Selection for fertility traits, such as daughter pregnancy rate (DPR), heifer conception rate (HCR), and cow conception rate, has been shown to improve pregnancy success within dairy herds. This study explored the relationship between fertility traits and embryo development in both in vitro and in vivo conditions in dairy cattle. Data from 2,408 in vitro-produced (IVP) and 1,801 in vivo-derived (IVD) embryo procedures were analyzed. Embryos produced in these procedures were classified by quality (grade 1, 2, and 3). We assessed the relationship between fertility traits (DPR and HCR) and various embryo development parameters, including the total number of structures collected, blastocyst rate, and embryo quality, using generalized linear models. The analysis focused on female Holstein donors aged 10 to 20 mo. Fertility traits did not show a significant association with in vitro embryo production. However, in vivo, higher fertility values for DPR and HCR were associated with improved blastocyst rates, suggesting that these fertility traits may have a greater influence on embryo development in vivo. In addition, higher HCR values in the embryo were associated with increased pregnancy per transfer by d 30 when embryos were transferred into both heifers and cows, resulting in more calves born. Higher DPR values were also associated with a higher proportion of calves born when embryos were transferred to cows, indicating the potential influence of DPR on pregnancy outcomes in lactating cows. Our findings show that fertility traits DPR and HCR are associated with improved embryo development and competence to establish pregnancy, highlighting the potential for selection for embryo developmental and reproductive success. Further research is needed to better understand the mechanisms underlying these effects and to identify genetic markers and individual genes that could enhance fertility outcomes.

Cattle have a synepitheliochorial type of placenta characterized by placental cotyledons and maternal endometrial caruncles forming placentomes that are essential for fetal development to term. As the elongated conceptus begins implantation, binucleated trophoblast giant cells (TGC) begin to appear in the chorion. The TGC are hypothesized to arise from progenitor UNC through endoreplication and express unique placenta-specific genes, including placental lactogen (CSH2), pregnancy-associated glycoproteins (PAGs), and prolactin like proteins (PRPs). Despite their essential role in placental cotyledon development and placental function, the cellular and molecular mechanisms regulating trophoblast differentiation in the bovine placenta remain undefined. Here, a differentiation protocol was developed that enabled bovine trophoblast stem cells (TSCs) to generate TGC. The morphologically distinct TGCs were binucleated with a cytoplasm containing abundant secretory granules. Expression of TGC marker genes (CSH2, PAGs, PRPs) was increased in differentiated TSCs. Single-cell transcriptome analysis revealed distinct developmental programs underlying TGC lineage specification. To interrogate regulatory mechanisms governing TGC differentiation, bovine TSCs were engineered to enable inducible GCM1 (glial cells missing 1) expression. Induction of GCM1 during TSC differentiation increased TGC number and TGC-specific gene expression. This robust and tractable in vitro TSC differentiation system is useful to explore trophoblast differentiation and provide fundamental insights into the cellular and molecular mechanisms regulating placenta development in cattle.

Abstract Paternal contributions to embryogenesis extend beyond DNA, yet the molecular cargo carried by sperm and its impact on development remain poorly defined. Aggresomes (AGG), cytoplasmic inclusions formed by misfolded proteins, are present in mammalian gametes, but their functional consequences are unclear. Here, we show that excessive AGG content in bovine sperm head compromises preimplantation embryo development. Using image-based flow cytometry, sperm from 32 sires were classified into low-, moderate-, and high-AGG groups. AGG levels were unrelated to sire age and did not affect in vitro capacitation or acrosome remodeling. However, embryos derived from high-AGG sires exhibited reduced blastocyst formation, delayed cleavage timing, and a higher incidence of developmental arrest at the 4-6 cell stage. Embryos from high-AGG sires also accumulated more AGG during development, showed elevated reactive oxygen species (ROS) levels, and displayed altered mitophagy dynamics. Supplementation with an ER stress inhibitor temporarily improved cleavage but did not enhance overall blastocyst formation, indicating a limited and stage-specific effect. In vivo, embryos from high-AGG sires showed lower transferable quality compared with those from low-AGG sires. These findings establish sperm head AGG content as a novel paternal determinant of embryo quality. By linking sperm-borne misfolded protein aggregates to disrupted developmental pathways in the resulting embryo, our study reveals a previously unrecognized mechanism of paternal influence on fertility and suggests new opportunities for molecular screening for male fertility. Significance Statement Sperm contribute more to the embryo than DNA alone, yet the consequences of sperm-borne molecular cargo for early development remain largely unknown. We show that aggregates of misfolded proteins in the sperm head, a marker of disrupted protein quality control, impair preimplantation embryo development in cattle. Sires with elevated sperm aggregate content produce embryos that cleave later, arrest more frequently, and reach the blastocyst stage at lower rates, both in vitro and in vivo. These embryos carry greater aggregate loads, show heightened oxidative stress, and display dysregulated mitochondrial clearance. Our findings establish paternal proteostasis as a determinant of embryo quality and identify a class of sperm defects invisible to conventional semen analysis, opening new avenues for molecular fertility screening.

Long-term trophectoderm (TE) cell culture provides a powerful model to investigate placenta-specific factors to better understand mechanisms relevant to pregnancy establishment and placental development. However, current TE culture systems rely on costly commercial media and extracellular matrix (ECM) components, which limit their scalability and accessibility. This study evaluated cost-effective alternatives to established conditions by testing modified DMEM/F-12 and a biphasic TE culture system as substitutes for commercial Advanced DMEM/F-12 and for continuous TE culture, and by assessing 0.1% gelatine as an ECM alternative to collagen IV. Trophectoderm outgrowths cultured on collagen IV or gelatine did not differ in attachment timing (P = 0.78) or in expression of placental and differentiation markers PLAC8 (P = 0.78), GATA2 (P = 0.18), and PAG10 (P = 0.39). Similarly, blastocysts cultured in commercial Advanced DMEM/F-12 or base DMEM/F-12 exhibited no differences in attachment day (P = 0.98), TE growth area from days 10-20 (P > 0.05), or expression of PLAC8 (P = 0.35) and PAG10 (P = 0.08), although PAG7 differed between treatments (P < 0.05). Embryos cultured in continuous TE attached later (P = 0.01) than those cultured in TE media, but no differences were observed in TE growth area or expression of PLAC8, PAG7, or PAG10 (P > 0.05). Collectively, these results indicate that affordable media formulations and gelatine-coated cultureware support TE attachment, proliferation, and differentiation. This cost-effective culture framework enables broader application of TE models and supports extended studies of trophoblast function, placental signalling, and early conceptus development.

In-vitro embryo production (IVP) in cattle is limited by the suboptimal competence of oocytes matured outside the follicular environment. While maturation is often achieved, incomplete cytoplasmic maturation may impair embryo development and survival. This study evaluated the influence of follicle size on oocyte cytoplasmic maturation, lipid metabolism, cumulus cell function, and embryo cryosurvival. Oocytes were collected from small (< 6 mm), medium (6-9 mm), and large (10-20 mm) follicles and evaluated at 0, 12, and 24 h of in-vitro maturation (IVM). Organelle distribution, lipid and mitochondrial content and distribution, cortical granule distribution, and transzonal projections (TZPs) were evaluated. Gene expression was assessed in surrounding cumulus cells, and embryo development and cryotolerance were evaluated following fertilization and culture. While organelle patterns did not differ among follicle sizes for mitochondria, lipids, or cortical granules, oocytes from large follicles had lower lipid accumulation by 24 h IVM, and fewer TZPs throughout maturation. Gene expression in cumulus cells from large follicles also had a greater abundance of transcripts involved in steroidogenesis, growth factor signaling, and glucose metabolism midway through maturation. Although cleavage and blastocyst development rates were similar across groups, embryos from large follicle oocytes had higher post-thaw survival and lower lipid content than those from small follicles. These findings demonstrate that follicle size affects key metabolic and structural aspects of oocyte maturation, ultimately influencing embryo quality. Selecting oocytes from larger follicles may improve embryo cryosurvival by promoting more complete cytoplasmic maturation and more efficient lipid regulation during IVM.

The aim of this study was to evaluate the effect of the linoleic–α-linolenic acid ratio (LA:ALA) on cyclicity, oocyte quality, early pregnancy parameters, milk yield, and composition. Holstein cows were randomized to a 6:1-LA:ALA diet (Low-OMG3: n = 3 pens; 11 primiparous, 14 multiparous) or a 2:1-LA:ALA diet (High-OMG3: n = 3 pens; 10 primiparous, 14 multiparous). Diets were isocaloric and isonitrogenous and fed between 15 and 140 days in milk (DIM). Data were compared using linear mixed models. As expected, omega-3 concentrations in milk and blood increased in the High- compared to Low-OMG3 cows. No effect of diet was observed on cyclicity by 45DIM or oocyte quality at 50DIM. High-OMG3 cows had larger corpus luteum size (11–32 d post-timed artificial insemination [TAI]) and greater blood flow (32–60 d post-TAI) than Low-OMG3 cows. However, there was no effect of diet on progesterone, pregnancy-associated glycoproteins, or conceptus size. High-OMG3 cows produced more milk throughout the study, had greater lactose, and tended to have greater protein yield at 50DIM. In conclusion, decreasing the LA:ALA dietary ratio in lactating dairy cows did not provide evidence of effects on cyclicity, oocyte quality, or other early pregnancy-related parameters, but affected corpus luteum size and blood flow, enhanced milk production, and partially increased protein and lactose yields.

In vitro embryo production (IVP) is now the predominant technique utilized to generate embryos for transfer in cattle. Although artificial insemination and natural mating remain the main breeding methods, the number of embryos produced through IVP exceeds those derived in vivo (IVD). Superstimulation with follicle-stimulating hormone (FSH) is necessary for IVD embryos and is frequently used in IVP to increase follicular development. However, despite the industry shift from IVD to IVP, the success of IVP embryos remains poor. This study systematically evaluated embryo production outcomes from in vivo and in vitro systems in Bos taurus beef and dairy cattle, with a focus on FSH superstimulation. Data from published studies were integrated to compare oocyte yield, embryo production, and developmental rates. Overall, OPU yields more oocytes in both beef and dairy cattle relative to recovered structures by uterine flush, but this did not consistently translate into higher embryo numbers. Importantly, embryo outcomes differed by both cattle purpose (beef/dairy) and whether embryo counts or rates were considered. Beef cattle produced more transferable embryos (counts) under IVD than IVP, whereas dairy cattle had comparable embryo counts between techniques. In contrast, embryo rates (percentage of embryos relative to oocytes/structures recovered) were similar between IVD and IVP in both beef and dairy. These findings demonstrate that embryo production outcomes depend on whether absolute counts or proportional success rates are evaluated. While FSH improves follicular response, it does not consistently increase embryo yield. Further refinement of reproductive strategies is needed to optimize embryo production efficiency across systems.

Elevated ambient temperatures disrupt fertility through coordinated effects on female and male gametes as well as early embryos. In females, heat stress alters hypothalamic-pituitary-ovarian signaling, reduces follicular perfusion, disrupts steroidogenesis, and induces oxidative and metabolic stress within the follicular microenvironment, resulting in compromised oocyte competence that can persist across multiple follicular waves. In males, elevated testicular temperature interferes with spermatogenesis in a stage-dependent manner, leading to delayed impairments in sperm chromatin integrity, motility, and fertilizing capacity that may not be detected by routine semen evaluation. Early embryos are particularly vulnerable to elevated temperature, with heat stress activating mitochondrial dysfunction, oxidative stress, apoptotic pathways, and impaired stress-response mechanisms before thermotolerance is acquired at later developmental stages. This review integrates systemic, cellular, and molecular evidence to describe how heat stress acts as a multilevel disruptor of reproduction in cattle, identifies key mechanisms underlying carryover effects on fertility, and highlights knowledge gaps critical for improving reproductive resilience under thermal stress.

Embryo development and pregnancy establishment require successful interaction between the oocyte and sperm and proper activation of developmental signaling pathways. In the bovine, the successful execution of this biological equation requires, on one hand, maternal determinants such as the intrinsic quality of the oocyte and molecules secreted by the endometrium epithelium critical to sustain embryo development. In contrast, paternal contributions encompass both visible sperm characteristics assessed by routine semen evaluation and hidden traits that escape conventional laboratory assessment of sperm quality. Paternal contributors extend beyond DNA delivery, as sperm also transfer a diverse array of molecules critical for reproduction. For instance, sperm can deliver proteins that could have short- and long-term consequences for sperm functionality, organelles that can be used to assemble the cytoskeleton machinery of the developing embryo, and other molecules that can regulate critical processes in reproduction to ensure the survival of different mammalian species. Taken together, paternal contributors can significantly influence fertility, with their impact manifesting at events occurring around fertilization with or without subsequent effects on embryo development and with or without consequences for pregnancy establishment, leading to the divergence in fertility observed among males.

The hypothesis was that early postpartum uterine disease would reduce the developmental capacity of oocytes thus contributing to the reduced fertility of dairy cows with uterine disease. Dairy cows were diagnosed healthy or with metritis at 7-10 d postpartum. The reproductive tract was collected at approximately 1 mo (Exp. 1) or approximately 80 or 165 d (Exp. 2) postpartum for the collection of cumulus-oocyte complexes (COC). The COC were matured, co-incubated with sperm for fertilization, and cultured to the blastocyst stage (8 d) in vitro. For Exp.1, the disease diagnosis (healthy or metritis) did not affect the number of collected COC or the subsequent embryo development to the blastocyst stage. The presence of purulent material in the uterine lumen (endometritis) at time of oocyte collection, however, was associated with a reduced cleavage rate evaluated 3 d following fertilization. For Exp. 2, there was no effect of disease diagnosis (healthy or metritis) on the number of COC or their subsequent development. Reduced cleavage rates were observed in COC retrieved from cows slaughtered at 80 d postpartum, but not at 165 d postpartum, and this reduction was associated with a vaginal microbiome indicative of uterine disease at 4-5 wk postpartum. Regression analyses that included plasma haptoglobin or energy metabolite concentrations or uterine bacterial genera abundance did not explain a large percentage of the variation in oocyte development in vitro. We conclude that there is an effect of uterine disease at one month postpartum on the oocyte and its capacity for development (Exp. 1) and this effect may be present at 80 d postpartum (Exp. 2). In later postpartum cows (165 d postpartum; Exp. 2) there was no effect of uterine disease on in vitro oocyte development.

A central determinant of successful reproduction is pregnancy establishment and maintenance that relies on proper development of the conceptus (embryo/fetus and associated extraembryonic membranes including the placenta). Pregnancy loss in cattle can be caused by inadequate development and differentiation of the placenta. However, the cellular and molecular mechanisms regulating bovine placenta development and, particularly, trophoblast differentiation are not well understood. Recent single-cell RNA-seq analyses revealed dynamic changes in cell populations and gene expression patterns during bovine placental development. Here, the chromatin accessibility landscape across diverse cell populations was determined in the developing (Day 40) and mature (Day 170) bovine placenta using the 10X Genomics multiome (snRNA-seq and snATAC-seq) platform. Analyses revealed distinct trophoblast, mesenchyme, endothelial, immune, and epithelial cell populations characterized by unique gene expression and chromatin accessibility signatures. ATAC-seq peaks defined open chromatin regions, facilitating the identification of transcription factor binding sites and candidate gene regulatory networks involved with trophoblast differentiation. Several transcription factors, known for their involvement in trophoblast differentiation in other mammalian species, were identified as candidate regulators of uninucleate to binucleate trophoblast differentiation. This study adds to our foundational understanding of gene regulation and expression in the placenta, offering insights into the mechanisms governing pregnancy loss in cattle.

Reproductive success is an essential component of profitable and sustainable dairy operations. Although selection for production traits such as milk yield has led to a decline in fertility in dairy cattle, strategies including assisted reproductive technologies such as in vitro fertilization and embryo transfer, as well as genomic selection for fertility traits, are being implemented to help mitigate this loss. Previous studies have identified genetic markers associated with fertility traits such as daughter pregnancy rate, conception rate, and interval to first conception. However, genetic markers associated with in vitro embryo development have yet to be explored. Therefore, this study aimed to identify genetic markers associated with in vitro embryo cleavage and blastocyst rates with the future goal of creating an index to select bulls with superior abilities to produce embryos in vitro. Rigorous in vitro fertilization trials identified Holstein bulls with divergent abilities to produce embryos that successfully cleave and develop into blastocysts in vitro. A total of 40 bulls with embryo cleavage and blastocyst rate phenotypes were whole-genome sequenced. Sequencing was performed on an Illumina platform with PE150 reads followed by cleaning, mapping to ARS-UCD1.2, variant calling with GATK HaplotypeCaller, and quality control following the 1000 Bulls Genomes Project best practices. Genome-wide association analyses identified 819 and 442 SNPs associated with embryo cleavage and blastocyst rates, respectively. Significant regions on chromosomes 15, 18, 21, 22, and 23 were in linkage disequilibrium with QTL previously associated with reproductive traits in cattle. Further, a region on chromosome 28 with the most significant variant associated with cleavage rate contained several synonymous variants in EGLN1, which is a critical component of the hypoxia inducible pathway. An additional region on chromosome 18 associated with cleavage rate contained a missense variant in SMG9, a gene involved in the nonsense-mediated mRNA decay pathway. A region on chromosome 21 associated with blastocyst rate also contained variants in regulatory regions downstream from AEN, a gene required for efficient DNA fragmentation during the p53-dependent apoptosis pathway. In summary, this study identified a preliminary collection of genomic regions associated with embryo cleavage and blastocyst rates in vitro that contain relevant genes and other QTL associated with reproductive traits in cattle. These regions, after validation, could contribute to a selection index for identification of bulls with genetic predispositions to produce a greater number of embryos in vitro for use in assisted reproduction techniques.

When related animals are mated to one another, genetic defects may become apparent if recessive mutations are inherited from both sides of the pedigree. The widespread availability of high-density DNA genotypes for millions of animals has made it possible to identify and track known defects as well as to identify and track previously unknown defects that cause early embryonic losses. Although the number of known defects has increased over time, the availability of carrier information has been used to dramatically reduce the frequency of many disorders. The economic impact of known genetic defects in the US dairy cattle population has decreased by ∼2/3 since 2016, due largely to the avoidance of carrier-to-carrier matings. Effective population management requires robust systems for reporting new defects, identification of causal mechanisms, and development of commercially available tests. The United States and Canada depend on informal cooperation among many groups, including farmers, purebred cattle associations, genetics companies, and researchers, to identify emerging and causal defects. The structure of a collaborative system including all key sectors of the dairy cattle industry to support long-term population management is described. This review provides a comprehensive overview of the landscape surrounding genetic defects in dairy cattle. Topics covered include current defects of relevance to commercial dairy producers, trends in carrier frequencies over time, how best to manage these defects, strategies for detecting emerging diseases, and marketing and trade considerations.

Abstract:The central goal of the following studies was to understand how FGF2, LIF, and IGF1, a cocktail called 'FLI', influence bovine embryo development by the degree of transcriptomic variation throughout preimplantation development. All embryos were produced in vitro with or without FLI supplementation at the beginning of culture. For each treatment, embryos were collected at the 4-6 cell, 9-16 cell, morula, or blastocyst stages, and RNA was isolated and sequenced at a depth of 50 million reads per sample. In the FLI group, at the 9-16 cell stage, there were seven upregulated and six downregulated differentially expressed genes (DEGs). At the morula stage, of the 1,856 DEGs, 580 were upregulated in FLI. Gene ontology analysis showed increased MAPK signaling, TGF-beta signaling, and Hippo signaling, which all help regulate cell adhesion, lineage commitment, and growth regulation in the developing embryo. In FLI blastocyst stage embryos, 199 upregulated and 545 downregulated DEGs revealed an increase in processes associated with interferon-gamma production and cell differentiation. Overall, FLI modulates many of the regulatory pathways in the developing embryo to drive increased cell survival, cell integrity, and overall embryo development. Lay summary:This study investigated whether adding three supportive proteins - FGF2, LIF, and IGF1 (together called FLI) - could improve the development of cow embryos grown in vitro. In cattle breeding, embryos are often produced outside the body to enhance fertility and support genetic selection. However, many embryos fail to develop properly under laboratory conditions. To address this, researchers tested whether FLI could create a more favorable environment for early embryo growth. Although embryos grown with and without FLI appeared similar under the microscope, gene expression analysis revealed important differences. Embryos exposed to FLI showed signs of improved cell survival, healthier growth, and reduced stress. These molecular changes suggest that FLI may help embryos become more resilient to key procedures such as freezing and transfer. The findings support the use of FLI as a culture supplement to improve the efficiency and success of in vitro embryo production systems used in livestock reproductive biotechnologies.

Context Sires differ in their ability to produce viable blastocysts, yet our understanding of the cellular mechanisms regulated by the sire during early embryo development is limited. Aims The first aim was to characterise autophagy and reactive oxygen species (ROS) in embryos produced by high and low performing sires under normal and stress culture conditions. The second aim was to evaluate DNA damage and lipid peroxidation as mechanisms that may be impacted by increased cellular stress, specifically oxidative stress. Methods Embryos were produced using four high and four low performing sires based on their ability to produce embryos. Autophagy and ROS were measured throughout development. To evaluate oxidative stress response, autophagy, and ROS were measured in 2–6 cell embryos exposed to heat stress. To understand how cellular stress impacts development, DNA damage and lipid peroxidation were assessed. Key results Under normal conditions, embryos from low performing sires had increased ROS and autophagy. Under heat stress, embryos from low performing sires had increased ROS, yet those from high performing sires had increased autophagy. There was no difference in DNA damage or lipid peroxidation. Conclusions Results suggest that embryos from low performing sires may begin development under increased cellular stress, and autophagy potentially increases to mitigate the impacts of stress. Implications There is potential for improving embryonic competence through selection of sires with lower stress-related markers.

The use of in vitro embryo production (IVP) has increased globally, particularly in the United States. Although maternal factors influencing embryo development have been extensively studied, the influence of the sire is not well understood. Sperm plays a crucial role in embryo development providing DNA, triggering oocyte maturation, and aiding in mitosis. Current sire fertility measurements do not consistently align with embryo production outcomes. Low-fertility sires may perform well in IVP systems but produce fewer pregnancies. Testing sires in vitro could identify characteristics affecting embryo development and pregnancy loss risk in IVP and embryo transfer programs.

In brief:The localization and abundance of the sperm BSP proteins correlate with in vitro fertility in domestic bulls used in artificial insemination service. Abstract:Binder of sperm (BSP) proteins, secreted mainly by the accessory sex glands, are the major protein family present in bovine seminal plasma and on the sperm surface after ejaculation. In vivo, BSP proteins facilitate sperm capacitation and sperm reservoir formation; however, their impact on sperm function within the in vitro systems is less clear. Therefore, this biomarker-based study aimed to characterize the localization and abundance of BSP proteins from in vitro processed frozen-thawed bovine spermatozoa. Using image-based flow cytometry and Western blotting, BSP protein localization, abundance, membrane and acrosomal integrity were investigated in the supernatant (nonmotile) and pellet (motile) fractions of gradient-separated bull spermatozoa. Spermatozoa from the supernatant fraction had high enrichment of all BSP proteins investigated (BSP1, BSP3, BSP5; P < 0.05) when compared to the pellet fraction. In the pellet fraction, BSP1 and BSP3 bound predominately to the acrosomal region, whereas BSP5 had a high affinity for the midpiece. However, in the supernatant fraction, BSP proteins predominately coated the entire sperm surface resulting in the loss of regional specificity. High BSP protein abundance in the spermatozoa also correlated with acrosome and membrane damage. Whereas a high abundance of BSP5 correlated with low embryo cleavage rates, high abundance of BSP1 on the sperm head coincided with a high blastocyst rate. Therefore, changes in the quantity and localization of specific BSP proteins could act as potential biomarkers of sperm quality and fertility.