International Journal of NeurosciencePublication details, including instructions for authors and subscription information:http://www.informaworld.com/smpp/title~content=t713644851

Short-Term Neurophysiologic Consequences of Intrapartum Asphyxia in PigletsBorn by Spontaneous ParturitionH. Orozco-Gregorio a; D. Mota-Rojas b; M. Alonso-Spilsbury b; A. Olmos-Hernandez b; R. Ramirez-Necoecheab; E. Y. Velazquez-Armenta c; A. A. Nava-Ocampo cd; R. Hernandez-Gonzalez e; M. E. Trujillo-Ortega f; D.Villanueva-Garcia g

a Postgraduate Division of Animal Production Sciences and Health Facultad de Medicina Veterinaria yZootecnia, Universidad Nacional Autónoma de México, México, DF, México b Department of AnimalProduction & Agriculture Área de Investigación: Ecodesarrollo de la Producción Animal, UniversidadAutónoma Metropolitana Xochimilco, México, DF, México c PharmaReasons, Toronto, ON, Canada d Divisionof Clinical Pharmacology & Toxicology, The Hospital for Sick Children, Toronto, ON, Canada e Department ofExperimental Research and Animal Resources, Instituto Nacional de Ciencias Médicas y Nutrición SalvadorZubirán, México, DF, México f Department of Animal Medicine and Production: Swine Facultad de MedicinaVeterinaria y Zootecnia, Universidad Nacional Autónoma de México, México, DF, México g Division ofNeonatology, Hospital Infantil de México Federico Gómez, México, DF, México

Online Publication Date: 01 September 2008

To cite this Article Orozco-Gregorio, H., Mota-Rojas, D., Alonso-Spilsbury, M., Olmos-Hernandez, A., Ramirez-Necoechea, R.,Velazquez-Armenta, E. Y., Nava-Ocampo, A. A., Hernandez-Gonzalez, R., Trujillo-Ortega, M. E. and Villanueva-Garcia,D.(2008)'Short-Term Neurophysiologic Consequences of Intrapartum Asphyxia in Piglets Born by SpontaneousParturition',International Journal of Neuroscience,118:9,1299 — 1315

To link to this Article: DOI: 10.1080/00207450701872846

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International Journal of Neuroscience, 118:1299–1315, 2008Copyright C© 2008 Informa Healthcare USA, Inc.ISSN: 0020-7454 / 1543-5245 onlineDOI: 10.1080/00207450701872846

SHORT-TERM NEUROPHYSIOLOGICCONSEQUENCES OF INTRAPARTUMASPHYXIA IN PIGLETS BORN BYSPONTANEOUS PARTURITION

H. OROZCO-GREGORIO

Postgraduate Division of Animal Production Sciencesand HealthFacultad de Medicina Veterinaria y ZootecniaUniversidad Nacional Autonoma de MexicoMexico, DF, Mexico

Received 5 March 2007.The study was supported by a grant from the Programa de Mejoramiento del Profesorado

(PROMEP-SEP) to the academic staff of Etologıa, Production Animal and Fauna Silvestre. HectorOrozco-Gregorio is member of the program of Maestria en Ciencias de la Produccion y de la

Salud Animal, Universidad Nacional Autonoma de Mexico (UNAM), and was supported by ascholarship from CONACYT, Mexico. Daniel Mota-Rojas, Rafael Hernandez-Gonzalez, MarıaAlonso-Spilsbury, and Marıa E. Trujillo were supported as members by the Sistema Nacional deInvestigadores in Mexico. The authors are indebted to Q. Antonio Campos Osorno, chief of thebranch of gasometry at I. L. Diagnosticos S.A. de C.V., Mexico, DF, for temporarily providingthe instruments and tools for blood gas and electrolyte analyses and to QFB Mario Alberto GarcıaPina for his professional support in the installation and verification of the instruments as well asfor the time he devoted to help us in using it. We are also thankful for the technical assistance ofgraduate students Pedro Sanchez, Miguel Gonzalez, Gloria Gonzalez, and Monica Zermeno. Theauthors are indebted to Ms. Olivia Tischler for her valuable assistance in editing the manuscript.

Address correspondence to Dr. Alejandro A. Nava-Ocampo, Division of Clinical Pharmacology& Toxicology, The Hospital for Sick Children, 555 University Avenue, Toronto, ON M5G 1×8,Canada. E-mail: navaocampo aa@yahoo.com

1299

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1300 H. OROZCO-GREGORIO ET AL.

D. MOTA-ROJASM. ALONSO-SPILSBURYA. OLMOS-HERNANDEZR. RAMIREZ-NECOECHEA

Department of Animal Production & AgricultureArea de Investigacion: Ecodesarrollo de laProduccion AnimalUniversidad Autonoma Metropolitana XochimilcoMexico, DF, Mexico

E. Y. VELAZQUEZ-ARMENTA

PharmaReasonsToronto, ON, Canada

A. A. NAVA-OCAMPO

Division of Clinical Pharmacology & ToxicologyThe Hospital for Sick ChildrenToronto, ON, CanadaandPharmaReasonsToronto, ON, Canada

R. HERNANDEZ-GONZALEZ

Department of Experimental Research and AnimalResourcesInstituto Nacional de Ciencias Medicas y Nutricion SalvadorZubiranMexico, DF, Mexico

M. E. TRUJILLO-ORTEGA

Department of Animal Medicine and Production: SwineFacultad de Medicina Veterinaria y ZootecniaUniversidad Nacional Autonoma de MexicoMexico, DF, Mexico

D. VILLANUEVA-GARCIA

Division of NeonatologyHospital Infantil de Mexico Federico GomezMexico, DF, Mexico

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INTRAPARTUM ASPHYXIA IN PIGLETS 1301

Piglets appear to be neurologically sensitive to intrapartum asphyxia. Our aim was toinvestigate the short-term neurophysiologic consequences of intrapartum asphyxiain piglets. We studied 10 piglets suffering intrapartum asphyxia and 10 controlpiglets. Glucose and blood gas levels, tympanic membrane temperature, and bodyweight were measured within the first 2 min after birth. Animals were followedup for a 5-day period. As surrogated markers of piglets’ neurological function, aviability score and the time elapsed from birth to the first contact with the maternaludder were recorded. In the control group, temperature and blood pH levels atbirth were significantly higher (p ≤ .001), whereas calcium, lactate and PCO2

levels were statistically lower (p ≤ .05) than in the piglets experiencing intrapartumasphyxia. Lower temperature and blood pH levels as well as higher blood PCO2

and lactate levels were observed in piglets with lower viability scores and in pigletswith prolonged times until first udder contact. At the end of the study, asphyxiatedpiglets weighed on average 200 g less (p = .023) than control piglets. In conclusion,intrapartum asphyxia in spontaneously born piglets was associated with signs ofacute neurological dysfunction and lower weight gain, supporting the hypothesisthat they may be used as a naturalistic model for the study of asphyxia in newborns.

Keywords acid-base equilibrium, neurological dysfunction, physiological adap-tation

INTRODUCTION

Almost 10% ofdeaths in children younger than 5 years old are intrapartum-related neonatal deaths (Lawn, Shibuya, & Stein, 2005). Asphyxia appearsto be the leading cause of intrapartum complications and neurodevelopmentsequels in the surviving children (Low, 2004; Shevell, 2004). Although there areseveral limitations to extrapolate the results from experimental animal modelsto humans, some similarities have been found in the response to asphyxiaamong different mammal neonates (Singer, 1999).

In pigs, intrapartum asphyxia is mainly responsible for intrapartumstillbirths (Alonso-Spilsbury et al., 2005; Randall, 1971; Sprecher, Leman,Dziuk, Cropper, & Dedecker, 1974). The major cause appears to be thepremature rupture of the umbilical cord during the delivery (Randall, 1989). Ina previous study that involved preventing breathing in piglets within the first 4min of their postnatal life, it was observed that the thermoregulatory capabilitiesof asphyxiated piglets were altered (Herpin et al., 1996). In addition, our recentstudy supported the hypothesis that the piglet naturally exposed to intrapartumasphyxia may be a suitable experimental model for further studies in the field,particularly due the similarities in the response to asphyxia between humansand piglets, including respiratory and metabolic acidosis (Trujillo-Ortega et al.,2007). Acute neurofunctional alterations evidenced by a prolonged latency until

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1302 H. OROZCO-GREGORIO ET AL.

contact with the maternal udder and lower viability scores were also observed.However, in that study we failed to investigate whether such alterations mayproduce any sequel. Therefore, the present study was designed in order toinvestigate, under highly controlled conditions, whether piglets surviving severeintrapartum asphyxia may have short-term sequels.

MATERIAL AND METHODS

Animals

The study was approved by the institutional review board of the veterinaryschool at the Universidad Autonoma Metropolitana-Xochimilco and the Insti-tutional Animal Care and Use Committee of the Department of ExperimentalResearch and Animal Resources at the Instituto Nacional de Ciencias Medicas yNutricion “Salvador Zubiran,” Mexico, DF, Mexico. The study was performedin a swine farm located in the State of Mexico, Mexico.

We previously reported that the median number of piglets born per sowis 10 out of which 4% are liveborn piglets with moderate to severe skinmeconium stain (Mota-Rojas et al., 2005). Therefore, during a 2-week period, arandom sample of 50 parturient hybrid Yorkshire-Landrace sows were housedin crates at room temperatures of 23◦C ± 2◦C. This sample size of parturientsows was enough to provide approximately 20 piglets surviving moderateor severe degrees of intrapartum asphyxia. No induction of parturition withprostaglandins F2-alfa or oxytocin was performed, and medical interventionwas minimal and did not include resuscitation of asphyxiated piglets. As eachpiglet was born, the time at birth, sex, status of umbilical cord, and skincoloration were registered.

The piglets were selected within the first 2 min after birth. The controlgroup included a random sample of 10 piglets, each obtained from differentlitters, with normal or adhered umbilical cord and with less than 40% of theskin surface stained by meconium, as judged by one of the investigators. Theasphyxiated group consisted of 10 piglets born with edematous or rupturedumbilical cord and with > 40% of their skin surface stained by meconium.Piglets were measured, weighed, and sampled, as will be described later, andreturned to the sow. Once they found the udder they were allowed to remainintermittently suckling for 60–120 min after birth. Thereafter, the piglets weredischarged from the swine farm in disinfected plastic cages specially designedfor transportation of small animals in an estimated space for free movementsof 0.32 m2 per animal.

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INTRAPARTUM ASPHYXIA IN PIGLETS 1303

Piglets were transferred to the Department of Experimental Research andAnimal Resources investigations at the Instituto Nacional de Ciencias Medicasy Nutricion “Salvador Zubiran.” On arrival to the laboratory, piglets weremaintained in a room under strict controlled environmental conditions including15–20 air exchanges per hour, incoming air crossing 0.3-µm particle filters,light intensity fixed at 150 lux, and room temperature maintained between 28◦Cand 32◦C. Contact with piglets was restricted to four of the investigators, whoused sterile clothes and gloves in the room, and were scheduled to assist thepiglets in rotating turns of 24 hrs until completion of the study.

Feeding was provided with a special milk formula for piglets (BirthrightMilk, Ralco Nutrition, Inc.), containing 24% proteins, 16% ethereal extract,and 0.15% fiber. The amount of milk the piglets were allowed to drink milkwas: 25 ml every 2 hrs on the first day, 40 ml every 2 hrs on the second day,50 ml every 2 hrs on the third and fourth days, and 80 ml every 2 hrs onthe fifth day. In order to avoid any possibility to introduce external microbialcontamination into the farm where piglets were obtained, they were not allowedto be returned to the site. Therefore, after completion of 5 days of the studyperiod, we considered it in the protocol to sacrifice all surviving piglets byasphyxia or CO2 inhalation in a special chamber for animal euthanasia withopaque lateral walls and a transparent cap. The euthanasia was performed inaccordance with the Mexican Official Norm NOM-062-ZOO-1999.

Study Outcomes

Body weight was recorded by a digital bascule (Salter Weight-Tronix Ltd.) atbirth and daily until the completion of the study. Temperature was registeredby means of a tympanic membrane thermometer (ThermoScan Braun GMBH)immediately following birth and at the end of the study period. If a blood samplewas going to be obtained, temperature was measured prior to blood sampling.

At birth, the viability of piglets was scored according to the scale describedby Zaleski and Hacker (1993) and modified by Trujillo-Ortega et al. (2007).Briefly, the heart rate was classified in categories as <110, between 121 and160, and >161 beats per min; the time interval between piglet’s birth and firstbreath as >1 min, between 16 s and 1 min, and <15 s; muzzle skin color aspale, cyanotic, and pink; time interval between birth (for purposes of this study,counted after the piglet was returned to the maternal vulva) and first stand onits four legs as >5 min, between 1 and 5 min, and <1 min; and the skin stainedwith meconium as severe, mild, and absent. Each criterion was then scoredfrom 0 (the worst) to 2 (the best),with an overall score of the scale rangingfrom 0 to 10 in each piglet.

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1304 H. OROZCO-GREGORIO ET AL.

The time elapsed after the piglet was returned close to the maternal vulvaand the first contact with the maternal udder was also registered. For the purposeof the study, the viability score and the time until first udder contact wereconsidered as surrogated markers of the neurofunctional status of the piglet.Except for the time to stand, the parameters integrating the viability score, asdescribed earlier, were measured before the first blood sample was obtainedat birth. However, the time to stand and the time until first udder contact wereregistered after blood sampling, starting when the piglets were returned to theirmothers, close to the vulva, and finishing when the animals reached the standingposition supported by their four legs or reached the udder for the first time.

Blood samples were collected by retro-orbital puncture by capillary tubescontaining 100 µL of lithium heparin. Sampling times were at birth and 6,12, 24, 48, 72, 96, and 120 hrs later (0, 0.25, 0.5, 1, 2, 3, 4, and 5 days,respectively). The investigators who sampled the piglets had the skills tocollect the blood at the first attempt in <25 s. Blood levels of hematocrit (%),glucose (mg/dL), sodium (mmol/L), potassium (mmol/L), calcium (mEq/L),bicarbonate (mmol/L), lactate (mg/dL), and pH and partial pressure of carbondioxide [PCO2 (mm Hg)] and oxygen [PO2 (mm Hg)] were measured bymeans of automatic blood gas and electrolyte analyzer (GEM Premier 3000,Instrumentation Laboratory Diagnostics).

Data Analysis

In the two groups, the piglets’ weight, tympanic membrane temperature,and glucose, electrolyte, and gas blood levels (except by pH levels) weresummarized as mean ± SD. Data obtained immediately after birth werecompared between groups by means of an unpaired Student’s t-test. Glucose,electrolytes, and blood gases were also compared between the two study groupsby means of a two-way analysis of variance (ANOVA) for repeated measures inorder to identify time–response differences. Since the blood pH levels representlog units, this parameter was summarized as a median (range), and comparisonbetween groups was performed by a Mann-Whitney U test. In addition, theoutcomes obtained at the end of the study were compared between the twostudy groups by means of either a Student’s t-test or a Mann-Whitney U test(blood pH levels).

In order to identify any relationship between biochemical alterationssecondary to intrapartum asphyxia and acute neurophysiologic alterations, asingle linear regression analysis was performed between each of the statisticallysignificant parameters evaluated at piglets’ birth (independent variables) and

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INTRAPARTUM ASPHYXIA IN PIGLETS 1305

either the viability score or the time until first udder contact (dependentvariables). When two or more independent variables were found to bestatistically significantly correlated to any of the dependent variables, a multiplelinear regression was performed.

The Student’s –t-test, Mann-Whitney-U test, and single and multiple linearregression analyses were performed using StatsDirect 2.4.5 (StatsDirect Ltd.).The two-way ANOVA was performed through WinSTAT for Microsoft Excel(A-Prompt Corp.). The limit of significance was fixed at a two-tailed p < .05.The statistical analyses were performed by one of the investigators ignoringdata allocation group.

RESULTS

All the evaluations were performed as planned per protocol in all the studiedpiglets. In the control group, temperature and blood pH levels at birth weresignificantly higher (p ≤ .001) and glucose, calcium, lactate, and PCO2 bloodlevels significantly lower (p ≤ .05) than the levels obtained in piglets bornwith evidence of asphyxia (Table 1). The other electrolyte and gas blood levelswere similar between the two study groups. The time course of changes intemperature and calcium blood levels was significantly different both betweenthe control group and the group with piglets born with evidence of asphyxia (p =.049 and p < .0001, respectively) and among the eight different samplingtimes (p = .049 and p = .03, respectively).The time course of the otheroutcomes of the study was similar between groups (Figures 1–3). However,in most of the parameters, the results were obtained at birth evidently 6 hrslater.

As evidenced by the linear regression analyses, lower temperature andblood pH levels and higher PCO2 and lactate blood levels were found in pigletswith lower viability scores as well as in piglets that took longer times to reachthe maternal udder (Table 2). The multiple linear regression analyses usingeither viability scores or time until first udder contact as the explained variablesdid not provide any equation, probably owing to the high colinearity observedamong the parameters.

At the end of the study period, the control group had gained an average 520g of body weight versus 168 g gained by the piglets surviving asphyxia (p =.023) (Table 3). However, the biochemical parameters were similar betweenthe two groups, except the blood pH levels that remained significantly higherin the control group. None of the piglets had evidence of eye damage secondaryto the blood sampling procedure. We did not observe seizures, loss of musculartone, or spastic contractions in any piglet.

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Tabl

e1.

Blo

odga

ses,

elec

trol

ytes

,and

gluc

ose

leve

lsat

birt

hin

pigl

ets

surv

ivin

gin

trap

artu

mas

phyx

ia

Con

trol

grou

pA

sphy

xia

grou

p95

%C

Ip

Val

ue

Bir

thW

eigh

t(g)

1,33

2±

213

1,48

1±

229

−59

to35

7.1

5B

irth

Tem

pera

ture

(◦C

)39

.3±

0.3

37.0

±0.

32.

0to

2.6

<.0

001

Neo

nata

lVia

bilit

y9.

0±

0.7

5.0

±1.

23.

1to

4.9

<.0

001

Tim

eun

tilFi

rstU

dder

Con

tact

(min

)30

.5±

4.6

60.6

±7.

2−3

5.8

to−2

4.4

<.0

001

Hem

atoc

rit(

%)

34.8

±5.

338

.9±

5.8

−9.5

to1.

3.1

3G

luco

se(m

g/dL

)89

.3±

11.4

95.2

±28

.6−2

6.3

to14

.5.5

6a

Sodi

um(m

mol

/L)

138.

1±

3.3

134.

2±

5.5

−0.4

to8.

2.0

7Po

tass

ium

(mm

ol/L

)7.

1±

2.4

7.0

±1.

5−1

.8to

2.0

.89

Cal

cium

(mm

ol/L

)1.

5±

0.2

1.9

±0.

3−0

.6to

−0.0

5.0

23pH

7.16

(7.0

0–7.

26)

6.97

(6.9

0–7.

16)

0.08

to0.

24.0

01L

acta

te(m

g/dL

)65

.3±

15.5

99.3

±23

.9−5

2.9

to−1

5.1

<.0

05B

icar

bona

te(m

mol

/L)

22.5

±3.

819

.7±

7.3

−2.6

to8.

3.2

9PO

2(m

mH

g)26

.4±

17.7

18.6

±14

.2−7

.3to

22.9

.29

PCO

2(m

mH

g)60

.4±

18.7

92.9

±13

.3−4

7.7

to−1

7.3

<.0

005

The

pHle

vels

wer

eex

pres

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asm

edia

ns(r

ange

s).T

here

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eona

talv

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was

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ing

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ale

repo

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byZ

ales

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al.(

2007

).Te

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the

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cm

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a Ass

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ianc

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1306

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INTRAPARTUM ASPHYXIA IN PIGLETS 1307

Glucose

0 1 2 3 4 5

mg

/dL

40

60

80

100

120

140 Sodium

0 1 2 3 4 5

0 1 2 3 4 5

mm

ol/L

124

128

132

136

140

144

148

Potassium

Time (Days)0 1 2 3 4 5

mm

ol/L

4

5

6

7

8

9

10Calcium

Time (Days)

mm

ol/L

1.2

1.4

1.6

1.8

2.0

2.2

2.4

*

Figure 1. The time course of changes in blood glucose, sodium, and potassium levels was notsignificantly different between the groups or among sampling times (two-way ANOVA; p > .05).Blood calcium levels were significantly different both between the control group and the groupwith piglets born with evidence of asphyxia (p = .049 and p < .0001, respectively) and among thedifferent sampling times (p = .049 and p = .03, respectively). Blood calcium levels immediatelyafter birth were significantly higher (∗p = .023) in asphyxiated piglets than in controls. Opencircles: control group. Closed circles: asphyxiated piglets.

DISCUSSION

In the present study, we observed a similar spontaneous recovery of theacid–base and electrolyte and glucose imbalance produced at birth inasphyxiated and control piglets. In addition, we did not observe any caseof severe neurological dysfunction including seizures, loss of muscular toneor spastic muscular contractions, and spontaneous death in the studied piglets.However, there was a clear difference between groups in their neurologicalachievement, as judged by the approximately 50% lower scores in the neonatalviability scale and the twice longer times to first udder contact in the asphyxiatedpiglets. In contrast, a previous study showed that piglets born with metabolicacidosis and hypercapnia secondary to intrapartum asphyxia have low ratesof survival (Randall, 1979). In addition, we recently reported an intrapartum

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1308 H. OROZCO-GREGORIO ET AL.

Control Group

Time (Days)

0 1 2 3 4 5

pH

Lev

els

6.8

6.9

7.0

7.1

7.2

7.3

7.4

7.5

7.6 Asphyxiated Group

Time (Days)

0 1 2 3 4 5

6.8

6.9

7.0

7.1

7.2

7.3

7.4

7.5

7.6

*

**

Figure 2. Time course of changes in blood pH levels in the two study groups. The pH levelswere significantly lower (∗p = .001; Mann-Whitney U test) and significantly higher (∗∗p = .023;Mann-Whitney U test) within the first minute after birth and at 5 days of age, respectively, inasphyxiated piglets compared to the control group. Open circles: control group. Closed circles:asphyxiated piglets.

mortality rate of 8.3% among piglets delivered by uninduced parturition(Trujillo-Ortega et al., 2007). Such difference may be explained by the postnatalcare provided to the piglets in our study, which is not typically provided underuncontrolled conditions.

Table 2. Statistically significant linear correlations between study outcomes and vitality andtime until first udder contact

Linear equation Correlation value p Value

Neonatal Viability (Y1)Temperature Y1 = 1.6 Temperature − 55.5 0.89 <.0001pH Y1 = 13.3 pH − 86.82 0.66 .0014PCO2 Y1 = −0.059 PCO2 + 11.5 −0.60 .005Lactate Y1 = −0.058 Lactate + 11.8 −0.68 .001

Time until First UdderContact (Y2)

Temperature Y2 = −12.4 Temperature + 518.6 −0.91 <.0001pH Y2 = −86.0 pH + 653.3 −0.58 .007PCO2 Y2 = 0.46 PCO2 + 10.3 0.64 .002Lactate Y2 = 0.42 Lactate + 11.4 0.66 .002

Neonatal viability was scored according to the scale reported by Zaleski and Hacker (1993) andmodified by Trujillo-Ortega et al. (2007). Temperature was obtained in the tympanic membrane.Blood samples for pH, PCO2 and lactate levels were obtained by retro-orbital puncture.

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INTRAPARTUM ASPHYXIA IN PIGLETS 1309

Lactate

0 1 2 3 4 5

mg

/dL

0

20

40

60

80

100

120

140Bicarbonate

0 1 2 3 4 5

mm

ol/L

10

15

20

25

30

35

PO2

Time (Days)

0 1 2 3 4 5

mm

Hg

0

10

20

30

40

50

60 PCO2

Time (Days)

0 1 2 3 4 5

mm

Hg

30

40

50

60

70

80

90

100

110

*

*

Figure 3. The time course of changes in the levels of lactate, bicarbonate, PO2, and PCO2 in theblood was similar between groups and among the different sampling times (two-way ANOVA;p > .05). However, significantly higher levels of lactate and PCO2 in the blood were observedwithin the first minute after birth in asphyxiated piglets than controls (∗p < .05 and ∗∗p < .0005,respectively). Open circles: control group. Closed circles: asphyxiated piglets.

The main aspects of monitoring fluid and electrolyte imbalance in humannewborns, as well as the reference ranges for sodium, potassium, chloride,total CO2, glucose, and total and ionized calcium serum levels were reviewedelsewhere (Lorenz, 1997). However, several pathologic processes may alterany of the levels mentioned, and therefore the evaluation of multiple markersmay be required for the diagnosis of intrapartum asphyxia (Shevell, 2004). Theblood pH levels are usually maintained within a narrow range by a numberof buffer systems in the body. An abnormal value is secondary to either apoorly compensated acid–base disturbance or a combination of metabolic andrespiratory alterations resulting in an imbalance in the same direction. In thepresent study, the blood samples were obtained at a time when the piglets hadnot yet initiated spontaneous breathing, resulting in evidently low blood pHlevels in the two study groups. The process was, however, compensated in both

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1310 H. OROZCO-GREGORIO ET AL.

Table 3. Blood gases, electrolytes, and glucose levels in piglets exposed to asphyxia 5 days afterbirth

Control group Asphyxia group 95%CI p Value

Weight (g) 1, 852 ± 100 1, 648 ± 227 39 to 369 .023Temperature (◦C) 38.1 ± 0.5 38.4 ± 0.4 −0.05 to 0.8 .08Hematocrit (%) 26.0 ± 4.2 28.0 ± 6.1 −7.0 to 3.0 .4Glucose (mg/dL) 60.6 ± 12.5 62.2 ± 5.6 −10.7 to 7.5 .7∗

Sodium (mmol/L) 138.7 ± 2.5 137.1 ± 5.8 −2.6 to 5.8 .4∗

Potassium (mmol/L) 6.5 ± 0.81 6.3 ± 0.99 −0.7 to 1.0 .8Calcium (mmol/L) 1.4 ± 0.09 1.4 ± 0.17 −0.1 to 0.1 .9pH 7.42 (7.39–7.42) 7.50 (7.42–7.54) −0.1 to −0.01 .023Lactate (mg/dL) 26.8 ± 8.0 31.3 ± 16.5 −16.7 to 7.7 .5a

Bicarbonate (mmol/L) 27.3 ± 2.9 27.3 ± 3.8 −3.2 to 3.2 .9PO2 (mm Hg) 36.0 ± 16.4 29.5 ± 4.7 −4.8 to 17.8 0.25a

PCO2 (mmHg) 41.4 ± 3.4 41.8 ± 4.0 −3.9 to 3.1 0.8

The pH levels were expressed as median (ranges). The rest of data were summarized as mean ±SD. Temperature was obtained in the tympanic membrane. Blood samples were obtained byretro-orbital puncture. aAssuming unequal variances.

groups once the piglets started to breath. These changes were in parallel witha drop in blood PCO2 and lactate levels.

In humans, O2 saturation levels in fetal blood can be used as prognosticfactors in the newborns, especially in the presence of meconium-stainedamniotic fluid (Grignaffini et al., 2004; Skoczylas & Laudanski, 2003). Theincreased levels of lactate reported in our study can be explained by an enhancedlactate production from skeletal muscle glycogen (Edwards & Silver, 1969)and a reduction of lactate utilization in the gluconeogenic pathway (Warnes,Seamark, & Ballard, 1977). Since a single lactate or base deficit level mayprovide only limited information about the duration of asphyxia and the qualityof surviving fetuses (Low, 1988), we performed serial determinations of lactatelevels and noticed a return to levels similar to those of controls within the first6 hrs postpartum.

It is naturally expected that human neonates born with intrapartumasphyxia have hypoglycemia. However, at birth, the increments in glucoseplasma levels may be explained by a release of catecholamines andstimulation of liver glycogenolysis secondary to the intrapartum asphyxia(Herpin et al., 1996; Randall, 1979). High glucose blood levels have alsobeen reported in stillborn and weak-born piglets (Lauterbach et al., 1987;Trujillo-Ortega et al., 2007), in piglets born during the later stages of parturition(Stanton, Brown, & Mueller, 1973), in highly asphyxiated piglets (Herpin

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INTRAPARTUM ASPHYXIA IN PIGLETS 1311

et al., 1996), and in piglets that died within the first 10 days of life (Tuchscherer,Puppe, Tuchscherer, & Tiemann, 2000). However, if asphyxia persists or thenewborn piglet is not fed soon after birth, the hepatic glycogen storageswould be depleted followed by a transient normalization of glucose levelsuntil reaching hypoglycemic values. In our study, glucose blood levels wereevidently normalized at 6 hrs after birth, a time when the overall status ofthe piglets was improved by initiation of respiration and feeding, reflecting anormalization of metabolic alterations secondary to the stress produced at birth.

With the abrupt termination of calcium transport across the placenta atbirth, total calcium concentrations in umbilical cord plasma fall, reaching anadir at 24–48 hr of postnantal age (Tsang, Chen, Friedman, & Chen, 1973).Secondary to a slow response in the parathyroid secretion during the postnatalfall in plasma calcium concentration, clinically significant hypocalcemia mayoccur in premature infants (Tsang et al., 1973) and asphyxiated newborns(Tsang et al., 1974). However, in this study, the calcium levels in pigletssurviving intrapartum asphyxia were significantly higher than those in controlpiglets. It is probable that there was an enhanced release of parathyroidhormones in response to asphyxia, favoring calcium mobilization from bonesin the newborn piglets. In addition, asphyxia produces rapid metabolic changes,alters intracellular oxygen supply, and favors oxidative stress. Since calciumand oxidative stress have been involved in the mechanisms of ischemic braininjury (Starkov, Chinopoulos, & Fiskum, 2004), these two factors could besignificant contributors to the acute neurological dysfunction identified in ourstudy.

During fetal hypoxia, blood redistribution increases intestinal peristalticrelaxation of the anal sphincter, leading to defecation of meconium into theamniotic fluid (Davis, Philips, Harris, Wilson, & Huddlestone, 1985). Undersuch circumstances, piglets are born meconium-stained. If asphyxia is severe,fetuses may gasp violently inside the amniotic sac, favoring the inhalationof amniotic fluid containing meconium, which may subsequently producesevere airway obstruction. However, we did not observe clinical signs ofrespiratory dysfunction or respiratory deterioration in any of the two studygroups suggesting that meconium aspiration syndrome was not present in thestudied piglets.

Birth temperature <36◦C was associated with 25% deaths in asphyxiatedhuman neonates (Manzar, 1999). The processes involved in fetal and neonatalthermoregulation were recently reviewed elsewhere (Asakura, 2004). Duringintrauterine life, the fetus is warmed by its own metabolic processes, andfetal temperature constantly remains 0.3◦C to 0.5◦C higher than the adults. At

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1312 H. OROZCO-GREGORIO ET AL.

birth, neonatal heat is rapidly lost, and increased neonatal heat production isessential for survival. In our study, at birth, the temperature of asphyxiatedpiglets was 2◦C lower than controls. Lower temperatures were associatedwith lower viability scores and prolonged times until first udder contact,indicating an impaired capability for postnatal thermoregulation in asphyxiatedpiglets.

Hypoxia, hypercarbia, and metabolic acidosis (conventionally measuredby base deficit) secondary to anaerobic metabolism are essential featuresof asphyxia. Proved many years ago, the evaluation of these parameters inblood samples obtained from the umbilical artery at birth reflects the severityof intrapartum asphyxia in animals and humans (Dawes, Jacobson, Mott,Shelley, & Stafford, 1963; James, Weisbrot, Prince, Holaday, & Apgar, 1985).In a retrospective study of full-term neonates with a significant degree ofintrapartum asphyxia, it was shown that the arterial lactate levels obtainedwithin 1 hr after birth, and before the metabolic acidosis was treated with sodiumbicarbonate, were important predictors of moderate to severe hypoxic ischemicencephalopathy (Shah, Tracy, & Smyth, 2004). However, base deficit may besimilarly recovered in few hours postpartum in neonates with different degreesof asphyxia, varying from mild asphyxia to hypoxic-ischemic encephalopathyand from good to severe adverse outcomes (Shah, Raju, Beyene, & Perlman,2003; Swanstrom & Bratteby, 1981). In comparison, children completelyrecovered from fetal asphyxia may have subsequent school-related dysfunctionsand certain degree of intellectual delay (D’Souza, McCartney, Nolan, & Taylor,1981; Robertson, Finer, & Grace, 1989).

In conclusion, intrapartum asphyxia was associated with an evidentcombination of metabolic and respiratory acidosis in piglets. Although thesealterations were rapidly reversed once the animals started to breath, the pigletssurviving severe intrapartum asphyxia showed signs of acute neurologicaldysfunction including longer times until first udder contact and lower viabilityscores at birth as well as lower body weights at 5 days postpartum. Althoughthere are several limitations within this experimental animal model, our resultsfurther support the hypothesis that piglets surviving intrapartum asphyxia mayprovide a naturalistic model for extending the knowledge of asphyxia in humanneonates.

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