glycerol. Cryobiology 28, 36–42.
Abdelhakeam, A.A., Graham, E.F., Vazquez, I.A., Chaloner, K.M., 1991b. Studies on the absence of glycerolin unfrozen and frozen ram semen. Cryobiology 28, 43–49.
Andersen, K., Aamdal, J., Fougner, J.A., 1973. Intra-uterine and deep cervical insemination with frozen semenin sheep. Zuchthygiene 8, 113–118.
Bernstein, A.D., Petropavlovsky, V.V., 1937. Effect of non-electrolytes on viability of spermatozoa. Bjull.Ž . w xEksp. Biol. Med. 3 1 , 41–43, in Russian .
Walton, J.S., 1992. A breeding trial using a transcervical technique for artificial insemination in sheep. In:Proc. 12th Int. Congr. Anim. Reprod., The Hague vol. 3 pp. 1531–1533.
Chang, M.C., Walton, A., 1940. The effects of low temperature and acclimatization on the respiratory activityand survival of ram spermatozoa. Proc. R. Soc. B 129, 517–527.
Colas, G., 1975. Effect of initial freezing temperature, addition of glycerol and dilution on the survival andfertilizing ability of deep-frozen ram semen. J. Reprod. Fertil. 42, 277–285.
Colas, G., Brice, G., 1975. Etude des principaux facteurs de survie et de fecondance du sperme de belier apres`congelation. Ceres 2, 253–260.´
Colas, G., Brice, G., 1976. Seasonal variations of the fertilizing capacity of deep-frozen ram semen. In: Proc.8th Int. Congr Anim. Reprod. A.I., Crakow vol. 4 pp. 977–980.
Colas, G., Guerin, Y., 1981. A new method for thawing frozen semen. Theriogenology 16, 623–630.´
Colas, G., Dauzier, I., Courot, M., Ortavant, R., Signoret, J.P., 1968. Resultats obtenus au cours de l’etude de´ ´Ž . Ž .quelques facteurs importants de l’insemination artificielle ovine. Ann. Zootech. Paris 16 1 , 47–57.´
Dauzier, L., 1956. Quelques resultats sur l’ insemination artificielle des brebis et des chevres en France. In:´ ´ `Proc. 3rd Int. Congr. Anim. Reprod. A.I., Cambridge vol. 3 pp. 12–14.
Eppleston, J., 1993. Studies on the fertility of frozen–thawed ram semen. Ph.D. Thesis, University of Sydney,Sydney.
Eppleston, J., Roberts, E.M., 1986. The effect of progestogen, PMSG and time of insemination on fertility inewes following intra-uterine insemination with frozen semen. Aust. Vet. J. 63, 124–125.
wErochin, A.S., Derjazencev, V.I., 1991. Peroxidation of lipids in ram semen. Ovtsevodstvo 3, 17–19, inxRussian .
Evans, G., 1991. Application of reproductive technology to the Australian livestock industries. Reprod. Fertil.Dev. 3, 627–650.
Evans, G., Maxwell, W.M.C., 1987. Salamon’s Artificial Insemination of Sheep and Goats. Butterworths,Sydney, 194 pp.
Evans, G., Holland, M.K., Nottle, M.B., Sharpe, P.H., Armstrong, D.T., 1984. Production of embryos in sheepŽ .using FSH preparations and laparoscopic intrauterine insemination. In: Lindsay, D.R., Pearce, D.T. Eds. ,
Reproduction in Sheep. Australian Academy of Science and Australian Wool Corporation, Canberra, pp.313–315.
Fahy, G.M., 1986. The relevance of cryoprotectant toxicity to cryobiology. Cryobiology 23, 1–13.Feredean, T., Barbulescu, I., Popovici, P., 1967. Studies on improvement of media for dilution of ram
w xspermatozoa. Lucr. Stiint. Inst. Cercet. Zootech. Bucharest 25, 459–469, in Rumanian .Findlater, R.C.F., Haresign, W., 1987. Effect of timing of intrauterine insemination with frozen–thawed semen
on conception rates in ewes. Proc. Brit. Soc. Anim. Prod., Scarborough, Paper No. 27.Findlater, R.C.F., Haresign, W., Curnock, R.M., Beck, N.F.G., 1988a. Effect of timing of intrauterine
insemination with frozen–thawed semen on fertility in ewes. In: Proc. 3rd World Congr. Sheep Beef CattleBreed., Paris vol. 1 pp. 194–196.
Findlater, R.C.F., Haresign, W., Curnock, R.M., Beck, N.F.G., 1988b. Effect of timing of intrauterineinsemination with frozen–thawed semen on fertility in ewes. Proc. 11th Int. Congr. Anim. Reprod. A.I.,Dublin vol. 3, Paper No. 242.
Findlater, R.C.F., Haresign, W., Curnock, R.M., Beck, N.F.G., 1991. Evaluation of intrauterine inseminationof sheep with frozen semen: effects of time of insemination and semen dose on conception rates. Anim.Prod. 53, 89–96.
Fischer, T., 1990. Computerized videomicrography applied to deep-frozen ram semen, with reference toosmolarity of the diluent and morpohology of the spermatozoal head cap. Inaugural-Dissertation, Fachbere-ich Veterinarmedizin, Freie Universitat, Berlin. A.B.A. No. 3712.¨ ¨
Fiser, P.S., 1975. New extenders for freezing ram semen. Cryobiology 16, 614–615.Fiser, P.S., Fairfull, R.W., 1984. The effect of glycerol concentration and cooling velocity on cryosurvival of
ram spermatozoa frozen in straws. Cryobiology 21, 542–551.Fiser, P.S., Fairfull, R.W., 1986. The effects of rapid cooling, cold shock of ram semen, photoperiod, and egg
yolk in diluents on the survival of spematozoa before and after freezing. Cryobiology 23, 518–524.Fiser, P.S., Ainsworth, L., Fairfull, R.W., 1987. Evaluation of a new diluent and different processing
procedures for cryopreservation of ram semen. Theriogenology 28, 599–607.Foote, R.H., Young, D.C., Dunn, H.O., 1958. Fertility of bull semen stored for one and two days at 58C in
20% yolk–citrate–glycine–glucose extenders. J. Dairy Sci. 41, 732.Fukui, Y., Hirai, H., Honda, K., Havashi, K., 1993a. Lambing rates by fixed-time intrauterine insemination
with frozen semen in seasonally anoestrous ewes treated with a progestogen-impregnated sponge orCIDRw device. J. Reprod. Dev. 39, 1–5.
Fukui, Y., Fuji, M., Tashiro, Y., 1993b. Insemination of frozen–thawed semen in seasonally anoestrous ewestreated with two different progestogen-impregnated devices. J. Reprod. Dev. 39, 269–273.
Garde, J., Guiterrez, A., Artiga, C.G., Vazquez, I., 1993. Influence of freezing process on in vitro capacitationŽ .of ram semen. Theriogenology 39, 225, Abstract .
Gillan, L., Maxwell, W.M.C., 1999. The functional integrity and fate of cryopreserved ram spermatozoa in thefemale tract. J. Reprod. Fertil., Suppl. 54, 271–283.
Gillan, L., Evans, G., Maxwell, W.M.C., 1997. The capacitation status and fertility of fresh and frozen–thawedram spermatozoa. Reprod. Fertil. Dev. 9, 481–487.
Gourley, D.D., Riese, R.L., 1990. Laparoscopic artificial insemination of sheep. Vet. Clin. North Am. FoodAnim. Prac. 6, 615–633.
Graham, E.F., Crabo, B.G., Pace, M.M., 1978. Current status of semen preservation in the ram, boar andŽ .stallion. J. Anim. Sci. 47 Suppl. 2 , 80–119.
Habibulin, I.H., 1963. The preservation of ram semen at temperatures above freezing point. Ovtsevodstvo 9Ž . w x7 , 28–29, in Russian .
Halbert, G.W., Dobson, H., Walton, J.S., Sharpe, P., Buckrell, B.C., 1990. Field evaluation of a technique fortranscervical intrauterine insemination of ewes. Theriogenology 33, 1231–1243.
Hammerstedt, R.H., Keith, A.D., Snipes, W., Amann, R.P., Arruda, D., Griel, L., 1978. Use of spin labels toevaluate effects of cold shock and osmolarity on sperm. Biol. Reprod. 18, 686–696.
Ž .Haresign, W., 1992. Manipulation of reproduction in sheep. J. Reprod. Fertil. Suppl. 45 , 127–139.Hinshelwood, E.A., Nelson, M., Burril, M.J., Drobnis, E.Z., 1980. Effects of diluent, washing and equilibra-
tion time on progressive motility and glutamic oxaloacetic transaminase levels for frozen ram semen. J.Ž .Anim. Sci. 51 Suppl. 1 , 440.
Hunter, R.H.F., Barwise, L., King, R., 1982. Sperm transport, storage and release in the sheep oviduct inrelation to the time of ovulation. Br. Vet. J. 138, 225–232.
Hunton, J.R., Flecker, S.E., Maxwell, W.M.C., 1987. Pregnancy rates following intra-uterine inseminationwith pellet or straw-frozen ram semen. J. Agric. Sci. 109, 189–191.
Ivakhnenko, V.K., Aibasov, M.M., 1982. The effect of electrolytes in sugar–egg yolk media on the quality offrozen ram semen. Breeding of Sheep and Goats and Wool Production. All-Union Research Institute for
w xSheep and Goat Breeding, Stavropol, pp. 77–79, in Russian .Ivanov, I.I., 1907. De la fecondation artificielle chez les mammiferes. Arch. Sci. Bio. Inst. Imp. Med. Exp., St´ ` ´
w xKasymov, K.T., 1984. A progressive method of storing semen. Ovtsevodstvo No. 7, 17–18, in Russian .Kasymov, K.T., Ashimov, Z.B., 1975. Fertilizing capacity of deep-frozen ram spermatozoa. Ovtsevodstvo No.
w x8, 9–11, in Russian .Killeen, I.D., Caffery, G.J., 1982. Uterine insemination of ewes with the aid of a laparoscope. Aust. Vet. J. 59,
95.Lardy, H.A., Phillips, P.H., 1939. Preservation of spermatozoa. Proc. Am. Soc. Anim. Prod. 32nd Ann. Meet.
pp. 219–221.Lightfoot, R.J., Salamon, S., 1969a. Freezing of ram semen by the pellet method. II. The effect of method of
dilution, dilution rate, glycerol concentration, and duration of storage at 58C prior to freezing on survival ofspermatozoa. Aust. J. Biol. Sci. 22, 1547–1560.
Lightfoot, R.J., Salamon, S., 1969b. Freezing of ram semen by the pellet method. III. The effect of pelletvolume, composition of the thawing solution, and reconcentration of the thawed semen on survival ofspermatozoa. Aust. J. Biol. Sci. 22, 1561–1572.
Lightfoot, R.J., Salamon, S., 1970a. Fertility of ram spermatozoa frozen by the pellet method. I. Transport andviability of spermatozoa within the genital tract of the ewe. J. Reprod. Fertil. 22, 399–408.
Lightfoot, R.J., Salamon, S., 1970b. Fertility of ram spermatozoa frozen by the pellet method. II. The effectsof method of insemination on fertilization and embryonic mortality. J. Reprod. Fertil. 22, 385–398.
Lopatko, M.I., 1976. A method of freezing animal semen in media without glycerol and egg yolk. Proc. 8thInt. Congr. Anim. Reprod. A.I., Crakow vol. 4 pp. 830–833.
w xLopyrin, A.I., 1971. Biology of Reproduction in Sheep. Kolos, Moscow, in Russian 320 pp.Ž .Lopyrin, A.I., Rabocev, V.K., 1968. The optimum time to use preserved semen. Ovtsevodstvo 14 7 , 20–22,
w xin Russian .Marinov, P., Zlatarev, S.T., Grudova, Ch.N., 1983. Semen diluents for freezing ram spermatozoa. Cryobiology
w xof Reproductive Cells. Bulgarian Academy of Science, Sofia, pp. 86–101, in Bulgarian .Maxwell, W.M.C., 1984. Current problems and future potential of artificial insemination programmes. In:
Ž .Lindsay, D.R., Pearce, D.T. Eds. , Reproduction in Sheep. Australian Academy of Science and AustralianWool Corporation, Canderra, pp. 291–298.
Maxwell, W.M.C., 1986a. Artificial insemination of ewes with frozen–thawed semen at a synchronised
oestrus. 1. Effect of time of onset of oestrus, ovulation and insemination on fertility. Anim. Reprod. Sci.10, 301–308.
Maxwell, W.M.C., 1986b. Artificial insemination of ewes with frozen–thawed semen at a synchronisedoestrus. 2. Effect of dose of spermatozoa and site of insemination on fertility. Anim. Reprod. Sci. 10,309–316.
Maxwell, W.M.C., Barnes, D.R., 1986. Induction of oestrus in ewes using a controlled internal drug releasedevice and PMSG. J. Agric. Sci. 106, 201–203.
Maxwell, W.M.C., Salamon, S., 1993. Liquid storage of ram semen: a review. Reprod. Fertil. Dev. 5,613–638.
Maxwell, W.M.C., Stojanov, T., 1996. Liquid storage of ram semen in the absence or presence of someantioxidants. Reprod. Fertil. Dev. 8, 1013–1020.
Maxwell, W.M.C., Watson, P.F., 1996. Recent progress in the preservation of ram semen. Anim. Reprod. Sci.42, 55–65.
Maxwell, W.M.C., Evans, G., Rhodes, S.L., Hillard, M.A., Bindon, B.M., 1993. Fertility of superovulatedewes after intrauterine or oviducal insemination with low numbers of fresh or frozen–thawed spermatozoa.Reprod. Fertil. Dev. 5, 57–63.
Maxwell, W.M.C., Landers, A.J., Evans, G., 1995a. Survival and fertility of ram spermatozoa frozen inpellets, straws and minitubes. Theriogenology 43, 1201–1210.
Maxwell, W.M.C., Robinson, S.J., Roca, J., Molinia, F.C., Sanchez-Partida, L.G., Evans, G., 1995b. Motility,´acrosome integrity and fertility of frozen ram spermatozoa treated with caffeine, pentoxifyline, cAMP,2-deoxyadenosine and Kallikrein. Reprod. Fertil. Dev. 7, 1081–1087.
Maxwell, W.M.C., Catt, S.L., Evans, G., 1996a. Dose of fresh and frozen–thawed spermatozoa for in vitrow xfertilization of sheep oocytes. Theriogenology 45, 261, Abstract .
Maxwell, W.M.C., Nebel, R.L., Lewis, G.S., 1996b. Survival and fertility of microencapsulated ramspermatozoa stored at 58C. Reprod. Domest. Anim. 31, 665–673.
Maxwell, W.M.C., Evans, G., Mortimer, S.T., Gillan, L., Gellatly, E.S., McPhie, C.A., 1999. Normal fertilityafter cervical insemination with frozen–thawed spermatozoa supplemented with seminal plasma. Reprod.Fertil. Dev. 11, 123–126.
Ž .Mazur, P., 1985. Basic concepts in freezing cells. In: Johnson, L.A., Larsson, K. Eds. , Deep Freezing ofBoar Semen. Proc. 1st Int. Conf. Deep Freez. Boar Semen Swedish Univ. Agr. Sciences, Uppsala, pp.91–111.
Milovanov, V.K., 1940. The present state of storage and transport of semen of farm animals. Trudy Lab.w xIskusst. Osem. Zivotn., Moscow 1, 85–107, in Russian .
Milovanov, V.K., 1951. Methods of storage of semen of ruminants. News in the Biology of Reproduction ofw xFarm Animals. Seljhozgiz, Moscow, pp. 139–165, in Russian .
Milovanov, V.K., 1962. Biology of Reproduction and Artificial Insemination of Animals. Seljhozizdat,w xMoscow, in Russian 696 pp.
Milovanov, V.K., Sokolovskaja, I.I., 1980a. Long-term storage of ram semen and new possibilities ofw xlarge-scale selection in sheep breeding. Vestn. Skh. Nauki 12, 122–132, in Russian .
Milovanov, V.K., Sokolovskaja, I.I., 1980b. The results and perspectives of scientific research. Zhivotnovod-w xstvo No. 10, 48–50, in Russian .
Milovanov, V.K., Varnavskaja, V.A., Shajdullin, I.N., 1985. Medium for deep freezing of semen. Zhivot-w xnovodstvo No. 7, 39–41, in Russian .
Molinia, F.C., Evans, G., Quintana Casares, P.I., Maxwell, W.M.C., 1994. Effect of monosaccharides anddisaccharides in Tris-based diluents on motility, acrosome integrity and fertility of pellet frozen ramspermatozoa. Anim. Reprod. Sci. 36, 113–122.
Molinia, F.C., Evans, G., Maxwell, W.M.C., 1996. Fertility of ram spermatozoa pellet-frozen in zwitterion-buffered diluents. Reprod. Nutr. Dev. 36, 21–29.
Moses, D., Martinez, A.G., Iorio, G., Valcarcel, A., Ham, A., Pessi, H., Castanon, R., Marcia, A., de las´ ˜ ´ ´Heras, M.A., 1997. A large-scale program in laparoscopic intrauterine insemination with frozen–thawedsemen in Australian Merino sheep in Agrentine Patagonia. Theriogenology 48, 651–657.
Nagase, H., Graham, E.F., 1964. Pelleted semen: comparison of different extenders and processes on fertilityof bovine spermatozoa. Proc. 5th Int. Congr. Anim. Reprod. A.I., Trento vol. 4 pp. 387–389.
Nauk, V.A., 1991. Structure and Function of Spermatozoa of Farm Animals During Cryopreservation. Stiinta,w xKishinev, Moldavia, in Russian 199 pp.
Nauk, V.A., Lansberg, E.S., Sherikin, V.N., 1970. Cryogenic changes in ram spermatozoa. Ovtsevodstvo No.w x10, 25–27, in Russian .
Nebel, R.L., Vishwanath, R., McMillan, W.H., Saacke, R.G., 1993. Microencapsulation of bovine spermato-zoa for use in artificial insemination: a review. Reprod. Fertil. Dev. 5, 701–712.
Nehring, H.P., Reinhardt, P., Fischer, P., Peter, W., Ehlert, F., 1989. Befruchtungsergebnisse beim Schaf nachInsemination gefrierkonservierten Spermas unter besonderer Berucksichtigung einer intrauterinen transmu-¨ralen Deponierung. Mh. Vet. Med. 44, 601–604.
Ostashko, F.I., Kantsedal, V.I., 1976. The influence of b-glucuronidase on survival and fertilizing ability offrozen ram semen. Proc. 8th Int. Congr. Anim. Reprod. A.I., Crakow vol. 5 pp. 1038–1040.
Patt, J.A. Jr., Nath, J., 1969. Effects of diluents, equilibration time and freezing rates on the storage of ramsemen. Cryobiology 5, 385–392.
Phillips, P.H., Lardy, H.A., 1940. A yolk–buffer pabulum for preservation of bull sperm. J. Dairy Sci. 23,399–404.
w xPlatov, E.M., 1977. Study on freezing ram semen. Ovtsevodstvo No. 9, 35–37, in Russian .Platov, E.M., 1988. Cryopreservation of ram spermatozoa. Cryoconservation of Spermatozoa of Farm
w xAnimals. Agropromizdat, Leningrad, pp. 161–195, in Russian .Polge, C., Smith, A.U., Parkes, A.S., 1949. Revival of spermatozoa after vitrification and dehydration at low
Ž .temperatures. Nature Lond. 164, 666.Quintana Casares, P.I., Maxwell, W.M.C., Wilson, H.R., Setchell, B.P., 1990. The effect of dose of motile
spermatozoa inseminated by intrauterine and cervical insemination on conception rate. Proc. Aust. Soc.w xReprod. Biol. 22, 68, Abstract .
Reinhold, G., Richter, A., Schulz, J., Menger, H., 1990. Ergebnisse der Besamung von Schafen mitgefrierkonserviertem Sperma unter Anwendung der Laparoskopie. Monatsh. Veterinarmed. 45, 452–456.¨
Rey, L.R., 1957. Studies on the action of liquid nitrogen on cultures in vitro of fibroblasts. Proc. R. Soc. Lond.Ser. B 147, 460–466.
Sadykov, R.E., Asanbekov, O.A., Uchaev, A.A., 1985. Results of introduction into practice of frozen ramw xsemen. Tr. Kirg. Nauchno. Proizv. Objed. Zhiv. No. 37, 72–81, in Russian .
Salamon, S., 1968. Deep freezing of ram semen: recovery of spermatozoa after pelleting and comparison withother methods. Aust. J. Biol. Sci. 21, 355–360.
Salamon, S., 1970. The survival of ram spermatozoa following pellet freezing below y798C. Aust. J. Biol.Sci. 23, 459–468.
Salamon, S., 1971. Fertility of ram spermatozoa following pellet freezing on dry ice at y798C and y1408C.Aust. J. Biol. Sci. 24, 183–185.
Salamon, S., 1972. Fertility of ram spermatozoa frozen–stored for three years. Proc. 7th Int. Congr. Anim.Reprod. A.I., Munich vol. 2 pp. 1493–1495.
Salamon, S., 1976. Artificial Insemination of Sheep. Publicity Press, Chippendale, N.S.W., 104 pp.Salamon, S., 1977. Fertility following deposition of equal numbers of frozen–thawed ram spermatozoa by
single and double insemination. Aust. J. Agric. Res. 28, 477–479.Salamon, S., Lightfoot, R.J., 1969. Freezing of ram spermatozoa by the pellet method. I. The effect of diluent
composition on survival of spermatozoa. Aust. J. Biol. Sci. 22, 1527–1546.Salamon, S., Lightfoot, R.J., 1970. Freezing of ram spermatozoa by the pellet method. III. The effect of
insemination technique, oxytocin and relaxin on lambing. J. Reprod. Fertil. 22, 409–423.Salamon, S., Maxwell, W.M.C., 1995a. Frozen storage of ram semen. I. Processing, freezing, thawing and
fertility after cervical insemination. Anim. Reprod. Sci. 37, 185–249.Salamon, S., Maxwell, W.M.C., 1995b. Frozen storage of ram semen. II. Causes of low fertility after cervical
insemination and methods of improvement. Anim. Reprod. Sci. 38, 1–36.Salamon, S., Visser, D., 1972. Effect of composition of tris-based diluent and of thawing solution on survival
of ram spermatozoa frozen by the pellet method. Aust. J. Biol. Sci. 25, 605–618.Salamon, S., Maxwell, W.M.C., Firth, J.H., 1979. Fertility of ram semen after storage at 58C. Anim. Reprod.
Sci. 2, 373–385.Salamon, S., Maxwell, W.M.C., Evans, G., 1985. Fertility of ram semen frozen–stored for 16 years. Proc.
w xAust. Soc. Reprod. Biol. 17, 62, Abstract .Salamon, S., Gillan, L., Evans, G., Maxwell, W.M.C., 1996. Fertility of ram semen after long-term
w xfrozen-storage. 13th Int. Congr. Anim. Reprod., Sydney vol. 3 pp. P24–25, Abstract .
Salisbury, G.W., VanDemark, N.L., 1961. Physiology of Reproduction and Artificial Insemination. W.H.Freeman, San Francisco, 639 pp.
Salisbury, G.W., Fuller, H.K., Willett, E.L., 1941. Preservation of bovine spermatozoa in yolk–citrate diluentand field results from its use. J. Dairy Sci. 24, 905–910.
Ž .Salisbury, G.W., Hart, R.G., Lodge, J.R., 1976. The fertile life of spermatozoa. Perspect. Biol. Med. 19 2 ,213–230.
Sanchez-Partida, L.G., Setchell, B.P., Maxwell, W.M.C., 1997. Epididymal compounds and antioxidants in´diluents for the frozen storage of ram spermatozoa. Reprod. Fertil. Dev. 9, 689–696.
wSmirnov, I.V., 1949. Storage of semen of farm animals by deep-freezing. Sov. Zootekh. No. 4, 63–65, inxRussian .
w xSmirnov, I.V., 1950. Deep freezing of semen of farm animals. Zh. Obshch. Biol. 3, 185–197, in Russian .Smirnov, I.V., Davidenko, V.M., Shinkarenko, I.S., Ignatenko, O.I., 1978. The effect of some physical and
w xbiological factors on freezing of ram semen. Zhivotnovodstvo No. 5, 58–60, in Russian .Sokolovskaja, I.I., Drozdova, L.P., Glysheva, M.G., Korotkov, A.I., Maksimov, Ju.L., Lebedeva, V., 1956. An
Ž . w ximproved synthetic medium for bull and ram semen. Zhivotnovodstvo 18 8 , 22–32, in Russian .Soderquist, L., Madrid-Bury, N., Rodriguez-Martinez, H., 1996. Influence of thawing procedure on membrane¨
damage in ram spermatozoa frozen in mini straws. 13th Int. Congr. Anim. Reprod., Sydney vol. 2 pp.w xP2–11, Abstract .
Stojanov, V.H., 1980. An experiment on deep-cervical insemination of sheep with frozen semen. Zhivotnovod-w xstvo No. 1, 45–46, in Russian .
Tekin, N., Gunzel, A.R., 1986. Investigations on the freezing of ram semen using different diluents and in¨w xvitro evaluation procedures. Veteriner Fakultesi Dergisi, Ankara Universitesi 33, 381–393, in Turkish .
Upreti, G.C., Jensen, K., Oliver, J.E., Duganzich, D.M., Munday, R., Smith, J.F., 1997. Motility of ramspermatozoa during storage in a chemically defined diluent containing antioxidants. Anim. Reprod. Sci. 48,269–278.
Varnavskij, A.N., Turbin, V.F., 1974. Some results of studies on freezing ram semen. Zhivotnovodstvo No. 6,w x65–67, in Russian .
Varnavskij, A.N., Varnavskaja, V.A., 1976. Results of artificial insemination with frozen semen. Ovtsevodstvow xNo. 9, 19–20, in Russian .
Visser, D., 1975. Studies of factors affecting the survival and fertility of ram and boar spermatozoa followingfreezing in Tris-based diluents. Ph.D. Thesis, University of Sydney, Sydney.
Visser, D., Salamon, S., 1973. Fertility of ram semen frozen in a tris-based diluent. Aust. J. Biol. Sci. 26,513–516.
Visser, D., Salamon, S., 1974a. Fertility following inseminations with frozen–thawed reconcentrated andunconcentrated ram semen. Aust. J. Biol. Sci. 27, 423–425.
Visser, D., Salamon, S., 1974b. The effect of pellet volume, dilution rates prefreezing and at thawing and ofthawing temperature on the survival and acrosome morphology of frozen ram spermatozoa. S. Afr. J.Anim. Sci. 4, 147–155.
Visser, D., Salamon, S., 1974c. The effect of freezing method on the survival of ram spermatozoa. S. Afr. J.Anim. Sci. 4, 157–163.
Volkov, A.S., 1974. Effect of number of active spermatozoa in the thawed semen on fertility of ewes.w xZhivotnovodstvo No. 10, 69–71, in Russian .
Walker, S.K., Smith, D.H., Little, D.L., Warner, S.M., Quinn, P., Seamark, R.F., 1984. Artificial inseminationŽ .and transfer of embryos by laparoscopy. In: Lindsay, D.R., Pearce, D.T. Eds. , Reproduction in Sheep.
Australian Academy of Science and Australian Wool Corporation, Canberra, pp. 306–309.Walker, S.K., Smith, D.K., Ancell, P., Seamark, R.F., 1989. Time of ovulation in South Australian Merino
ewes following synchronization of oestrus. 2. Efficacy of GnRH treatment and its relevance to insemina-tion programs utilizing frozen–thawed semen. Theriogenology 31, 741–752.
Watson, P.F., 1995. Recent developments and concepts in the cryopreservation of spermatozoa and theassessment of their post-thawing function. Reprod. Fertil. Dev. 7, 871–891.
Watson, P.F., Martin, I.C.A., 1974. Regions of the freezing curve causing changes in structure and viability ofŽ .ram sperm. Nature Lond. 251, 315–316.
Willett, E.L., Salisbury, G.W., 1942. The effect of various diluters, cooling rate, temperature of storage, and
some other factors, on the livability of spermatozoa in stored samples of bull semen. Cornell Univ. Agric.Exp. Sta. Mem. 249.
Wilson, H.R., Maxwell, W.M.C., 1989. The use of CIDRs and progestagen sponges in sheep AI programmes.w xProc. Aust. Assoc. Anim. Artif. Breed. 2, 13, Abstract .
Windsor, D.P., Szell, A.Z., Buschbeck, C., Edward, A.Y., Milton, J.T.B., Buckrell, B.C., 1994. Transcervicalartificial insemination of Australian Merino ewes with frozen–thawed semen. Theriogenology 42, 147–157.
Yoshioka, Z., Inudo, Y., Torizuka, T., 1951. Experiments on the storage of semen and insemination of thestored semen in sheep and goats. Bull. Natl. Inst. Agric. Sci. Ser. G No. 1, 53–60.
Zamfirescu, S., Vicovan, A., Barbulescu, I., 1980. Results of artificial insemination in sheep using frozenw xsemen. Rev. Cresterea Anim. 30, 11–15, in Rumanian .
Zheltobrjuk, N.A., Ivakhnenko, V.K., Aibasov, M.M., 1981. Freezing of ram semen in dextran containingdiluents. Breeding of Sheep and Goats and Wool Production. All-Union Research Institute for Sheep and
w xGoat Breeding, Stavropol, pp. 56–60, in Russian .Zheltobrjuk, N.A., Ivakhnenko, V.K., Aibasov, M.M., 1987. Fertilizing capacity of ram spermatozoa frozen in
raffinose and lactose–gum arabic media. In: Methods and Processes of Effective Selection of Sheep. pp.w x79–84, in Russian .
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