Oocyte developmental potential and embryo production before puberty in cattle.

With the development of in vitro technologies, embryos can be produced using oocytes retrieved directly from the ovaries, i.e., regardless of ovulation. This has allowed the use of different animal categories as oocyte donors, including prepubertal cattle. The advantages of using this strategy to shorten the generation interval and accelerate genetic gain over time were soon recognized, and the first offspring generated using oocytes collected from calves were born in the early 1990s. Nevertheless, embryo production from calves and prepubertal heifers remains a challenge. The oocytes collected before puberty present low in vitro developmental potential, and the subsequent blastocyst rates are consistently lower than those from pubertal females. The acquisition of developmental competence by the oocytes occurs progressively throughout the prepubertal period, which can be subdivided into an early, intermediate, and late prepubertal (or peripubertal) phases, each characterized by different physiological and endocrine features. Therefore, embryo yield increases with age but will only achieve its maximum after puberty. The most common strategy to improve oocyte developmental potential before puberty is the use of gonadotrophic stimulation prior to oocyte retrieval. The results with superstimulation, however, vary among studies, depending on the source, dose, and length of FSH treatment, as well as the age and breed of the donors. The use of calves and prepubertal heifers as oocyte donors should also consider the possible impacts of the oocyte retrieval technique (LOPU or OPU) and the use of exogenous hormones on their subsequent fertility and productive life.

Is it safe to use ovarian follicular fluid from cows seropositive for brucellosis in the production of in vitro embryos?

Animal reproduction biotechniques are important tools for the technological advancement of livestock, as they allow the selection of the reproductive potential of superior quality females and males; however, infectious diseases that have a predilection for the reproductive system can be a hindrance for the use of these technologies. Therefore, the present study aimed to detect Brucella spp. in the ovarian follicular fluid of brucellosis-positive bovine cows. A total of 47 bovine ovarian follicular fluid aspirates from cows, positive in tests for brucellosis and from Brucella-positive herd, were submitted to PCR. The primers used in the PCR were specific to the genus Brucella (bcsp31 gene). All 47 bovine aspirates were negative for Brucella spp. 0.00% (95% CI: 0.00-4.00%). Our results demonstrated that Brucella spp. was absent in the ovarian follicular fluid from seropositive cows, which indicates that Brucella spp.-infected cows could be used for reproductive biotechnologies carried out with follicular aspirates. Future studies are needed to more precisely evaluate the feasibility and safety of using these oocytes from brucellosis-seropositive cows to transfer embryos to heifers/cows not infected by Brucella, aiming to produce calves free of the infection.

Extracellular vesicles derived from bovine adipose-derived mesenchymal stromal cells enhance in vitro embryo production from lesioned ovaries.

BACKGROUND AND AIMS: Ovum pick-up (OPU) is an intrinsic step of in vitro fertilization procedures. Nevertheless, it can cause ovarian lesions and compromise female fertility in bovines. Recently, we have shown that intraovarian injection of adipose-derived mesenchymal stromal cells (AD-MSCs) effectively preserves ovarian function in bovines. Given that MSC-derived extracellular vesicles (MSC-EVs) have been shown to recapitulate several therapeutic effects attributed to AD-MSCs and that they present logistic and regulatory advantages compared to AD-MSCs, we tested whether MSC-EVs would also be useful to treat OPU-induced lesions. METHODS: MSC-EVs were isolated from the secretome of bovine AD-MSCs, using ultrafiltration (UF) and ultracentrifugation methods. The MSC-EVs were characterized according to concentration and mean particle size, morphology, protein concentration and EV markers, miRNA, mRNA, long noncoding RNA profile, total RNA yield and potential for induction of the proliferation and migration of bovine ovarian stromal cells. We then investigated whether intraovarian injection of MSC-EVs obtained by UF would reduce the negative effects of acute OPU-induced ovarian lesions in bovines. To do so, 20 animals were divided into 4 experimental groups (n = 5), submitted to 4 OPU cycles and different experimental treatments including vehicle only (G1), MSC-EVs produced by 7.5 × 106 AD-MSCs (G2), MSC-EVs produced by 2.5 × 106 AD-MSCs (G3) or 3 doses of MSC-EVs produced by 2.5 × 106 AD-MSCs, injected after OPU sessions 1, 2 and 3 (G4). RESULTS: Characterization of the MSC-EVs revealed that the size of the particles was similar in the different isolation methods; however, the UF method generated a greater MSC-EV yield. MSC-EVs processed by both methods demonstrated a similar ability to promote cell migration and proliferation in ovarian stromal cells. Considering the higher yield and lower complexity of the UF method, UF-MSC-EVs were used in the in vivo experiment. We evaluated three therapeutic regimens for cows subjected to OPU, noting that the group treated with three MSC-EV injections (G4) maintained oocyte production and increased in vitro embryo production, compared to G1, which presented compromised embryo production following the OPU-induced lesions. CONCLUSIONS: MSC-EVs have beneficial effects both on the migration and proliferation of ovarian stromal cells and on the fertility of bovines with follicular puncture injury in vivo.

Supplementation with MitoTEMPO before cryopreservation improves sperm quality and fertility potential of Piedmontese beef bull semen.

The purpose of this study was to improve the quality of frozen-thawed Piedmontese bull semen by incorporating MitoTEMPO (MT) in extended semen before cryopreservation. Semen was collected from 4 fertile bulls, using an artificial vagina, once weekly for 6 consecutive weeks. Semen samples were pooled, diluted with Bullxcell® extender, and supplemented with different concentrations of MT (0 as control, 5, 10, 20, 40, and 80 µM) before cooling, equilibration, and freezing procedures. The frozen-thawed semen was assessed for motility, vitality, acrosome intactness, plasma membrane integrity, DNA integrity, apoptosis, mitochondrial membrane potential, intracellular ROS level and in vitro fertilizing capability. The results showed that MT at concentrations of 10, 20, and 40 µM improved the total, progressive, and rapid motility directly after thawing while, at the highest tested concentration (80 µM), it decreased the progressive and rapid motility after 1, 2, and 3 h of incubation. The sperm kinetics including STR and LIN were noticeably increased at concentrations of 10, 20, and 40 µM directly after thawing (0 h), whereas the MT effect was variable on the other sperm kinetics during the different incubation periods. MitoTEMPO improved the sperm vitality at all tested concentrations, while the acrosomal and DNA integrity were improved at 20 µM and the mitochondrial membrane potentials was increased at 80 µM. The cleavage and blastocyst formation rates were significantly increased by using semen treated with 20 µM MT compared with controls. These findings suggest a potential use of MT mainly at a concentration of 20 µM as an additive in the cryopreservation media of bull semen to improve sperm quality.

Use of assisted reproductive technologies (ARTs) to shorten the generational interval in ruminants: current status and perspectives.

The challenges posed by climate change and increasing world population are stimulating renewed efforts for improving the sustainability of animal production. To meet such challenges, the contribution of genomic selection approaches, in combination with assisted reproductive technologies (ARTs), to spreading and preserving animal genetics is essential. The largest increase in genetic gain can be achieved by shortening the generation interval. This review provides an overview of the current status and progress of advanced ARTs that could be applied to reduce the generation time in both female and male of domestic ruminants. In females, the use of juvenile in vitro embryo transfer (JIVET) enables to generate offspring after the transfer of in vitro produced embryos derived from oocytes of prepubertal genetically superior donors reducing the generational interval and acceleration genetic gain. The current challenge is increasing in vitro embryo production (IVEP) from prepubertal derived oocytes which is still low and variable. The two main factors limiting IVEP success are the intrinsic quality of prepubertal oocytes and the culture systems for in vitro maturation (IVM). In males, advancements in ARTs are providing new strategies to in vitro propagate spermatogonia and differentiate them into mature sperm or even to recapitulate the whole process of spermatogenesis from embryonic stem cells. Moreover, the successful use of immature cells, such as round spermatids, for intracytoplasmic injection (ROSI) and IVEP could allow to complete the entire process in few months. However, these approaches have been successfully applied to human and mouse whereas only a few studies have been published in ruminants and results are still controversial. This is also dependent on the efficiency of ROSI that is limited by the current isolation and selection protocols of round spermatids. In conclusion, the current efforts for improving these reproductive methodologies could lead toward a significant reduction of the generational interval in livestock animals that could have a considerable impact on agriculture sustainability.

The effects of a glyphosate-based herbicide on the bovine gametes during an in vitro embryo production model.

Roundup® (R), while it is the most used herbicide globally, and its residues are ubiquitous in urban and suburban areas, its impact on vertebrates' safety remains highly debated. Here, in three in vitro experiments, we investigated the effects of a very low dose (1 ppm) of R on the fertilization capacity and embryo development in cattle. In the first experiment, frozen-thawed bull semen exposed to R for 1 h exhibited reduced motility parameters but unaffected fertilization ability. However, after in vitro fertilization, the rates of embryo formation were significantly lower compared to the untreated controls. In the second experiment, oocytes exposed to R during in vitro maturation showed reduced cleavage rates, and the embryo yield on days 7, 8, and 9 of embryo culture was significantly lower than that of the controls. In the third experiment, oocytes were matured in the presence of R and in a medium containing both R and Zinc, chosen to offer antioxidant protection to the oocytes. Day-7 blastocysts were analyzed for the expression of genes associated with oxidative stress, apoptosis, and epigenetic reprogramming. Exposure to R markedly suppressed embryo formation rates compared to the controls. The combination of R with Zinc restored the blastocyst yield, which on days 8 and 9 was comparable to that of the controls and higher than the groups exposed only to R on all days. The gene expression analysis revealed that R promotes oxidative stress development, triggers apoptosis, and induces epigenetic changes in developing embryos, while zinc presence alleviates these adverse effects of R. These findings imply that even at very low doses, R could be highly toxic, leading to functional abnormalities in both gametes, potentially affecting fertility in both genders.

Seminal cell-free DNA as a potential marker for in vitro fertility of Nellore bulls.

PURPOSE: This study aimed to identify a marker for freezability and in vitro fertility of sperm samples before freezing. METHODS: Semen was collected from nine Nelore bulls; half of the ejaculate was used for seminal plasma cell-free DNA (cfDNA) quantification, and the other half was cryopreserved. Evaluation of sperm movement using computer-assisted semen analysis and plasma membrane integrity and stability, acrosomal integrity, apoptosis, and mitochondrial potential using flow cytometry were performed on fresh and frozen/thawed semen at 0, 3, 6, and 12 h after thawing. Frozen/thawed sperm was also used for in vitro embryo production. cfDNA was extracted from each bull, and the total DNA and number of cell-free mitochondrial DNA (cfmtDNA) copies were quantified. Semen from each animal was used for IVF, and cleavage, blastocyst formation, and cell counts were evaluated. RESULTS: Two groups were formed and compared based on the concentrations of cfDNA and cfmDNA present: low-cfDNA and high-cfDNA and low-cfmtDNA and high-cfmtDNA. Up to 12 h post-thawing, there were no differences between the groups in the majority of the sperm parameters evaluated. Cleavage, day 6 and 7 blastocyst rates, and the number of cells were higher in the high cfDNA group than in the low cfDNA group. Similar results were observed for cfmtDNA, except for the number of cells, which was similar between the groups. CONCLUSION: The concentration of cfDNA and the relative number of copies of cfmtDNA in seminal plasma cannot predict the freezability of semen but can be used to predict in vitro embryo production.

Effect of IGFBP-4 during In Vitro Maturation on Developmental Competence of Bovine Cumulus Oocyte Complexes.

Insulin-like growth factors (IGFs) are essential for oocyte maturation. Their bioavailability is regulated by their respective binding proteins (IGFBPs) and proteases. IGFBP-4 blocks the biological effects of IGFs. High IGFBP-4 expression has been associated with follicle atresia. We hypothesized that IGFBP-4 affects oocyte developmental competence during maturation. Therefore, the aim of this study was to examine the effect of IGFBP-4 on the developmental rate of bovine cumulus-oocyte complexes (COCs) during in vitro embryo production. Abattoir-derived COCs were matured with rbIGFBP-4 (2000, 540, and 54 ng/mL) compared to a control. Cumulus expansion, oocyte maturation, cleavage, blastocyst, and hatching rates were evaluated. Furthermore, blastocyst gene expression of SOCS2, STAT3, SLC2A1, SLCA3, BAX, and POU5F1 transcripts were quantified using RT-qPCR. No statistical differences were detected among the groups for cumulus expansion, maturation, cleavage, blastocyst rates, or all gene transcripts analyzed. However, at day 8 and 9, the number of total hatching and successfully hatched blastocysts was lower in 2000 ng/mL rbIGFBP-4 compared to the control (day 8: total hatching: 17.1 ± 0.21 vs. 31.2 ± 0.11%, p = 0.02 and hatched blastocyst 6.7 ± 0.31 vs. 21.5 ± 0.14%, p = 0.004; day 9 total hatching 36.4 ± 0.18 vs. 57.7 ± 0.10%, p = 0.009 and hatched blastocyst 18.2 ± 0.21 vs. 38.1 ± 0.11%, p = 0.004). We concluded that high concentrations of rbIGFBP-4 might negatively affect the subsequent ability of the embryo to hatch and possibly compromise further elongation.

Fertilization with follicular fluid reduces HSP70 and BAX expression on bovine in vitro embryos.

The in vivo fertilization process occurs in the presence of follicular fluid (FF). The aim of this study was to evaluate the effect of in vitro fertilization medium supplementation with 5% or 10% bovine follicular fluid (BFF) on the production of in vitro bovine embryos. FF was collected from ovarian follicles with a diameter of 8-10 mm, and cumulus-oocyte complexes (COCs) were co-incubated with sperm for 24 h in the commercial medium BotuFIV® (BotuPharma©), being distributed among the experimental groups: oocytes fertilized in a control medium; oocytes fertilized in a medium supplemented with 5% BFF; and oocytes fertilized in a medium supplemented with 10% BFF. After fertilization, the zygotes were cultured in vitro for 8 days. Embryo development was assessed through cleavage rates (day 2) and blastocyst formation rates (day 8). The relative expression of the genes OCT4, IFNT2, BAX, HSP70 and SOD2 was measured using the real-time polymerase chain reaction method. There was no difference (p > .05) among the different experimental groups in terms of cleavage rates and blastocyst formation rates. Regarding the gene expression results, only the blastocysts from oocytes fertilized with 10% BFF showed significantly lower expression of IFNT2 (p = .003) and SOD2 (p = .01) genes compared to blastocysts from oocytes fertilized in control medium alone, while there was no difference between blastocyst from oocytes fertilized in control medium and the ones from oocytes fertilized with 5% BFF. In addition to this, the blastocysts from oocytes fertilized with 5% BFF showed significantly reduced levels of expression of the heat shock protein HSP70 (p < .001) and the pro-apoptotic protein BAX (p = .015) compared to blastocysts from oocytes fertilized with control medium. This may indicate that lower supplementation of BFF to the IVF medium creates a more suitable environment for fertilization and is less stressful for the zygote.

Interleukin-6 supplementation improves bovine conceptus elongation and transcriptomic indicators of developmental competence†.

A high incidence of pregnancy failures occurs in cattle during the second week of pregnancy as blastocysts transition into an elongated conceptus. This work explored whether interleukin-6 supplementation during in vitro embryo production would improve subsequent conceptus development. Bovine embryos were treated with 0 or 100 ng/mL recombinant bovine interleukin-6 beginning on day 5 post-fertilization. At day 7.5 post-fertilization, blastocysts were transferred into estrus synchronized beef cows (n = 5 recipients/treatment, 10 embryos/recipient). Seven days after transfer (day 14.5), cows were euthanized to harvest reproductive tracts and collect conceptuses. Individual conceptus lengths and stages were recorded before processing for RNA sequencing. Increases in conceptus recovery, length, and the proportion of tubular and filamentous conceptuses were detected in conceptuses derived from interleukin-6-treated embryos. The interleukin-6 treatment generated 591 differentially expressed genes in conceptuses (n = 9-10/treatment). Gene ontology enrichment analyses revealed changes in transcriptional regulation, DNA-binding, and antiviral actions. Only a few differentially expressed genes were associated with extraembryonic development, but several differentially expressed genes were associated with embryonic regulation of transcription, mesoderm and ectoderm development, organogenesis, limb formation, and somatogenesis. To conclude, this work provides evidence that interleukin-6 treatment before embryo transfer promotes pre-implantation conceptus development and gene expression in ways that resemble the generation of a robust conceptus containing favorable abilities to survive this critical period of pregnancy.

The ability of donkey sperm to induce oocyte activation and mule embryo development after ICSI.

Members of the Equus genus exhibit a fascinating capacity for hybridization, giving rise to healthy offspring. Mules, resulting from the mating of a mare with a jack, represent the most prevalent equid hybrid, serving diverse roles in our society. While in vitro embryo production, particularly through Intracytoplasmic Sperm Injection (ICSI), has rapidly gained significance in domestic horses, the in vitro production in other equids remains largely unexplored. Utilizing donkey sperm for fertilizing horse oocytes not only addresses this gap but also provides an opportunity to investigate donkey sperm's fertilization capability in vitro to further improve donkey ICSI. In this work, we initially studied the localization of donkey sperm Phospholipase C zeta (PLCζ) and assessed the sperm's capacity to induce pronuclear formation and maternal SMARCA4 recruitment upon injection into pig oocytes through ICSI. Subsequently, we investigated the injection of donkey sperm into horse oocytes, evaluating in vitro production up to the blastocyst stage using sperm from different jacks, including frozen and refrigerated samples. Distinct patterns of PLCζ localization were observed for donkey sperm cells compared to their horse counterparts. Additionally, donkey sperm exhibits a reduced ability to induce porcine oocyte activation. However, when injected into horse oocytes, donkey sperm demonstrated sufficient capability to induce oocyte activation as no discernible differences in cleavage or blastocyst rates are observed between in vitro produced mules and horse ICSI embryos. Our study not only delineates PLCζ localization in donkey sperm but also suggests potential differences in the ability to induce oocyte activation in pigs compared to horses while observing no distinctions in pronuclear recruitment of SMARCA4. Interestingly, donkey sperm remains sufficiently capable of inducing horse oocyte activation for in vitro mule blastocyst production.

Effect of split weaning on follicle development and oocyte quality in multiparous sows.

Increasing piglet weaning age while maintaining the reproductive efficiency of the breeding herd depends on being able to stimulate sows to ovulate during lactation without reducing subsequent pregnancy rates and litter sizes. Embryo survival is affected by the quality of the oocytes shed at ovulation, and oocyte quality is profoundly impacted by the follicular environment in which the oocyte matures. This study determined the effect of reducing suckled litter size from 11 to 7 piglets on day 18 of lactation on the ovarian follicular environment and oocyte developmental competence at day 21 of lactation. Thirty-nine, Large White X Landrace sows (parity 3.2 ± 0.2; mean ± SEM; range 2-6) had their litter size either maintained at 11 piglets (control); or reduced to seven piglets on day 18 of lactation (split wean (SW)). Sows were slaughtered on day 21 of lactation and ovaries were collected for analysis of follicular fluid composition and in vitro blastocyst development rates. There was no effect of split weaning on fertilisation rate and development to blastocyst stage; however, a greater proportion of blastocysts from control sows were classified as early blastocyst stage. Furthermore, follicular fluid concentrations of oestradiol were higher in SW sows. Together, these results indicate split weaning prior to mating in lactation alters the ovarian follicular environment and while blastocyst development rates were unaffected, embryos from control sows may be of poorer quality as indicated by a delay in development.

Field outcomes of laparoscopic ovum pick-up combined with in vitro embryo production in sheep: Effects of long-acting recombinant ovine FSH pre-stimulation, collection frequency, and donor breed.

Laparoscopic ovum pick-up (LOPU) combined with in vitro embryo production (IVEP) is a technology platform that improves the utilization rate of the elite ewe's ovarian oocytes and increases the number of obtained offspring. This study aimed to evaluate the effects of FSH pre-stimulation, serial oocyte collection, and breed on LOPU-IVEP under field conditions. Donors were randomly assigned to five groups (group A: decreasing doses of pituitary FSH (p-FSH); group B: constant doses of p-FSH; group C: two doses of long-acting recombinant ovine FSH (ro-FSH); group D: single administration of a long-acting ro-FSH in; group E: no FSH stimulation). Oocyte yield following LOPU (average recovered oocytes: 20.9 ± 0.5; average viable oocytes: 17.2 ± 0.4) and oocyte developmental competence (average blastocysts: 7.0 ± 0.2) in group C were significantly better than these of group D and group E, and similar to these of groups A and B. Meanwhile, there were no differences in oocyte yield and developmental capacity using repeated LOPU session at 1-, 2-, and 3-month intervals (p > 0.05). Finally, we compared LOPU-IVEP outcomes among five sheep breeds. The results indicated that East Friesian × Chinese Mongolian crossbred sheep and purebred East Friesian sheep had the more recovered oocytes and viable oocytes compared with the Suffolk, Dorper, and Texel breeds, and average number of blastocysts in East Friesian × Chinese Mongolian sheep group was also highest among the groups (8.1 ±0.3, p < 0.05). In summary, the results of this study indicate long-acting ro-FSH pre-stimulation combined with 12 times LOPU sessions over one year maximizes embryo production of elite donor ewes under field conditions.

Manual versus deep learning measurements to evaluate cumulus expansion of bovine oocytes and its relationship with embryo development in vitro.

Cumulus expansion is an important indicator of oocyte maturation and has been suggested to be indicative of greater oocyte developmental capacity. Although multiple methods have been described to assess cumulus expansion, none of them is considered a gold standard. Additionally, these methods are subjective and time-consuming. In this manuscript, the reliability of three cumulus expansion measurement methods was assessed, and a deep learning model was created to automatically perform the measurement. Cumulus expansion of 232 cumulus-oocyte complexes was evaluated by three independent observers using three methods: (1) measurement of the cumulus area, (2) measurement of three distances between the zona pellucida and outer cumulus, and (3) scoring cumulus expansion on a 5-point Likert scale. The reliability of the methods was calculated in terms of intraclass-correlation coefficients (ICC) for both inter- and intra-observer agreements. The area method resulted in the best overall inter-observer agreement with an ICC of 0.89 versus 0.54 and 0.30 for the 3-distance and scoring methods, respectively. Therefore, the area method served as the base to create a deep learning model, AI-xpansion, which reaches a human-level performance in terms of average rank, bias and variance. To evaluate the accuracy of the methods, the results of cumulus expansion calculations were linked to embryonic development. Cumulus expansion had increased significantly in oocytes that achieved successful embryo development when measured by AI-xpansion, the area- or 3-distance method, while this was not the case for the scoring method. Measuring the area is the most reliable method to manually evaluate cumulus expansion, whilst deep learning automatically performs the calculation with human-level precision and high accuracy and could therefore be a valuable prospective tool for embryologists.

Phenotypic and Genetic Analyses of In Vitro Embryo Production Traits in Chinese Holstein Cattle.

Ovum pick up and in vitro embryo production (OPU-IVEP) is an essential technique in the dairy industry. The production efficiency of OPU-IVEP is significantly influenced by various factors, and phenotypic and genetic characteristics are highly variable in different populations. The objectives of this study were (1) to reveal the phenotypic characteristics, including population distribution, and impacts of donor age and month on in vitro embryo production and (2) to estimate genetic parameters for five in vitro embryo production traits in Chinese Holstein cattle. A total of 7311 OPU-IVEP records of 867 Holstein heifers from August 2021 to March 2023 were collected in this study. Five in vitro embryo production traits were defined, including the number of cumulus-oocyte complexes (NCOC), the number of cleaved embryos (NCLV), the number of grade I embryos (NGE), and the proportion of NCLV to NCOC (PCLV) and NGE to NCOC (PGE). A univariate repeatability animal model was employed to estimate heritability and repeatability, and a bivariate repeatability animal model was employed to estimate the genetic correlations among five in vitro embryo production traits. It was found that the in vitro embryo production traits were significantly influenced by season, as the NGE and PGE were significantly decreased from June to August. In addition, the production efficiency of OPU-IVEP was also influenced by donor age. On the observed scale, the estimates of heritability were 0.33 for NCOC, 0.24 for NCLV, 0.16 for NGE, 0.06 for PCLV, and 0.10 for PGE, respectively. On the log-transformed scale, the estimates of heritability of NCOC, NCLV, and NGE were 0.34, 0.18, and 0.13. The genetic correlations among NCOC, NCLV, and NGE ranged from 0.61 (NCLV and NGE) to 0.95 (NCOC and NCLV), considering both scales. However, there were low genetic correlations between NCOC and proportion traits (PCLV and PGE) on both the observed scale and the log-transformed scale. In the end, the variation in Chinese Holstein cattle was found to be considerable. The EBV value and average NCOC, NGE, and PGE for the top 10% donors presented extreme differences to those for the bottom 10% donors for NCOC (24.02 versus 2.60), NGE (3.42 versus 0.36), and PGE (30.54% versus 3.46%). Overall, the results of this study reveal that in vitro embryo production traits are heritable with low to high heritability, and the count traits (NCOC, NCLV, and NGE) and proportion traits (PCLV and PGE) reflect different aspects of in vitro embryo production and should be incorporated into genetic selection for improving the embryo production efficiency of dairy cattle.

Developmental Hurdles That Can Compromise Pregnancy during the First Month of Gestation in Cattle.

Several key developmental events are associated with early embryonic pregnancy losses in beef and dairy cows. These developmental problems are observed at a greater frequency in pregnancies generated from in-vitro-produced bovine embryos. This review describes critical problems that arise during oocyte maturation, fertilization, early embryonic development, compaction and blastulation, embryonic cell lineage specification, elongation, gastrulation, and placentation. Additionally, discussed are potential remediation strategies, but unfortunately, corrective actions are not available for several of the problems being discussed. Further research is needed to produce bovine embryos that have a greater likelihood of surviving to term.

Transcriptome Signature of Immature and In Vitro-Matured Equine Cumulus-Oocytes Complex.

Maturation is a critical step in the development of an oocyte, and it is during this time that the oocyte advances to metaphase II (MII) of the meiotic cycle and acquires developmental competence to be fertilized and become an embryo. However, in vitro maturation (IVM) remains one of the limiting steps in the in vitro production of embryos (IVP), with a variable percentage of oocytes reaching the MII stage and unpredictable levels of developmental competence. Understanding the dynamics of oocyte maturation is essential for the optimization of IVM culture conditions and subsequent IVP outcomes. Thus, the aim of this study was to elucidate the transcriptome dynamics of oocyte maturation by comparing transcriptomic changes during in vitro maturation in both oocytes and their surrounding cumulus cells. Cumulus-oocyte complexes were obtained from antral follicles and divided into two groups: immature and in vitro-matured (MII). RNA was extracted separately from oocytes (OC) and cumulus cells (CC), followed by library preparation and RNA sequencing. A total of 13,918 gene transcripts were identified in OC, with 538 differentially expressed genes (DEG) between immature OC and in vitro-matured OC. In CC, 13,104 genes were expressed with 871 DEG. Gene ontology (GO) analysis showed an association between the DEGs and pathways relating to nuclear maturation in OC and GTPase activity, extracellular matrix organization, and collagen trimers in CC. Additionally, the follicle-stimulating hormone receptor gene (FSHR) and luteinizing hormone/choriogonadotropin receptor gene (LHCGR) showed differential expressions between CC-MII and immature CC samples. Overall, these results serve as a foundation to further investigate the biological pathways relevant to oocyte maturation in horses and pave the road to improve the IVP outcomes and the overall clinical management of equine assisted reproductive technologies (ART).

Effect of use and dosage of p-follicle-stimulating hormone for ovarian superstimulation before ovum pick-up and in vitro embryo production in pregnant Holstein heifers.

The benefit of ovarian superstimulation using exogenous FSH before ovum pick-up (OPU) and in vitro embryo production (IVEP) has been the subject of conflicting results. The objective of the present study, therefore, was to evaluate the effect of use and dose of porcine FSH (p-FSH) before OPU/IVEP on ovarian response and embryo production in pregnant heifers. Pregnant Holstein heifers (n = 48) were randomly assigned to receive 0, 160, or 300 mg NIH-FSH-P1 in a crossover design. Ovum pick-up was performed at 49, 63, and 77 d of gestation with a 14 d "washout" between OPU sessions. Follicle ablation was performed on D 0 (p.m.) and p-FSH treatments, consisting of 4 decreasing dose injections administered 12 h apart, were initiated 36 h after follicle ablation (d 2 a.m.). Heifers underwent OPU on D 5 (a.m.), 40 h after the last p-FSH treatment, and cumulus oocyte complexes (COC) were subjected to IVEP procedures. Differences between treatment groups were evaluated using generalized linear mixed models. There were quadratic effects of treatment on both number and percentage of small (<6 mm), medium (6-10 mm), and large (>10 mm) follicles. Number and percentage of medium follicles increased with increasing p-FSH dosages, although the magnitude of the change was greater between 0 and 160 mg, than between 160 and 300 mg of p-FSH. Total number of follicles, number of COC recovered and number of viable COC increased linearly with increasing p-FSH dose. Conversely, there was no evidence for an effect of p-FSH dose on COC recovery percentage nor the percentage of viable COC. Cleavage percentage, number of cleaved oocytes, blastocyst percentage, and number of blastocysts increased linearly with increasing p-FSH dose. In conclusion, use of p-FSH before OPU resulted in greater superstimulatory response and oocyte competence which in turn increased IVEP. Furthermore, these effects were dose dependent such that use of a greater dose of p-FSH up to 300 mg progressively increased embryo yield.

Evaluation of the effects of the recommended oral dose of diflubenzuron on bovine sperm and oocyte quality using CASA and OPU-IVEP.

Introduction: The aim of this study was to evaluate potential effects of diflubenzuron on the production and quality of gametes, and on in vitro embryo production (IVEP) outcomes, in cattle. Methods: Two experiments were performed, the first to evaluate effects on semen, and the second on cumulus-oocyte complexes (COC) and on IVEP. Nelore (Bos taurus indicus) bulls (n = 14) or heifers (n = 16) were allocated into control (CG) or treatment (DIF) groups. All groups received a mineral mix supplement added (DIF) or not (CG) with diflubenzuron (30 mg/head/day), during 8 weeks. Animals were weighed and blood samples were collected throughout the experimental period. Every other week, bulls were subjected to semen collection and heifers to transvaginal ultrasound-guided follicle aspiration sessions. Semen underwent physical and morphological evaluation, and samples were stored for further computer-assisted sperm analysis. The COC recovered were evaluated according to morphology and those classified as viable were sent to an IVEP laboratory. Results: Diflubenzuron had no effect (P > 0.05) on average body weight or in any blood hematological or biochemical endpoints, regardless of gender. In experiment 1, there was no difference (P > 0.05) between DIF and CG groups for sperm concentration, morphology, or kinetics. In experiment 2, there was also no effect of diflubenzuron on the number of total, viable, or grade I oocytes, as well as on cleavage or blastocyst rates (P > 0.05). Discussion: In summary, the oral administration of diflubenzuron, within the recommended dose, has no short-term negative effects on sperm production and quality or on oocyte yield and developmental potential in vitro, in cattle.

Zinc oxide nanoparticles functionalized with curcumin supplementation during in vitro embryo culture impaired in a concentration-dependent-manner blastocyst production in cattle.

This work investigated the effect of zinc oxide nanoparticles functionalized with curcumin (ZnO(np) + CUR) supplementation during the in vitro embryo culture (IVC) on the bovine in vitro embryo production, and the cellular antioxidant response. The cumulus-oocyte complexes (COCs) were matured, fertilized and then the presumptive zygotes were cultured in the medium in the absence (0 µM-control) or presence of different concentrations of ZnO(np) + CUR (3, 6 or 12 µM). After IVC, the embryos were destined either to assay intracellular ROS levels and mitochondrial membrane potential. The results demonstrated that only the addition of 12 µM ZnO(np) + CUR during IVC decreased intracellular ROS production and the rate of blastocyst production when compared to the control (p < .05). In conclusion, ZnO(np) + CUR addition during the IVC impaired in concentration-dependent-manner bovine in vitro embryo production.