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Department of Animal Production: Swine, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México, México
1 Department of Animal Production and Agriculture, Research Area: Ecodesarrollo de la Producción Animal, School of Veterinary Medicine, Universidad Autónoma Metropolitana-Xochimilco, México
2 Departamento de Investigación Experimental y Bioterio, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, México
3 Division of Clinical Pharmacology and Toxicology, The Hospital for Sick Children, 555 University Ave., Toronto, Ontario M5G 1X8, Canada
4 EIAH-Benemérita Universidad Autónoma de Puebla, San Juan Acateno, Teziutlan, Puebla, México
(Requests for offprints should be addressed to A A Nava-Ocampo; Email: navaocampo_aa{at}yahoo.com)
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animals
Twenty crossbred Large White x Landrace gilts were bred with Landrace boars by artificial insemination at the time of their second estrus. The day of insemination was considered as the first day of gestation. During gestation, sows were housed in individual stalls with plasticized slatted-floors and water was available ad libitum. Sows received 2.5 to 3 kg/day of a corn-soybean diet balanced to meet current National Research Council requirements. The diet provided 14% crude protein, 0.70% lysine, and 14 MJ ME/kg of food, and remained similar throughout the pregnancy period.
Sows were housed in individual crates with concrete floors from the 4th day before the expected delivery date until the 21st postpartum day, a period that covered parturition and piglets’ weaning. Delivery time was controlled by intramuscular injection of prostaglandins (Lutalyse; Pharmacia & Upjohn, Mexico DF, Mexico) administered 36 h prior to the expected delivery date at 114 days of gestational age.
Treatments
Sows were randomly assigned to receive daily intramuscular injections in the neck region of either 1 ml saline (n=10 sows) or 6 mg/day recombinant porcine ST (rpST) (Reporcin, Alpharma Animal Health Pty. Ltd, Victoria, Australia) in 1 ml saline (n=10 sows) from days 80 to 114 of gestation. The dose of rpST had previously been proved to increase fetal growth (Rehfeldt et al. 2001).
Outcomes in the sows
On days 80 and 114 of pregnancy, control and rpST-treated sows were weighed (kg) and their dorsal subcutaneous fat (mm) was measured by ultrasound (Renco Preg-Alert, Minneapolis, MN, USA). During parturition, the overall duration of parturition was registered as the time elapsed (min) from the rupture of amniotic membranes to the expulsion of the placenta. The time to deliver each piglet (time interval between piglets in min) was measured and averaged for each sow.
Within the first minute at the start of parturition, a blood sample was collected from any of the sow’s ear veins for quantitation of glucose concentrations by a digital glucometer (Acuu-CheK; Active Roche Diagnostics GmbH), blood lactate by enzymatic fotometry reflexion using a portable lactate analyzer (Accutrend Lactate, Sports Resource Group Inc, Hawthorne, NY, USA), and pH by a Ciba-Corning 278 Blood Gas System (Diamond Diagnostic Inc, Holliston, MA, USA); pH was corrected for body temperature. Body temperature was obtained instantaneously by a tympanic membrane thermometer (ThermoScan Braun GmbH, Kronberg, Germany). Finally, the litter size and placental weight were registered.
Outcomes in the newborn piglets
Each piglet was clinically examined in order to evaluate any objective evidence of external malformation. The following somatometric parameters were obtained: weight (kg), crown-to-rump length (cm), height (cm), skull and hip width (cm), thoracic circumference (cm), and the hind limb circumference (cm) measured in either of the hind limbs. Widths were obtained by a vernier caliper whereas the other somatometric parameters were obtained by standard bascules and metric tapes.
Piglets born dead were classified according to the criteria previously described in detail, as type I and type II piglets (Randall 1972, Mota-Rojas et al. 2002). Briefly, type I or antepartum stillbirths had a rather characteristic edematous and hemorrhagic appearance, sometimes a grayish-brown decoloration due to early mummification and, if the process was advanced then the fetuses were found dehydrated and losing hair. Type II or intrapartum stillbirths had the exact appearance of their normal littermates with the exception that they did not breathe. We also reported the number of live-born, meconium-stained piglets in each group.
Once the umbilical cord was ruptured, a blood sample was collected within the first minute after birth for biochemical evaluations. During sampling, no distinction was made between arterial and venous blood. Glucose, lactate, partial pressure of CO2 (pCO2) and pH blood levels were obtained as described above. Body temperature was obtained instantaneously by a tympanic membrane thermometer within the first minute of birth.
Statistical analysis
Data were summarized as means ± S.D. or as median (and ranges). The sows’ weight and back fat were compared between groups by a two-way analysis of variance (ANOVA) with repeated measures (with evaluation times and treatment as the between- and within-factors respectively). In this kind of analysis the total variation in the scores is broken down into 5 main sources of variation: (1) between-subjects factor (i.e. independent variable), (2) between-subjects error, (3) within-subjects factor (i.e. repeated measures), (4) within-subjects error, and (5) interaction of between- and within-subjects factors (Cramer 1998). The F ratio for determining whether there is a significant effect for between-subjects factor is the between-subjects mean square divided by the between-subjects error mean square. The F ratio for finding out whether the within-subjects factor is significant is the within-subjects factor mean square divided by the within-subjects error mean square, while the F ratio for seeing whether the interaction effect is significant is the interaction mean square divided by the within-subjects error mean square.
A Student’s t-test for unpaired samples was used to compare the following variables between the two study groups in sows: duration of parturition, time interval between birth of piglets, placental weight, tympanic membrane temperature, glucose blood levels, and lactate blood levels, and in piglets: birth weight, crown-rump length, height, skull width, hip width, and thoracic and hind-limb circumference.
The litter size, pH blood levels in the parturient sows, and body temperature, glucose, pH, lactate and pCO2 blood levels in the newborn piglets were compared between the two study groups by the Mann-Whitney U test, a method defined by:
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where samples of size n1 and n2 are pooled and Ri are the ranks. The test can be resolved as the number of times observations in one sample precede observations in the other sample in the ranking. The alternative hypothesis was that values in one group tended to be distributed differently compared with the values in the other group.
As categorical variables, the number of intrapartum deaths (% of all piglets) and meconium-stained piglets (% of live born piglets) were compared between groups by 2x2 
2 test, defined by:
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where for 2 rows (r) and 2 columns (c) of n observations (4 cells in total), O is an observed frequency and E is an estimated expected frequency. The expected frequency for any cell is estimated as the row total times the column total divided by the grand total.
In order to evaluate whether the variations in the birth weight increased with piglet number in the two study groups, a two-way ANOVA for repeated measures (with piglet number and treatment as the between- and within-factors respectively) was performed as described above. In addition, a survival plot with comparison between groups of probability of survival was performed by the Cox-Mantel test as follows. The observations Xia for K=2 were considered as a series of contests between the two samples:
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where the rows corresponded to the two samples (a=1, 2), the first column had the numbers of animals in each sample who were observed at time ti, and the row sums were the number of animals in each sample who were ‘at risk’ at time ti, and the second column had the number of animals who were ‘ at risk’ at time ti but did not die. The Cox-Mantel type of statistic was based on the score:
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where the observed deaths in the K samples occurred at r distinct times 0=t0 
t1<t2 ...<tr, di was the total number of observed deaths at time ti in the two samples, Ni was the size of the total risk set at time ti, and wi was an arbitrary weight equalling 1. The null hypothesis was that the risk of death/event was the same in the two groups.
The investigators who did the evaluation and collected the study outcomes were not aware of the treatments and did not participate in the selection of animals or in the data analysis. The investigator who did the analyses was not aware of the treatments. A two-tailed P<0.05 was considered as significant in every test.
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Results |
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The body weight at the start of administration of rpST was similar in the two groups (Table 1
). On day 114 of gestation, there was an increment of approximately 10 kg in the rpST group and 20 kg in the control group. Statistical differences were observed between the two gestational days (P<0.0001) but not between treatments (P>0.05). The back fat in the two groups was similar at the start of the study (Table 1). At 114 days of gestation, dorsal fat loss decreased in the rpST-treated group whereas in the control group it remained unchanged; statistical differences were observed between the two gestational days (P<0.0001) but not between treatments (P>0.10).
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12, 6 and 3 piglets respectively were born in the rpST-treated group, while 7 and 4 piglets respectively were born in the control group. In addition, placental weights were on average approximately 20% heavier in the treated group. Of relevance, duration of parturition was approximately 30 min shorter in the rpST-treated group (Table 1
). Furthermore, the sows in the rpST-treated group showed higher glucose blood levels, lower venous pH blood levels and higher venous lactate blood levels than the control group (P<0.005). Outcomes in the newborn piglets
In total, 111 piglets were born to controls and 109 piglets to treated sows. Of these, 9 (8.1%) and 19 (17.4%) piglets respectively were classified as intrapartum deaths (2=4.3; P=0.04) (Table 2). In the control group, the survival analysis showed that, starting at piglet number 7, as the piglet number increased, the survival probability decreased (Fig. 1). However, in the rpST-treated group, the curve was shifted to the left and survival probability decreased at piglet number 1. The survival analysis was different between the groups (P=0.04).
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). Glucose blood levels were approximately 5 mg/dl higher than controls. However, lactate and pCO2 levels were significanlty higher in piglets born in the control group than in piglets born in the rpST-treated group.
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), whereas the stillborn piglets were 1.4 times heavier in the former group than in the latter. In both cases, the difference was statistically significant (P<0.001). The birth weight from all the piglets born in the control group decreased with piglet number whereas in the rpST-treated group birth weight remained constant (Fig. 3). The two-way ANOVA for repeated measures was significant for changes according to both the piglet number and the treatment group (P<0.0001 and P<0.01 respectively). The other somatometric parameters collected from the live-born and the intrapartum death piglets were also significantly higher in the rpST-treated group (Table 2), further evidencing that piglets born to rpST-treated sows were significantly larger than controls. None of control or treated sows died during the study period or parturition, and none of the piglets was born with external evidence of birth defects.
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Discussion |
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Fetal growth seems to be affected not only by the amount of protein included in the maternal diet but also by the balance of macronutrients (Moore & Davies 2005); fetal growth rate is sensitive to the supply of nutrients from maternal to fetal blood. This supply varies with the availability of circulating nutrients in the mother and the efficiency of their transfer across the placenta into the fetal circulation. Conditions that reduce the availability of substrates or those which reduce nutrient delivery across the placenta can retard fetal growth (Gluckman & Harding 1997, Pardi et al. 2002), and severe maternal undernutrition reduces birth weight in humans (Stein et al. 1995, Godfrey et al. 1996). Conversely, maternal hyperglycemia increases placental glucose transfer and birth weight in humans and rodents (Herrera et al. 1985, Hauguel et al. 1986). In this study, significantly higher glucose blood levels were observed in rpST-treated sows than in controls and larger piglets were born in the former group, providing further support for the role of maternal hyperglycemia in fetal growth in mammals.
Somatotropin is an important modulator of insulin sensitivity, and both hormones are important regulators of cellular and whole body metabolism as well as somatic growth and body composition. The relationship between these two hormones was recently thoroughly reviewed by Dominici et al.(2005); ST counteracts the action of insulin on glucose homeostasis in both humans and animals, and a significant impairment of glucose metabolism may be observed after exogenous administration of ST in physiological or pharmacological doses in humans. In addition, both ST and insulin-like growth factor (IGF) have been linked to diabetic retinopathy (Frystyk 2005). In the present study, glucose blood levels in rpST-treated sows were 1.7 times higher than in controls at the start of parturition. Although they reached a statistical difference, glucose blood levels in the piglets born to treated sows were only 1.1 higher than the levels observed in the piglets born to control animals. The differences in the evident hyperglycemic effects observed in sows treated with rpST in late pregnancy and the modest rise in glucose blood levels in piglets can be explained by direct hormonal effects in the former and by a negligible effect in the latter due to the lack of placental passage of ST (Fholenhag et al. 1994). We failed to evaluate whether the hyperglycemia observed in sows treated with rpST from 80 to 114 days of gestation was promptly reversed. However, the higher glucose blood levels observed in their offspring was clinically insignificant and further studies are needed to clarify all the potential benefits and disadvantages that exogenous administration of rpST during pregnancy may provide as a therapeutic alternative for intrauterine growth retardation both in humans and animals.
Fetal availability of nutrients is, however, regulated by a more complex hormone regulation than the interaction between ST and insulin. Maternal ST and IGF seem to be key determinants of fetal growth and birth weight (Chellakooty et al. 2004). In addition, the placenta produces a variant of ST that progressively replaces pituitary ST in the maternal circulation from midgestation (Frankenne et al. 1988), and has been correlated to birth weight in humans (Evain-Brion et al. 1994, McIntyre et al. 2000). In addition, fetal growth induced by maternal treatment with ST during early to mid-gestation may not necessarily be maintained to term (Gatford et al. 2004), whereas treatment during the late or greater part of gestation has been related to increased piglet size at birth, as occurred in the present study.
In our study, despite the fact that the diet was not adjusted according to the sows’ gestational age, it provided 35 to 42 MJ ME/day, and weight gain was observed in both the control and the rpST-treated groups. However, weight gain was associated with back fat loss in the latter group. These changes were previously reported in other studies and may be explained by the repartitioning effect of rpST (energy from fat to muscle) observed in growing-finishing pigs and in ST-treated sows (Cromwell et al. 1992, Rehfeldt et al. 2001).
Human fetuses with growth restriction have an increased risk of neonatal mortality (Yanney & Marlow 2004). This relationship was evident in the control group where the intrapartum death piglets had, on average, birth weights 15% lower than the live-born piglets. However, intrapartum death piglets in the rpST-treated group were, on average, 100 g (approximately 7%) heavier than live-born piglets in the same group. It may be hypothesized that such differences in birth weight were large enough to present difficult parturition in this group of sows.
During fetal hypoxia, blood redistribution may increase intestinal peristaltic relaxation of the anal sphincter leading to defecation of meconium into the amniotic fluid (Davis et al. 1985). In our study, more meconium-stained piglets were born in the rpST-treated group, which may reflect the difficulties in delivering them. However, piglets born in the control group were more susceptible to metabolic acidosis, judged by their lower pH, and higher lactate and blood levels at birth than those reported in the pCO2 piglets born to treated sows. These contrasting results may support the view that rpST produces a combination of indirect effects in the piglets with a certain degree of relevance at birth that deserve to be explored in future studies.
Higher mortality rates have been reported with a larger number of piglets and at more advanced stages of parturition (Mota-Rojas et al. 2002, Borges et al. 2005). In the present study, the litter size did not differ between groups. Furthermore, although in the control group the number of intrapartum deaths increased with the piglet number, in the rpST-treated group the intrapartum deaths were mostly observed in the animals born in the first half of the litter. The contribution of weight at birth cannot explain this variability. In the control group, birth weight was lower in the last born piglets whereas the birth weight of piglets in the rpST-treated group did not vary throughout the progeny order. Therefore, the contribution of other mechanisms to the rate of intrapartum deaths in response to metabolic alterations induced in sows by rpST administration, cannot be ignored.
Previous studies evaluating rpST administration during pregnancy have observed a certain number of deaths in sows before and during parturition and lactation (Smith et al. 1991, Cromwell et al. 1992). The mortality seems to be dose-related, with death rates of 0, 6, 26 and 100% in sows injected with 0, 4, 8 and 16 mg rpST respectively (Smith et al. 1991). We did not observe any case of maternal death in control or treated sows with 6 mg rpST. However, sows treated with rpST had more metabolic acidosis compared with control sows, but the opposite relationship was observed in their offspring. These contrasting results suggest that whereas the sows were exposed to all the effects secondary to exogenous ST administration, different processes would be responsible for the changes observed at birth in the neonatal piglets.
In summary, 6 mg rpST/day administered to pregnant sows from days 80 to 114 of gestation resulted in higher blood glucose levels in the sows and their offspring and in an increased rate of intrapartum deaths. In both study groups, piglets were born without external evidence of malformations. Considering the lack of transplacental rpST passage, whether the higher mortality rate observed in the offspring of rpST-treated sows was secondary to difficulties during parturition due to the increased piglets’ size or to alterations in the metabolism of piglets secondary to maternal exposure to rpST, remains to be elucidated in future studies.
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Acknowledgements |
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References |
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Received in final form 14 March 2006
Accepted 16 March 2006
Made available online as an Accepted Preprint 31 March 2006
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) were constant across the piglet number at parturition whereas in the control group (•) they decreased (P<0.0001 for changes according to piglet number and P<0.01 between treatment groups; two-way ANOVA for repeated measures). Data are means±S.D.