Invitro-produced Bovine Embryos Research Articles

In vitro developmental competence of bovine demi-embryos generated by blastomere separation and blastocyst bisection.

The efficiency of bovine invitro embryo production can be significantly improved by splitting embryos at different stages. However, the blastocyst quality of invitro-produced demi-embryos remains unexplored. The objective of this research was to compare embryo developmental rates and quality of bovine demi-embryos produced by two different strategies: (a) embryo bisection (BSEC) and (b) 2-cell blastomere separation (BSEP). To determine demi-embryos quality, we evaluated total blastocyst cell number and proportion of SOX2+ cells. Additionally, the expression of SOX2, NANOG, OCT4, CDX2, IFNT, BAX and BCL genes and let-7a and miRNA-30c Micro RNAs was analysed. BSEP resulted in improved blastocyst development, higher ICM cells and a significantly higher expression of IFNΤ than demi-embryos produced by BSEC. Let-7a, which is associated with low pregnancy establishment was detected in BSEC, while miRNA-30c expression was observed in all treatments. In conclusion, BSEP of 2-cell embryos is more efficient to improve invitro bovine embryo development and to produce good quality demi-embryos based on ICM cell number and the expression pattern of the genes explored compared to BSEC.

Oviduct and endometrial epithelium improve in vitro produced bovine embryo developmental kinetics.

Standard in vitro produced (IVP) bovine embryo culture media limit embryonic development. Culturing IVP bovine embryos in standard IVP bovine embryo culture media conditioned with oviduct and/or endometrial cells improves blastocyst formation and reduces the time to formation. In vitro embryo production in cattle greatly impacts blastomere biochemistry, embryo rate of development and pre- and post-transfer survival. In vivo, the bovine embryo migrates through the oviduct isthmus before entering the uterus on approximately day 4 of development where it remains unattached within the uterine lumen until day 20 of gestation. During this time, the embryo is sequentially exposed to oviduct followed by endometrial secretions that support embryonic development. Considering this, we tested the effect of culturing in vitro produced (IVP) bovine embryos sequentially in oviduct epithelial- (OEp; days 1-3) followed by endometrial epithelial- (EEp) or EEp and fibroblast cell (EEp/F; days 4-8)-conditioned media on embryonic development using a time-lapse monitoring system. Compared to control, culturing IVP embryos in EEp- or EEp/F-conditioned media without prior culture in OEp-conditioned media increased blastocyst formation (P < 0.05) and reduced the time to blastocyst formation (P < 0.05). Culturing IVP bovine embryos in OEp-conditioned media followed by EEp- or EEp/F-conditioned media, however, had the greatest impact on embryo developmental kinetics and increased morula and blastocyst formation (P < 0.05) and reduced time to formation (P < 0.05). Day 8 blastocyst cell numbers, diameter and quality were not significantly different, although, blastocyst quality scores were less (indicative of better quality) for all cell-conditioned media compared to control. In conclusion, IVP bovine embryo development may be improved using a sequential embryo culture system involving bovine oviduct followed by endometrial cell-conditioned media.

298 Interleukin-6 Family Members Improve Pre-Implantation Bovine Blastocyst Composition

Abstract Inner cell mass (ICM) size is a predictor of bovine blastocyst quality and post-transfer pregnancy success. In vitro produced (IVP) embryos have reduced ICM cell numbers compared with in vivo produced embryos, and we propose this is one reason pregnancy success is compromised in IVP embryos. Our recent work found that human recombinant interleukin-6 (IL6) supplementation during in vitro embryo culture increased ICM cell numbers. We hypothesized that other human recombinant members of the IL6 cytokine family, specifically interleukin-11 (IL11) and leukemia inhibitory factor (LIF), will have a similar response. In vitro maturation, fertilization and culture procedures were used to produce embryos that were treated with 100 ng/mL recombinant human recombinant IL6, IL11, LIF, or carrier [control; 1% (wt/vol) SOF-BSA] beginning at day 6 post-fertilization (25 embryos/50 mL drop; 3 drops/treatment; 5 replicates). A subset of day 8 blastocysts (n = 28 to 29) was fixed and processed for cell counting. Analyses were performed by least-squares ANOVA using the general linear model. No treatment differences in overall blastocyst rates, trophectoderm (TE) cell numbers, and total cell numbers were observed at day 8. Treatment with IL6 (P = 0.09) and LIF (P = 0.06) tended to increase ICM cell numbers when compared with the control, whereas IL11 was not different from the control. Treatment with IL6 increased the ICM:TE ratio (P &amp;lt; 0.05) but the other cytokines did not influence this ratio. Several treatment by stage interactions were observed. In regular day 8 blastocysts (n = 10 to 14), none of the treatments affected ICM numbers or the ICM:TE ratio, but LIF increased TE number (P &amp;lt; 0.05) and IL6 increased total cell number (P &amp;lt; 0.05). When grouping expanded and hatched blastocysts together (n = 14 to 18), each treatment increased ICM cell numbers and IL6 increased the ICM:TE ratio (P &amp;lt; 0.05). Furthermore, both IL11 (P = 0.08) and LIF (P = 0.08) tended to increase the ICM:TE ratio. Only LIF increased total cell numbers (P &amp;lt; 0.05). None of the other treatments affected total cell numbers. These observations suggest that IL6, IL11, and LIF are capable of influencing ICM cell numbers in IVP bovine embryos, but they may possess different abilities in affecting TE and overall cell numbers. These findings differ from previous findings from our lab. We suspect that transitioning from bovine recombinant preparations with undefined purity and bioactivity to using purified human recombinant proteins with high bioactivity allowed us to detect these responses for each cytokine. Collectively, this work implicates several members of the IL6 cytokine family as embryokines that may be used to improve bovine IVP embryo quality. Funding for this work was provided by USDA-NIFA (2017-67015-2646 and 2021-67015-34485) and by NIH (R21-OD026516-01).

Modulating the lipid profile of blastocyst cell membrane with DPPC multilamellar vesicles

The aim of this study was to evaluate the effect of multilamellar vesicles (MLVs) of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in co-culture with in vitro-produced bovine embryos (IVPEs). The stability of five concentrations of MLVs (1.0, 1.25, 1.5, 1.75, and 2.0 mM) produced using ultrapure water or embryonic culture medium with 24 or 48 h of incubation at 38.5 °C with 5% CO2 was assessed. In addition, the toxicity of MLVs and their modulation of the lipid profile of the plasma membrane of IVPEs were evaluated after 48 h of co-culture. Both media allowed the production of MLVs. Incubation (24 and 48 h) did not impair the MLV structure but affected the average diameter. The rate of blastocyst production was not reduced, demonstrating the nontoxicity of the MLVs even at 2.0 mmol/L. The lipid profile of the embryos was different depending on the MLV concentration. In comparison with control embryos, embryos cultured with MLVs at 2.0 mmol/L had a higher relative abundance of six lipid ions (m/z 720.6, 754.9, 759.0, 779.1, 781.2, and 797.3). This study sheds light on a new culture system in which the MLV concentration could change the lipid profile of the embryonic cell membrane in a dose-dependent manner.

51 Comparison of SexedULTRA 4M and conventional semen for invitro production of bovine embryos using two bulls

SexedULTRA 4M™ (Select Sires) is made using an improved method of sex-sorting sperm in a less damaging environment for retaining sperm integrity through the sorting process. The combination of invitro embryo production (IVP) and sexed semen technologies has been successful for intensity selection in the cattle industry. The objective of this research was to compare conventional (CONV) and SexedULTRA 4M (ULTRA-4M) semen for bovine IVP using 2 bulls. The research was carried out in the reproduction laboratory at Palominos Ranch (Jalisco, México). The IVP was performed with a continuous invitro culture system. Ovaries from commercial cattle (n=213) were collected from a slaughterhouse (León, México) and transported to the laboratory within 2h in physiological saline solution (0.9% NaCl) supplemented with penicillin G (100IUmL−1) and streptomycin sulphate (100µg mL−1). For IVP, IVF Bioscience™ media were used for IVM, IVF, and invitro culture (IVC). For the IVM, the cumulus–oocyte complexes (COCs) were selected (grades 1 and 2) and matured for 24h at 38.5°C in 5% CO2 in air and 100% humidity. Matured oocytes (n=1200, divided equally into 3 replicates) were divided into 2 groups, the CONV group and the ULTRA-4M group. The IVF process was conducted with both CONV and ULTRA-4M semen from the 2 bulls, separately, at an adjusted concentration of 2×106 and 0.5×106 sperm mL−1, respectively, for 18h in 38.5°C, 5% CO2 in air, and 100% humidity. The presumptive zygotes were denuded by pipetting and set in IVC until Day 7 at 38.5°C, 5% CO2, 5% O2 and 90% N2 at 100% humidity. The cleavage rate, embryos with more than 6 cells, and blastocysts on Day 7 of culture were evaluated. Statistical analysis was carried out with the GLM procedure of SAS (version 9.3; SAS Institute Inc.) to evaluate the results of CONV versus ULTRA-4M for each bull (α level=0.05). Cleavage rates were 57.1%±1.5 and 59.4%±1.5, respectively in CONV and ULTRA-4M groups with Bull 1 and 43.3%±1.5 and 45.2%±1.5 with Bull 2. The percentages of embryos with more than 6 cells were 51.4%±1.0 and 53.9%±1.0, respectively, with CONV and ULTRA-4M with Bull 1 and 30.4%±1.0 and 33.4%±1.0 with Bull 2. The percentage of blastocysts on Day 7 with Bull 1 was 34.4%±1.7 for CONV and 36.2%±1.7 for ULTRA-4M; for Bull 2, the results were 26.5%±1.7 for CONV and 29.5%±1.7 for ULTRA-4M. There were no significant differences between the CONV and ULTRA-4M groups (P&amp;gt;0.05) for all variables analysed for each bull; however, Bull 1 was significantly superior to Bull 2 for all variables analysed. In conclusion, under the conditions of this research, ULTRA-4M and CONV semen produced similar bovine IVP results.

Effect of vitrification on global gene expression dynamics of bovine elongating embryos.

Embryo vitrification involves exposure to high concentrations of cryoprotectants and osmotic stress during cooling and warming in the cryopreservation process. Many of these factors can potentially affect gene expression. In this study, invitro-produced bovine embryos at the blastocyst stage were subjected to vitrification. Four recipients each were used for transferring non-vitrified (n=80) and vitrified (n=80) embryos. A total of 12 non-vitrified and 9 vitrified viable day-14 (D14) embryos were recovered by uterine flushing. RNA-seq analysis of the whole embryo or isolated trophectoderm (TE) from vitrified and fresh recovered D14 embryos revealed a total of 927 and 4376 genes with changed expression in embryos and TE isolates, respectively, as a result of vitrification. In addition, we found 671 and 61 genes commonly up- or downregulated in both vitrified whole embryos and TE. Commonly upregulated pathways by vitrification included epithelial adherens junctions, sirtuin signalling, germ cell-sertoli cell junction, ATM signalling, NER and protein ubiquitination pathways. The commonly downregulated pathways included EIF2 signalling, oxidative phosphorylation, mitochondrial dysfunction, regulation of eIF4 and p70S6K signalling and mTOR signalling pathways. Our analysis identified specific pathways and implicated specific gene expression patterns affecting embryo developmental competence that are important to cryopreservation.

62 Investigating differences in gene expression between invitro-produced bovine embryos and parthenotes

Parthenogenic activation allows for the development of an oocyte without male gamete contribution and may serve as a suitable model for understanding maternal and paternal contributions during development. In the bovine, parthenotes lack the ability to survive to term once transferred into females. The goal of this study was to investigate the gene expression profile at the blastocyst stage in parthenotes and embryos by characterising expression of developmentally important genes, such as markers for pluripotency (OCT4, NANOG), hypoblast (GATA6), epiblast (SOX2), trophectoderm (CDX2), and maternal-embryo communication (IFNT2). To test this, IVM oocytes were either fertilized to a bull with known fertility invitro or activated. To activate, oocytes were denuded, and placed in ionomycin calcium salt for 5min. They were incubated for 3h in 6-(dimethylamino)purine (6-DMAP) and placed in synthetic oviductal fluid (SOF). Putative zygotes and activated oocytes were cultured in SOF for 8 days. Cleavage (at least one cellular division) was recorded on Day 3 and development to the blastocyst stage was recorded on Day 8 after insemination or activation. Cleavage rate was 85.9% and 83.2% for parthenotes and embryos, respectively. Both groups produced a similar blastocyst rate, 32.3% for parthenotes and 33.7% for embryos. On Day 8, blastocyst stage parthenotes and embryos were collected. Data were analysed by ANOVA (Tukey HSD post hoc test) using the GLM procedure of SAS version 9.4. Pools of 5 embryos or parthenotes (3 replicates) were flash frozen and stored until RNA isolation (PicoPure™ RNA Isolation Kit). Real-time PCR was used for quantification of gene expression. Genes were analysed relative to a housekeeping gene, GAPDH. There was no difference in gene expression detected for OCT4 (P=0.25), NANOG (P=0.11), GATA6 (P=0.32), SOX2 (P=0.25), or IFNT2 (P=0.52). Expression of CDX2 was lower in the parthenotes than the embryos (P=0.05). In a second experiment, the proteins GATA6, NANOG, and CDX2 were immunolocalized in 17 parthenotes and 15 embryos. Fixed embryos were permeabilized, blocked, and placed in primary antibodies overnight. After, they were placed into the secondary antibody for 1h, followed by nuclear stain. There was a decreased mean intensity of CDX2 in the parthenotes compared to embryos (P=0.005). No difference (P&amp;gt;0.05) in GATA6 or NANOG was observed between the 2 groups. The ratio of inner cell mass to trophectoderm was higher (P=0.04) in the parthenotes (2.5±0.23) than in the embryos (1.7±0.25). Here we analysed and confirmed the expression of developmentally important genes at the blastocyst stage in embryos and parthenotes. CDX2, a marker of the trophectoderm that will later give rise to the placenta, was downregulated in parthenotes. This highlights the importance of the contribution of the paternal genome to development. Further research is necessary to elucidate the ability of the parthenotes to establish and maintain pregnancy. This research was supported by the National Needs Fellowship funded by USDA NIFA Grant 2019-38420-28972.

136 GameteGuard in maturation medium improves bovine embryo quality and quantity

The number of invitro-produced (IVP) bovine embryos generated in the United States increased 11% from 2017 to 2018, demonstrating a demand for new technology to rapidly and efficiently improve herd genetics. Unfortunately, embryo production on a per oocyte basis remains low, limiting the number of high-quality commercial embryos available for transfer or freezing. Reactive oxygen species (ROS) are a major contributor to low embryo production rates of invitro gametes and are especially problematic in maturation media. This is due to high numbers of mitochondria in the bovine oocyte producing ROS exacerbated by the invitro environment. GameteGuard® is an organic plant-based additive that provides protection against reactive oxygen species. The aims of this research were to evaluate the effects of GameteGuard addition to invitro bovine embryo production media to (1) mitigate the impact of ROS on oocyte maturation and competence, and (2) increase the production of high-quality embryos in our IVP system. Four rounds of invitro embryo production (Colorado State University protocol and media) with or without maturation medium supplemented with GameteGuard were performed (n=1175 total oocytes). Oocytes were aspirated from abattoir ovaries; those with homogeneous ooplasm and multiple layers of even cumulus cells were matured using control (n=585) or GameteGuard-supplemented (n=590) maturation medium. Oocytes were fertilized with frozen/thawed semen from proven IVF bulls (n=4) and then cultured in a two-step media system to Day 7. Blastocysts were evaluated on Day 7 according to IETS embryo grading standards. A subset of 80 blastocysts were fixed and stained after grading using an In Situ Cell Death Detection Kit (Fluorescein; Millipore Sigma) and Hoechst 33342 (Invitrogen) as apoptotic and counter-stains, respectively. Stained blastocysts were assessed by immunofluorescence microscopy to obtain the percentage of apoptotic cells and total cell count. Because multiple bulls were used, a linear mixed model was required for data analysis. Treatment was considered the fixed effect and bulls as a random effect to determine the impact of the treatment on stage and quality of blastocysts. Cleavage rates were not different (P&amp;gt;0.5) but GameteGuard supplementation of maturation medium resulted in a 22% increase in grade-1 embryos per total blastocysts produced (P&amp;lt;0.01; n=79/106 control vs. n=120/137 GameteGuard grade 1 blastocysts). Additionally, the percent of grade 2 and 3 embryos per total blastocysts decreased, (P&amp;lt;0.01; 27%, n=27/106 control and 11%, n=14/137 GameteGuard grade 2 and 3 blastocysts), suggesting that supplementation with GameteGuard promoted development of better-quality embryos overall. The percentage of apoptotic cells was also significantly higher in control blastocysts compared with treated blastocysts (P&amp;lt;0.02; 11.4%, n=38 control and 12.8%, n=42 GameteGuard). GameteGuard supplementation of the maturation medium significantly increased the production of high-quality IVP bovine embryos, thereby providing potential marketable gain for embryo producers.

120 High variation in prediction of developmental competence of bovine oocytes based on visual examination

The quality of an oocyte is often represented by its morphological characteristics. Stringent selection of bovine cumulus–oocyte complexes (COCs) for invitro embryo production (IVP) is regularly based on parameters such as colour and granulation of the ooplasm, density of the surrounding cumulus cells, brightness of the zona pellucida, among other less common parameters. The link between developmental competence and morphology of the COC has been demonstrated in literature in group culture system. However, this has not been yet examined in an individual invitro production system. This study investigates the ability of IVP researchers to predict whether an oocyte will develop into a blastocyst at Day 8 of invitro culture in an individual production system. A set of 29 pictures of immature bovine COCs was presented in duplicates to eight bovine IVP researchers with different institutional backgrounds. The observers were asked if they would select each oocyte for further IVP processing, assuming that only the oocytes with the highest developmental potential can proceed, and thus a stringent selection is warranted. These 29 immature oocytes were matured, fertilized, and cultured individually invitro (using conventional methods) so that their ability to develop into a blastocyst at Day 8 post-fertilization was known. The ability to reach the blastocyst stage was stated as the gold standard and compared with the answers of the observers using kappa statistics and sensitivity (Se) and specificity (Sp) tests. Kappa (κ) for interobserver agreement was κ=0.01 (Se=50%, Sp=53%; positive predictive value=14%, negative predictive value=87%) with 50% accuracy of prediction. The intraobserver agreement was κ=0.69, with κ=1 referring to 100% agreement (Se=87%, Sp=83%), with an accuracy of 85%. The ability of trained embryologists to predict the fate of oocytes’ developmental competence was accurate for only half of the presented oocytes. Additionally, the proportion of false positive (1 − Sp) and false negative (1−Se) predictions was equally distributed. The low positive predictive value might be due to the lower development rates in individual culture systems versus group culture systems, whereas observers are mainly trained to select oocytes for group culture. This study demonstrated the subjective nature of the oocyte selection process that is based on visual examination of COC morphology because interobserver agreement was almost nonexistent. Interpretation of the morphology within an observer remains consequent, represented by the high intraobserver agreement. Consequently, there is a need to develop a new model for the objective determination of oocyte quality for individual IVP. The implementation of machine learning algorithms could objectively enhance oocyte selection and artificial reproductive technologies in extension.

149 Uptake of C18:0 from culture media during invitro culture decreases cryosurvival rates of bovine embryos

Cryosurvival of invitro-produced bovine embryos is lower than that of invivo-produced embryos, limiting their usability in the field. Previous work showed that the embryo’s lipid composition relates to its quality and cryosurvival. The present study aimed to investigate the effects of free fatty acid (FA) additions to embryo culture media during the oviduct phase of embryonic development on the improvement of cryosurvival of invitro-produced blastocysts. Bovine cumulus–oocyte complexes (n=1675, 3 replicates) were harvested from slaughterhouse ovaries, invitro matured (23h), and subsequently fertilized (18–20h). Embryos were cultured until Day 5 post-fertilization in synthetic oviductal fluid (SOF) with (1) bovine serum albumin (BSA; control, n=253); (2) delipidified BSA (&amp;gt;96% FA free, n=460); (3), delipidified BSA complexed with 25µM unsaturated oleic acid (C18:1, n=455); or (4) with saturated stearic acid (C18:0, n=507) with a stoichiometry of 5:1. At Day 5, SOF was refreshed and embryos were cultured without supplementation. At Days 7 and 8, blastocyst rates were determined. Blastocysts were LD540 stained for lipid droplets (LD), and the LD number and size were analysed by ANOVA. Cryosurvival%, defined by re-expansion of the blastocoel, was analysed by logistic regression. Additionally, fresh and frozen–thawed blastocysts were stained for apoptosis (terminal deoxynucleotidyl transferase dUTP nick-end labelling, TUNEL), necrosis (EthD-1), and DNA (Hoechst 33342) and analysed using negative binomial regression. Group differences were tested using a post hoc Tukey test. Statistical analysis was performed in R Studio (version 3.4.2), and P-values &amp;lt;0.05 were considered significant. FA-free culture delayed and decreased blastocyst rates to 19% compared with any FA supplementation: 35%, 27%, and 29% for control, C18:1, and C18:0, respectively (P&amp;lt;0.04). Cryosurvival doubled with culture in FA-free SOF (58%) and C18:1 (63%) compared with C18:0 (23% P=0.01 and P&amp;lt;0.01, respectively) and control (29%; P=0.15 and P&amp;lt;0.02, respectively), approaching cryosurvival rates of donated multiple ovulation embryo transfer (MOET) embryos (CRV Company; 67%). C18:0 exposure also resulted in elevated necrosis levels after cryopreservation (5–8% of cells), compared with all groups (2–4%; P&amp;lt;0.016). The LD size increased in blastocysts cultured with C18:1 compared with all groups (3.1µm2 vs. 2.4–2.7µm2; P&amp;lt;0.016). C18:0 addition to SOF during embryo culture invitro, as well as a mixture of FA in control SOF (including C18:0), caused a reduction of ∼50% in blastocyst cryosurvival compared with MOET blastocysts. Interestingly, either C18:1 addition or the complete omission of FA in SOF during embryo culture invitro restored the cryosurvival of blastocysts to the level of MOET blastocysts. Currently, we are investigating whether the free FA conditions in the oviduct endorse the distinct quality between invivo- and invitro-produced embryos.

Upregulation of interferon-alpha gene in bovine embryos produced in vitro in response to experimental infection with noncytophatic bovine-viral-diarrhea virus.

In-vitro fertilization is a routine livestock-breeding technique widely used around the world. Several studies have reported the interaction of bovine viral-diarrhea virus (BVDV) with gametes and in-vitro-produced (IVP) bovine embryos. Since, gene expression in BVDV-infected IVP bovine embryos is scarcely addressed. The aim of this work was to evaluate the differential expression of genes involved in immune and inflammatory response. Groups of 20–25 embryos on Day 6 (morula stage) were exposed (infected) or not (control) to an NCP-BVDV strain in SOF medium. After 24 h, embryos that reached expanded blastocyst stage were washed. Total RNA of each embryo group was extracted to determine the transcription levels of 9 specific transcripts related with antiviral and inflammatory response by SYBR Green real time quantitative (RT-qPCR). Culture media and an aliquot of the last embryos wash on Day 7 were analyzed by titration and virus isolation, respectively. A conventional PCR confirmed BVDV presence in IVP embryos. A significantly higher expression of interferon-α was observed in blastocysts exposed to NCP-BVDV compared to the controls (p < 0.05). In this study, the upregulation of INFα and TLR7 genes involved in inflammatory and immune response in BVDV-infected IVP bovine embryos is a new finding in this field. This differential expression suggest that embryonic cells could function in a manner like immune cells by recognizing and responding early to interaction with viral pathogens. These results provide new insights into the action of BVDV on the complex molecular pathways controlling bovine early embryonic development.

Inverse relationship between autophagy and CTSK is related to bovine embryo quality.

Improving the quality and the developmental competence of in vitro produced (IVP) embryos is an indispensable goal for assisted reproductive technology. Autophagy is a major protective mechanism for intracellular degradation of unnecessary cytoplasmic components. Autophagy ends by the fusion between autophagic vacuoles and lysosomes, allowing the degradation of the cargo by lysosomal enzymes, especially the cathepsins (CTSs). However, it is still unclear how autophagy and cathepsin K (CTSK) relate to embryo development. This study evaluated (1.) the activities of autophagy and CTSK in relation to bovine embryo quality and (2.) the effect of autophagy induction and/or CTSK inhibition on preimplantation embryo development and quality. We show here that good-quality embryos exhibited a greater autophagic activity and less CTSK activity compared to poor-quality embryos. Blastomeres of an individual embryo may vary in their quality. Good quality blastomeres showed an increased autophagic activity and decreased CTSK activity compared to poor-quality blastomeres within the same embryo at different developmental stages. Importantly, induction of autophagy and/or inhibition of CTSK improved the developmental rate (increased blastocyst and hatching rates) and the quality (increased total cell number and decreased the percentage of apoptotic cells) of IVP bovine embryos. These results demonstrate a promising approach to selectively isolate good-quality embryos and improve the efficiency of IVEP of cattle embryos.

Recent progress in bovine in vitro-derived embryo cryotolerance: Impact of in vitro culture systems, advances in cryopreservation and future considerations.

Cryopreservation of in vitro-derived bovine embryos is a crucial step for the widespread reproduction and conservation of valuable high-merit animals. Given the current popularity of bovine in vitro embryo production (IVP), there is a demand for a highly efficient ultra-low temperature storage method in order to maximize donor ovum pickup (OPU) turn-over, recipient availability/utilization and domestic/overseas commercial trading opportunities. However, IVP bovine embryos are still very sensitive to chilling and cryopreservation, and despite recent progress, a convenient (simple and robust) protocol has not yet been developed. At the moment, there are two methods for bovine IVP embryo cryopreservation: slow programmable freezing and vitrification. Both of the aforementioned techniques have pros and cons. While controlled-rate slow cooling can easily be adapted for direct transfer (DT), ice crystal formation remains an issue. On the other hand, vitrification solved this problem but the possibility of successful DT commercial incorporation remains to be determined. Moreover, simplification of the vitrification protocol (including warming) through the use of an in-straw dilution without the use of a microscope is a prerequisite for its use under farm conditions. This review summarizes the bovine IVP embryo cryopreservation achievements, strengths and limitations of both freezing systems and prospective improvements to enhance cryosurvival, as well as perspectives on future directions of this assisted reproductive technology.

Cryopreservation of in vitro-produced bovine embryos by vitrification: In pursuit of a simplified, standardized procedure that improves pregnancy rates to promote cattle industry use

The goal of cryopreservation is to retain the original stage of gametes and embryos after they have endured cooling and warming. Slow freezing is a standard method for in vivo-derived bovine embryo cryopreservation, threefifths of such embryos being frozen by this method globally. However, it is evident that slow freezing is not efficient for cryopreserving in vitro-produced bovine embryos. Hence, only one-third of in vitro-produced bovine embryos are cryopreserved. Vitrification is a preferred method for storage of human embryos; consequently, it has been explored as a novel means to store in vitro-produced bovine embryos, for which it shows considerable promise as an alternative to slow freezing. This is due to several reasons: vitrification is often less time-consuming than slow freezing; it does not need expensive slow rate freezing machines; and it has been proven to have comparatively higher survival rates. Yet, in the cattle industry vitrification continues to present shortcomings, such as possible toxicity of vitrification solutions and failure to standardize methods, which pose a challenge for its application to in vitro-produced bovine embryos. Therefore, determining the most suitable procedure is crucial to make vitrification more practical in commercial settings.

91 Invivo- and invitro-produced bovine embryos have different microRNA profiles after invitro individual culture

Invivo- and invitro-produced bovine embryos have different metabolic characteristics, embryonic development, and gene transcription. Additionally, pregnancy rates at 30 days (on average 51% and 34% when using fixed-time AI and invitro production, respectively) are different in beef cattle. Between Days 8 and 17 of the oestrous cycle, concurrent with embryo-maternal recognition, is when 40% of embryonic losses occur. These losses may occur due to altered embryo-maternal cross-talk. MicroRNA (miRNA) can be involved in this communication; however, its potentially regulated pathways in invivo and invitro embryos on Day 9 are unknown. Our hypothesis is that bovine embryos produced invivo and invitro contain different miRNA profiles, even after invivo bovine embryo were invitro cultured. Cows had the follicular wave synchronized and were superovulated to produce invivo or invitro bovine embryos. For the invitro group, on Day −8 of the protocol, the dominant follicles were recovered by ovum pickup, and invitro embryo production was performed to obtain embryos. For the invivo group, on Day −8, the cows were inseminated 12 and 24h after GnRH analogue application and on Day 7 after expected oestrus, uterine flushing was performed to obtain the embryos. Embryos from both groups were individually cultured for 48h. Three pools (of 5 embryos each) per group were used for reverse transcription of miRNAs from total RNA using miScript II RT Kit (Qiagen). Relative levels of 383 bovine miRNAs were determined using the geometric mean of miR-99b, RNU43 snoRNA, and Hm/Ms/Rt U1 snRNA by RT-qPCR. Differences in relative levels of miRNAs were determined by Student's t-test. A total of 210 miRNAs were detected in invivo and invitro embryos, and 13 out of 210 were differently identified between the groups. In invivo embryos, 6 miRNAs were up-regulated, whereas 7 miRNAs were up-regulated in invitro embryos. TARGETSCAN software was used to identify genes predicted as modulated by each miRNA. The top 100 genes predicted were used to identify enriched pathways according to DAVID Bioinformatics Resources. The miRNAs (miR-129, miR-132, miR-155, miR-192, miR-215, and miR-377) up-regulated in invivo embryos modulated pathways that include signaling pathways regulating pluripotency of stem cells (16 genes), TGF-β (11), hippo (10), oestrogen (8), and cell cycle (7). Moreover, miR-23a, miR-338, miR-34a, miR-491, miR-92b, miR-940, and miR-1271, which were increased in invitro embryos, regulate PI3K-Akt (17 genes), signaling pathways regulating pluripotency of stem cells (10), oestrogen (9), toll-like receptor (9), Wnt (9), and HIF-1 (7). The results demonstrate that even after 48h of invitro culture, bovine embryos produced invivo and invitro have different miRNA profiles that modulate pathways associated with embryonic development on Day 9. Furthermore, these results suggest that bioactive molecules, such as miRNAs, can modify embryo-maternal cross-talk, depending on the environment where the embryos are produced. Funding was provided by FAPESP 2017/19681-9, 2014/22887-0, and 2018/13155-6.

110 Absence of transmission of Mycoplasma bovis via naturally contaminated semen during invitro fertilization

Mycoplasma bovis (Mbo) has been isolated from genital tracts of bulls, and it can survive in processed semen. Experimental studies have shown that Mbo inoculation into the uterus or insemination with Mbo-infected semen can cause bursitis, salpingitis, abortion, and infertility. The control of Mbo is very difficult because of latent carrier animals, increasing resistance to antibiotics, and unavailability of effective vaccines. The aim of this study was to follow the passage of Mbo infection from naturally contaminated semen to transferable embryos during bovine invitro embryo production (IVP). (Unless otherwise stated, all chemicals used were purchased from Sigma-Aldrich.) Two batches of slaughterhouse-derived oocytes were matured in tissue culture medium 199 (TCM-199) with glutamax-I (Gibco™; Invitrogen Corporation) supplemented with 0.25mM sodium pyruvate, 100IUmL−1 penicillin, 100µgmL−1 streptomycin, 2ngmL−1 FSH (Puregon, Organon), 1µgmL−1 β-oestradiol (E-2257), and 10% heat-inactivated fetal bovine serum (FBS; Gibco™) for 24h at 38.5°C in maximal humidity in 5% CO2 in air. Matured oocytes were fertilized for 20h in IVF-TL medium supplemented with 10µgmL−1 of heparin and 2mM of PHE at 38.5°C in maximal humidity in 5% CO2 in air, using spermatozoa per mL as a final concentration. The batches of oocytes were divided between uninfected IVP bull (N=205) and naturally Mbo-infected AI bull (N=690). Zygotes were cultured in G1/G2 media (Vitrolife) supplemented with bovine serum albumin, fatty acid free (4mgmL−1), at 38.5°C in maximal humidity in 5% O2, 5% CO2, and 90% N2. Blastocysts were collected for Mbo cultures on Days 7 and 8 (IVF=Day 0). Samples of washed semen, fertilization medium, cumulus cells, culture medium, all wash media, and transferable embryos (with and without zona pellucidae) were collected for Mbo cultures. Half of the embryos were treated with trypsin according to IETS standards after the collections. The Mbo cultures were performed in accordance with procedures previously described by Bölske (1988 Zentralbl. Bakteriol. Mikrobiol. Hyg. A 69, 331-340), followed by detection with real-time PCR. Infection with Mbo does not seem to have negative effects on fertilization (cleavage rates: 77.1% and 89.0% for IVP and Mbo AI bulls, respectively) or embryo development rates (blastocyst rate: 26.3% and 32.5% for IVP and Mbo AI bulls, respectively). Following Mbo cultures, only washed semen was found to be Mbo positive via real-time PCR. We conclude that M. bovis is not likely transmitted in bovine IVP when using naturally infected semen. We acknowledge Tiina Kortelainen for technical assistance and the Ministry of Agriculture and Forestry for funding.

72 Invitro embryo production using prepubertal calf oocytes with conventional semen and sexed semen ULTRA-4M

Invitro embryo production (IVP) can increase the reproductive potential and genetic quality of cattle, as well as other species. This powerful assisted reproduction tool can be used to produce embryos from prepubertal calves, reducing the generation interval. A recent sexed semen technology known as ULTRA (ST Genetics), completely modified the technique, the media and sperm concentration. In field trials with AI there was no difference between conventional semen (CONV) and ultra-sexed semen at a concentration of 4 million per straw (ULTRA-4M). The combined use of IVP and ULTRA-4M can decrease the selection time for improving dairy and beef cattle herd genetics. The objective of this research was to compare the CONV and ULTRA-4M semen using bovine IVP and prepubertal calves. The research was carried out in the reproduction laboratory of the Facultad de Estudios Superiores Cuautitlán - Universidad Nacional Autónoma de México (FESC-UNAM). The IVP was performed with a continuous invitro culture (IVC) system. The ovaries were collected in Campeche, México, from Bos indicus×Bos taurus crossbred calves (6 months old) using surgical castration (for export to the United States) and transported to the laboratory (FESC-UNAM) in BO-HEPES-IVM (Bioscience™), in an oocyte transporter (WTA). Vitrogen media were used for IVF and IVC. For IVM, the cumulus-oocyte complexes (COCs) were selected (only grades 1 and 2) and matured for 24h at 38.5°C. Matured oocytes (n=600, divided equally into five replicates) were divided into 2 groups, the CONV group and the ULTRA-4M group. The IVF process was conducted with CONV and ULTRA-4M semen from the same bull (Holstein) at a concentration of 2×106 and 0.5×106 spermatozoamL−1, respectively, for 18h in 38.5°C, 5% CO2, 95% air, and 100% humidity. The presumptive zygotes were denuded by pipetting and set in IVC until Day 7 at 38.5°C, 5% CO2, 5% O2, and 90% N2 at 100% humidity. The cleavage results were recorded 56h after the beginning of IVC. The cleavage rate, embryos with more than 6 cells, and blastocysts on Day 7 of culture were evaluated. The statistical analysis was carried out with the GLM procedure of the SAS software (version 9.3; SAS Institute Inc.) to evaluate the results of CONV vs. ULTRA-4M (α level=0.05). The percentage of cleavage for CONV was 46%±1.4 and 43.2%±1.4 for ULTRA-4M. The results for embryos with more than 6 cells in the CONV and ULTRA-4M groups were 16%±0.6 and 14%±0.6, respectively. The percentage of blastocysts on Day 7 for CONV was 9%±0.6 and 8%±0.6 for ULTRA-4M. There were no significant differences between groups (P&amp;gt;0.05) for all variables analysed. In conclusion, under the conditions of this research the ULTRA-4M and CONV produced similar results for IVP.

89 Embryonic metabolism orchestrates epigenetic mechanisms: What can we anticipate from the first cleavages?

Intermediates of the energy metabolism (as acetyl Co-A) can donate their acetyl group to introduce acetylation in histones, establishing a relationship between metabolism and epigenetic control in somatic and embryonic stem cells. Embryos with different kinetics during the first cleavages also have alterations in epigenetic profile as well as in metabolism and energy substrate consumption during invitro culture. The aim of this work was to verify if and how this relation between metabolism and epigenetic parameters was also presented in invitro-produced bovine embryos. For that, we first characterised the pattern of H3K9ac, and the molecular pattern of enzymes involved with histone acetylation and acetyl-CoA production in female blastocysts derived from fast and slow cleavage embryos. To validate the results, we also produced bovine embryos cultured with an inhibitor of the pyruvate production, and consequently the acetyl Co-A generation to check if this could interfere in the H3K9ac pattern. For this, embryos were invitro produced following standard protocol and classified at 40 hours post-insemination as fast (4 or more cells) or slow (2 cells) and collected at the blastocyst stage. Blastocysts were immunostained to H3K9ac and the fluorescence intensity of each nucleus was quantified using ImageJ and analysed by Student's t-test. For transcript quantitation, RNAseq data were accessed from a previous report using the same kinetics classification model (Milazzotto et al. 2016Mol. Rep. Dev. 83, 324-336; https://doi.org/10.1002/mrd.22619) and analysed using limma-voom on Galaxy 3.38.3. To validate the results, bovine embryos were produced and cultured until Day 4 and then incubated until the blastocyst stage with different doses of iodoacetate (IA; 2 and 5mM) to reduce the intracellular levels of acetyl CoA. These blastocysts were also assessed by H3K9 acetylation. Slow blastocysts presented higher fluorescence intensity for H3K9ac than fast blastocysts (fast 13.33±0.37 AU vs. slow 38.14±1.17 AU; P&amp;lt;0.0001). Despite the fact that there were no differences in transcripts related to this acetylation (ELP3 and HAT2), slow blastocysts presented higher levels of transcripts for PDHB1 and PDHA1, responsible for acetyl-CoA production (PDHB1: fast 11.6±0.2CPM vs. slow 13.1±0.2 counts per million; P&amp;lt;0.01 PDHA1: fast 12.6±0.2 CPM vs. slow 13.2±0.3 CPM; P&amp;lt;0.01). The reduction of acetyl-CoA in blastocysts induced by IA led to lower levels of H3K9ac in 1 and 2mM doses when compared with the control (control: 43.8±0.7 AU; 1 mM: 34.7±0.5 AU; 2 mM: 30.1±0.6 AU; P&amp;lt;0.0001). Interestingly, H3K9ac levels were similar for 5mM IA and control group (5 mM: 41.2±1.4; P&amp;gt;0.05), suggesting a compensatory mechanism in extreme cases to maintain the histone acetylation. As far as we know, this is the first work that describes a relation between metabolism and epigenetics in bovine embryos. Although the pattern of genes related to acetylation seems to be unaltered, changes in acetyl Co-A production pathway exert an influence on H3K9ac status. Grant support was provided by grant 2017/18384-0 and 2018/11668-6 from FAPESP and CAPES.

65 Effect of oviductal fluid extracellular vesicle supplementation during invitro culture on development and quality of bovine embryos

Recently, it has been postulated that oviductal extracellular vesicles (oEV) might act as natural nanoshuttles bringing key components (small noncoding RNAs and proteins) of the oviduct into gametes and embryos. Furthermore, co-incubation of frozen-thawed oEV with invitro-produced bovine embryos was reported to increase blastocyst rate and quality (Almiñana et al. 2017 Reproduction 154, 153-168). The objective of this study was to determine the dose-dependent effect of oEV supplementation of embryo culture medium on the invitro development and cryotolerance of embryos. Briefly, oEV were isolated by ultracentrifugation from a pool of oviductal fluids (8 cows/sample) collected at the slaughterhouse at the post-ovulatory stage and ipsilateral to ovulation and stored at −80°C until used. Slaughterhouse-derived bovine oocytes were invitro matured and fertilised with frozen-thawed semen from one bull (4 replicates; 194 presumptive zygotes per group), according to our standard procedures. After IVF, groups of presumptive zygotes (n=20/drop) were cultured under humidified air with 5% CO2, 5% O2 at 38.8°C for 7 days in 30µL of synthetic oviductal fluid-bovine serum albumin supplemented with oEV at different protein concentrations: 0.5, 0.05, or 0.005mgmL−1 and without (control). Cleavage rates were evaluated on Day 2 and blastocyst rates were assessed on Days 6 and 7 (IVF as Day 0). At Day 7, expanded grade 1 blastocysts were evaluated (International Embryo Technology Society classification) and embryos at the expanded grade 1 blastocyst stage were slow frozen in 1.5M ethylene glycol + 0.1M sucrose and stored in liquid nitrogen. For cryotolerance evaluation, embryos were thawed and cultured for 48h in synthetic oviductal fluid-bovine serum albumin + 1% estrous cow serum. Hatching rates were assessed at 48h post-thawing. Data were analysed by a logistic regression mixed model (SAS, SAS Institute Inc.; Glimmix procedure) followed by post-hoc Tukey for multiple comparisons. Differences were considered significant at P&amp;lt;0.05. No differences were observed among the different oEV concentrations tested for cleavage and Day 6 blastocysts. A tendency (P=0.0535) was observed for Day 7 blastocyst rates (19.1±2.8, 29.4±3.3, 16.0±2.6, and 20.6±2.9 for 0.5, 0.05, 0.005mgmL−1, and control, respectively) in favour of the 0.05mgmL−1 group. However, a significant difference (P&amp;lt;0.0288) for Day 7 grade 1 expanded blastocyst rates in favour of the 0.05mgmL−1 group was observed (5.2±1.6, 12.9±2.4, 3.1±1.2, and 9.8±2.2 for 0.5, 0.05, 0.005mgmL−1, and control, respectively). For cryopreserved embryos, hatching rates of frozen-thawed embryos were not significant among experimental groups (81.6±10.2 (n=19), 89.6±6.6 (n=27), 77.2±12.2 (n=10), and 60.2±13.6 (n=23) for 0.5, 0.05, 0.005mgmL−1, and control, respectively). In conclusion, under our experimental conditions, the supplementation of the embryo culture medium with frozen-thawed post-ovulatory oEV at the protein concentration of 0.05mgmL−1 increased the Day 7 grade 1 expanded blastocyst rate. Moreover, we showed a tendency to improve Day 7 blastocyst rates but with no apparent effects on the cryotolerance of embryos. This work was supported by APIS GENE.

71 Comparison of sexed semen ULTRA-4M with conventional semen for the invitro production of bovine embryos

The first commercial production of sexed semen was at the Cogent company in the United Kingdom. Since then millions of offspring have been born using sexed semen produced by flow cytometry. Sexed semen technology has recently been modernized to what is now known as ULTRA (ST Genetics), completely modifying the technique, medium, and sperm concentration. In field trials using AI, there was no difference between conventional semen (CONV) and ultra-sexed semen at a concentration of 4 million sperm per straw (ULTRA-4M). The combination of invitro embryo production (IVP) and sexed semen technologies has been successful for intensity selection in the cattle industry. The objective of this work was to compare the CONV and ULTRA-4M semen for bovine IVP. The research was carried out in the reproduction laboratory at the Facultad de Estudios Superiores Cuautitlán-Universidad Nacional Autónoma de México (FESC-UNAM). The IVP was performed with a continuous invitro culture (IVC) system. Ovaries (n=213) were collected from a slaughterhouse (Querétaro, México) and transported to the laboratory within 2h (FESC-UNAM) in physiological saline solution (0.9% NaCl) supplemented with penicillin G (100IUmL−1) and streptomycin sulfate (100µgmL−1). For IVP, VITROGEN media were used for IVM, IVF, and IVC. For the IVM, the cumulus-oocyte complexes were selected (only grades 1 and 2) and matured for 24h at 38.5°C in 5% CO2, 95% air, and 100% humidity. Matured oocytes (n=1000) were divided into two groups, the CONV group and the ULTRA-4M group. The IVF process was developed with CONV and ULTRA-4M semen from the same bull (Holstein) at a concentration of 2×106 and 0.5×106 spermmL−1, respectively, for 18h in 38.5°C, 5% CO2, 95% air, and 100% humidity. The presumptive zygotes were denuded by pipetting and left in IVC until Day 7 at 38.5°C, 5% CO2, 5% O2, and 90% N2 at 100% humidity. The cleavage rate, embryos of more than 6 cells, and blastocysts on Day 7 of culture were evaluated. The statistical analysis was carried out with the GLM procedure of the SAS software (version 9.3; SAS Institute Inc.) to evaluate the results of CONV vs. ULTRA-4M (α level=0.05). Percent cleavage for CONV was 72.2%±2.53 and 75.6%±2.53 for ULTRA-4M. For embryos with more than 6 cells, the results for CONV and ULTRA-4M were 59.8%±5.61 and 62.8%±5.61, respectively. The percentage of blastocysts on Day 7 was 37.8%±5.39 for CONV and 43.6%±5.39 for ULTRA-4M. There were no significant differences between the groups (P&amp;gt;0.05) for all variables analysed. Although the number of blastocysts on Day 7 were numerically higher in the ULTRA-4M, differences were not significant. In conclusion, under the conditions of this research the ULTRA-4M had similar results as CONV for bovine IVP.