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Effect of recombinant porcine somatotropin on fetal and placental growthin gilts with reduced uterine capacity^1,2,3,4

J. A. Sterle^*,5, T. C. Cantley^*, R. L. Matteri^,6, J. A. Carroll, M. C. Lucy^* and W. R. Lamberson^*,7

^* Department of Animal Sciences, University of Missouri, Columbia 65211 and and USDA, ARS, Animal Physiology Research Unit, Columbia, MO 65211

⁷ Correspondence:
159 Animal Science Research Center, University of Missouri, Columbia 65211 (fax: 573-882-2687; E-mail:
LambersonW{at}missouri.edu).

	Abstract

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Crowded uterine conditions were induced by unilateral hysterectomy-ovariectomy (UHO) in 42 gilts to determine the effect of recombinant porcine somatotropin on fetal and placental growth. Gilts were randomly assigned across three replicates to one of three treatments: Control (C; n = 14), daily injections of 1 mL saline from d 0 to 64 of gestation, Early (E; n = 12), 5 mg of rpST/d from d 0 to 30, followed by 1 mL saline from d 31 to 64, and Late (L; n = 16), 1 mL saline/d from d 0 to 29, followed by 5 mg of rpST/d from d 30 to 64 of gestation. Blood was collected from each gilt via jugular venipuncture at d 0 and every 15 d thereafter. Gilts were hysterectomized on d 65 of gestation. Length of placental attachment and fetal crown-rump length were measured. Placentas and fetuses were weighed. Placental length, wet weight, and dry weight were recorded. Treatment with rpST (either E or L) increased (P < 0.0001) maternal plasma IGF-I concentrations relative to controls. Treatment with rpST did not affect placental wet weight or placental DNA content. However, E and L treatments increased the percentage of placental protein (P = 0.01) and placental dry matter (P = 0.10) and increased contact area of uterine-placental interface (P = 0.01). Despite changes in placental composition and morphology, weights of fetuses collected from L-treated gilts did not differ from controls, whereas weights of fetuses collected from E-treated gilts tended to be less than controls (P < 0.06). Administration of rpST increased maternal IGF-I concentrations and placental surface area but failed to increase fetal growth in the UHO model. Therefore, mechanisms that are independent of maternal IGF-I or placental contact area may control early fetal growth under crowded uterine conditions.

Key Words: Fetal Growth • Insulin-like Growth Factor • Pigs • Placenta • Pregnancy • Somatotropin

	Introduction

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Uterine capacity is a measurement of the ability of the uterus to support embryos and fetuses through gestation. Physical, biochemical, and morphological limitations to uterine capacity include space, nutrients, gaseous exchange, and placental surface area. Litter size is thus determined by either ovulation rate (adjusted for potential embryonic viability) and/or uterine capacity (Bennett and Leymaster, 1989). Increasing ovulation rate has had limited effects on litter size because increased fetal death occurs if uterine capacity is exceeded during gestation. Unilateral hysterectomy-ovariectomy (UHO; removal of one ovary and the ipsilateral uterine horn) results in ovarian hypertrophy and compensatory ovulation (Staigmiller et al., 1972, 1974; Monk and Erb, 1974). The UHO model forces uterine capacity to be limiting to litter size and has been used to evaluate uterine capacity in swine under a variety of experimental conditions (Christenson et al., 1987).

One potential method for partitioning nutrients to fetuses is to treat pregnant sows with rpST (Etherton et al., 1989). Administration of rpST during gestation increased fetal and placental weight at d 44 of gestation (Sterle et al., 1995). Numerically greater weights at d 40 of gestation (Kelley et al., 1992) and an increased weight and advanced maturity at birth (Rehfeldt et al., 1993) have also been reported.

The objective of this study was to determine the effects of rpST on fetal and placental growth to d 65 of gestation under crowded uterine conditions. The UHO model was used to restrict space, thereby creating crowded conditions within the uterus. Two intervals of treatment (d 0 to 30 and d 30 to 64 of gestation) were chosen for rpST administration to evaluate effects relative to our previous window of treatment (d 30 to 43, Sterle et al., 1995). Physical, histological, and endocrine data were collected to evaluate mechanisms controlling fetal and placental growth in swine.

	Materials and Methods

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Animals and Experimental Design

All animal procedures were reviewed and approved by the University of Missouri Animal Care and Use Committee. Unilateral hysterectomy-ovariectomy was performed at approximately 155 d of age on 56 prepubertal gilts. Gilts were immobilized by administration of 0.2 mL/kg of 5% thiopental sodium injected into an ear vein. Anesthesia was maintained with a closed circuit system of halothane and oxygen. The reproductive tract was exposed, and one ovary and the ipsilateral uterine horn were removed. Gilts were artificially inseminated at first estrus and randomly assigned across three replicates to one of three treatments: Control (C; n = 14), daily injections of 1 mL saline from d 0 to 64 of gestation, Early (E; n = 12), daily injections of 5 mg of rpST (Monsanto, St. Louis, MO) from d 0 to 30, followed by 1 mL saline from d 31 to 64 of gestation, and Late (L; n = 16), daily injections of 1 mL saline from d 0 to 29, followed by 5 mg of rpST from d 30 to 64 of gestation. All injections were given intramuscularly in the trapezius.

Gilts were maintained in three adjacent pens in a modified open-front confinement building with a gutter flush waste removal system and natural ventilation. Pens had partially slatted floors with a total space of approximately 1.5 m² per gilt. Gilts were assigned so that each treatment was represented in each pen. Gilts were limit-fed (2.5 to 3 kg/gilt per day) a corn-soybean meal based diet balanced to meet current NRC requirements. This diet contained 14% crude protein, 0.70% lysine, and 3,358 kcal/kg digestible energy. Gilts were allowed ad libitum access to water. Detection of estrus was performed with a mature boar once daily in a neutral pen. Gilts were artificially inseminated every 12 h during estrus. Gilts were treated in their respective pens and continued to be checked daily for return to estrus until they were transported to the surgical unit between d 60 and 64 of gestation. Immediately before hysterectomy gilts were weighed and evaluated for pregnancy with the use of a transabdominal ultrasound scan. Gilts detected as having returned to estrus or determined open via ultrasonography were excluded from the experiment.

At surgery the uterus and ovary were removed and weighed, and corpora lutea were counted to determine ovulation rate. The broad ligament was removed from the uterus, and the uterus was opened. Care was taken not to puncture the fluid-filled extra-embryonic membranes. The number of fetuses was recorded. Each fetus was removed from its amniotic sac, and the umbilical cord was clamped and severed. Sex and weight of the fetus were recorded. Fetal heart, liver, kidneys, and semitendinosis muscle were collected and weighed. The percentage of viable fetuses [(number of viable fetuses/total number of fetuses) x 100] and the percentage survival [(number of viable fetuses/number of corpora lutea) x 100] were calculated.

Implantation length, measured as the length of the vascular area of the uterus associated with each placenta, was measured and recorded after removal of the fetuses. Placentas were stripped from the endometrium and weighed. Dry weight was determined after incubating placentas at 55°C until no change in weight was observed between two consecutive days. Dried placentas were then ground into a fine powder and stored for DNA and protein analyses.

Placental DNA and Protein Quantification

Placental DNA was extracted from the dried samples using the procedures of Welsh et al. (1991). The amount of extracted DNA was determined using Hoechst 33258 dye and fluorometric procedures (Cesarone et al., 1979). Total protein content was determined by thermal conductivity nitrogen analysis (Leco, 1994).

Histology

Placental samples from replicate three were not weighed but instead were used for histological analyses. Three placental-uterine samples from each viable conceptus were taken from random locations and placed in neutral buffered formalin. Fixed samples were embedded in paraffin, sectioned (5 µm), mounted on slides, and stained with hematoxylin and eosin. Uterine-placental interface sections were microscopically examined in order to determine surface contact area. Regions (3.7 mm²) of the uterine-placental interface were digitally captured, and the contact area converted to pixels. The pixel density (number of occupied pixels within a defined area) was determined. A mean value was calculated by averaging ten regions from each section.

Blood Sampling and Hormone Analysis

Maternal blood was collected on d 0, 15, 30, 45, and 60 of gestation via jugular venipuncture and temporarily stored on ice. Plasma was harvested after centrifugation (1,200 x g for 15 min) and stored at -20°C until assay. Plasma somatotropin concentrations were determined by RIA (Matteri et al., 1994). Intraassay coefficient of variation was 3.27%. All samples were run in a single assay; therefore, there was not an interassay coefficient of variation. Plasma IGF-I concentrations were determined by using procedures for extraction and assay as described by Lee et al. (1991). The IGF-I assay used recombinant IGF-I for iodination and standards (UBI-01-141; Upstate Biotechnology, Lake Placid, NY). A polyclonal antiserum for IGF-I RIA (UB3-189) was provided by the National Hormone and Pituitary Program (NIDDK, A. F. Parlow, Scientific Director). Intra- and interassay coefficients of variation for IGF-I were 5.3% and 10.8%, respectively.

Statistical Analyses

Fetal and placental variables were fitted to a model in PROC GLM of SAS (SAS Inst. Inc., Cary, NC) that included the effects of treatment, replicate, sex, and sex by treatment interaction, with treatment by replication or gilt nested within treatment and replicate, identified as the error term for treatment. The model for maternal variables with single observations included effects of treatment with treatment by replicate as the error term. The model for hormone concentrations in maternal plasma included effects of treatment, day, and treatment by day, with gilt nested within treatment designated as the error term for treatment. Orthogonal contrasts of C vs E and L and E vs L were conducted when the P-value for the treatment effect was less than or equal to 0.10 to further detect differences between treatments. Categorical data (pregnancy rate at d 65) were analyzed using a chi-square model.

	Results

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Maternal Variables

The proportion of gilts maintaining pregnancy to d 65 postbreeding was greater for L gilts than for E gilts (P < 0.025; Table 1). Gilts returning to estrus (n = 6) or determined open via transabdominal ultrasonography (n = 8) were excluded from the experiment.

View larger version (38K): [in this window] [in a new window]   Figure 1. Plasma concentrations of somatotropin in unilaterally hysterectomized-ovariectomized gilts [C = daily injections of 1 mL saline from d 0 to 64 of gestation, E = daily injections of 5 mg rpST d 0 to 30, followed by 1 mL of saline daily from d 31 to 64, L = daily saline injections from d 0 to 29, followed by daily injections of 5 mg rpST d 30 to 64; treatment x day P = 0.01].

View larger version (32K): [in this window] [in a new window]   Figure 2. Concentrations of IGF-I in plasma of unilaterally hysterectomized-ovariectomized gilts [C = daily injections of 1 mL saline from d 0 to 64 of gestation, E = daily injections of 5 mg rpST d 0 to 30, followed by 1 mL of saline daily from d 31 to 64, L = daily saline injections from d 0 to 29, followed by daily injections of 5 mg rpST d 30 to 64; treatment x d P = 0.0001].

Early rpST treatment decreased (P = 0.06) fetal weight relative to L at d 65 of gestation with C being intermediate (Table 3). Fetal crown-rump length was similar across treatments (P = 0.17). There was no difference in fetal heart, liver, kidney, or semitendinosis muscle weight between treatments. Placental wet weight also did not differ among treatments; however, E and L treatments tended to increase percentage placental dry matter (P = 0.10). Placental protein content (percentage DM) was increased in E and L relative to control (P = 0.01). There was no effect of treatment on placental DNA content (P = 0.30). The E and L treatments increased uterine-placental interface contact area (as estimated by pixel density of a standard area), compared to control (0.37 ± 0.04 vs 0.56 ± 0.10, 0.55 ± 0.03 mm², C vs E, L, respectively; P = 0.01; Figure 3).

View larger version (130K): [in this window] [in a new window]   Figure 3. Cross-sections of uterine-placental interface at d 65 of gestation in gilts. Contact area determined by pixel density of a standard area. A. Interface from a control gilt (daily injection of 1 mL saline from d = 0 to 64 of gestation). B. Same section as A after density fill. C. Interface from a gilt treated with 5 mg rpST from d 0 to 30 of gestation, followed by 1 mL saline from d 31 to 63 of gestation (E rpST). D. Same section as C after density fill. E. Interface from a gilt treated with 1 mL saline from d 0 to 29 of gestation, followed by daily injections of 5 mg rpST from d 30 to 64 of gestation (L rpST). F. Same section as E after density fill (0.37 ± 0.04 vs 0.56 ± 0.10, 0.55 ± 0.03 mm2, C vs E, L, respectively; P = 0.01).

	Discussion

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The results from the present study suggest that the insufficiency associated with a smaller placenta may be partially compensated for with administration of rpST by its increasing uterine-placental contact via increased complexity of the opposing surfaces without an increase in placental size. This effect, however, was not adequate to increase fetal weight under these conditions. The suppression of fetal growth in the E treatment group suggests that the increased placental growth cannot be maintained after cessation of exogenous somatotropin. The fetuses in the E group may have experienced suppression in growth after the end of somatotropin treatment.

Musser et al. (1997) reported an increase in maternal IGF-I concentrations when sows were fed in excess of established requirements from d 29 to 45 of gestation. The fetuses from sows on the high plane of nutrition did not exhibit the expected negative relationship between number of fetuses per sow and fetal weight. Therefore, increased maternal IGF-I concentrations, or other responses to high feed intake during this period of gestation, may overcome the detrimental effects of fetal crowding when a large number of fetuses occupy the uterus. Gatford et al. (2000) reported an increase in fetal body weight, length, and skull width at d 51 of pregnancy in gilts injected from d 25 to 51 with somatotropin and restricted to approximately 30% of ad libitum intake. Glucose, therefore, was not limiting fetal growth in undernourished pregnant pigs during this time of gestation and the increased fetal growth was not caused by improved placental glucose transport. These data imply that the availability of amino acids is limiting in underfed conditions in the first half of pregnancy. The gilts in the present study were fed traditional diets formulated to meet NRC requirements. Perhaps the rpST treatment along with pregnancy increased nutrient demands beyond what was provided in the diet. Determination of nutritional requirements under these conditions and subsequent formulation and feeding of these diets may provide information on possible nutritional interactions as well as potential effects on fetal growth.

The additional somatotropin and IGF-I in blood may have had a direct endocrine action on the pregnant uterus. A second possibility is that the additional somatotropin in blood caused a local increase in IGF-I gene expression within the pregnant uterus. The local increase in IGF-I mRNA could have led to an increase in IGF-I protein that mediated the effects that were observed in the present study. In a previous study, it was demonstrated that rpST increased maternal liver IGF-I mRNA in pregnant gilts treated from d 30 to d 44 of gestation (Sterle et al., 1998). The amount of IGF-I mRNA within maternal reproductive tissues, however, was not increased after rpST. Relative to liver, somatotropin receptor concentration in reproductive tissues is low (Sterle et al., 1998). A local IGF-I response to rpST may not exist because there are too few somatotropin receptors in reproductive and fetal tissues. Other mechanisms besides a local IGF-I response, therefore, lead to placental, fetal, and uterine changes in rpST-treated gilts.

The results of the present study indicate a stimulation of placental growth as a result of exogenous rpST administration. However, it appears that these effects are not continued after cessation of treatment and rpST may in fact be detrimental to fetal growth later in gestation. While the effects at term have not been investigated here based on previous reports and the present data, we would not predict a beneficial effect at birth. The numerical increase in number of pregnancies lost after d 35 of gestation also may indicate a detrimental effect of rpST on fetal survivability.

	Implications

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Developing an effective means of increasing uterine capacity may increase litter size. Administration of recombinant porcine somatotropin during gestation has had variable effects that may be dependent on stage of gestation, duration of administration, reproductive age of female, and environmental conditions. It has not been established whether these effects will persist to parturition.

	Footnotes

 
¹ Journal paper no. 13,198 of the Missouri Agric. Exp. Sta., Columbia 65211.

² Appreciation is extended to Betty Nichols, August Rieke, Steve Sterle, Carol Okamura, Cindy Boyd, Kurt Holiman, Paul Little, Clyde Morgan, Ron Christenson, Jeff Vallet, Ron Belyea, and Bill Trout for their technical expertise and assistance.

³ Mention of a trade name or proprietary product does not constitute a guarantee or warranty of the product by the USDA and does not imply its approval to the exclusion of other products that may also be suitable.

⁴ The recombinant porcine somatotropin used for these studies was generously provided by the Monsanto Co. (St. Louis, MO).

⁵ Current address: Extension Swine Specialist, 212 Kleberg Center, Texas A&M Univ., College Station 77843-2471.

⁶ Current address: Assistant Area Director, 800 Buchanan St., Room 2034, Albany, CA 94710-1105.

Received for publication December 21, 2001. Accepted for publication October 1, 2002.

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