The Impact Of Induced Stress During Days 13 And 14 Of Pregnancy On The Composition Of Allantoic Fluid And Conceptus Development In Sows

Theriogenology 61 (2004) 757–767

The impact of induced stress during Days 13 and 14 of pregnancy on the composition of allantoic fluid and conceptus development in sows Pia Razdanb,*, Padet Tummaruka, Hans Kindahlb, Heriberto Rodriguez-Martinezb, Fredrik Hulte´nb, Stig Einarssonb a

Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Sciences, Chulalongkorn University, Bankok, Thailand b Department of Obstetrics and Gynaecology, Faculty of Veterinary Medicine, Centre for Reproductive Biology in Uppsala (CRU), Swedish University of Agricultural Sciences, SLU, P.O. Box 7039, SE-750 07 Uppsala, Sweden Received 17 April 2003; received in revised form 19 June 2003; accepted 22 June 2003

Abstract Stress due to regrouping of breeding females is difficult to avoid completely in loose-housing systems. The effects of stress during the maternal recognition of pregnancy on fetal development and survival at Day 30 of pregnancy was, therefore, studied in 17 sows allocated into one control (C-) group, one group deprived of food during Days 13 and 14 (FD-), and one group (A-), which was treated with ACTH (0.01 mg/kg body weight of Synacthen1 Depot) every sixth hour during the same period. Total number of fetuses, fetal survival rate, volume of allantoic fluid, and the weight and length of total fetal unit, placentas, allantochorion and fetuses were determined. The concentrations of progesterone (P4), PGFM, PGF2, PGE, estrone-sulfate, and estradiol-17b in the allantoic fluid were analyzed. No significant differences between groups were found for any parameter measured except for P4. Food deprivation increased P4 concentration in the allantoic fluid, and there was a positive correlation between the P4 concentration and the weight of the placenta. It is, therefore, suggested that P4 influences the placenta size among food-deprived sows. # 2003 Elsevier Inc. All rights reserved. Keywords: Stress; Sow; Conceptus development; Progesterone

* Corresponding author. Tel.: þ46-18-671342; fax: þ46-18-673545. E-mail address: [email protected] (P. Razdan).

0093-691X/$ – see front matter # 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0093-691X(03)00252-8

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1. Introduction For practical reasons, it is not possible to completely avoid regrouping of breeding females within a group-housing system. Regrouping leads to fighting for the establishment of hierarchy within the newly formed female group and it takes approximately 48 h before a new rank-order is settled [1]. Regrouping is stressful for the animals and due to competition, low-ranked individuals are deprived of food from time-to-time. Both stressful stimuli in the form of food deprivation and the activation of the HPA axis by repeated injections with ACTH immediately after ovulation, have a negative impact on early embryo development [2–6]. However, there is still a lack of knowledge regarding how stress influences reproduction during the early pregnancy period. In a previous study, it was demonstrated that both food deprivation and repeated injections of ACTH for 48 h during Days 13 and 14 of pregnancy (Day 1: first day of standing estrus) affected the hormonal status of the sows in different ways during and after the treatment period [7]. The food-deprived sows had significantly elevated levels of progesterone during the treatment period and the ACTH-treated sows had a delay in the rise of estrone, from Day 19 of pregnancy to Day 21. About Day 12 of pregnancy, pig conceptuses signal their presence by releasing estrogens and possibly other factors that prevent the corpus luteum (CL) from regressing [8]. Estradiol secretion has been found to correlate better with the size of the embryo than with the age of the embryo [9]. It might, therefore, be hypothesized that a delayed rise of fetal estrone in peripheral blood after Day 19 of pregnancy, as seen in ACTH-treated sows, reflects an impeded development of the fetuses. Previously described effects of elevated levels of progesterone on fetal development and survival, both caused by food restriction or exogenous treatment during early pregnancy in sows are contradictory [10–12], which suggests that further studies are needed. The purpose of the present study was to investigate whether repeated administration of ACTH, or food deprivation, during Days 13 and 14 of pregnancy, might influence the hormonal concentration of the allantoic fluid and fetal development and survival by Day 30 of pregnancy. Day 30 of pregnancy was chosen as the time of slaughter both because the major incidence of embryo loss could be expected to have occurred at that time and because each conceptus, being a distinct entity, can be easily evaluated for morphological normality [13–15].

2. Materials and methods 2.1. Animals The research plan and all procedures involving the use of animals were reviewed and approved by the Ethical Committee for Experimentation with Animals, Sweden. From the day of arrival to the second estrus after weaning, the health status (appetite and general condition) of the sows was carefully monitored. In three cases of persistent clinical infections—one endometritis, one lameness, and one unknown cause of illness—the sows were excluded from the study. The experiment was performed with 17 healthy crossbred (Swedish Landrace  Swedish Yorkshire) sows in their second to fourth parity. The sows were brought from one commercial farm on the day of weaning and weighed at between

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180 and 230 kg. They were housed in individual pens on straw, in the vicinity of a boar, at the Department of Obstetrics and Gynaecology, SLU, Uppsala, Sweden. Sows were fed 2.9 kg of a commercial ration divided into two meals at 7.00 a.m. and 3.00 p.m., according to Swedish standards [16]. Water was provided ad libitum. The sows were randomly divided into three groups, one control group (C, n ¼ 6), one group that was deprived of food (FD, n ¼ 5), and one group treated with synthetic ACTH (A, n ¼ 6). One of the sows in the A-group returned to estrus at Day 28. She was not excluded from the study, since it was assumed that the abortion was an effect of treatment. However, the statistical significance of the results was the same despite her inclusion. 2.2. Estrous detection and ovulation Heat detection was performed by using the back-pressure test in front of a boar twice daily (morning and evening). Standing estrus was defined when sows responded with the standing reflex to the back-pressure test in the presence of a boar. First day of standing estrus was defined as Day 1 of pregnancy. The time of ovulation was determined through transrectal ultrasonography using an annular array sector scanner (type Scanner 250, Pie Medical b.v., Maastricht, the Netherlands) with a 5 MHz multiple-scan angle transducer as described by Mburu et al. [17]. Sows were monitored during the first estrus after weaning to predict the interval from onset of standing estrus to ovulation in the second estrus of each sow. From about 20 h after the onset of estrus, ultrasonography was performed every fourth hour until ovulation had occurred. Time of ovulation was set as the time when all large follicles had collapsed. The sows were inseminated twice 18–8 h prior to ovulation in the second estrus after weaning. The animals were inseminated with 100 ml of semen extended with BTS (Beltsville Thawing Solution [18]) to a total of 10  109 spermatozoa pooled from two boars with proven fertility. 2.3. Treatment To achieve easy and stress-free treatment, a jugular vein catheter was inserted under general anesthesia [19] a few days before the second estrus after weaning was expected (Days 15–18). One permanent Silastic tubing was inserted into the right jugular vein, passed subcutaneously to the back, and exteriorized through the skin where it was connected to a cannula. The tubing was flushed once a day with heparinized saline solution (25 IE/ml) until the treatment began. The treatment was performed during Days 13 and 14, defining the first day of standing estrus as Day 1 of pregnancy. The A-group sows were given intravenous injections of tetracosactide (Synacthen1 Depot), a synthetic ACTH, at a dose of 0.01 mg/kg body weight, diluted with saline solution to a total volume of 5 ml. Catheters were flushed with 5 ml of saline solution after each injection. Injections were given every sixth hour from 6.00 a.m. on Day 13 until 6.00 a.m. on Day 15 of pregnancy. FD-sows were deprived of food from the morning of Day 13 of pregnancy until the evening meal of Day 14. C-group sows were fed and given intravenous injections of 10 ml saline solution every sixth hour from 6.00 a.m. on Day 13 until 6.00 a.m. on Day 15 of pregnancy.

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2.4. Slaughter and recovery of the genital tract and fetuses The sows were slaughtered at Day 30  2 of pregnancy and the internal genital tract was immediately recovered and examined. The total number of CL was counted to determine ovulation rate. Fetal survival rate was determined by dividing the total number of viable fetuses with the total number of CL. The fetuses were classified as presumably viable or nonviable. Fetuses classified as nonviable were characterized by hemolyzed amniotic fluid, resorbing fetal membrane, degenerated fetus or no fetus present. The position of each fetoplacental unit within a uterine horn was recorded and the number of allantoic sacs was counted by palpation of the uterine horns. The allantoic sacks were then separated from each other by a pair of forceps and examined one by one, beginning with the sack situated most closely to the ovary in the left uterine horn (L1). A sample of 10 ml of allantoic fluid was taken from each allantoic sac with sterile syringes, the fluid was immediately transferred to plastic tubes and chilled on ice after which it was stored in a standard freezer (20 8C) for hormonal analyses. The examination of each fetal unit included weight of total fetal unit, volume of allantoic fluid, weight and length of placenta, weight of allantochorion, and weight and length of fetus. Material for histological examination was taken from the fetal sacs nearest to the ovaries and the uterine body, respectively. The samples were numbered accordingly; tissues taken from the fetal sac nearest to the left ovary (no. 1), the left horn nearest the uterine body (no. 2), the right horn nearest to the uterine body (no. 3), and from the fetal sac closest to the right ovary (no. 4). For light microscopy examination, samples were placed in 10% neutral buffered formaline and 5 mm paraffin-embedded sections were stained in Hematoxylin–Eosin (HE). Histological examination was performed to determine differences regarding the number of blood vessels in the placentae and fetal membranes, erythropoietic cells in the liver and any change in the appearance of the kidneys. A mean value of the area of erythropoietic cells in the liver of the fetuses was calculated by image analysis (TZ-240 Easy Image, Tekno Optik AB, Huddinge, Sweden) using Nikon Microphot-FXA, Japan. 2.5. Hormone assays 2.5.1. Progesterone The concentration of progesterone in the allantoic fluid was determined using a solidphase radioimmunoassay (Coat-A-Count1Progesterone, Diagnostic Products Corporation, LA). The kit was used according to the manufacturer’s instructions. The coefficient of variation was 5.4, 11.4, and 6.1% for low, medium, and high controls, respectively. The average detection limit of the assay was 0.27 nmol/l. 2.5.2. Estrone-sulfate Allantoic fluid estrone-sulfate (E1SO4) was determined using a radioimmunoassay kit (DSL-5400, Diagnostic Systems Laboratories, Inc., Webster, USA) providing materials for the quantitative measurement of estrone-sulfate in serum or plasma. The kit was used according to the manufacturer’s instructions. The coefficient of variation was 4.4% at 2.0 nmol/l and 13.3% at 22.2 nmol. The average detection limit of the assay was 14 pmol/l.

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2.5.3. Estradiol-17b The concentration of estradiol-17b in the allantoic fluid was determined using a solidphase radioimmunoassay (Coat-A-Count1, Diagnostic Products Corporation, LA). The kit was used according to the manufacturer’s instructions but the samples and standards were extracted prior to analysis. The coefficient of variation was 8.7, 0.7, and 0.2% for low, medium, and high controls, respectively. The average detection limit of the assay was 9.2 pmol/l. 2.5.4. Prostaglandins The primary prostaglandins F2a and E2 were analyzed in the amniotic fluid by a modified method of Lindgren et al. [20]. The samples were diluted in different steps in 0.25% bovine g-globulin buffer. The dilutions aimed to fit the standard curves of the prostaglandin radioimmunoassay. The antiserum cross-reacted 75% against PGF1a, 2% against PGF2b, 1.5% against PGF1b, and <0.1% for PGE2, PGE1, PGD2, TXB2, and 15-ketodihydroPGF2a. First, the samples were analyzed directly for the contents of PGF2a and then after reduction of the samples with sodiumborohydride [20]. Sodiumborohydride reduces PGE2 to a mixture of PGF2b and PGF2a. With samples containing a known amount of PGE2, the ratio between the two isomers was found to be around 1. To simplify calculations, the increase in PGF2a contents after reduction was multiplied by 2 to obtain the PGE2 content. The practical limits of detection for PGF2a and PGE2 were 0.3 and 0.5 nmol/l, respectively. The inter-assay coefficient of variation was 16% and the intra-assay coefficients of variation varied between 6 and 14% at the optimal ranges of the standard curve. 2.6. Statistical analysis Statistical analyses were carried out using the mixed procedure in the SAS software package [21]. The statistical model used to compare differences in ovulation rate, total number of fetuses, embryo survival rate, day of slaughter, weight of total fetal unit, volume of allantoic fluid, weight of placenta, weight of allantochorion, weight of fetuses, crown to rump length, size of erythropoietic area of the fetal liver, and hormonal concentration in the allantoic fluid included the fixed effect of treatment (three groups). To evaluate differences between the treatment groups regarding diversity in size within litter, the within-litter standard deviation for each parameter was calculated and included in the same model as mentioned above. Sow was set as a random factor to account for repeated sampling within sow. The relationship between the concentration of progesterone in the allantoic fluid and placenta size at the time of slaughter was analyzed using Pearson’s correlation analysis in the SAS package.

3. Results 3.1. Morphological examination There was no significant difference in the number of CL, number of fetuses, or embryo survival rate between the groups (Table 1). The A-group sow, no. 7138, returned to estrus at Day 28 of pregnancy. She had no fetuses present at the time of slaughter but there were scars in the endometrial mucosa (presumably implantation sites), indicating that

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Table 1 Day of slaughter, number of corpora lutea (CL), number of fetuses, and embryo survival rate (no. of viable fetuses/no. of CL) (means  S:D:) in control (C-), food-deprived (FD-), and ACTH-stimulated (A-) sows during Days 13 and 14 of pregnancy C-group (means  S.D.) Day of slaughter Total no. of CL Total no. of viable fetuses Total no. of nonviable fetuses Embryo survival rate (%)

30.3 19.0 14.3 0.7 75.6

    

0.4 1.3 2.0 0.2 11.2

FD-group (means  S.D.) 30.8 20.6 15.2 0.0 75.7

    

0.5 1.4 2.2 0.0 12.2

A-group (means  S.D.) 30.0 18.5 11 0.5 62.0

    

0.5 1.3 2.0 0.3 11.2

fertilization had occurred and that the fetuses had been lost after Day 12 of pregnancy. Postmortem examination of the genital tract and fetuses revealed no significant differences in weight of total fetal unit, weight of fetuses, crown to rump length, weight of placenta, weight of allantochorion, and volume of allantoic fluid between the groups (Table 2). No differences in the diversity of size within litter could be found in any of the measured parameters (Table 3). The histological examination illustrated that there were no detectable Table 2 Weight of total fetal unit, weight of fetuses, crown to rump length, weight of placenta, length of placenta, weight of allantochorion, volume of allantoic fluid (means  S:D:) in control (C-), food-deprived (FD-), and ACTHstimulated (A-) sows during Days 13 and 14 of pregnancy C-group (means  S.D.) Weight of total fetal unit (g) Weight of fetuses (g) Crown to rump length (mm) Weight of placenta (g) Length of placenta (mm) Weight of allantochorion (g) Volume of allantoic fluid (ml)

341 1.3 23.5 107 108 21.7 208

      

27 0.1 0.7 10 15 2.6 19

FD-group (means  S.D.) 370 1.4 24.6 120 126 23.1 225

      

30 0.1 0.8 10 17 2.9 20

A-group (means  S.D.) 315 1.1 22.6 103 105 19.3 195

      

30 0.1 0.8 10 17 2.9 20

Table 3 Diversity within litter in weight of total fetal unit, weight of fetuses, crown to rump length, weight of placenta, length of placenta, weight of allantochorion, volume of allantoic fluid (means  S:D:) in control (C-), fooddeprived (FD-), and ACTH-stimulated (A-) sows during Days 13 and 14 of pregnancy C-group (means  S.D.) Weight of total fetal unit (g) Weight of fetuses (g) Crown to rump length (mm) Weight of placenta (g) Length of placenta (mm) Weight of allantochorion (g) Volume of allantoic fluid (ml)

92 0.2 1.2 23 2.4 8.0 52

      

12 0.04 0.3 5.8 3.2 1.2 8.8

FD-group (means  S.D.) 70 0.2 1.0 22 8.0 6.8 56

      

13 0.04 0.3 5.8 3.5 1.3 9.6

A-group (means  S.D.) 67 0.3 1.1 33 2.2 5.6 46

      

13 0.04 0.3 5.8 3.5 1.3 9.6

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Table 4 Concentration of progesterone, estrone-sulphate (E1SO4), estradiol-17b, prostaglandin F2a-metabolite (PGF2amet), prostaglandin F2a (PGF2a), prostaglandin E (PGE) in the allantoic fluid (means  S:D:) of control (C-), food-deprived (FD-), and ACTH-stimulated (A-) sows during Days 13 and 14 of pregnancy C-group (means  S.D.) Progesterone (nmol/l) E1SO4 (nmol/l) Estradiol-17b (pmol/l) PGF2a-met (nmol/l) PGF2a (nmol/l) PGE (nmol/l)

6.6 932 6942 7.5 0.8 0.5

     

0.5a 178a 1431a 0.5a 0.4a 0.04a

FD-group (means  S.D.) 8.2 1020 7571 6.6 1.3 0.5

     

0.6b 195a 1568a 0.6a 0.4a 0.05a

A-group (means  S.D.) 7.1 895 7876 7.2 1.1 0.5

     

0.6a,b 195a 1568a 0.6a 0.4a 0.05a

Rows with different superscripts (a, b) differ (P < 0:05).

differences in the number of blood vessels in the placentas and fetal membranes, or any changes in the appearance of the kidneys among groups. Image analysis of the area of erythropoietic cells in the fetal livers showed that the fetuses of the C-group had a larger area of erythropoietic cells in their livers (3:2  2:1) compared with the A-group (2:4  1:4) and the FD-group (2:6  1:2), but the differences were not significant (P ¼ 0:4). 3.2. Hormonal levels in allantoic fluid 3.2.1. Progesterone The concentration of progesterone in the allantoic fluids of the FD-sows was significantly higher (P < 0:05) compared with the C-group sows (Table 4). The progesterone concentration in the allantoic fluids of the A-group did not differ from either the FD-group (P ¼ 0:2) or the C-group (P ¼ 0:5). Within the FD-group, the concentration of progesterone was positively correlated (P < 0:05) with the placental size, which was not the case within the C- or A-group (P ¼ 0:7). 3.2.2. Estrone-sulfate and estradiol-17b There was no significant difference (P > 0:05) between the groups in the concentration of estrone-sulfate and estradiol-17b in the allantoic fluid (Table 4). 3.2.3. Prostaglandin There was no significant difference (P > 0:05) between groups in the concentrations of PGF2a-metabolite, PGF2a, and PGE in the allantoic fluid at the time of slaughter (Table 4).

4. Discussion There were no significant differences in fetal development, survival rate, and conceptus diversity within litter revealed by the post-mortem examination at Day 30 of pregnancy, while some numerical differences were observed between the groups. These differences

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will, to some extent, be discussed as observations but only when they are supported by the literature. The progesterone concentration was significantly higher in the allantoic fluid of the FD-group sows compared with the C-group sows and the progesterone concentration was positively correlated with placental size in the FD-group. In an earlier study, a significantly increased level of progesterone in peripheral blood plasma was seen when the sows were deprived of food during Days 13 and 14 [7]. Exogenous administration of progesterone and estrone during the first 30 days of pregnancy elevates placental growth [22]. The FD-group had, on average, larger placentas (120 g) than both the C- and A-group (107 and 103 g respectively), even though the differences were not significant, while there were almost no differences in the size and weight of the embryos between the groups. This could indicate a compensation for placenta inefficiency. At this stage of pregnancy, embryo survival is not affected by uterine space [23]. However, it can be speculated that if placentas remain large later in pregnancy, fetal death may occur due to a crowding effect. On the other hand, it may be a disadvantage for an embryo to develop with a small area of placental attachment in the early stages of pregnancy, because it is probably not possible to increase its relative placenta size later. The size of the placenta and the relative placental efficiency may, to some extent, be determined by the level of estrogens and possibly other uterine luminal growth factors at the time of elongation [24]. Moreover, Hunter et al. [25] suggested that the higher fetal survival rate and larger litters seen in Meishan gilts is partly due to a reduction in placental estradiol synthesis, which limits conceptus growth and development compared with Large White hybrids. Neither food deprivation nor ACTH treatment had any significant effects on the levels of estrone-sulfate and estrone in peripheral blood plasma during the time of elongation [7]. Moreover, no significant differences in the levels of estradiol-17b and estrone-sulfate in the allantoic fluid at the time of slaughter were observed between groups in the present study. This could explain why the placenta size did not differ significantly between groups despite the high concentration of progesterone seen in both the allantoic fluid and peripheral blood plasma of the FD-sows. Another reason might be that the progesterone receptors are undetectable in uterine luminal epithelium on Days 12–18 of pregnancy, yet remain in the underlying stroma and myometrium [26]. Thus, stromalderived factors, expressed under the influence of progesterone (progestomedins), may exert paracrine effects on the luminal and glandular epithelia to achieve the differentiation that is necessary for implantation, such as the formation of the epitheliochorial placenta. More highly developed embryos within one litter might, by synthesizing more estradiol than their less advanced littermates, increase uterine secretion and thereby enhance the asynchrony between the less developed embryos and the uterine environment [27]. A vicious circle begins, which leads to an increased diversity in size within the litter that could finally kill the less developed fetuses. A-group sows had the largest diversity in placenta size within litter (33 g compared with 23 and 22 g, C- and FD-group, respectively) although the difference was not significant. It has been illustrated that ACTH treatment during Days 13 and 14 of pregnancy delayed the rise of estrone in the peripheral blood plasma until Day 22, compared with Day 19 in control sows [7]. A disturbance in the fetal production of estrogens after Day 13 of pregnancy, when the second phase of fetal estrogen production occurs [28], could lead to a large diversity in placenta size.

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The A-group sow, no. 7138, returned to estrus at Day 28 of pregnancy and autopsy revealed scars from the implantation sites, which indicates a loss of fetuses after Day 12 of pregnancy. This is consistent with the observations of Glossop and Foulkes [29] who reported two distinct periods when sows return to estrus after service; the first one was about Day 20 (range 17–23) and presumably represents animals in which fertilization failed or in which embryos were lost before Day 12; the second one occurs at about Day 26 (range Days 24–31), indicating exposure to estrogens during the first phase of embryonic estrogen production but not during the second one. According to Geisert et al. [30], this delayed return to estrus would represent a short delay, but not total absence of luteolysis, and therefore a delayed return to estrus as seen in sow no. 7138. Supporting the suggestion of Bazer [31] that endometrial synthesis of PGF2a is not inhibited during pregnancy, Razdan et al. [7] described a significant increase in the peripheral blood plasma concentration of PGF2a-metabolite between Days 11 and 12 of pregnancy. Conceptus synthesis and release of estrogen diverts secretion of PGF2a into the uterine lumen (exocrine) rather than towards the stroma (endocrine) [32]. It has been speculated that prostaglandin enhances nutrient availability to the developing conceptuses by affecting blood flow and increasing permeability of the endometrial vascular bed supplying the uterus [33]. In the present study, there were no significant differences in the concentration of PGF2a, PGF2a-metabolite, or PGE in the allantoic fluid between the treatment groups, which partly explains the absence of significant differences in embryo growth rates observed between the groups. The production of nucleated erythroid precursors in the yolk sac (primitive erythropoiesis) continues until ‘‘seeding’’ of the newly formed liver by circulating erythroid cells. After formation, the liver is the major site for definitive erythropoiesis in the fetuses. The transition from primitive to definitive erythropoiesis occurs on approximately Day 20 in the pig, after the formation of the hepatic anlage on Day 18 [34]. The erythropoiesis in swine fetuses undergoes dramatic maturation during Days 24–30 of gestation, as indicated by both a dramatic increase in circulating blood cell populations and a change from primary primitive precursors to almost entirely mature erythrocytes [35]. This study revealed a larger area of erythropoietic cells in the livers of the C-group fetuses compared with the treated groups, but the difference was not significant. Treatments performed at the time of implantation will not directly affect fetal erythropoiesis because it is still poorly developed during this period of time. On the other hand, it has been suggested that placenta efficiency, uterine crowding, and size of fetuses might affect the hematocrits in Days 40 and 105 fetuses [35,36]. These parameters might all constitute a link between the uterine environment at the time of implantation and erythropoiesis in the fetal liver after Day 20 of pregnancy. Stress in the form of food deprivation and treatment with synthetic ACTH for 48 h during the time of maternal recognition of pregnancy seemed to have no effect on fetal survival up to Day 30 of pregnancy. Food deprivation increased progesterone concentration in the allantoic fluid measured at Day 30 of pregnancy. The positive correlation noted between the progesterone concentration in allantoic fluid and the weight of the placenta suggest that progesterone influences placenta size among food-deprived sows. The stress that the sows are exposed to during a regrouping situation is probably more harmful than the effect of simulated stress, as achieved in the present study, and needs to be further evaluated.

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