In Vitro Embryo Production Research Articles

Nutritional Strategies to Promote Bovine Oocyte Quality for In Vitro Embryo Production: Do They Really Work?

The ability of bovine oocytes to reach the blastocyst stage (i.e., embryo with around 150 cells in cattle) in vitro can be affected by technical (e.g., culture medium used) and physiological factors in oocyte donors (e.g., age, breed). As such, the nutritional status of oocyte donors plays a significant role in the efficiency of in vitro embryo production (IVEP), and several nutritional strategies have been investigated in cattle subjected to ovum pick-up (OPU). However, there is no clear consensus on the reliability of nutritional schemes to improve IVEP in cattle. Available evidence suggests that a moderate body condition score (i.e., 3 in a 1-5 scale) in cattle is compatible with a metabolic microenvironment in ovarian follicles that will promote embryo formation in vitro. The usefulness of fatty acid and micronutrient supplementation to improve IVEP in cattle is debatable with the current information available. Overall, the supply of maintenance nutritional requirements according to developmental and productive stage seems to be enough to provide bovine oocyte donors with a good chance of producing embryos in vitro. Future nutrition research in cattle using OPU-IVEP models needs to consider animal well-being aspects (i.e., stress caused by handling and sampling), which could affect the results.

Can in vitro embryo production be estimated from semen variables in Senepol breed by using artificial intelligence?

Thoroughly analyzing the sperm and exploring the information obtained using artificial intelligence (AI) could be the key to improving fertility estimation. Artificial neural networks have already been applied to calculate zootechnical indices in animals and predict fertility in humans. This method of estimating the results of reproductive biotechnologies, such as in vitro embryo production (IVEP) in cattle, could be valuable for livestock production. This study was developed to model IVEP estimates in Senepol animals based on various sperm attributes, through retrospective data from 290 IVEP routines performed using 38 commercial doses of semen from Senepol bulls. All sperm samples that had undergone the same procedure during sperm selection for in vitro fertilization were evaluated using a computer-assisted sperm analysis (CASA) system to define sperm subpopulations. Sperm morphology was also analyzed in a wet preparation, and the integrity of the plasma and acrosomal membranes, mitochondrial potential, oxidative status, and chromatin resistance were evaluated using flow cytometry. A previous study identified three sperm subpopulations in such samples and the information used in tandem with other sperm quality variables to perform an AI analysis. AI analysis generated models that estimated IVEP based on the season, donor, percentage of viable oocytes, and 18 other sperm predictor variables. The accuracy of the results obtained for the three best AI models for predicting the IVEP was 90.7, 75.3, and 79.6%, respectively. Therefore, applying this AI technique would enable the estimation of high or low embryo production for individual bulls based on the sperm analysis information.

Sex ratio and conception rates of fresh/vitrified embryos at different developmental stages by ovum pick up in Hanwoo cows.

Embryo transfer plays a crucial role in enhancing the breeding value of livestock; it has been applied in Hanwoo cattle, which is a popular breed for beef production in Korea. Both in vivo-derived (IVD) and in vitro-produced (IVP) embryos are used for this purpose; however, IVP embryos have been preferred recently owing to advancements in ovum pick-up (OPU) technology and genomic selection. Despite technological advancements, comprehensive data on large-scale OPU/IVEP/embryo transfer in Hanwoo cows are lacking. In this study, 16 elite Hanwoo donor cows were selected on the basis of specific criteria. Oocytes were retrieved from 241 cows using OPU. The collected cumulus-oocyte complexes (COCs) were matured, fertilized, and cultured in vitro to produce transferable embryos. Embryos were classified according to their developmental stage and then transferred to 675 recipient cows. A total of 3,317 COCs were collected, with an average of 13.76 COCs per cow. The number of transferable embryos produced per cow was 3.7. Hanwoo OPU-derived IVP embryos exhibited a higher production yield than the global average, indicating a stable IVEP environment. Both fresh and frozen IVP embryos yielded similar conception rates; hence, the use of vitrified-thawed embryos in transfer plans feasible. However, frozen-thawed embryos at Stage 7 had a lower conception rate than those at earlier stages. There was no significant difference between the conception rates of sexually mature heifers and postpartum cows used as recipients. The male-to-female offspring ratio increased as the developmental stage progressed. Seasonal effects on conception rates were not observed; however, higher abortion rates and a higher proportion of male offspring were observed during winter. This study provides valuable data for the Korean embryo transfer industry, enabling more strategic growth of the domestic Hanwoo embryo industry.

Improving Cryopreservation Efficiency and Pregnancy Rate through Superovulation with Follicle-Stimulating Hormone in Korean Hanwoo Cows via Ovum Pick Up.

The aim of this study was to devise an efficient technique for generating embryos from high-quality bovine females. Oocytes were collected from 20 control and 15 Hanwoo (Bos taurus coreanae) females treated with the FSH. A combination of decreasing FSH doses (36, 36, 24, and 24 mg, 12 h apart), progesterone, estrogen, and prostaglandins were administered to synchronize and mildly stimulate the animals. The FSH-treated group (1125 oocytes) and control group (1022 oocytes) exhibited a higher proportion of grade A and B oocytes (88.2%) than the other grades (p < 0.05), with most at the germinal vesicle 2 stage (64.0%). Moreover, the FSH-treated group achieved a notably higher blastocyst rate (44.7%) compared to the control group (31.1%) (p < 0.05). After undergoing vitrification and in vitro culture (IVC) warming, embryos in the FSH group exhibited higher re-expansion rates (grade 1: 86.9%; grades 2 and 3: 57.9%) compared to those in the control (p < 0.05). This highlights the positive impact of FSH treatment on in vitro embryo production (IVEP) and the OPU rate.

Current Advances in Bovine In Vitro Maturation and Embryo Production Using Different Antioxidants: A Review.

In vitro maturation (IVM) is one of the most important steps in in vitro embryo production (IVEP). It is a complicated procedure in which nuclear and cytoplasmatic changes in oocytes appear. In order to carry out the in vitro maturation procedure correctly, it is necessary to provide the oocytes with as close to a natural (in vivo) environment as possible. Many factors contribute to the overall poor quality of in vitro-matured oocytes. One important factor may be oxidative stress (OS). The generation of oxidants, such as reactive oxygen species, is common under culture conditions. The solution for OC treatment and prevention is antioxidants. In the last 5 years, many studies have examined different antioxidants and their effects on in vitro maturation of oocytes and embryo production. The aim of this systematic review was to present the achievements of scientific research in the last five years, in which the effects of many antioxidants were tested on bovine oocyte maturation and embryo production.

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 assigned randomly 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 (D2 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.

Health assessment of Holstein calves born after in vitro fertilization, biopsy-based genotyping at the blastocyst stage and subsequent embryo transfer

Establishing methods for evaluating genomic estimated breeding values of bovine embryos can potentially increase the efficiency of breeding programs by transferring only embryos with a high genomic estimated breeding value. This may be achieved by analyzing DNA from trophectoderm biopsies. However, manipulation of bovine embryos is associated with a risk of impaired conceptus health. More knowledge on the health implications of embryonic handling procedures is required. In this study, we followed pregnancies after transfer of in vitro-produced (IVP) embryos and assessed the health of the offspring during the first 2 weeks of life. Three groups of calves were studied: i) freshly transferred non-biopsied embryos (39 transfers, 17 calves; Group B-/C-); ii) biopsied and freshly transferred IVP embryos (42 transfers, 21 calves; Group B+/C-); iii) biopsied and cryopreserved IVP embryos (17 transfers, 6 calves; Group B+/C+). Blood biochemical and hematologic values were compared between groups and to a control group of 13 calves produced by conventional artificial insemination. The pregnancy rate on day 50 and the calving rate did not differ among the groups, but the average gestation length of the B+/C+ group was significantly shorter and with wider variation than the two other groups. There was a tendency toward a higher average body weight at birth in group B+/C+ (45.1 kg) and the standard deviation in body weight was larger (11.7 kg) compared to the B-/C- (39.5 kg; 3.2 kg) and B+/C- (41.8 kg; 6 kg) groups. Body weight on day 14 was higher in the B+/C+ calves compared to the other groups. There was no difference in the biochemical and hematological values at birth between the groups and these were within the normal range. However, when compared to a group of calves produced by standard artificial insemination, significantly higher concentrations were found for the hepatic-related enzymes ALAT, ASAT, ALP, and GGT in group B-/C-and B+/C-, while only higher ALP concentrations were found in B+/C+ calves. The biochemical findings indicate higher heterogeneity in IVP calves compared to calves produced by artificial insemination. The more manipulated IVP embryos also showed increased heterogeneity in body weight at birth, with a shift toward heavier calves, which calls for closer attendance at parturition to handle dystocia in a timely manner and minimize fetal losses.

The Role of Ionomycin in the Improvement of in vitro Fertilization and Increasing the Rate of Bovine Embryo Development

Background: The utilization of in vitro fertilization (IVF) and in vitro embryo production (IVEP) has increased significantly as an effective tool of assisted reproductive technology. Sperm capacitation is a process of membrane maturation allowing the acrosome reaction to take place. Ionomycin is a calcium ionophore that induces the acrosome reaction by increasing calcium cycling across phospholipid membranes. The aim of this study was to enhance the cleavage rate and development of bovine embryos. Methods: Bovine ovaries were collected from the central slaughter house. Oocytes were obtained from follicles and were let to mature for 24 h. Frozen bull semen thawed in 37°C water and transferred to a discontinuous percoll gradient (Sigma GE17-0891-01). The spermatozoon pellet was separated into four groups after resuspension in bovine in vitro fertilization media (BO-IVF): control without additives; with DMSO and with ionomycin (25 and 50 nM) and kept in an incubator for three hours. The oocytes matured under commonly used media (TCM-199) for 24 h. The mature oocytes were then fertilized for 18-24h. The embryos were cultured in SOF medium until the seventh day to study cleavage rate and blastocyst rate. Result: The results indicated that 50 nM ionomycin supplementation considerably reduced cleavage while DMSO and 25 nM ionomycin supplementation had no effect on cleavage percentage. Moreover, when compared to the other groups, ionomycin at 25 nM significantly increased blastocyst development. In conclusion, the highest rate of blastocyst development was observed in the capacitation medium supplemented with 25 nM ionomycin, apparently due to the importance of calcium and metabolism in spermatozoa for fertilization and embryo development.

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

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. 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. 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). 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.

Relationship of field and in vitro fertility of dairy bulls with sperm parameters, including DAG1 and SERPINA5 proteins

IntroductionSperm interacts with the female reproductive tract and oocyte through proteins, and these cell-to-cell interactions may play a role in sperm fertility. For consideration of a protein as a potential marker of fertility, there must be variability expressed among animals. The proteins dystroglycan (DAG1) and plasma serine protease inhibitor (SERPINA5) have been reported to play a role in cell-to-cell interactions. Thus, the objectives of this study were to characterize the localization and abundance variability of DAG1 and SERPINA5 in bovine sperm, and to investigate the relationship of DAG1 and SERPINA5 with field fertility (i.e., sire conception rate; SCR), in vitro embryo production (IVP), and sperm parameters.Material and methodsDairy bulls (n = 22) were classified as high-SCR (SCR &amp;gt; 1.0) or low-SCR (SCR &amp;lt; –4.0), and good [blastocyst (BL)-by-cleavage (CL) ratio (BL/CL) &amp;gt; 39%] or poor (BL/CL &amp;lt; 38%) BL/CL. Sperm was evaluated for DAG1 and SERPINA5 immunolocalization, and concentration in two separate ejaculates. Variance between bulls compared with within bulls was evaluated using a generalized linear model (GLM) procedure. The relationship of SCR and IVP classification on DAG1 and SERPINA5 concentrations, percentage of tail labeled for SERPINA5, SCR, sperm total and progressive motility, sperm plasma membrane integrity (PMI), CL, BL, and BL/CL were evaluated with the GLIMMIX procedure, and the correlations between these variables were evaluated.ResultsBoth proteins were localized on the sperm head; however, SERPINA5 was also localized on the sperm tail. There was greater variance in concentration among bulls than within bulls for DAG1 (P &amp;lt; 0.0001; 69.4 vs. 49.1, respectively) and SERPINA5 (P &amp;lt; 0.0001; 325.8 vs. 285.4, respectively). There was a positive correlation between the concentrations of DAG1 and of SERPINA5 (P = 0.01; r = 0.54). In addition, the percentage of tail labeled for SERPINA5 was correlated with PMI (P = 0.05; r = 0.44). There was no relationship between SCR and IVP classifications and DAG1 (P ≥ 0.55), SERPINA5 (P ≥ 0.54), or the percentage of sperm tail labeled for SERPINA5 (P ≥ 0.22).DiscussionIn conclusion, DAG1 and SERPINA5 were localized to the sperm head, and SERPINA 5 was also localized to the tail. Concentrations of DAG1 and SERPINA5 on the sperm head were correlated with each other. The percentage of tail labeled for SERPINA5 was correlated with sperm PMI; however, neither protein was associated with SCR or IVP. Thus, when evaluated by immunofluorescent microscopy, DAG1 and SERPINA5 concentrations are variable and are not good fertility markers for bull sperm.

The impact of in vitro embryo production on placental and umbilical cord vascularization is minimized by the addition of reproductive fluids

Animals born from in-vitro-produced (IVP) embryos show changes in the placenta and umbilical cord vascularization. This study compares the placental and umbilical vascular morphometry in pigs (n = 19) born through artificial insemination (AI group) or after transfer of IVP embryos cultured with (RF-IVP group) or without (C-IVP group) reproductive fluids. The relationship between vascular parameters and animal growth during the first year of life was also analyzed. Samples were collected at birth, fixed, paraffin-embedded, cut in sections, stained, and photographed for vascular and morphometric analysis with ImageJ® and Slide Viewer®. The average daily weight gain was individually scored from birth to the first year of life. No differences were found in placental vascular morphometry among groups, except for the vascular area of small vessels (arterioles, venules, and small vessels) that was higher in the C-IVP group. Regarding the umbilical cord, the values for perimeter (AI: 26.40 ± 3.93 mm; IVP: 30.51 ± 4.74 mm), diameter (AI: 8.35 ± 1.01 mm; IVP: 10.26 ± 1.85 mm), area (AI: 43.18 ± 12.87; IVP: 56.61 ± 14.89 mm2), and Wharton's jelly area (AI: 36.86 ± 12.04 mm2; IVP 48.88 ± 12.80 mm2) were higher in IVP-derived than AI-derived animals, whereas arterial and venous morphometric data were similar between groups. A correlation study showed that placental and umbilical cord vascular phenotypes affect the further growth of pigs. In conclusion, assisted reproductive technologies impact small caliber vessels in the placenta and morphometric parameters in the umbilical cord. The addition of reproductive fluids in IVP-embryo contributes to reduce the differences with in vivo-derived animals.

A study on methods for preimplantation genetic diagnosis on equine embryos

Determination of embryonic sex or the presence of alleles associated with genetic conditions is commonly applied in equine reproduction. To date, two methods for preimplantation genetic diagnosis (PGD) have been described for equine embryos: trophoblast cell biopsy (TCB) or blastocele fluid aspiration (BFA). While TCB has been described in both in vivo- (IVV) and in vitro-produced (IVP) embryos, BFA has been reported only for IVV embryos. Alternative methods for PGD, including analysis of cell-free DNA (CFD) in the medium where IVP has been cultured, are commonly used in humans, but not for equine embryos. In the current study, three experiments were performed to determine the agreement among methods for PGD on both IVV and IVP equine embryos. In Experiment 1, IVV (n = 10) and IVP (n = 13) embryos were subjected to BFA, cultured for 24 hours, subjected to TCB, and then cultured for additional 24 hours. No detrimental effect on embryonic diameter was observed for either embryo group (IVV: before BFA: 740 ± 618 µm vs. after TCB: 718 ± 526 µm; IVP: before BFA: 174 ± 8 µm vs. after TCB: 176 ± 10 µm; P > 0.05). In Experiment 2, the agreement among BFA, TCB, and the whole embryo (Whole) was studied by detecting the genes SRY (Y chromosome) and AR (X chromosome) using PCR. For IVV embryos (n = 14),there was a 100% agreement (14/14 embryos) between Whole and TCB for both SRY and AR, while a 64% agreement (9/14 embryos) between Whole and BFA, or between BFA and TCB. On the other hand, for IVP embryos (n = 13), there was a 75% agreement (10/13 embryos) between Whole and TCB for both SRY and AR, while a 31% agreement (4/13 embryos) between Whole and BFA, and a 46% agreement (6/13 embryos) between BFA and TCB. In Experiment 3, CFD was performed in IVP embryos (n = 10), to determine embryonic sex by means of whole genome amplification (WGA) followed by detection of the genes SRY (Y chromosome) and AMEL (X and Y chromosome) using PCR. For this, WGA was performed in both Whole, CFD samples, and Control medium (negative control). Overall, CFD samples yielded results for SRY and AMEL in 6/10 embryos (60%); the sex determined in CFD samples corresponded to the same results for the Whole group. None of the Control samples amplified any DNA. In conclusion, equine embryos can be subjected to 2 biopsy procedures without apparent detrimental effects on embryonic size. For IVV, but not IVP embryos, BFA can be considered a potential alternative to TCB for PGD. Finally, CFD can be further explored as a non-invasive method for PGD in IVP equine embryos.

Simultaneous genome-wide haplotyping and copy number detection enabling PGT-A and PGT-M testing in equine in vitro produced embryos

The number of equine embryos being produced increases yearly (37,094 +10% in 2021; newsletter IETS December 2022). The reasons include the growing popularity of in vitro embryo production (IVP), which increases the genetic yield from high quality mares, maximizes the use of expensive straws of semen from high quality stallions and enables IVP to be performed outside the reproductive season. On the 4th October 2022, a record price of €95,000 was paid for an embryo. However, despite the embryos’ promising pedigrees, it remains unknown if transfer of such embryos will result in a healthy foal with the aspired phenotypical characteristics. This is largely because in horses, preimplantation genetic testing for aneuploidies and monogenic diseases or traits (PGT-A and PGT-M) remains underdeveloped especially when compared to humans. In addition, equine IVP has been reported to double early pregnancy losses, possibly because of an increased incidence of genetic errors. Here, we explored the possibility of concurrent genome-wide PGT-A and PGT-M in horses and assessed the incidence of chromosomal aberrations in IVP arrested or blastocyst stage embryos. We used GGP Equine SNP arrays (Illumina) with over 70,000 SNP positions to analyze whole-genome amplified DNA (Repli-G Single cell kit, Qiagen) from 14 trophectoderm biopsies and corresponding whole blastocysts and 26 individual cells from six embryos arrested at cleavage stage following IVP. Haplarithmisis (Zamani–Esteki et al. Am. J. Hum.Genet. 2015; 96:894-912) was applied to simultaneously determine the genome-wide copy number and haplotype. Five biopsies and seven single cells failed to give a result. From six arrested embryos, five had chromosomal errors in most of their cells, including (combinations of) triploidy and one or more monosomies and trisomies of meiotic and / or mitotic origin. Two biopsies showed a loss of maternal chromosome 28 and 31 respectively, which was confirmed in the corresponding blastocysts, suggesting meiotic errors. In one of those biopsies, additional complex aneuploidies not present in the blastocyst were found, suggesting a mitotic origin. Thus, aneuploidy and ploidy errors are highly prevalent in IVP arrested embryos compared to blastocysts. Like other mammals, these errors may contribute to embryonic arrest before or after clinical pregnancy detection. As a proof of principle, we determined the inherited haplotypes for four common disease-associated genes with high carrier frequency in different horse breeds (GBE1, PLOD1, B3GALNT2, MUTYH), and for one color coat-associated gene (STX17) in one biopsy-embryo combination, confirming the potential of generic genome-wide PGT-M concurrent with PGT-A testing. Similar to humans, we postulate that embryo selection will improve embryo transfer success rates in horses and enable selection for traits and against diseases thus accelerating breeding programs.

Molecular effect of holding equine oocytes: a road to cytoplasmic maturation?

The quality of oocytes has a direct effect on the outcome of in vitro embryo production (IVP). For successful IVP, oocytes need to undergo maturation, which requires coordinated nuclear (NM) and cytoplasmic maturation (CM). The lack of adequate cytoplasmic maturation has been proposed to be associated with a lower developmental competence of the oocyte. The improvement of in vitro techniques relies upon mirroring in vivo conditions. Therefore, acquiring a deeper knowledge of the process involved in CM and pathways leading to germinal vesicle breakdown and maturation can provide the necessary foundation to improve IVP results. Holding oocytes in commercial embryo holding media for 24hrs at room temperature has become a routine procedure for oocyte shipment and laboratory scheduling. However, the effects of this procedure on oocyte CM remain unresolved. In this study, we analyzed the changes induced by the holding period on the transcriptome of equine oocytes (OC) and the surrounding cumulus cells (CC). Cumulus-oocyte-complexes (COCs) were collected from six mares using transvaginal aspiration. Only non-expanded, non-degenerated COCs were randomly allocated to one of two groups: no holding (NH) or 24hrs holding (H). The COCs were denuded, and each single OC and CC were stored individually in RNAlater® at -20°C until further processing. RNA was extracted from 24 samples (NH/CC=6; NH/OC=6; H/CC=6; H/OC=6), followed by librarypreparation and sequencing (150 pb-PE). Reads were trimmed and mapped to the equine reference genome (Ecab 3.0). Mapped reads were quantified using featureCount and analysis of differentially expressed genes (DEGs) was performed using DESeq2 (FDR< 0.1 and log fold change >2). Overall, there were 26,077 genes expressed in CCs and 17,930 in OCs. When compared by holding time, there were 660 DEGs between the CCs (NH vs. H) and 205 between the OCs. In CC, 74 genes had a higher expression and 586 had a lower expression in H than NH groups. In OC, 7 genes were upregulated, and 198 were downregulated in H compared to NH group. Gene ontology analysis showed changes in genes related to glutathione (GSH) synthesis, AP-1 transcription factor complex and mitogen-activated protein kinase (MAPK) pathway in CC. In OC, DEGs were involved in α-tubulin detyrosination and tight junction proteins involved in magnesium transport. Overall, our results suggest modification in processes related to CM, including post-translational changes in tubulin, GSH composition, and activation of MAPK pathways during oocyte holding. Meanwhile, genes considered markers of NM and meiotic resumption remained unchanged, suggesting pre-maturation like changes occur prior to germinal vesicle breakdown. Further studies to understand the implications of this pre-maturation step in the developmental competence of oocytes can help improve the outcome of equine IVP. Funding: Center for Equine Health

In vitro-produced equine blastocysts exhibit dispersed ICM cell allocation

Although in vitro production (IVP) of horse embryos has increased markedly in clinical practice, pregnancy rates after transfer are lower, and the incidence of pregnancy loss and monozygotic twins, higher than for in vivo-derived (IVD) embryos. Cell lineage specification is critical to normal early embryo development, and starts with differentiation of trophectoderm (TE) from inner cell mass (ICM) cells, followed by division of the ICM into primitive endoderm (PE) and epiblast (EPI). We examined the influence of developmental stage and speed, IVP and culture (in vivo versus in vitro) on expression of markers for the 3 early cell lineages, namely CDX-2 (TE), GATA-6 (PE) and SOX-2 (EPI). Day 7 IVD blastocysts (n=3 early; n=3 expanded), and IVP embryos identified as blastocysts on day 7 (n=5) or day 9 (n=9) were examined using confocal microscopy to compare total cell number, proportions and distribution of cells expressing CDX-2, GATA-6 and SOX-2. Additional day 7 IVP blastocysts were cultured for 2 days, either in vitro (n=5) or in vivo (after transfer into recipient mares; n=3), to examine the impact on subsequent development. Numerical data were analyzed using T- or Mann-Whitney U-tests. In IVD embryos, CDX-2 expression was exclusive to the TE, whereas SOX-2 expression was detected in both ICM and TE cells in early blastocysts but only thepresumed EPI in expanded blastocysts. GATA-6 was co-expressed with SOX-2 in outer ICM cells in early blastocysts but specific to PE cells lining the EPI and TE in expanded blastocysts. In IVP blastocysts, CDX-2 was also specific to the TE. However, SOX-2 and GATA-6 positive cells were intermingled in the ICM, and both SOX-2 and GATA-6 were co-expressed in TE cells. Strikingly, the EPI cells of IVP blastocysts were more dispersed, with larger mean inter-EPI cell distances (day 7 blastocysts, 52±6µm; day 9, 68±9µm), than in IVD embryos (35±3µm). Total cell number was higher in day 7 IVD (486±81) than IVP (day 7, 317±21; day 9, 377±104) blastocysts, but proportions of the different lineages didn't differ. Cell distribution in day 7 IVP embryos didn't change during an extra 2-day in vitro culture whereas, after 2 days in a mare's uterus, the SOX-2 (EPI) cells had aggregated and compacted, and segregated from PE cells. Overall, with respect to cell lineage allocation, IVP embryos resemble early IVD blastocysts in co-expressing SOX-2 and GATA-6 in TE cells, but differ by having a more dispersed ICM/EPI. While the EPI cells do compact following transfer to a recipient mare, the initial dispersal might contribute to both lower developmental competence (inadequate EPI aggregation) and a higher incidence of monozygotic twins (separated EPIs).

Effect of pre-maturation oocyte timing on equine ICSI embryo development rates

Within the last 5 years, client adoption of ovum-pick-up (OPU) paired with equine intracytoplasmic sperm injection (ICSI) and in vitro embryo production (IVP) in Brazil has been rapid. Equine ICSI has facilitated the use of expensive semen be it imported, rare, poor-quality or low motility for IVP. A pre-maturation step occurs between OPU on the farm and transport to the lab. Oocytes are kept in 5 ml vials of pre-maturation medium, held at 22°C until placement in maturation medium. Oocytes in pre-maturation medium are kept at this low temperature to inhibit the onset of germinal vesicle breakdown and the onset of oocyte maturation. If maturation begins during transport, oocyte maturation rates and oocyte quality would be severely reduced. Recently, (Merlo, B et al 2022 Anim Reprod Sci 107071) reported different rates of equine ICSI embryo development based on timing of oocyte pre-maturation and maturation. In our IVP system, oocyte maturation time is tightly controlled between 28 and 32 hours, so we decided to evaluatethe timing of pre-maturation since it can vary between 0 and 24 hours. This variation is based on the time required for OPU completion by multiple teams, aspirating on the same day in different locations. We tested the potential impact of oocyte pre-maturation in 3-time intervals 0-8, 9-16 and 17-24 hours, in the 2021-2022 breeding season. The average blastocyst formation rate based on total oocytes collected was 17%. Embryo development from oocytes aspirated in the 3 pre-maturation time intervals was: 0-8 hours, 522 embryos produced from 3413 oocytes aspirated (15.3%); 9-16 hours, 471 embryos produced from 2667 oocytes aspirated (17.7%) and 17-24 hours, 3931 embryos produced from 22,896 oocytes aspirated (17.2%). There were no significant differences observed (Mann-Whitney test p >0.05), demonstrating that up to 24 hours, the holding time of pre-maturation oocytes, held at 22.5°C had no significant impact on embryo development in our equine ICSI embryo production system.

In Vitro-Produced Equine Blastocysts Exhibit Greater Dispersal and Intermingling of Inner Cell Mass Cells than In Vivo Embryos.

In vitro production (IVP) of equine embryos is increasingly popular in clinical practice but suffers from higher incidences of early embryonic loss and monozygotic twin development than transfer of in vivo derived (IVD) embryos. Early embryo development is classically characterized by two cell fate decisions: (1) first, trophectoderm (TE) cells differentiate from inner cell mass (ICM); (2) second, the ICM segregates into epiblast (EPI) and primitive endoderm (PE). This study examined the influence of embryo type (IVD versus IVP), developmental stage or speed, and culture environment (in vitro versus in vivo) on the expression of the cell lineage markers, CDX-2 (TE), SOX-2 (EPI) and GATA-6 (PE). The numbers and distribution of cells expressing the three lineage markers were evaluated in day 7 IVD early blastocysts (n = 3) and blastocysts (n = 3), and in IVP embryos first identified as blastocysts after 7 (fast development, n = 5) or 9 (slow development, n = 9) days. Furthermore, day 7 IVP blastocysts were examined after additional culture for 2 days either in vitro (n = 5) or in vivo (after transfer into recipient mares, n = 3). In IVD early blastocysts, SOX-2 positive cells were encircled by GATA-6 positive cells in the ICM, with SOX-2 co-expression in some presumed PE cells. In IVD blastocysts, SOX-2 expression was exclusive to the compacted presumptive EPI, while GATA-6 and CDX-2 expression were consistent with PE and TE specification, respectively. In IVP blastocysts, SOX-2 and GATA-6 positive cells were intermingled and relatively dispersed, and co-expression of SOX-2 or GATA-6 was evident in some CDX-2 positive TE cells. IVP blastocysts had lower TE and total cell numbers than IVD blastocysts and displayed larger mean inter-EPI cell distances; these features were more pronounced in slower-developing IVP blastocysts. Transferring IVP blastocysts into recipient mares led to the compaction of SOX-2 positive cells into a presumptive EPI, whereas extended in vitro culture did not. In conclusion, IVP equine embryos have a poorly compacted ICM with intermingled EPI and PE cells; features accentuated in slowly developing embryos but remedied by transfer to a recipient mare.

Application of preimplantation genetic diagnosis in 91 equine ICSI embryos

Preimplantation genetic diagnosis (PGD) of in vitro-produced (IVP) equine embryos has been reported in research and clinical settings. In the previous reports, 4-16% biopsied samples failed to yield PCR results of disease-causing genes. There is little information on the feasibility of a second biopsy and refreezing of those PGD-failed frozen IVP equine embryos. In this report, we evaluated biopsy results from 91 IVP equine embryos produced from 2018 to 2022 and tested for hereditary equine regional dermal asthenia (HERDA), glycogen branching enzyme deficiency (GBED) and hyperkalemic periodic paralysis (HYPP) genes. We also examined the pregnancy and foaling rates of twice biopsied-frozen (2BF) embryos by comparing them with fresh or once biopsied-frozen (1BF) embryos. IVP embryos were produced by intracytoplasmic sperm injection and in vitro culture of shipped immature oocytes. Biopsy of embryos was performed as previously reported with minor modifications (Choi et al., Reprod Fertil Dev 2016;28:1382-1389). Biopsies were kept frozen for a maximum of 7 days and shipped to the University of California, Davis for genetic analysis. A total of 15 fresh, 39 1BF and 5 2BF IVP embryos were transferred to recipients at embryo transfer facilities. The non-transferred vitrified embryos, 12 1BF and 1 2BF IVP embryos, were kept frozen accordingto owners’ instructions. Among the 91 IVP biopsies, 7 (8%) failed to yield PCR results and 84 samples were genetically analyzed; 14 (24%) were homozygous for HERDA in 58 samples, 4 (21%) were homozygous for GBED in 19 samples and 0 were homozygous for HYPP in 7 samples. The molecular sexes of 84 samples were 44 males and 40 females. Among the 7 2BF IVP biopsies, 1 failed to yield PCR results and 6 were genetically analyzed; 1 was homozygous for GBED. The molecular sexes of 6 2BF samples were 1 male and 5 females. The pregnancy rates of fresh, 1BF and 2BF IVP embryos were not significantly different among groups (60% (9/15), 64% (25/39) and 80% (4/5), respectively, Fisher's exact test). Of these, 3 pregnancies from 1BF were removed from the foaling rate calculation because of unknown and ongoing pregnancies. The foaling rates of fresh, 1BF and 2BF IVP embryos were not significantly different among groups (47% (7/15), 33% (12/36) and 60% (3/5), respectively, Fisher's exact test). Out of 22 foals analyzed, all matched with molecular genders. In summary, PGD for disease-causing genes can be further improved by a second biopsy and refreezing of frozen IVP equine embryos. In addition, two biopsy-freezing cycles of IVP equine embryos were not detrimental to in vivo embryo development.

Effects of anethole supplementation on bovine embryo production and quality

Improvements are necessary to further expand the potential of in vitro embryo production (IVP). Anethole, a natural antioxidant, was shown to be a low-cost alternative with an important role in the gene modulation of oxidative stress, and it can be used to produce higher quality embryos. The aim of this study was to evaluate the anethole effect during in vitro maturation (IVM) on the oocyte nuclear maturation, and then, during IVM and/or in vitro culture (IVC) on the production and quality of bovine embryos. In Experiment 1, cumulus-oocyte complexes (COCs) were matured in IVM medium in the following groups: control group (MC); supplemented with 300 µg/ml anethole (M300); and supplemented with 3,000 µg/ml anethole (M3000). The three groups were matured for 24 h to assess nuclear maturation (metaphase II) and gene expression of cumulus cells (CCs). The group that obtained the best response was M300, which was chosen for Experiment 2, together with MC. In this experiment, maturation was carried out either in IVM medium with 300 µg/ml anethole (M300) or not (MC). Subsequently, the COCs were submitted to in vitro fertilization and then divided into four groups for IVC: MCCC (IVM and IVC controls); MCC300 (control IVM and IVC with 300 µg/ml); M300-CC (IVM with 300 µg/ml and control IVC); and M300-C300 (IVM and IVC with 300 µg/ml anethole each). On the third day (D3) of the culture, cleavage was evaluated, and on D8, the production and classification of blastocysts were assessed. It was observed that 3,000 µg/ml anethole reduced the percentage of COCs that had reached nuclear maturation. The genes GREM1, GSTA1, SOD1, and CAT, related to oxidative stress resistance, and COX2 and HAS, involved in cell metabolism, showed increased abundance in CCs in the anethole presence. Regarding embryo production, the use of 300 µg/ml anethole during IVM and IVC improved the percentage of cleaved embryos. In addition, anethole treatment during IVM and IVC increased the expression of GPX1 gene while decreasing IFNT2α gene in the embryos produced. In conclusion, the addition of 300 µg/ml anethole during IVM modulated the expression of genes related to oxidative stress and oocyte development, and its use during IVM and IVC modulated the gene expression related to embryo quality. However, further studies need to be performed to better understand anethole's impact on embryo development.

145 Specific Gravity as an Assessment of Embryonic Viability to Improve Implantation Rates for in Vitro Produced and in Vivo Derived Bovine Embryos

Abstract The objective of this study was to evaluate the ability of a specific gravity chamber (SGC) to identify embryos most likely to result in successful implantation following embryo transfer. In this study, 19 in vitro produced (IVP) and 15 in vivo derived (IVD) Grade bovine embryos were allowed to descend through media in an SGC. Descent time through a 1-cm timing region was recorded, and embryos were retrieved from the chamber and transferred into recipient females or cryopreserved and transferred at a later date. To determine if chamber evaluation had an effect on pregnancy status, 27 IVP and 12 IVD embryos that were not evaluated with the SGC were also transferred alongside project embryos. Pregnancy was determined via transrectal ultrasound palpation and recorded. There was no significant difference in conception rates between embryos evaluated with the SGC and those not, indicating that chamber analysis had no negative impact on viability of both IVP (P = 0.54) and IVD (P = 0.93) embryos. Twenty-two of the 34 total project embryos resulted in successful implantation and pregnancy, with 13 pregnancies (68.4%) by IVP and 9 (60%) by IVD. There was no difference in average descent time of pregnant and non-pregnant embryos for IVP (P = 0.54) or IVD embryos (P = 0.24). When divided into groups according to descent time, 32% of all observations for IVP embryos occurred between 30 to 50 seconds with a 66.7% conception rate, and 42% occurred between 70 to 90 seconds with a 75% conception rate (P = 0.85). For IVD, 60% of all observations occurred between 90 to10 seconds, with a conception rate of 66.7% (P = 0.35). Though not significant, the numeric trends highlighted in these data lend support to previous research utilizing specific gravity. Further research is needed to thoroughly evaluate the potential of this technology as a method of noninvasive assessment of embryo viability.