Fabrice Reigner

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.

The Effects of Weaning Methods on Gut Microbiota Composition and Horse Physiology

Weaning has been described as one of the most stressful events in the life of horses. Given the importance of the interaction between the gut-brain axis and gut microbiota under stress, we evaluated (i) the effect of two different weaning methods on the composition of gut microbiota across time and (ii) how the shifts of gut microbiota composition after weaning affect the host. A total of 34 foals were randomly subjected to a progressive (P) or an abrupt (A) weaning method. In the P method, mares were separated from foals at progressively increasing intervals every day, starting from five min during the fourth week prior to weaning and ending with 6 h during the last week before weaning. In the A method, mares and foals were never separated prior to weaning (0 d). Different host phenotypes and gut microbiota composition were studied across 6 age strata (days −30, 0, 3, 5, 7, and 30 after weaning) by 16S rRNA gene sequencing. Results revealed that the beneficial species belonging to Prevotella, Paraprevotella, and Ruminococcus were more abundant in the A group prior to weaning compared to the P group, suggesting that the gut microbiota in the A cohort was better adapted to weaning. Streptococcus, on the other hand, showed the opposite pattern after weaning. Fungal loads, which are thought to increase the capacity for fermenting the complex polysaccharides from diet, were higher in P relative to A. Beyond the effects of weaning methods, maternal separation at weaning markedly shifted the composition of the gut microbiota in all foals, which fell into three distinct community types at 3 days post-weaning. Most genera in community type 2 (i.e., Eubacterium, Coprococcus, Clostridium XI, and Blautia spp.) were negatively correlated with salivary cortisol levels, but positively correlated with telomere length and N-butyrate production. Average daily gain was also greater in the foals harboring a community type 2 microbiota. Therefore, community type 2 is likely to confer better stress response adaptation following weaning. This study identified potential microbial biomarkers that could predict the likelihood for physiological adaptations to weaning in horses, although causality remains to be addressed.

In vitro maturation affects chromosome segregation, spindle morphology and acetylation of lysine 16 on histone H4 in horse oocytes.

Implantation failure and genetic developmental disabilities in mammals are caused by errors in chromosome segregation originating mainly in the oocyte during meiosis I. Some conditions, like maternal ageing or in vitro maturation (IVM), increase the incidence of oocyte aneuploidy. Here oocytes from adult mares were used to investigate oocyte maturation in a monovulatory species. Experiments were conducted to compare: (1) the incidence of aneuploidy, (2) the morphology of the spindle, (3) the acetylation of lysine 16 on histone H4 (H4K16) and (4) the relative amount of histone acetyltransferase 1 (HAT1), K(lysine) acetyltransferase 8 (KAT8, also known as MYST1), histone deacetylase 1 (HDAC1) and NAD-dependent protein deacetylase sirtuin 1 (SIRT1) mRNA in metaphase II stage oocytes that were in vitro matured or collected from peri-ovulatory follicles. The frequency of aneuploidy and anomalies in spindle morphology was increased following IVM, along with a decrease in H4K16 acetylation that was in agreement with our previous observations. However, differences in the amount of the transcripts investigated were not detected. These results suggest that the degradation of transcripts encoding for histone deacetylases and acetyltransferases is not involved in the changes of H4K16 acetylation observed following IVM, while translational or post-translational mechanisms might have a role. Our study also suggests that epigenetic instabilities introduced by IVM may affect the oocyte and embryo genetic stability.

Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes

The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted reproduction also allows for the production of embryos from high performers without interrupting their sports career and contributes to an increase in the number of foals from mares of high genetic value. The present manuscript describes the procedures used for collecting immature and mature oocytes from horse ovaries using ovum pick-up (OPU). These oocytes were then used to investigate the incidence of aneuploidy by adapting a protocol previously developed in mice. Specifically, the chromosomes and the centromeres of metaphase II (MII) oocytes were fluorescently labeled and counted on sequential focal plans after confocal laser microscope scanning. This analysis revealed a higher incidence in the aneuploidy rate when immature oocytes were collected from the follicles and matured in vitro compared to in vivo. Immunostaining for tubulin and the acetylated form of histone four at specific lysine residues also revealed differences in the morphology of the meiotic spindle and in the global pattern of histone acetylation. Finally, the expression of mRNAs coding for histone deacetylases (HDACs) and acetyl-transferases (HATs) was investigated by reverse transcription and quantitative-PCR (q-PCR). No differences in the relative expression of transcripts were observed between in vitro and in vivo matured oocytes. In agreement with a general silencing of the transcriptional activity during oocyte maturation, the analysis of the total transcript amount can only reveal mRNA stability or degradation. Therefore, these findings indicate that other translational and post-translational regulations might be affected. Overall, the present study describes an experimental approach to morphologically and biochemically characterize the horse oocyte, a cell type that is extremely challenging to study due to low sample availability. However, it can expand our knowledge on the reproductive biology and infertility in monovulatory species.

Preimplantation genetic diagnosis in Welsh pony embryos after biopsy and cryopreservation

Preimplantation genetic diagnosis and embryo cryopreservation are important tools to improve genetic management in equine species with marked consequences on the economic value, health, biodiversity, and preservation of the animals. This study aimed to develop a biopsy method at the blastocyst stage that provides viable genotyped cryopreserved Welsh pony embryos. Embryos were collected at d 6.75 to 7 after ovulation. Biopsies were performed with either a microblade or a micropipette. After biopsy, embryos were cryopreserved. The survival rate of biopsied embryos was evaluated on fresh and cryopreserved embryos either 24 h after in vitro culture or after transfer to recipients. Fresh and nonbiopsied embryos were used as controls. Sex, coat color genes, myotony (neuromuscular disorder) diagnosis, and markers of parentage were investigated using PCR on biopsied cells after whole-genome amplification and on remaining embryos. The embryo survival rate after transfer was not affected by the micropipette biopsy (50%, = 8; 43%, = 7; and 50%, = 12, at d 30 for fresh biopsied embryos, vitrified biopsied embryos, and control embryos, respectively) but was significantly reduced by the use of microblade biopsy: 9 ( = 11) vs. 67% ( = 12) for control embryos. Successful sex determination was achieved for 82% ( = 28) of the micropipette biopsies and 100% ( = 50) of the microblade biopsies. Sex determined on biopsied cells was found to correspond completely (100%) with that determined on the remaining embryo ( = 37). More than 90% of the parentage checking markers, coat color, and myotony diagnosis were successfully determined on biopsies obtained with either a micropipette or a microblade. Mendelian incompatibility (7.5 and 5.5%) and embryo genotyping errors (6.6 and 8.6%) were low and not significantly different between the 2 methods. In conclusion, for the first time, pregnancy at Day 30 was obtained after transfer of Welsh pony biopsied and vitrified embryos >300 μm in diameter to recipient pony mares. The biopsied cells collected enabled multigenetic embryo diagnoses to be performed to a high degree of accuracy. The micropipette biopsy is the better method to apply on Welsh pony embryos.

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.

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.

Economic assessment of FEC-based targeted selective drenching in horses.

In the face of an increased prevalence of drug-resistant cyathostomin populations, a targeted selective treatment (TST) strategy based on Faecal Egg Counts (FECs) has been proposed as an alternative management strategy. However, associated costs may be a barrier to the uptake of this strategy. Our study aims to provide an economic assessment of FEC-based TST. FECs were determined in a Welsh pony herd thrice a year from 2010 to 2014. This database was used to explore the impact of FEC price, sampling strategy (individual or pooled) and labour-associated costs. Drug price was set at the cheapest level, hence providing a conservative framework to determine the maximum viable FEC price in the context of a cost-driven horse industry. The maximum viable FEC price for a cost-efficient individual based strategy was determined by an in silico bootstrap approach consisting of randomly sampling 1000 virtual pony herds of various sizes (1 to 100 ponies) from the available database and estimating the associated costs (FEC price ranging from € 1 to € 10, anthelmintic costs and labour-associated costs). The costs and benefits of the pooling strategy that consists of basing the decision to treat on group FEC values were also investigated. This is thought to reduce FEC-based costs but may result in highly infected individuals being left undrenched, i.e. in false-negatives, as a result of FEC overdispersion. For various pool-sizes (1-20 ponies) and various cut-off thresholds (50-200 eggs/g), we sampled 1000 pony herds in silico to estimate the associated costs and determine the number of positive ponies within a negative pool. Following these simulations, pool-based FECs of various sizes were performed on 40 ponies to compare predictions with real data. Within 4 years, anthelmintic costs were cut by 80%, albeit with free FECs. In silico estimations suggested that an individual FEC-based TST would not be cost-efficient in this context for an FEC price above € 5. With a pooled FEC strategy, the proportion of false-negatives never exceeded 15% of the pool size. The combination of a 14-pony pool and a cut-off value of 150 eggs/g minimized total costs while keeping the number of false negatives to a reasonable level. Real data obtained from infected ponies however, suggested that pool size should not exceed 10 individuals, since the inhomogeneous mixing of faeces of larger pools probably reduced the correlation between average pooled FECs and the mean of individual FECs. Our study provides an economic framework that could be valuable for emphasizing the use of FEC-based approaches in the field.

Exposure to follicular fluid during oocyte maturation and oviductal fluid during post-maturation does not improve in vitro embryo production in the horse

Most wild equids and many domestic horse breeds are at risk of extinction, so there is an urgent need for genome resource banking. Embryos cryopreservation allows the preservation of genetics from male and female and is the fastest method to restore a breed. In the equine, embryo production in vitro would allow the production of several embryos per cycle. Intracytoplasmic sperm injection (ICSI) is used to generate horse embryos, but it requires expensive equipment and expertise in micromanipulation, and blastocyst development rates remain low. No conventional in vitro fertilization (IVF) technique for equine embryo production is available. The development of culture conditions able to mimic the maturation of the oocyte in preovulatory follicular fluid (pFF) and the post-maturation in oviductal fluid (OF) may improve embryo production in vitro. Our aim was to analyse the effect of in vitro maturation in pFF and incubation in OF on in vitro maturation of equine oocytes, fertilization using conventional IVF or ICSI, and embryo development after culture in synthetic oviductal fluid (SOF) or DMEM-F12. Oocytes collected from slaughtered mares or by ovum pick up were matured in vitro in pFF or semi-synthetic maturation medium (MM). The in vitro maturation, fertilization and development rates were not statistically different between pFF and MM. After in vitro maturation, oocytes were incubated with or without OF. Post-maturation in OF did not significantly improve the fertilization and development rates. Thus, in our study, exposure to physiological fluids for oocyte maturation and post-maturation does not improve in vitro embryo production in the horse.

Ivermectin failure in the control of Oxyuris equi in a herd of ponies in France.

Drug resistance in equine gastro-intestinal parasitic nematodes has been reported throughout the world. While the focus is usually put on cyathostomins, observations of macrocylic lactone failure against Oxyuris equi have accumulated over the last decade. Here we report the failure of ivermectin in the control of O. equi in an experimental Welsh pony herd. In a first trial, 6 ponies previously drenched with moxidectin and showing patent O. equi infections were administered ivermectin and subsequently monitored for O. equi egg excretion over one month. This trial demonstrated a failure of ivermectin to control O. equi egg excretion as half of ponies demonstrated recurrent egg excretion in the peri-anal region during 21days after treatment. One year later, six female Welsh ponies drenched with moxidectin demonstrated signs of itching and scratching in their peri-anal region with worms being found transiently in fecal materials three weeks later. Ponies were allocated to three treatment groups, i.e. ivermectin, pyrantel embonate and fenbendazole and monitored for egg excretion over five weeks. Fenbendazole and pyrantel embonate broke ivermectin suboptimal efficacy as soon as 8 and 14days respectively after treatment, while egg excretion remained constant throughout the 41-day long trial in the ivermectin-treated ponies. This is the first report of ivermectin failure against O. equi in France. In the absence of critical efficacy test, it remains unclear whether true resistance is at stake or if these observations confound a constitutive suboptimal efficacy of ivermectin against O. equi.