Bovine embryo in vitro production (IVP) is characterised by low efficiency and variable outcomes. Monitoring early embryonic development by videomicroscopy revealed substantial morphokinetic heterogeneity in the first four embryonic cycles (EC, conventionally referred to as the 2-, 4-, 8- and 16-cell stages). Morphokinetic analysis offers a promising approach to characterize divergent developmental trajectories and provide a better understanding of underlying molecular mechanisms. We developed a Random Forest classification system (Bovine Embryo Analyser based on Morphokinetics: BEAM) to predict embryo phenotype. It is based on morphokinetic variables collected from the 1st to the 4th EC and predicts four blastocyst categories (EHB: Early Hatching Blastocyst, HB: Hatching Blastocyst, SSB: Subtle Developmental Shift Blastocyst, ADB: Arrhythmic Development Blastocyst). Classification performance on an independent dataset was good (F1 score = 0.59; Accuracy = 0.78), indicating that the BEAM can be useful for embryo development studies. The BEAM was further applied to embryos submitted to 4.3 days of culture and having completed the 4th EC (16-32 cells). RNA sequencing was performed on sixteen samples (4 x 8 pooled embryos/category). The ADB category was significantly enriched in transcripts involved in the regulation of transcriptional activity compared to the EHB category. In addition, in the ADB category, 22.7% (n = 185/816) of the upregulated genes were of maternal origin while only 6.2% were of embryonic origin (n = 54/816). In conclusion, despite being at a comparable developmental stage and transcriptionally competent, the ADB category showed delayed maternal transcript degradation suggesting delayed transition to embryonic transcriptional autonomy.

Accurate selection of bovine embryos is a challenging task, as current practice relies on a single expert assessment on the seventh day after insemination, resulting in high rates of pregnancy loss. Time-lapse videomicroscopy provides detailed information on early development, but is difficult to exploit because of complex motion patterns and time-consuming analysis. We propose TransFACT, a transformer-based framework for modeling early developmental stages and embryo transferability using 2D time-lapse videos from the first four days of development. TransFACT combines frame-level temporal features with stage-level representations, using developmental stages as auxiliary supervision to predict transferability on day four. Our experiments demonstrate that TransFACT, by leveraging an existing method designed for action recognition, achieves superior performance than its competitor in predicting embryo transferability.

Pluripotent stem cells have long been used to produce knockout mice via germline chimera technology. However, aside from the rat, this approach has not been successfully applied to other mammals. Here, we demonstrate that rabbit induced pluripotent stem cells (iPSCs) can be reprogrammed using KLF2, ERAS and PRMT6, enabling them to efficiently colonize embryos. These chimeric embryos can develop into fetuses and newborn rabbits, with iPSCs contributing up to 100 % to certain organs. Notably, female rabbits generated through this method are healthy and transmit the iPSC genome to their offspring with a high efficiency, demonstrating germline chimerism. This advancement establishes a foundation for developing rabbit models of human disease with complex genetic traits.

Little is known about the molecular underpinnings of pluripotent stem cells’ (PSCs) ability to colonize the epiblast of preimplantation embryos and generate chimeras. In our study, using rabbit PSCs as a model system, we conducted unbiased screening of a cDNA library that encodes a panel of 36 pluripotency factors. From this screening, we identified KLF2, ERAS and PRMT6, whose overexpression confers the ability for self-renewal in a KOSR/FGF2-free culture medium supplemented with LIF, activin A, PKC and WNT inhibitors. The reprogrammed cells acquired transcriptomic and epigenetic features of naive pluripotency, including the reactivation of the 2nd X-chromosome. Leveraging these PSC lines, we determined the transcriptomic signature of embryonic colonization-competence, demonstrating transcriptional repression of genes involved in MAPK, WNT, HIPPO, and EPH signaling pathways, alongside the activation of genes involved in amino-acid metabolism, NF-kB signaling, and p53 pathway. Remarkably, a subset of reprogrammed cells, expressing CD75 at a high level, gained the ability to produce chimeric fetuses with a high contribution from PSCs in all lineages.

How living systems achieve precision in form and function despite their intrinsic stochasticity is a fundamental yet ongoing question in biology. We generated morphomaps of preimplantation embryogenesis in mouse, rabbit, and monkey embryos, and these morphomaps revealed that although blastomere divisions desynchronized passively, 8-cell embryos converged toward robust three-dimensional shapes. Using topological analysis and genetic perturbations, we found that embryos progressively changed their cellular connectivity to a preferred topology, which could be predicted by a physical model in which actomyosin contractility and noise facilitate topological transitions, lowering surface energy. This mechanism favored regular embryo packing and promoted a higher number of inner cells in the 16-cell embryo. Synchronized division reduced embryo packing and generated substantially more misallocated cells and fewer inner-cell-mass cells. These findings suggest that stochasticity in division timing contributes to robust patterning.

Coprophagia by suckling rabbits, i.e. ingestion of feces from their mother, reduces mortality after weaning. We hypothesized that this beneficial effect of coprophagia is immune-mediated at the intestinal level. Therefore, this study investigated immune development after weaning by analyzing the ileal transcriptome at day 35 and 49 in rabbits with differential access to coprophagia in early life. Rabbit pups had access between day 1 and 15 to (i) no feces (NF) or (ii) feces from unrelated does (Foreign Feces, FF) or (iii) feces from unrelated does treated with antibiotics (FFab). 350 genes were differentially expressed between day 35 and day 49 in suckling rabbits with access to coprophagia. These genes coded for antimicrobial peptides, a mucin, cytokines and chemokines, pattern recognition receptors, proteins involved in immunoglobulin A secretion and in interferon signaling pathway. Strikingly, prevention of coprophagia or access to feces from antibiotic-treated does in early life blunted immune development between day 35 et 49 in the ileum of rabbits. Thus, coprophagia might be crucial for the maturation of intestinal immunity in rabbits and could explain why this behavior improves survival.

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

The maternal metabolic environment can be detrimental to the health of the offspring. In a previous work, we showed that maternal high-fat (HH) feeding in rabbit induced sex-dependent metabolic adaptation in the fetus and led to metabolic syndrome in adult offspring. As early development representing a critical window of susceptibility, in the present work we aimed to explore the effects of the HH diet on the oocyte, preimplantation embryo and its microenvironment. In oocytes from females on HH diet, transcriptomic analysis revealed a weak modification in the content of transcripts mainly involved in meiosis and translational control. The effect of maternal HH diet on the embryonic microenvironment was investigated by identifying the metabolite composition of uterine and embryonic fluids collected in vivo by biomicroscopy. Metabolomic analysis revealed differences in the HH uterine fluid surrounding the embryo, with increased pyruvate concentration. Within the blastocoelic fluid, metabolomic profiles showed decreased glucose and alanine concentrations. In addition, the blastocyst transcriptome showed under-expression of genes and pathways involved in lipid, glucose and amino acid transport and metabolism, most pronounced in female embryos. This work demonstrates that the maternal HH diet disrupts the in vivo composition of the embryonic microenvironment, where the presence of nutrients is increased. In contrast to this nutrient-rich environment, the embryo presents a decrease in nutrient sensing and metabolism suggesting a potential protective process. In addition, this work identifies a very early sex-specific response to the maternal HH diet, from the blastocyst stage.

Animal toxicological studies often fail to mimic the complexity of the human exposome, associating low doses, combined molecules and long-term exposure. Since the reproductive potential of a woman begins in the fetal ovary, the literature regarding the disruption of its reproductive health by environmental toxicants remains limited. Studies draw attention to follicle development, a major determinant for the quality of the oocyte, and the preimplantation embryo, as both of them are targets for epigenetic reprogramming. The “Folliculogenesis and Embryo Development EXPOsure to a mixture of toxicants: evaluation in the rabbit model” (FEDEXPO) project emerged from consideration of these limitations and aims to evaluate in the rabbit model the impacts of an exposure to a mixture of known and suspected endocrine disrupting chemicals (EDCs) during two specific windows, including folliculogenesis and preimplantation embryo development. The mixture combines eight environmental toxicants, namely perfluorooctanesulfonic acid (PFOS), perfluorooctanoic acid (PFOA), dichlorodiphenyldichloroethylene (DDE), hexachlorobenzene (HCB), β-hexachlorocyclohexane (β-HCH), 2,2′4,4′-tetrabromodiphenyl ether (BDE-47), di(2-ethylhexyl) phthalate (DEHP) and bisphenol S (BPS), at relevant exposure levels for reproductive-aged women based on biomonitoring data. The project will be organized in order to assess the consequences of this exposure on the ovarian function of the directly exposed F0 females and monitor the development and health of the F1 offspring from the preimplantation stage. Emphasis will be made on the reproductive health of the offspring. Lastly, this multigenerational study will also tackle potential mechanisms for the inheritance of health disruption via the oocyte or the preimplantation embryo.

Airborne pollution is a rising concern in urban areas, particularly diesel engine exhaust (DE), known to have major health effects on directly exposed subjects. The intergenerational consequences of indirect exposure to DE, however, are poorly characterized. The study aimed at exploring intergenerational effects of maternal exposure to DE in a rabbit model. Pregnant females were exposed to diluted (1 mg/m3), filtered DE (Nanoparticle diameter ≈ 69 nm) or clean air (controls) for 2 h/day, 5 days/week by nose-only exposure (total exposure: 20 days). They were allocated to one of two groups, respectively to evaluate the effects of in utero exposure on feto-placental development and to explore offspring phenotype at adulthood. Feto-placental development and growth were monitored by ultrasound. At 28 days post-coitum (dpc), dams were euthanized and feto-placental units were measured and collected. The second group of females gave birth to pups that were raised to adulthood. Their cardio-metabolic phenotype was explored, including body weight, food intake, fasting biochemistry, body composition, cardiovascular parameters and glucose tolerance. At 6.5 months of age, females were mated to control males. Second generation feto-placental units were collected at 28 dpc to analyze placental structure and gene expression. At mid gestation, DE exposure induced early signs of fetal growth retardation with decreased head length and umbilical pulse. Near term, fetal head length, plasma insulin and IGF1 concentrations were reduced. Placental function was also affected, with reduced placental efficiency, decreased placental blood flow and fetal vessel volume. Non-aggregated and "fingerprint-like" nanoparticles were observed in maternal blood space, in both trophoblastic cells and fetal blood, demonstrating transplacental transfer. In adult offspring, in utero exposure to DE led to blood pressure increase, hyperglycaemia, and increased perirenal fat mass, together with reduced plasma HDL-cholesterol and peripheral fat-to-body weight ratio in males, altogether suggesting a metabolic syndrome. Only trends were observed in adult exposed females with higher plasma triglyceride concentrations and decreased bone density compared to controls. Once pregnant, however, these F1 females developed fatty liver. Although second generation 28dpc fetoplacental biometry was not affected, fetal lipid metabolism was altered with decreased fetal plasma cholesterol and increased triglyceride concentrations. Placental function (explored using gene set enrichment analyses of placental transcriptomic data) was altered in the "exposed" group with over-representation of proteasome complex and ubiquitin pathway gene sets together with under-representation of genes sets involved in ion channel function and inflammation pathways. Using this rabbit model, repeated daily gestational exposure to DE at levels close to urban pollution can affect feto-placental development in both first and second generation and predispose offspring to cardiometabolic disorders in a sex-specific manner. These results demonstrate that DE exposure and in utero indirect exposure in particular should be considered as a programming factor within the context of the Developmental Origins of Health and Disease leading to intergenerational transmission.

This study aimed to evaluate gene expression for embryos collected 8±1.5 days after ovulation, of different sizes. Non-lactating Saddlebred mares located on two experimental farms were inseminated with semen from one stallion per farm after hCG induction. Mares were examined for ovulation at 24 or 48 h after hCG, depending on the farm. Twenty-eight embryos (11 and 17, respectively) were collected 8 days after ovulation was confirmed, with each embryo from a different dam. Embryos were measured and bisected to obtain trophoblast (TE) or inner cell mass enriched trophoblast (TE-ICM). Paired end, non-oriented RNA sequencing was performed (Illumina, NextSeq500) on both samples of embryos of different diameters: Small (<700µm, mean of 560±85µm, n=9), Medium (700-1200µm, 886±160µm, n=11) and Large (>1,200µm, 1,719±488µm, n=8). For TE-ICM data, deconvolution (DeMixT) was used to discriminate gene expression in ICM vs TE. Differential expression was analyzed (DESeq2) with farm and embryo sex as cofactors (false discovery rate (FDR) <0.05 cutoff). Overrepresentation tests were performed on differentially expressed genes (DEG) using PANTHER software with GObp database. Within the 14,249 and 13,406 genes expressed in ICM and TE, respectively, 642 in ICM and 123 DEG in TE were observed in Small vs Medium while 1,228 in ICM and 443 DEG in TE were identified in Large vs Medium. Of particular interest, in the ICM and TE, when compared to Medium embryos, Insulin Like Growth Factor(IGF)2 was more expressed in Small (log2 Fold Change, log2FC=1.9 and 2.0 for ICM and TE, respectively, FDR<0.05) while IGF1 was up-regulated in ICM (log2FC=2.0, FDR<0.05) and down-regulated in TE (log2FC=-1.2, FDR<0.05) in Large embryos. IGF1 is known to stimulate estrogen production in pig embryos. Here, several P450 cytochromes including cytochrome P450 aromatase (CYP19A1, log2FC=4.46, FDR<0.0001) associated with 17β-Hydroxysteroid dehydrogenase (HSD17B1, log2FC=0.58, FDR<0.05) were upregulated in the ICM in Large vs Medium. Moreover, Nanog Homeobox gene (log2FC=-3.0, FDR<0.0001) and several SRY-Box Transcription Factors (SOX), including SOX2 (log2FC =-2.3, FDR<0.01) were downregulated in the ICM of Large vs Medium. In ICM, upregulated DEGs in Small vs Medium were principally involved in cell proliferation and downregulated DEGs in mitochondria and regulation of apoptosis while in upregulated DEGs Large vs Medium, steroid biosynthesis, cell growth and migration and lipid transport were overrepresented. In TE, no gene set was overrepresented in Small vs Medium, while downregulated DEGs in Large vs Medium were involved in DNA methylation and amino acid metabolism. In conclusion, equine embryos of different diameter differ in gene expression associated with developmental parameters. Differences in gene expression between ICM and TE suggest increased secretory activity in ICM with embryo growth.

Background Breeding a mare until she is not fertile or even until her death is common in equine industry but the fertility decreases as the mare age increases. Embryo loss due to reduced embryo quality is partly accountable for this observation. Here, the effect of mare's age on blastocysts' gene expression was explored. Day 8 post-ovulation embryos were collected from multiparous young (YM, 6-year-old, N = 5) and older (OM, > 10-year-old, N = 6) non-nursing Saddlebred mares, inseminated with the semen of one stallion. Pure or inner cell mass (ICM) enriched trophoblast, obtained by embryo bisection, were RNA sequenced. Deconvolution algorithm was used to discriminate gene expression in the ICM from that in the trophoblast. Differential expression was analyzed with embryo sex and diameter as cofactors. Functional annotation and classification of differentially expressed genes and gene set enrichment analysis were also performed. Results Maternal aging did not affect embryo recovery rate, embryo diameter nor total RNA quantity. In both compartments, the expression of genes involved in mitochondria and protein metabolism were disturbed by maternal age, although more genes were affected in the ICM. Mitosis, signaling and adhesion pathways and embryo development were decreased in the ICM of embryos from old mares. In trophoblast, ion movement pathways were affected. Conclusions This is the first study showing that maternal age affects gene expression in the equine blastocyst, demonstrating significant effects as early as 10 years of age. These perturbations may affect further embryo development and contribute to decreased fertility due to aging.