Sylvaine Camous

Review: Placental perturbations induce the developmental abnormalities often observed in bovine somatic cell nuclear transfer

Since the first success in cloning sheep, the production of viable animals by somatic cell nuclear transfer (SCNT) has developed significantly. Cattle are by far the most successfully cloned species but, despite this, the technique is still associated with a high incidence of pregnancy failure and accompanying placental and fetal pathologies. Pre- and early post-implantation losses can affect up to 70% of the pregnancies. In the surviving pregnancies, placentomegaly and fetal overgrowth are commonly observed, but the incidence varies widely, depending on the genotype of the nuclear donor cell and differences in SCNT procedures. In all cases, the placenta is central to the onset of the pathologies. Although cellular organisation of the SCNT placenta appears normal, placental vascularisation is modified and fetal-to-maternal tissue ratios are slightly increased in the SCNT placentomes. In terms of functionality, steroidogenesis is perturbed and abnormal estrogen production and metabolism probably play an important part in the increased gestation length and lack of preparation for parturition observed in SCNT recipients. Maternal plasma concentrations of pregnancy-associated glycoproteins are increased, mostly due to a reduction in turnover rate rather than increased placental production. Placental glucose transport and fructose synthesis appear to be modified and hyperfructosemia has been observed in neonatal SCNT calves. Gene expression analyses of the bovine SCNT placenta show that multiple pathways and functions are affected. Abnormal epigenetic re-programming appears to be a key component of the observed pathologies, as shown by studies on the expression of imprinted genes in SCNT placenta.

DIAGNOSIS OF PREGNANCY BY RADIOIMMUNOASSAY OF A PREGNANCY-SPECIFIC PROTEIN IN THE PLASMA OF DAIRY COWS

The accuracy and efficiency of progesterone (P4) and bovine pregnancy-specific protein B (bPSPB) radioimmunoassays (RIA) in detecting pregnant and nonpregnant dairy cows were compared at different stages of pregnancy. The study included 145 French Friesian heifers and cows from a single herd. A total of 175 artificial insemination (A.I.) and blood sampling procedures were performed. Animals were bled 24 d post AI for P4 RIA. They were bled at 24, 26, 30 to 35, and 70 +/- 9 after AI for bPSPB RIA. Females were declared nonpregnant when plasma P4 concentrations were lower than 1.5 ng/ml. With the bPSPB RIA, cows were nonpregnant when at least one of the B Bo x 100 replicates was higher than 95% in the RIA. When compared with palpations per rectum at 70 d, the accuracy of positive diagnoses (no. positive and pregnant/no. positive diagnoses) by P4 RIA at Day 24 was 67.2% (82 122 ). The accuracy of negative diagnoses was 98% (52 53 ). Accuracy of positive diagnoses by bPSPB RIA increased with gestation age (P<0.05) from 86.2% (50 58 ) on Day 24 to 98.8% (83 84 ) at time of palpation per rectum. Accuracy of negative diagnoses increased (P< 0.001) from Day 24 (71.8%; 84 117 ) to Days 30 to 35 (100%, 83 83 ). Efficiency in detecting nonpregnant females was much higher (P < 0.001) with the bPSPB RIA on Days 30 to 35 (90.2%; 83 92 ) than with the P4 RIA on Day 24 (56.5%, 52 92 ). It is concluded that 30 days after AI, the bPSPB RIA is an efficient test both for pregnancy prediction and detection of nonpregnant dairy cows.

Effects of moderate amounts of barley in late pregnancy on growth, glucose metabolism and osteoarticular status of pre-weaning horses

In stud management, broodmares are commonly fed concentrates in late pregnancy. This practice, however, was shown to correlate with an increased incidence of osteochondrosis in foals, which may be related to insulin sensitivity. We hypothesized that supplementation of the mare with barley in the last trimester of pregnancy alters the pre-weaning foal growth, glucose metabolism and osteoarticular status. Here, pregnant multiparous saddlebred mares were fed forage only (group F, n=13) or both forage and cracked barley (group B, n=12) from the 7th month of pregnancy until term, as calculated to cover nutritional needs of broodmares. Diets were given in two daily meals. All mares and foals returned to pasture after parturition. Post-natal growth, glucose metabolism and osteoarticular status were investigated in pre-weaning foals. B mares maintained an optimal body condition score (>3.5), whereas that of F mares decreased and remained low (<2.5) up to 3 months of lactation, with a significantly lower bodyweight (-7%) than B mares throughout the last 2 months of pregnancy. B mares had increased plasma glucose and insulin after the first meal and after the second meal to a lesser extent, which was not observed in F mares. B mares also had increased insulin secretion during an intravenous glucose tolerance test (IVGTT). Plasma NEFA and leptin were only temporarily affected by diet in mares during pregnancy or in early lactation. Neonatal B foals had increased serum osteocalcin and slightly increased glucose increments and clearance after glucose injection, but these effects had vanished at weaning. Body measurements, plasma IGF-1, T4, T3, NEFA and leptin concentrations, insulin secretion during IVGTT, as well as glucose metabolism rate during euglycemic hyperinsulinemic clamps after weaning, did not differ between groups. Radiographic examination of joints indicated increased osteochondrosis relative risk in B foals, but this was not significant. These data demonstrate that B or F maternal nutrition has very few effects on foal growth, endocrinology and glucose homeostasis until weaning, but may induce cartilage lesions.

Enhanced or Reduced Fetal Growth Induced by Embryo Transfer into Smaller or Larger Breeds Alters Post-Natal Growth and Metabolism in Pre-Weaning Horses

In equids, placentation is diffuse and nutrient supply to the fetus is determined by uterine size. This correlates with maternal size and affects intra-uterine development and subsequent post-natal growth, as well as insulin sensitivity in the newborn. Long-term effects remain to be described. In this study, fetal growth was enhanced or restricted through ET using pony (P), saddlebred (S) and draft (D) horses. Control P-P (n = 21) and S-S (n = 28) pregnancies were obtained by AI. Enhanced and restricted pregnancies were obtained by transferring P or S embryos into D mares (P-D, n = 6 and S-D, n = 8) or S embryos into P mares (S-P, n = 6), respectively. Control and experimental foals were raised by their dams and recipient mothers, respectively. Weight gain, growth hormones and glucose homeostasis were investigated in the foals from birth to weaning. Fetal growth was enhanced in P-D and these foals remained consistently heavier, with reduced T3 concentrations until weaning compared to P-P. P-D had lower fasting glucose from days 30 to 200 and higher insulin secretion than P-P after IVGTT on day 3. Euglycemic clamps in the immediate post-weaning period revealed no difference in insulin sensitivity between P-D and P-P. Fetal growth was restricted in S-P and these foals remained consistently lighter until weaning compared to S-D, with elevated T3 concentrations in the newborn compared to S-S. S-P exhibited higher fasting glycemia than S-S and S-D from days 30 to 200. They had higher maximum increment in plasma glucose than S-D after IVGTT on day 3 and clamps on day 200 demonstrated higher insulin sensitivity compared to S-D. Neither the restricted nor the enhanced fetal environment affected IGF-1 concentrations. Thus, enhanced and restricted fetal and post-natal environments had combined effects that persisted until weaning. They induced different adaptive responses in post-natal glucose metabolism: an early insulin-resistance was induced in enhanced P-D, while S-P developed increased insulin sensitivity.

Altered secretion of pregnancy-associated glycoproteins during gestation in bovine somatic clones

Somatic nuclear transfer (NT) in cattle is often accompanied by severe placental anomalies, hypertrophy, and hydrallantois, which induce a high rate of pregnancy losses throughout gestation. These placental deficits are associated with an abnormal increase of the maternal plasma levels of pregnancy-associated glycoprotein (PAG), produced by the trophoblastic binucleate cells (BNC) of the placenta. The objective of this study was to analyze the origin of the abnormally elevated PAG concentrations in the peripheral circulation of NT recipients during pathological pregnancies. Concentrations of PAG were measured both in maternal blood, in chorionic and cotyledonary tissular extracts from control recipients (after artificial insemination, AI, or in vitro fertilization, IVF) and clone recipients on Day 32, Day 62, and during the third trimester of gestation. Three different radioimmunoassay (RIA) systems were used. One homologous RIA for PSP60, similar to bovine PAG-1 (PAG(67 kDa)), and two heterologous RIA with PAG(67 kDa) as standard and tracer, and antisera anti-caprine PAG (AS#706 and AS#708). Circulating and tissular concentrations of bovine placental lactogen (bPL), a glycoprotein also produced by BNC, were determined by RIA at the same stages. The number of BNC in the placental tissues was determined by cell counting after immunostaining with anti PSP60 antibody on tissue sections from control and NT pregnancies. Maternal plasma PAG concentrations were not different among groups on Day 32, but they were significantly higher in NT than in control pregnancies on Day 62 with all three RIA and during the third trimester with two RIA (RIA-PSP60 and RIA with AS#708). Circulating bPL concentrations were undetectable on Days 32 and 62 and were not different in the third trimester between NT and control pregnancies. Tissular amounts of PAG on total proteins were not different between the two groups at all stages studied. No difference was determined in the percentage of PSP60-positive BNC in placental tissues between controls and NT on Day 62 and during the third trimester of pregnancy. Western blots of tissular extracts from placenta showed no major molecular weight changes of PAG in NT pregnancies compared to controls. No differences in maternal circulation concentrations or tissular content of bPL were observed between control and NT pregnancies. In conclusion, the specific increase of PAG in maternal plasma concentrations during abnormal NT pregnancies do not result from a higher proportion of BNC, or an increased protein expression of PAG and could be due to changes in the composition of terminal glycosylation which result into a clearance decrease of PAG from the circulation.

Nutritional programming and the reproductive function of the offspring

There is ample evidence on the importance of maternal nutrition during pregnancy on fetal and offspring development. In ruminant females, the pool of oocytes is complete and definitive before birth, based on the resting reserve of primordial follicles established during fetal life, which represent the lifespan supply for the female’s fertilisable oocytes, whereas in males, although the production of spermatozoa is a continuous process throughout post-pubertal life. Sertoli cells, which play a central role in the development of a functional testis, proliferate during pre- and post-natal life, coordinating testicular development. Both male and female fertility may, therefore, be affected by the maternal environment, but studies on the effects of developmental nutritional conditions on reproductive function and fertility, both in males and females, are relatively scarce. In humans, intrauterine growth retardation has been associated with abnormal ovarian development, characterised by a decreased volume of primordial follicles in the ovarian cortical tissue in girls, and a higher incidence of cryptorchidism in boys, with subsequent low sperm counts in adulthood. Age at puberty and gonadotropin and inhibin B plasma concentrations are also affected. Animal studies suggest both in males and females that maternal undernutrition during pregnancy may affect pituitary response to GnRH and gonadal development and function, depending on the timing and magnitude of the undernutrition. Excess nutrition, which is often associated with intrauterine growth retardation in domestic species, induces effects on the onset of puberty and both testicular and ovarian function, maybe through the observed reduction in fetal growth. This review addresses the influence of maternal nutrition on offspring reproductive function using examples in humans and animals, with particular focus on ruminants.

Long-term consequences of feed restriction during late pregnancy in goats on feeding behavior and emotional reactivity of female offspring.

Feed restriction during pregnancy can have detrimental effects on offspring development both during the juvenile period and during adult life. Long-term effects of maternal feed restriction during the last third of pregnancy on growth, metabolism and behavior of female kids, with a focus on feeding behavior and emotional reactivity, were studied in goats. Female kids born to control (CONT, n=17) or born to feed restricted goats (REST, n=15) were artificially reared and monitored from birth to 24 months of age. Maternal feeding restriction globally reduced live weight (P<0.001) and body condition score (P=0.02) of REST compared to CONT offspring. Females from the REST group had a higher daily feed intake (P=0.05) and tended to eat more rapidly (P=0.09) than females from the CONT group at 12 months of age. One year later, REST goats still ate more than CONT goats (P=0.05). Glucose metabolism did not appear to be modified as no differences were observed in glucose or insulin responses to an intravenous glucose tolerance test. No differences in time budget were found at 12 months of age. However, the HPA axis response to an ACTH injection was greater in REST than in CONT goats: higher peak cortisol concentration (P=0.02) and a greater area under the curve were found (P=0.01) at 14 months of age. In conclusion, maternal feed restriction during late pregnancy modified both feeding behavior and the stress physiology of female offspring for up to 2 years of age. However, the changes observed were small.

Enhanced or Reduced Fetal Growth Induced by Embryo Transfer Into Smaller or Larger Breeds Alters Postnatal Growth and Metabolism in Weaned Horses

&NA; Embryo transfer between breeds of different sizes impacts fetal growth in horses. We have shown that it elicits various postnatal adaptations in terms of growth and glucose metabolism until weaning. Postweaning effects remain to be described. Pony (P), saddlebred (S), and draft (D) horses were used. Control Pony‐in‐Pony (P‐P; n = 21) and Saddlebred‐in‐Saddlebred (S‐S; n = 28) pregnancies were obtained by artificial insemination. Enhanced and restricted pregnancies were obtained by transferring P or S embryos into D mares (Pony‐in‐Draft [P‐D], n = 6 and Saddlebred‐in‐Draft [S‐D], n = 8) and S embryos into P mares (Saddlebred‐in‐Pony [S‐P], n = 6), respectively. Control and experimental foals were raised by their dams and recipient mothers, respectively and weaned on day 180. Weight gain, growth hormones, and glucose metabolism were investigated in foals between days 180 and 540. Pony‐in‐Draft (P‐D) remained heavier than P‐P on days 180, 360, and 540, with lower glucose and higher non‐esterified fatty‐acids on days 180, 360, and 540 and higher T3 on day 180. Insulin sensitivity was similar between pony groups on days 200 and 540. S‐P were lighter than S‐D on day 180 but caught up by day 540. S‐P had higher glucose than S‐D on days 180, 360, and 540, as well as lower non‐esterified fatty‐acids and higher T3 on day 180. Insulin sensitivity was higher in S‐P than in S‐D on day 200. No difference was observed between saddlebred groups thereafter. In conclusion, in horses, fetal growth is determinant for postnatal metabolism, especially for energy availability. HighlightsET into larger or smaller breeds is used to achieve phenotypic changes in equids.Genetic considerations apart, mares are determinant in foals growth and metabolism.The maternal impact extends beyond weaning time, at least until 1 1/2 years of age.