Allantoic Fluid Volume Research Articles

Canine and feline foetal fluids: Volume, hormonal and biochemical characterization during pregnancy.

This study aimed to evaluate the volume, the concentration of steroid hormones, and biochemical composition of the foetal fluids at different gestational ages in dogs and cats. Following the ovariohysterectomy, the allantoic and amniotic fluid samples were collected from pregnant bitches and queens and were assigned to different groups according to their gestational age. The canine and feline allantoic fluid volume increased during pregnancy, reached its maximum values on days 40-49 and then decreased. The canine and feline amniotic fluid volume increased steadily by the last days of pregnancy. In spite of significant changes of sex hormones in the foetal fluids, their concentration and ratios were not significantly different between male and female fetuses. The canine amniotic cortisol concentration increased until days 40-49 and decreased significantly afterwards. The maximum cortisol concentrations in the feline allantoic and amniotic fluids were observed on days 50-60 and 40-49, respectively. During the canine pregnancy, the concentrations of calcium, phosphorus, chloride, sodium, triglyceride, cholesterol, total protein, albumin and the activities of aminotransferase (AST), alkaline phosphatase (ALP), amylase and gamma-glutamyl transferase (GGT) in the amniotic fluid were higher than the allantoic fluid. The magnesium, potassium, lactate dehydrogenase (LDH) activity, creatine and lipase were higher in the allantoic fluid. In the feline allantoic fluid, potassium, magnesium, phosphorus, creatinine, albumin and glucose concentrations and the activities of creatine kinase (CK), GGT, LDH and lipase were higher. The ALP, AST activities, sodium and calcium concentrations were higher in the amniotic fluid (p<0.05). Volume of foetal fluids was determined in dogs and cats. Concentration of sex hormones did not different between male and female fetuses.

Dietary supplementation with L-citrulline improves placental angiogenesis and embryonic survival in gilts.

This study was conducted with gilts as an animal model to test the hypothesis that dietary supplementation with L-citrulline (Cit) improves placental angiogenesis and embryonic survival. Between Days 14 and 25 of gestation, each gilt was fed a corn- and soybean-meal-based diet (2 kg/day) supplemented with 0.4% Cit or an isonitrogenous amount of L-alanine (Control). On Day 25 of gestation, gilts were hysterectomized to obtain conceptuses. Amniotic and allantoic fluids and placentae were analyzed for NOx [stable oxidation products of nitric oxide (NO)], polyamines, and amino acids (AAs). Placentae were also analyzed for syntheses of NO and polyamines; concentrations of AAs and related metabolites; and the expression of angiogenic factors and aquaporins (AQPs). Compared to the control group, Cit supplementation increased (P < 0.01) the number of viable fetuses by 2.0 per litter, the number and diameter of placental blood vessels (21% and 24%, respectively), placental weight (15%), and total allantoic and amniotic fluid volumes (20% and 47%, respectively). Cit supplementation also increased (P < 0.01) enzymatic activities of GTP-cyclohydrolase-1 (32%) and ornithine decarboxylase (27%) in placentae; syntheses of NO (29%) and polyamines (26%); concentrations of NOx (19%), tetrahydrobiopterin (28%), polyamines (22%), cAMP (26%), and cGMP (24%) in placentae; total amounts of NOx (22-40%), polyamines (23-40%), AAs (16-255%), glucose (22-44%), and fructose (22-43%) in allantoic and amniotic fluids. Furthermore, Cit supplementation increased (P < 0.05) placental mRNA levels for angiogenic factors (eNOS [84%], GTP-CH1 [55%], PGF [61%], VEGFA120 [26%], and VEGFR2 [137%], as well as AQPs - AQP1 [105%], AQP3 [53%], AQP5 [77%], AQP8 [57%], and AQP9 [31%]). Collectively, dietary Cit supplementation enhanced placental NO and polyamine syntheses as well as angiogenesis to improve conceptus development and survival.

Dietary supplementation with 0.4% L-arginine between days 14 and 30 of gestation enhances NO and polyamine syntheses and water transport in porcine placentae

BackgroundMost embryonic loss in pigs occurs before d 30 of gestation. Dietary supplementation with L-arginine (Arg) during early gestation can enhance the survival and development of conceptuses (embryo/fetus and its extra-embryonic membranes) in gilts. However, the underlying mechanisms remain largely unknown.MethodsBetween d 14 and 30 of gestation, each gilt was fed daily 2 kg of a corn- and soybean-meal based diet (12% crude protein) supplemented with either 0.4% Arg (as Arg-HCl) or an isonitrogenous amount of L-alanine (Control). There were 10 gilts per treatment group. On d 30 of gestation, gilts were fed either Arg-HCl or L-alanine 30 min before they were hysterectomized, followed by the collection of placentae, embryos, fetal membranes, and fetal fluids. Amniotic and allantoic fluids were analyzed for nitrite and nitrate [NOx; stable oxidation products of nitric oxide (NO)], polyamines, and amino acids. Placentae were analyzed for syntheses of NO and polyamines, water and amino acid transport, concentrations of amino acid-related metabolites, and the expression of angiogenic factors and aquaporins (AQPs).ResultsCompared to the control group, Arg supplementation increased (P < 0.05) the number of viable fetuses by 1.9 per litter, the number and diameter of placental blood vessels (+ 25.9% and + 17.0% respectively), embryonic survival (+ 18.5%), total placental weight (+ 36.5%), the total weight of viable fetuses (+ 33.5%), fetal crown-to-rump length (+ 4.7%), and total allantoic and amniotic fluid volumes (+ 44.6% and + 75.5% respectively). Compared to control gilts, Arg supplementation increased (P < 0.05) placental activities of GTP cyclohydrolase-1 (+ 33.1%) and ornithine decarboxylase (+ 29.3%); placental syntheses of NO (+ 26.2%) and polyamines (+ 28.9%); placental concentrations of NOx (+ 22.5%), tetrahydrobiopterin (+ 21.1%), polyamines (+ 20.4%), cAMP (+ 27.7%), and cGMP (+ 24.7%); total amounts of NOx (+ 61.7% to + 96.8%), polyamines (+ 60.7% to + 88.7%), amino acids (+ 39% to + 118%), glucose (+ 60.5% to + 62.6%), and fructose (+ 41.4% to + 57.0%) in fetal fluids; and the placental transport of water (+ 33.9%), Arg (+ 78.4%), glutamine (+ 89.9%), and glycine (+ 89.6%). Furthermore, Arg supplementation increased (P < 0.05) placental mRNA levels for angiogenic factors [VEGFA120 (+ 117%), VEGFR1 (+ 445%), VEGFR2 (+ 373%), PGF (+ 197%), and GCH1 (+ 126%)] and AQPs [AQP1 (+ 280%), AQP3 (+ 137%), AQP5 (+ 172%), AQP8 (+ 165%), and AQP9 (+ 127%)].ConclusionSupplementing 0.4% Arg to a conventional diet for gilts between d 14 and d 30 of gestation enhanced placental NO and polyamine syntheses, angiogenesis, and water and amino acid transport to improve conceptus development and survival.

Effect of dietary protein and energy intake on embryonic survival and gene expression in the uterine endometrium of early pregnant gilts

Porcine embryonic loss during early gestation is a serious problem in swine production. Improving embryonic survival can be achieved by maternal manipulation. Protein and energy are two major components of the diet, which play decisive roles in embryonic survival. This study was performed to evaluate the effects of enhancing maternal protein or energy intake on embryonic survival during early gestation in gilts and to explore the underlying mechanism. From day (d) 0 to 30 of gestation, 40 gilts (Landrace × York) were randomly allocated to 5 diets according to daily intake of low (L, National Research Council (NRC) recommendation for gestation gilts), medium (M, 20% higher than NRC) or high (H, 40% higher than NRC) CP or metabolisable energy (ME) (LCPLME, MCPLME, HCPLME, LCPHME, HCPHME). Gilts were sacrificed on d 30 of gestation, and number of foetuses and corpora lutea, embryonic survival rate, uterine weight, and total volume of allantoic fluid were recorded or calculated. Gene expression was determined by Quantitative Real-time PCR (qPCR), western blot or immunohistochemistry. Results showed that increasing protein or ME intake significantly increased embryonic survival rate. Compared with diet LCPLME, plasma progesterone (P4) concentration in diet LCPHME increased at d 14 and d 30 of gestation. Progesterone receptor (PGR) was found not to be expressed in the epithelia but was strongly expressed in the stroma of the endometrium. Increasing protein or ME intake did not alter PGR expression in the endometrium. There was also no change in the amount of P4, hepatocyte growth factor, and fibroblast growth factor-7 in the endometrium. The mRNA abundance of cationic amino acid transporter 1 in the endometrium in diet LCPHME and HCPHME was significantly lower than in diet LCPLME. Diet HCPLME showed a tendency to increase neutral amino acid transporter 1 mRNA expression in the endometrium compared to diet LCPLME (P = 0.087). In conclusion, increasing maternal protein or ME intake had a positive effect on the embryonic survival. Increased protein intake by 20 or 40% did not alter plasma P4 level, but increasing ME intake by 40% improved plasma P4 concentration at d 14 and 30 of gestation. Increasing maternal protein or ME intake did not induce PGR expression in the endometrium. Maternal protein and energy intake likely mediate transportation of cationic and neutral amino acids from mother to foetus to affect embryonic survival and development.

120 Reproductive Performance, Early Embryo Survival, Fetal Characteristics, and Tissue Fatty Acid Profiles of Sows Fed Linoleic or α-Linolenic Acid

Abstract The objective of this study was to determine the potential reproductive benefits of supplementing sows with linoleic (LA) or α-linolenic acid (α-LA) throughout the preceding lactation and subsequent gestation until 30 d post-rebreeding. Thirty-three second parity sows were allotted to either a control (0.88% LA, 0.10% α-LA), high LA (3.3% LA, 0.10% α-LA,) or high α-LA (0.88% LA, 0.60% α-LA) diet fed throughout lactation and early gestation. At 30 d post-breeding, a subset of sows (n = 18) was harvested to determine embryo survival and fatty acid composition of reproductive and fetal tissues. Number of pigs weaned per litter (P = 0.77) and litter average daily gain (P = 0.34) did not differ among treatments; however, litters from sows supplemented with LA tended to be heavier at weaning (P = 0.09; 55.3, 61.3, and 57.1 kg for control, LA, and α-LA, respectively). Sows supplemented with LA had higher embryo survival (P &amp;lt; 0.01; 57.2, 79.5, and 58.7% for control, LA, and α-LA, respectively), heavier feto-placental units (P &amp;lt; 0.01; 210.3, 279.4 and 235.0 g for control, LA, and α-LA, respectively), and elevated volume of allantoic fluid (P &amp;lt; 0.01; 185.9, 247.9, and 200.8 mL for control, LA, and α-LA, respectively). Concentrations of EFA increased in maternal serum by the dietary EFA supplemented (P &amp;lt; 0.05). Supplementation of LA did not impact linoleic acid or arachidonic acid concentrations in endometrium, chorioallantoic membrane, or fetus; however, α-LA increased (P &amp;lt; 0.05) concentrations of α-linoleic acid in chorioallantoic membrane and eicosapentaenoic acid in serum and endometrium. Supplementation of LA, but not α-LA, throughout lactation and early subsequent gestation was beneficial for embryo survival. The lesser influence of dietary LA and α-LA in fetal tissues may be associated with the intake, transfer, and metabolism of EFA in the chorioallantoic membrane and endometrium.

Nutritional and Physiological Regulation of Water Transport in the Conceptus.

Water transport during pregnancy is essential for maintaining normal growth and development of conceptuses (embryo/fetus and associated membranes). Aquaporins (AQPs) are a family of small integral plasma membrane proteins that primarily transport water across the plasma membrane. At least 11 isoforms of AQPs (AQPs 1-9, 11, and 12) are differentially expressed in the mammalian placenta (amnion, allantois, and chorion), and organs (kidney, lung, brain, heart, and skin) of embryos/fetuses during prenatal development. Available evidence suggests that the presence of AQPs in the conceptus mediates water movement across the placenta to support the placentation, thehomeostasis of amniotic and allantoic fluid volumes, as well as embryonicand fetal survival,growth and development. Abundances of AQPs in the conceptus can be modulated by nutritional status and physiological factors affecting the pregnant female. Here, we summarize the effects of maternal dietary factors (such as intakes of protein, arginine, lipids, all-trans retinoic acid, copper, zinc, and mercury) on the expression of AQPs in the conceptus. We also discuss the physiological changes in hormones(e.g., progesterone and estrogen), oxygensupply, nitric oxide,pH, and osmotic pressure associated with the regulation of fluid exchange between mother and fetus. These findings may help to improve the survival, growth, and development of embryo/fetus in livestock species and other mammals (including humans).

227 Awardee Talk: Advances in Lipid Nutrition of Modern Sows

Abstract The modern lactating sow is a highly productive female capable of producing large quantities of milk to support large, rapidly growing litters. The associated high energy and nutritional demands are challenging to meet, especially during heat stress, when tissue loss is extensive. Supplemental lipids are efficient energy sources with a high energy density and low heat increment associated with digestion and metabolism. Besides serving as concentrated energy sources, unsaturated lipids are important sources of essential fatty acids (EFA; linoleic acid, C18:2n-6; and α-linolenic acid, C18:3n-3). These EFA are precursors of various prostanoids that are important regulators of reproductive processes. Lactating sows fed diets devoid of supplemental lipids exhibited poor reproduction but was significantly improved by inclusion of only 2% unsaturated lipids. It was shown that lactation diets were deficient in both linoleic and α-linolenic acid and tissue reserves were inadequate in mature sows. A multi-trial meta-analysis showed that more sows were successfully bred and maintained pregnancy when they consumed supplemental linoleic acid. Further, increased linoleic acid intake during lactation progressively reduced the number of sows removed from the herd due to reproductive failure (sows not returning to estrus, returning to estrus after breeding, pregnancy loss). Based on dose-response curves, the near maximum response was achieved when sows consumed a minimum of 125 g/d of linoleic acid and 10 g/d of α-linolenic acid during lactation. Recent studies also demonstrated improved subsequent reproduction with supplemental linoleic and α-linolenic acid during lactation, but not during the post-weaning period. Responses appeared to be related to heavier fetoplacental units, elevated allantoic fluid volume, and higher embryo survival measured on day 30 of gestation in sows supplemented with linoleic acid during lactation and early gestation. Research demonstrated the importance of both EFA in lactating sows and their critical role in improving reproduction, thus resolving seasonal anestrus.

Dietary supplementation with l-arginine between days 14 and 25 of gestation enhances embryonic development and survival in gilts

Embryonic loss is a major problem in mammals, but there are few effective ways to prevent it. Using a porcine model, we determined effects of dietary L-arginine supplementation between days 14 and 25 of gestation on embryonic growth and survival. Gilts were checked daily for estrus with boars in the morning and bred at onset of the second estrus and 12 h later (the time of breeding = day 0 of gestation). Between days 14 and 25 of gestation, 15 gilts/treatment were housed individually and fed twice daily 1 kg of a corn- and soybean meal-based diet supplemented with 0.0, 0.4, or 0.8 % L-arginine. All diets were made isonitrogenous by addition of L-alanine. On day 25 of gestation, gilts were hysterectomized to obtain conceptuses. Compared with controls, dietary supplementation with 0.4 or 0.8 % L-arginine increased (P ≤ 0.05) arginine concentrations in maternal plasma, total volume of amniotic fluid; total amounts of arginine in allantoic and amniotic fluids; total amounts of fructose and most amino acids in amniotic fluid; placental growth; and the number of viable fetuses per litter by 2. The numbers of total fetuses, fetal weight, corpora lutea, volume of allantoic fluid, maternal circulating levels of progesterone and estrogen, or total amounts of hormones in allantoic fluid did not differ among the three treatment groups. Reproductive performance of gilts did not differ between the 0.4 and 0.8 % L-arginine groups. Thus, dietary supplementation with 0.4 or 0.8 % L-arginine between days 14 and 25 of gestation enhances embryonic/fetal survival in swine.

Dietary Supplementation with L‐Arginine between Days 14 and 25 of Gestation Enhances Litter Size in Gilts

Gilts were checked daily for estrus with boars in the morning and bred at onset of the second estrus and 12 h later (the time of breeding = d 0 of gestation). Between d 14 and 25 of gestation, 15 gilts per treatment were housed individually and fed twice daily 1 kg of a corn‐and soybean meal‐based diet supplemented with 0.0%, 0.4%, or 0.8% L‐arginine. All diets were made isonitrogenous by addition of L‐alanine. At d 25 of gestation, gilts were hysterectomized to obtain conceptuses. Compared with control gilts, dietary supplementation with 0.4% or 0.8% L‐arginine increased (P ≤ 0.05) concentrations of arginine in maternal plasma, total volume of amniotic fluid, total amounts of arginine in allantoic and amniotic fluids, total amounts of fructose and most amino acids in amniotic fluid, placental growth, and the number of viable fetuses per litter by 2. The numbers of total fetuses or corpora lutea, total fetal weight, total volume of allantoic fluid, maternal circulating levels of progesterone and estrogen, or total amounts of the hormones in allantoic fluid did not differ among the three treatment groups. Reproductive performance of gilts did not differ between the 0.4% and 0.8% L‐arginine groups. These results indicate that dietary supplementation with 0.4% or 0.8% L‐arginine between d 14 and 25 of gestation can enhance embryonic/fetal survival in gilts. Supported by USDA grants.

Arginine nutrition and fetal brown adipose tissue development in diet-induced obese sheep

The global incidence of human obesity has more than doubled over the past three decades. An ovine model of obesity was developed to determine effects of maternal obesity and arginine supplementation on maternal, placental, and fetal parameters of growth, health, and well being. One-hundred-twenty days prior to embryo transfer, ewes were fed either ad libitum (n = 10) to induce obesity or 100% National Research Council-recommended nutrient requirements (n = 10) as controls. Embryos from superovulated ewes with normal body condition were transferred to the uterus of control-fed and obese ewes on day 5.5 post-estrus to generate genetically similar singleton pregnancies. Beginning on day 100 of gestation, obese ewes received intravenous administration of saline or L-arginine-HCl three times daily (81 mg arginine/kg body weight/day) to day 125, whereas control-fed ewes received saline. Fetal growth was assessed at necropsy on day 125. Maternal obesity increased (1) percentages of maternal and fetal carcass lipids and (2) concentrations of leptin, insulin, glucose, glutamate, leucine, lysine and threonine in maternal plasma while reducing (1) concentrations of progesterone, glycine and serine in maternal plasma and (2) amniotic and allantoic fluid volumes. Administration of L-arginine to obese ewes increased arginine and ornithine concentrations in maternal and fetal plasma, amniotic fluid volume, protein content in maternal carcass, and fetal brown adipose tissue (+60%), while reducing maternal lipid content and circulating leptin levels. Fetal or placental weight did not differ among treatments. Results indicate that arginine treatment beneficially reduces maternal adiposity and enhances fetal brown adipose tissue development in obese ewes.

Growth and development of the ovine conceptus1

This study was designed to characterize development of the ovine conceptus throughout gestation to establish the temporal relationships in metabolites, electrolytes, fluid volumes within the placenta, and hormonal changes with fetal growth. Length and weight of placentae, weight of cotyledons, and uterine weight increased between d 25 and 80 of gestation in advance of increases in fetal growth between d 80 and 140 of gestation. Allantoic fluid volumes changed (P < 0.01) between d 25 (21 mL) and 40 (91 mL), decreased to d 70 (32 mL), and then increased to d 140 (438 mL). Concentrations and total amounts of proteins in allantoic fluid were reduced between d 25 and 50, but total protein increased (P < 0.01) from d 40 (63 mg) to d 140 (2,991 mg). Concentrations of fructose in allantoic fluid varied between 2 and 6 mg/mL throughout gestation, but total fructose increased (P < 0.01) between d 25 (46 mg) and d 120 (679 mg). Concentrations of glucose ranged from 0.1 to 0.3 mg/mL, and total glucose increased (P < 0.05) from d 25 (3 mg) to d 140 (63 mg) of gestation. Amniotic fluid volume increased (P < 0.01) between d 30 and 140. Concentrations of estrogens in allantoic fluid, maternal uterine artery, and uterine vein increased (P < 0.01) with advancing pregnancy, and concentrations of progesterone in allantoic fluid (P < 0.07) and plasma (P < 0.05) were affected by day of gestation. Concentrations of glucose were greater (P < 0.05) in uterine artery than uterine vein, but concentrations of electrolytes and osmolarity of plasma were not affected by day of gestation. Increases in weights of fetal organs were proportional to increases in fetal weight during gestation. Results of the present study of conceptus growth and development highlight areas of needed research and provide benchmarks for comparisons when evaluating effects of various treatments, environmental conditions, and epigenetics on successful outcomes of pregnancy in sheep.

Intrauterine Infusion of Latency-Associated Peptide (LAP) During Early Porcine Pregnancy Affects Conceptus Elongation and Placental Size1

In the pig, transforming growth factor beta (TGFB), TGFB receptors (TGFBRs), and integrins are present during the peri-implantation period. Latency-associated peptide (LAP), a part of latent TGFB, can bind to integrin heterodimers via its Arg-Gly-Asp (RGD) sequence; therefore, ligand-receptor interactions between TGFB and TGFBRs, along with LAP and integrin heterodimers, may be functional in mediating events supporting conceptus elongation and attachment. With the use of surgically implantable osmotic pumps, we were able to maintain pregnancy with the aim of mechanistically altering in vivo receptor-ligand interactions involving TGFB with TGFBRs and LAP with integrins during porcine pregnancy. Day 9 pregnant gilts received intrauterine infusions of LAP-RGD, a recombinant mutant of LAP (LAP-RGE), or vehicle control and were ovariohysterectomized on Day 13 or 24 of pregnancy. We hypothesized that intrauterine infusion of LAP-RGD would decrease downstream signaling of TGFB while increasing LAP-integrin interactions and that net effect would enhance conceptus survival and attachment early in the peri-implantation period but possibly increase the chance of abnormal placentation later in pregnancy. Additionally, we hypothesized that infusion of LAP-RGE would disrupt TGFB signals but not alter integrin signaling, and thus the net result would be decreased conceptus survival and abnormal development. Unexpectedly, LAP-RGD intrauterine infusions resulted in a reduction of conceptus elongation, whereas infusions of LAP-RGE permitted implantation and placentation but resulted in larger fetal weight, allantois length, and allantoic fluid volume. Results suggest TGFB and integrins are contributing factors in the regulation of conceptus elongation and placental and fetal size.

Conceptus-related measurements during the first trimester of bovine pregnancy

In order to determine the variability inherent in conceptus-related measurements in first trimester bovine pregnancies, conceptus and fetometric parameters from beef cattle pregnancies ( n = 103) estimated to be between Days 36 and 103 of gestation were examined. During this period, the protein concentration of amniotic fluid ranged between 0.181 and 0.501 mg/mL. The amniotic fluid volume gradually increased from <1 mL at Day 36 to 950 mL at Day 103 ( R 2 = 0.9275) and amniotic compartment dimensions (length, R 2 = 0.9713; width, R 2 = 0.9802) increased predictably with fetal growth. Conversely, allantoic fluid protein concentration and volume correlated weakly with fetal age. A significant linear correlation existed between fetal crown rump length (CRL) and crown nose length ( R 2 = 0.9899) confirming that either measurement can be employed in the ultrasonographic estimation of fetal age. The amniotic compartment and fetometric data presented here have both research and clinical value, particularly in relation to fetal development evaluation and pregnancy viability diagnosis.

Lack of effect of metyrapone and exogenous cortisol on early porcine conceptus development

A study was conducted to evaluate the influence of maternal cortisol on early conceptus development in pigs (Sus scrofa). The corticosteroid synthesis inhibitor metyrapone was injected daily during days 14-19 of pregnancy, without (n = 6) and with commensurate administration of cortisol (n = 6). Blood samples were taken via an indwelling jugular catheter on days 14 and 18, and conceptuses were harvested during surgery on day 20. Compared with vehicle-injected control dams (n = 7) plasma cortisol and aldosterone concentrations were decreased (P < 0.01) by 52 and 29%, respectively, by metyrapone treatment. Cortisol administration reversed decreases in plasma cortisol by day 18. There were no treatment-associated effects on conceptus survival or size. Nor were there treatment-associated effects on allantoic fluid volume or content. Trophodermal glucocorticoid receptor (GR) mRNA expression decreased by 34% (P < 0.05) in metyrapone-treated pigs, and was not further influenced by concomitant administration of cortisol, thereby suggesting an influence of aldosterone on GR mRNA expression. Also, when all pigs were considered, there were treatment-independent second-order polynomial regressions (P < 0.05) between maternal plasma cortisol concentrations and embryonic weight, allantoic size and allantoic glucose concentrations, and between plasma aldosterone concentrations and trophodermal GR mRNA expression. Such biphasic corticosteroid concentration versus tissue parameter curves are noteworthy, but difficult to interpret validly. They may suggest that an appropriate corticosteroid environment is necessary for optimal porcine embryonic development during this stage of gestation, but cannot overshadow the absence of treatment effects on the porcine embryonic measures evaluated.

Reduction of amniotic and allantoic fluid volume in an ovine model of fetal growth restriction

Reduction of amniotic and allantoic fluid volume in an ovine model of fetal growth restriction

Perturbations in the Biochemical Composition of Fetal Fluids Are Apparent in Surviving Bovine Somatic Cell Nuclear Transfer Pregnancies in the First Half of Gestation1

Amniotic and allantoic fluid volumes and composition change dynamically throughout gestation. Cattle that are pregnant with somatic cell nuclear transfer (NT) fetuses show a high incidence of abnormal fluid accumulation (particularly hydrallantois) and fetal mortality from approximately midgestation. To investigate fetal fluid homeostasis in these pregnancies, Na, K, Cl, urea, creatinine, Ca, Mg, total PO(4), glucose, fructose, lactate, total protein, and osmolalities were measured in amniotic and allantoic fluids collected at Days 50, 100, and 150 of gestation from NT pregnancies and those generated by the transfer of in vitro-produced embryos or by artificial insemination. Deviations in fetal fluid composition between NT and control pregnancies were apparent after placental and fetal organ development, even when no gross morphological abnormalities were observed. Individual NT fetuses were affected to varying degrees. Elevated allantoic Na was associated with lower K and increased allantoic fluid volume or edema of the fetal membranes. Total PO(4) levels in NT allantoic and amniotic fluid were elevated at Days 100 and 150. This was not accompanied by hypophosphatemia at Day 150, suggesting that PO(4) acquisition by NT fetuses was adequate but that its readsorption by the kidneys may be impaired. Excessive NT placental weight was associated with low allantoic glucose and fructose as well as high lactate levels. However, the fructogenic ability of the NT placenta appeared to be normal. The osmolality of the fetal fluids was maintained within a narrow range, suggesting that the regulation of fluid composition, but not osmolality, was impaired in NT pregnancies.

52GREATER PHENOTYPIC VARIABILITY IN CLONED CATTLE FETUSES FROM ONE CELL LINE THAN CONTEMPORARY HALF-SIBS GENERATED BY ARTIFICIAL INSEMINATION OR IN VITRO EMBRYO PRODUCTION

The cloning of cattle by somatic cell nuclear transfer (NT) is associated with considerable variation in developmental abnormalities. Some of this variation may be due to the cell types/lines used and the specific production and culture methods for NT embryos. Fetal and placental development were studied in 24 pregnancies generated by NT (Wells et al., 2003 Theriogenology 59, 45–59) from a granulosa cell line. The controls consisted of 11 and 14 pregnancies resulting from in vitro-produced embryos (IVP) and artificial insemination (AI), respectively. All fetuses shared the same Friesian sire; oocytes for the derivation of NT or IVP embryos were obtained from abattoir-derived ovaries of Friesian cows. Morphometric measurements were made on the fetuses, fetal membranes, fluid volumes and placentomes at Days 50, 100 and 150 of gestation after slaughter. Pairwise comparisons of within group variances between the treatment groups were made using the F-test. The pregnancy loss from AI or embryo transfer to Day 150 was ∼40% for the AI and IVP groups. However, 60% of NT embryos transferred had been lost by Day 150. NT fetal weights at Day 100 were more variable than for the AI (P&amp;lt;0.001) and IVP (P&amp;lt;0.05) groups. The NT crown-rump lengths were also more variable compared with the IVP (P&amp;lt;0.05) but not the AI group. At Day 100, NT heart (P&amp;lt;0.01), kidney (P&amp;lt;0.01) and liver (P&amp;lt;0.05) weights were more variable compared with the AI group; both liver (P&amp;lt;0.05) and kidney (P&amp;lt;0.01) weights were similarly more variable in the Day 150 NT group. Part of this variability may be due to disproportionate organ growth in NT fetuses. Increased variability was most evident in the utero-placental tissues. At Day 50, the total fetal membrane weights were more variable in the NT compared with the AI (P&amp;lt;0.001) but not the IVP group. The Day 50 IVP membrane weights were also more variable (P&amp;lt;0.05) than for the AI group. The greater variability of the Day 150 NT membrane weights, when compared with AI (P&amp;lt;0.05) or IVP (P&amp;lt;0.01), could be due partly to greater variation in the allantoic and amniotic fluid volumes. Placentome numbers were significantly lower in the surviving Day 100 NT pregnancies but the weights of the total maternal caruncular tissue of the placentomes were significantly higher and more variable in the NT group (P&amp;lt;0.05 for both AI and IVP). Placentome numbers in surviving NT pregnancies at Day 150 were similar to the controls. However, the total caruncle weights in both the NT and IVP groups were more variable when compared with the AI group (P&amp;lt;0.05 for both). Thus, there is greater variability in fetal membrane and placental development in NT and, to a certain extent, IVP fetuses, when compared with the AI group. The increased variability within this NT group suggests that epigenetic effects arising from incomplete reprogramming of the donor genome and embryo culture can override genetic traits to a certain extent. Supported by FRST C10X0018.

164BOVINE PLACENTAL LACTOGEN (BPL) AND BOVINE PREGNANCY-SPECIFIC PROTEIN B (BPSPB) AS INDIRECT MEASURES OF PLACENTAL FUNCTION IN IN VITRO-DERIVED BOVINE PREGNANCIES

Associations between abnormal placental and fetal development after in vitro embryo manipulations have been suggested to play a key role in the occurrence of high birth weights. This study was designed to investigate bovine conceptus development in in vivo- (controls) and in vitro-produced (IVP) concepti and newborn calves, and its association with specific placental proteins usually present in maternal, fetal and neonatal plasma and fetal (amniotic and allantoic) fluids. Females were superovulated to obtain control embryos, whereas IVP embryos were derived from established in vitro embryo production procedures (Bertolini et al., 2002 Theriogenology 58,973). Pregnant animals from each group were slaughtered on Days 90 and 180 of gestation (n=4 control, n=5 IVP pregnancies/day), or allowed to develop to term (n=6/group). Conceptus and neonatal physical traits were recorded after slaughter or delivery (Bertolini et al., 2001 Theriogenology 55,347; 2002 57,361; 2002 58,973). Maternal and fetal plasma and fluids were sampled after slaughter; maternal blood was sampled weekly from Day 30 of pregnancy to term. Neonatal blood samples were collected at 10min, and at 1, 6, 12 and 24h after birth. Bovine placental lactogen (bPL) and pregnancy-specific protein B (bPSPB) concentrations were determined in plasma and fluid samples, according to Wallace (1993 Dom. Anim. Endocrinol. 10, 67) for bPL, and by a commercial service for bPSPB (BioTracking, Moscow, ID, USA.). Data were compared by Proc GLM of SAS and Pearson’s correlation test (SAS Institute, Cary, NC, USA.). No major physical differences in conceptus traits were observed between groups on Day 90, but concentrations of bPL and bPSPB were higher in fetal plasma (8.1±0.5 v. 10.7±0.5ngmL−1 for bPL, and 4.4±8.2 v. 32.5±5.8ngmL−1 for bPSPB) and allantoic fluid (bPL only; 3.6±1.1 v. 7.8±1.0ngmL−1) of IVP concepti (P&amp;lt;0.05). Day-180 IVP pregnancies displayed larger uterine and conceptus traits (P&amp;lt;0.05), and bPSPB concentrations were 2.9-fold lower (84.3±22.4 v. 20.5±22.4ngmL−1) in the allantoic fluid (P&amp;lt;0.05) of IVP pregnancies, for a 2- to 3-fold larger allantoic fluid volume than controls (P&amp;lt;0.07). Concentrations of bPL in fetal plasma and fluids were higher than in maternal plasma, but no differences in bPSPB concentrations were observed across fluid types. Newborn IVP calves and fetal membranes were larger, displaying 3- to 4-fold higher concentrations of plasma bPL (P&amp;lt;0.05) and bPSPB (P&amp;lt;0.08) than controls (10 and 60min after birth) and maternal plasma (at delivery). Maternal concentrations of bPL in IVP pregnancies were lower than controls during the last 8 weeks of gestation (P&amp;lt;0.05), to become similar as parturition approached. Generally, concentrations of bPL and bPSPB in plasma were correlated with physical traits (0.750&amp;gt;r&amp;gt;0.958, P&amp;lt;0.001) and with one another in plasma and fluids (0.715&amp;gt;r&amp;gt;0.938, P&amp;lt;0.001). Our results indicated that differential patterns of secretion of bPL and bPSPB into the maternal and fetal systems occurred at distinct stages of gestation, which were associated with altered conceptus development after in vitro embryo manipulations, indirectly demonstrating deviations in placental function in IVP pregnancies.

The impact of induced stress during Days 13 and 14 of pregnancy on the composition of allantoic fluid and conceptus development in sows

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 Synacthen ® 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-17β 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.

Regulatory response of intramembranous absorption of amniotic fluid to infusion of exogenous fluid in sheep.

Six fetal sheep were operated on at 118 to 121 days of gestation. The pulmonary end of the trachea was connected to the gastric end of the esophagus with a section of tubing. This left urine as the only source of amniotic fluid and intramembranous absorption as sole exit. Multiple indwelling fetal vascular, intra-amniotic, allantoic, and a fetal bladder catheter were placed. Beginning 5 days after surgery, all urine was drained from the bladder and immediately reinfused into the amniotic sac to monitor urine production rate. After 4 days of urine infusion alone, the urine infusion was augmented for 6 days with an intra-amniotic infusion of Ringer solution. Amniotic and allantoic fluid volumes were measured at autopsy. During the period of Ringer infusion, intramembranous absorption of amniotic fluid increased by more than 1,191 +/- 186 (SE) ml/day (P < 0.002) and the rates of Na(+) and Cl(-) absorption increased to more than five times (P < 0.005) and eight times (P < 0.005) their initial values. Only one of six fetuses had polyhydramnios. It is concluded that intramembranous absorption of amniotic fluid makes a strong regulatory adjustment in response to an abnormal increase in inflow of exogenous fluid.