Michèle Dahirel

Maternal exposure to diluted diesel engine exhaust alters placental function and induces intergenerational effects in rabbits.

BackgroundAirborne pollution is a rising concern in urban areas. Epidemiological studies in humans and animal experiments using rodent models indicate that gestational exposure to airborne pollution, in particular diesel engine exhaust (DE), reduces birth weight, but effects depend on exposure duration, gestational window and nanoparticle (NP) concentration. Our aim was to evaluate the effects of gestational exposure to diluted DE on feto-placental development in a rabbit model.Pregnant females were exposed to diluted (1xa0mg/m3), filtered DE (NP diameteru2009≈u200969xa0nm) or clean air (controls) for 2xa0h/day, 5xa0days/week by nose-only exposure (total exposure: 20xa0days in a 31-day gestation).ResultsDE exposure induced early signs of growth retardation at mid gestation with decreased head length (pu2009=u20090.04) and umbilical pulse (pu2009=u20090.018). Near term, fetal head length (pu2009=u20090.029) and plasma insulin and IGF1 concentrations (pu2009=u20090.05 and pu2009=u20090.019) were reduced. Placental function was also affected, with reduced placental efficiency (fetal/placental weight) (pu2009=u20090.049), decreased placental blood flow (pu2009=u20090.009) and fetal vessel volume (pu2009=u20090.002). Non-aggregated and “fingerprint” NP were observed at various locations, in maternal blood space, in trophoblastic cells and in the fetal blood, demonstrating transplacental transfer. Adult female offspring were bred with control males. Although fetoplacental biometry was not affected near term, second generation fetal metabolism was modified by grand-dam exposure with decreased plasma cholesterol (pu2009=u20090.008) and increased triglyceride concentrations (pu2009=u20090.015).ConclusionsRepeated daily gestational exposure to DE at levels close to urban pollution can affect feto-placental development in the first and second generation.

Maternal high-fat diet induces follicular atresia but does not affect fertility in adult rabbit offspring.

Alterations to the metabolic environment in utero can have an impact on subsequent female reproductive performance. Here, we used a model of rabbits receiving a high-fat diet (H diet; 7.7% fat and 0.2% cholesterol) or a control diet (C diet; 1.8% fat, no cholesterol) from 10 weeks of age up to mating at 27 weeks and throughout gestation and lactation. At weaning at 5 weeks of age, F1 female offspring were placed on either C or H diet, resulting in a total of four groups C/C, C/H, H/C and H/H diet. Female offspring were mated between 18 and 22 weeks of age and euthanized at 28 days of gestation. A few days before mating and/or just before euthanasia, F1 female rabbits were fasted overnight, weighed, and blood sampled for steroids and biochemistry. Organs were weighed at euthanasia and the ovaries were collected. C/H and H/H F1 offspring had higher cholesterol and high-density lipoprotein plasma concentrations, together with a higher fat mass compared with C/C does, reflecting the effect of the postnatal diet; however, no effect of the antenatal diet was observed on most parameters. The number of primordial, primary and secondary follicles were not different between the groups, but a significantly higher number of atretic follicles was observed in the C/H (P<0.001) and in the H/C (P<0.001) compared with control C/C ovaries, demonstrating both an effect of prenatal and postnatal maternal nutrition. These data indicated that both maternal and postnatal high-fat diet may induce follicular apoptosis; however, in this model, the reproduction was not affected.

Impact of maternal hyperlipidic hypercholesterolaemic diet on male reproductive organs and testosterone concentration in rabbits.

The concept of Developmental Origins of Health and Disease initially stemmed from the developmental programming of metabolic diseases. Reproductive functions and fertility in adulthood may also be programmed during foetal development. We studied the impact of dietary-induced maternal hyperlipidaemia and hypercholesterolaemia (HH), administered at 10 weeks of age and throughout the gestation and lactation, on male reproductive functions of rabbit offspring. Male rabbits born to HH dams and fed a control diet had significantly lighter testes and epididymes compared with rabbits born to control dams at adulthood. No significant changes in sperm concentration, sperm DNA integrity and sperm membrane composition were observed, but serum-free testosterone concentrations were decreased in HH males. This study confirms the importance of maternal metabolic status for the development of male reproductive organs.

Maternal parity affects placental development, growth and metabolism of foals until 1 year and a half

Primiparous mares are known to produce smaller foals than multiparous mares. This difference seems to be partly explained by the reduced exchange surface and volume of the placental villi in primiparous compared to multiparous placentas. The effect of maternal parity on foals post-natal growth, metabolism and sexual maturation, however, has been given little consideration. The objectives of this work were to analyse placental biometry and structure at term, growth of foals and yearlings, their metabolism and testicular maturation at one year of age. Twenty multiparous mares (M), aged over 6 years and 12 primiparous mares (P), aged up to 5 years were artificially inseminated with the same stallion and monitored the same way until foaling. At birth, foals and placentas were measured and placentas were sampled above at the umbilical cord insertion, as well as in the pregnant and the non-pregnant horn to perform stereological analyses. Foals were weighed and measured until 540 days of age. At 120 and 360 days of age, an Intravenous Glucose Tolerance Test was performed on foals and yearlings. At 360 days of age, the males were castrated and testicular maturation analysed by RT-qPCR. At birth, P dams produced lighter and smaller foals and placentas. The foal birth weight to placental surface ratio was lower in the P compared to the M group. P Foals remained lighter than M foals until 360 days of age and smaller until at least 540 days of age. At 120 days of age, P foals had a higher glucose tolerance than M foals, and then may be less mature than M foals in terms of the control of their glucose homeostasis. At 360 days of age, the testicles of prepubertal P stallions were less mature in the P vs the M group. In conclusion, primiparous dams produce intrauterine growth restricted, less mature and smaller foals compared to multiparous dams with altered metabolism and growth until at least 540 days of age. These differences could affect the sport career of these foals, especially if it begins at an early age.

Placental structure and function in different breeds in horses

Ponies and sometimes draft horses are often used as experimental models for horses although size and metabolic parameters are known to vary between horse breeds. So far, there is little information about differences of placental structure and no information about differences of placental function between breeds. The aim of this study was to investigate differences in placental size, structure and function at birth in relation to foal size and weight in ponies, Saddlebred and draft horses. Pony, Saddlebred and draft horse pregnancies were obtained by artificial insemination over 2 successive breeding seasons. Foals and total fetal membranes (TFM) were weighed and placentas measured for surface area at term. Placentas were sampled above the umbilical cord insertion. Surface density and volume fraction of the different cellular components of the placenta were measured on histological sections using stereology. The expression of genes involved in growth and development, nutrient transfer and vascularization was compared between groups. Foals and TFM were lighter at birth in ponies than Saddlebred horses, and both were lighter compared to draft horses. The surface density and volume fraction of microcotyledonary vessels was increased in pony compared to Saddlebred placentas. The relative expression of genes involved in growth and development was different between breeds and increased with maternal, fetal and placental weight. Primiparous dams produced lighter foals and smaller placentas, associated with a decreased volume fraction of microcotyledonary vessels and genes involved in growth and development and vascularization. Foal sex had little effect on placental structure and function as the expression of only one gene differed according to sex, with EGFR expression being decreased in placentas of females compared to males. In conclusion, foal and placental weight, as well as placental expression of genes involved in growth and development were correlated with maternal size. Placental structure also differed between breeds, with a stronger difference between ponies and both breeds of horses.

Placental alterations in structure and function in intra-uterine growth-retarded horses

BACKGROUNDnFollowing embryo transfer (ET), the size and breed of the recipient mare can affect fetal development and subsequent post natal growth rate and insulin sensitivity in foals.nnnOBJECTIVESnTo investigate placental adaptation in pregnancies where increased or restricted fetal growth was induced through ET between Pony, Saddlebred and Draught horses.nnnSTUDY DESIGNnIn vivo experiment.nnnMETHODSnControl Pony (P, nxa0=xa021) and Saddlebred (S, nxa0=xa028) pregnancies were obtained by artificial insemination. Increased pregnancies were obtained by transferring Pony (P-D, nxa0=xa06) and Saddlebred (S-D, nxa0=xa08) embryos into Draught mares. Restricted pregnancies were obtained by transferring Saddlebred embryos into Pony mares (S-P, nxa0=xa06). Placental weight and surface were recorded and samples collected for stereology and analysis of expression of genes involved in placental growth, vascularisation and nutrient transport. Data were analysed by linear model.nnnRESULTSnS-P foals were growth retarded when compared with controls despite increased gestational length. Placental weight was reduced but placental surface density and volume fraction were increased. Placental expression of genes involved in growth and development and nutrient transfer was strongly reduced. In contrast, placental size and weight were increased in enhanced growth P-D and S-D foals. The trophoblastic surface density and the allantoic vessels surface density were decreased in P-D and S-D, respectively, both with very few modifications in gene expression.nnnMAIN LIMITATIONSnControl embryos were produced by artificial insemination whereas experimental embryos were produced by ET.nnnCONCLUSIONSnPlacental structure and gene expression are modified after ET into a smaller or larger breed than that of the embryo. These adaptations contribute to the observed phenotype of foal growth restriction or enhanced growth at birth.

Placental function and structure at term is altered in broodmares fed with cereals from mid-gestation

INTRODUCTIONnFeeding pregnant broodmares with cereal concentrates has been shown to increase maternal insulin resistance and affect foal metabolism in the short and long-term. These effects are likely to be mediated by the placenta. Here, we investigated feto-placental biometry and placental structure and function at term in mares fed with or without cereals concentrates.nnnMATERIAL AND METHODSnFrom 7 months of gestation, 22 multiparous mares were fed forage only (group F (nu202f=u202f12)) or received forage and cracked barley (group B (nu202f=u202f10)) until foaling. Foals and placentas were weighed and placental samples were collected above the umbilical cord insertion at birth. Placental histological structure was studied by stereology. A RNAseq analysis was performed on 9 placentas of each group. Enrichment of gene sets was analysed using the Gene Set Enrichment Analysis (GSEA) software using the KEGG and GO databases.nnnRESULTSnNo difference in feto-placental biometry was observed between groups. The volume of microcotyledonary vessels was decreased in B placentas and the vascular wall of allantoic arterioles was thickened. Gene sets involved in neutral amino acids, folate and anions transport and fatty acids, cholesterol and folate degradation were down-regulated while gene sets involved in RNA expression, inflammation and vascularisation were up-regulated in B placentas.nnnCONCLUSIONnFeeding pregnant mares with concentrates from mid-gestation alters the placental function and structure as observed in other species in cases of maternal insulin resistance.

A short periconceptional exposure to maternal type-1 diabetes is sufficient to disrupt the feto-placental phenotype in a rabbit model

Tight metabolic control of type-1 diabetes is essential during gestation, but it could be crucial during the periconception period. Feto-placental consequences of maternal type-1 diabetes around the time of conception need to be explored. Using a rabbit model, type-1 diabetes was induced by alloxan 7 days before mating. Glycemia was maintained at 15-20u202fmmol/L with exogenous insulin injections to prevent ketoacidosis. At 4 days post-conception (dpc), embryos were collected from diabetic (D) or normoglycemic control (C) dams, respectively, and transferred into non-diabetic recipients. At 28dpc, D- and C-feto-placental units were collected for biometry, placental analyses and lipid profiles. D-fetuses were growth-retarded, hyperglycemic and dyslipidemic compared to C-fetuses. The efficiency of D-placentas was associated with an increased gene expression related to nutrient supply and lipid metabolism whereas volume density of fetal vessels decreased. Fetal plasma, placental and fetal liver membranes had specific fatty acid signatures depending on embryonic origin. Tissues from D-fetuses contained more omega-6 polyunsaturated fatty acids. The concentrations of docosahexaenoic acid decreased while linoleic acid increased in the heart of D-fetuses. This study demonstrates that a short exposure to maternal type-1 diabetes in the periconception window, until the blastocyst stage, is able to irreversibly malprogram the feto-placental phenotype, through precocious and persistent structural and molecular adaptations of placenta.

67 MATERNAL OBESITY AT CONCEPTION AND INSULIN SENSITIVITY IN LATE GESTATION ALTERS PLACENTAL STRUCTURE BUT NOT FETAL BIOMETRY AT BIRTH IN THE HORSE

Obesity is a major health issue in the horse industry, often associated with insulin resistance. This study aimed to analyse effects of maternal obesity at insemination and insulin resistance during late gestation on term placenta and foals at term. A total of 24 multiparous saddlebred mares were allocated to 1 of 2 groups at insemination: group obese (O) had a body condition score (BCS, French scale 1-5) >4 (N=15), and group normal (N) had a BCS=4 (N=9). From insemination until the sixth month of gestation, all the mares had access to pasture. From wintering, they were housed in box stalls and fed the same amount of energy, proteins, and fibre as a percentage of their body weight. During all the gestation, BCS, basal blood glucose, insulin, triglycerides, and nonesterified fatty acid plasma concentrations were measured each month. At 300 days of gestation, a frequent sampling glucose tolerance test, enabling the simultaneous evaluation of insulin sensitivity (IS) and glucose tolerance, was performed. At birth, placentas and foals were measured. Placentas were sampled around the umbilical cord to perform structural and functional analyses by stereology and RT-qPCR. Results were analysed using a type 3 ANOVA taking into account mare group and foal sex. Effects were considered significant when P<0.05. At 300 days of gestation, the frequent sampling glucose tolerance test indicated that half of the O mares were insulin resistant (IS <1; N=8), whereas the other half were insulin sensitive (IS >1; N=7). Based on these results, O mares were subdivided in 2 groups: obese resistant and obese sensitive (OS). Obese resistant mares were 61% less insulin sensitive than N mares and 59% less insulin sensitive than OS mares (P<0.0001, P<0.01, respectively). There was no difference for IS between N and OS mares. All analyses were thus performed comparing the 3 groups. In the N group, 3 mares were insulin resistant (one-third of the mares of the N group). Feed intake during wintering was not different between groups. Mares of both O groups maintained a high BCS (>4) during pregnancy, whereas N mares lost BCS down 2.75 at birth (<0.001). Basal glucose, insulin, triglycerides, and nonesterified fatty acid plasma were not different during gestation. At birth, no difference was observed for placental weight, surface and volume, nor for foal weight and withers height. The volume of allantoic vessels was reduced in placentas of OS mares compared with those of OR (P=0.03; 78%) and N mares (P=0.005; 65%). Moreover, placentas from OS mares had an increased volume of haemotrophic trophoblast (P=0.03) and microcotyledonary vessels (P=0.03) compared with N mares. No difference of expression was observed for 11 genes related to nutrient transfer, vascularization, growth, and development. Mare that are obese at insemination and insulin sensitive in late gestation appear to develop placental structural adaptations during gestation, possibly to increase fetoplacental exchanges, compared with obese, insulin resistant mares and lean mares. The monitoring of foal development (growth, metabolism, and osteoarticular status) is ongoing.

Effects of a preconceptional and gestational multi-vitamin-mineral-omega3 supplementation on fetoplacental development in a rabbit model

The developmental origins ofhealth and disease concept stipulates that nutritional imbalance in the preconceptional and gestational periods induces risks for the development and long-term health of the individual. In industrialized countries, most women take nutritional supplements before and during pregnancy, regardless of their diet. Potentially harmful embtyonic and fetal effects of an excess multi-micronutrient supplementation, however, are not known. The objective of this study was to assess, using a rabbit mode!, the effects of a multi-vitamin-mineral-mnega 3 supplementation (Gestarelle® G) administered in slight excess during preconceptional and gestational periods. Twenty-seven New Zealand does were used. Supplementation contained vitamins (Bl, B2, BS, B6, B8, B9, B12, C, E), minerais (magnesium, iron, copper, iodine), and n-3 polyunsaturated fatty acids (n-3 PUFA; namely DHA and EPA) in the form of oral capsules administered daily from 10 days before mating until the end ofpregnancy. Group lX posology allometrically matched thatltsedinhumans (n = 10), representing an excess of6 to 50% ofrabbit needs. Group 3X rabbits received 3 times the recommended posology (n = 9; 18 to 150% ofrabbit needs), whereas the control group received capsules containing only excipient (n = 8). Embryo and fetoplacental developmentwas monitored bytransabdominal two- and three-dimensional ultrasound and Doppler on Days 5, 7, 14, 21, and 28 with 5 conceptuses perdoe examined each time. Does were killed on Day 28 (term = 31 days), 24 h after the last capsule administration. Maternai and fetal blood, as weil as placenta and fetal organs, were collected. Data were analysed by ANOVA and Kruskall-Wallis as appropriatc. The n-3 PUFA plasma concentrations increased in the 3X does (P < 0.01). Fetal plasma concentrations were increased ont y in males in the lX group (P < 0.001) and bath in females and males in the 3X group (P < le- 15 . No statistical differences wcre observed between treatments for any of the ultrasound or Doppler analyses. In total, 208 fetuses were collected with no difference in litter size, sex ratio, nor biometrie measurements between groups. No structural anomaly was observed by histological analysis of fetal organs. In conclusion, multi-vitamin mineral-omega 3 supplementation administered dming the preconceptional and gestational periods in a rabbit mode! in the present study and within the measured parameters had no deleterious effect for dam and conceptuses at 1 and 3 times the recommended posology. Further worlc is on-going to study the effects on fetal brain development. Further studies are needed to evaluate putative post-natal effects.