Cattle Oocytes Research Articles

Sperm vitrification in horses and donkeys.

Sperm vitrification is an alternative freezing method, which includes high cooling rates and non-permeable cryoprotectants agents. The first attempt in equids was using the spheres technique by directly dropping small volumes of the sperm into liquid nitrogen. Later, vitrification was developed using 0.25 mL straws with outer covers, which resulted in similar progressive motility when compared to conventional freezing in donkeys (44.3 ± 15.0 % vs. 44.7 ± 18.2 %) or even higher in horses (48.2 ± 2.3 % vs. 37.3 ± 2.2 %). Subsequently, the vitrification of larger volumes of sperm in 0.5 mL straws was evaluated, but the results showed poor sperm quality after different warming procedures. Finally, fertility was assessed in vitro and in vivo. In horses, the sperm fertilizing capacity was assessed utilizing heterologous in vitro fertilization (IVF) and vitrified sperm showed the ability to penetrate cattle oocytes, leading to pronuclear formation and hybrid embryo cleavage. In donkeys, fertility trials were conducted in vivo on a small group of jennies, showing no statistically significant difference in pregnancy rates between artificial insemination (AI) with vitrified semen (22%) or frozen semen (10%); however, the uterine inflammatory response after AI with vitrified semen solved faster than frozen semen. In conclusion, sperm vitrification has been optimized in horses and donkeys using 0.25 mL straws with outer covers and can be considered as an alternative to conventional freezing.

190 Evaluation of the toxicity of different concentrations of dimethyl sulfoxide and ethylene glycol cryoprotectants on immature cattle oocytes selected by Brilliant cresyl blue stain and subsequent to polar body extrusion

190 Evaluation of the toxicity of different concentrations of dimethyl sulfoxide and ethylene glycol cryoprotectants on immature cattle oocytes selected by Brilliant cresyl blue stain and subsequent to polar body extrusion

Effect of Different Growth Factors on in vitro Developmental Competence and Quality of Cattle Oocytes

Effect of Different Growth Factors on in vitro Developmental Competence and Quality of Cattle Oocytes

Effect of supplementing epinephrine in maturation media on in-vitro developmental competence of cattle and buffalo oocytes

During in-vitro maturation, the oocyte experiences stressful conditions that likely compromise its development. Epinephrine is a catecholamine that plays a vital role during cellular stress by scavenging free radicals. The hypothesis is that epinephrine addition in maturation media improves the developmental competence of oocytes in cattle and buffalo. The objectives of the experiments were to investigate the effect of epinephrine addition in maturation media on nuclear maturation, developmental competence, and oocyte mRNA abundance of genes related to antioxidants and growth pathways in cattle and buffalo. In experiment 1, cattle oocytes were matured for 24 h in maturation media supplemented with increasing concentrations of epinephrine 0, 0.01, 1.0, and 100 μM. Oocytes were cultured to assess cleavage at 48 h and blastocyst on day 7 of the culture. The cumulus-oocyte complexes (COCs) expansion, nuclear maturation, and oocyte mRNA abundance of genes (SOD1, GPX4, GDF9, CASP9) were evaluated. In experiment 2, buffalo oocytes were matured and assessed for development and mRNA abundance as described for cattle. In addition, the blastomere number was counted in the hatched blastocyst. The data were analyzed using GLIMMIX and MIXED procedures of SAS. Results revealed that the supplementation of epinephrine increased (P ≤ 0.03) the COCs expansion, nuclear maturation, and developmental competence of oocytes in cattle. Interestingly, all the responses were maximized (quadratic effect; P ≤ 0.08) at 1 μM concentrations. The mRNA abundance of genes in cattle oocytes was not affected by the treatment. The experiment in buffalo revealed that epinephrine increased blastocyst formation without affecting COCs expansion, and nuclear maturation. The higher blastocyst was achieved at 0.01 μM concentrations of epinephrine. Interestingly, the addition of epinephrine increased the mRNA abundance of genes related to antioxidant pathways (SOD1, GPX4). Moreover, supplementation of epinephrine increased the blastomere count of the hatched blastocyst in buffalo. In conclusion, epinephrine addition in maturation media benefited oocyte development in cattle and blastocyst yield in buffalo at 1 and 0.01 μM concentrations, respectively. It appears that the addition of epinephrine affected different cellular pathways, COCs expansion, and nuclear maturation in cattle and increased antioxidant genes for buffalo.

Effects of donor age and reproductive history on developmental potential of ovum pickup oocytes in Japanese Black cattle (Wagyu)

The objectives of this study were to analyze the (1) effects of donor age and multiparity on development of in vitro fertilization (IVF) embryos after ovum pickup (OPU), (2) effects of repeated and consecutive OPU-IVF procedures on embryo development, and (3) embryo production from OPU-IVF in donors with differing embryo yields after multiple ovulation and embryo transfer technology (MOET) in Japanese Black cattle (Wagyu). Donors were pre-treated with low-dosage follicle-stimulating hormone (FSH; 200 IU total), and oocytes were collected via OPU and fertilized by IVF to generate blastocysts. The number of oocytes collected per OPU session per donor was lower in heifers (2–4 years old, 5.3 oocytes) than in primiparous and pluriparous cows (2–10 years old, 13.6–19.1 oocytes; P < 0.05). Rates of blastocyst development for oocytes from heifers (33.1%) were lower than for those from cows (2–10 years old, 44.1–54.3%; P < 0.05), and average blastocyst yield/OPU/animal was lower in heifers (3.7) than in 5–6 years old cows (10.1; P < 0.05). Donors undergoing five consecutive OPU-IVF sessions after low-dosage FSH showed similar oocyte retrieval (12.2–15.1 oocytes per OPU/animal), blastocyst development rates (35.6–45.0%), and embryo yield/OPU/animal (4.8–5.8; P > 0.05) across sessions. Additionally, embryo yield from OPU-IVF was significantly improved in animals with previous low embryo yield from MOET (5.9 vs. 2.6, respectively, P < 0.05). These results indicate that Wagyu cows with previous births can be more productive as OPU-IVF donors than heifers, and oocytes from donors undergoing to five consecutive OPU-IVF cycles are competent for embryo development without loss of embryo yield/OPU/animal. Moreover, OPU-IVF can be used for embryo production and breeding from all elite Japanese Black cattle, regardless of previous low embryo yield in routine MOET.

Impact of heat stress on genetic evaluation of oocyte and embryo production in Gir dairy cattle.

Identifying and selecting genotypes tolerant to heat stress might improve reproductive traits in dairy cattle, including oocyte and embryo production. The temperature-humidity index (THI) was used, via random regression models, to investigate the impact of heat stress on genetic parameters and breeding values of oocyte and embryo production in Gir dairy cattle. We evaluated records of total oocytes (TO), viable oocytes (VO), cleaved embryos (CE), and viable embryos (VE) from dairy Gir donors. Twenty-four models were tested, considering age at ovum pick-up (AOPU) and THI means as a regressor in the genetic evaluation. We computed THI in eight periods, from 0 to 112days before ovum pick-up, which were adjusted by different orders of Legendre polynomials (second, third, and fourth). The best-fit model according to Akaike's information criterion (AIC) and Model Posterior Probabilities (MPP) considered Legendre polynomials of third order and THI means of 112days for TO, fourth order and 56days for VO, second order and 28days for CE, and second order and 42days for VE, respectively. The heritability (h2) estimates across AOPU and THI scales ranged from 0.34 to 0.62 for TO, 0.31 to 0.58 for VO, 0.26 to 0.39 for CE, and 0.15 to 0.26 for VE, respectively. The fraction of the phenotypic variance explained by the permanent environment in different AOPU and THI scales ranged from 0.03 to 0.25 for TO, 0.05 to 0.26 for VO, 0.09 to 0.36 for CE, and 0.15 to 0.27 for VE, respectively. Spearman's rank correlation between the estimated breeding values in different AOPU and THI scale from the top 5% sires and females ranged from 0.18 to 0.90 for TO, 0.31 to 0.95 for VO, 0.14 to 0.85 for CE, and 0.47 to 0.94 for VE, respectively. The h2estimates for all evaluated traits varied from moderate to high magnitude across AOPU and THI scales, indicating that genetic selection can result in rapid genetic progress for the evaluated traits. There was a reranking among the best animals in different AOPU and THI. It is possible to select dairy Gir cattle tolerant to heat stress to improve oocyte and embryo production.

Pre-implantation development of cattle embryos produced from fresh bull semen enriched for X- chromosome-bearing spermatozoa using a monoclonal antibody.

Immunological approaches are gaining attention as a convenient and economical method for sex-sorting mammalian spermatozoa. A monoclonal antibody (WholeMom™) has previously been reported to cause agglutination of Y-chromosome-bearing spermatozoa in frozen-thawed semen for gender preselection. However, its usefulness for gender preselection in fresh semen and subsequent in vitro fertilization (IVF) after freeze-thawing has not been reported. This study investigated the in vitro development of cattle embryos produced from fresh bull semen pre-treated with WholeMom™ monoclonal antibody. Results showed that antibody-treated, non-agglutinated spermatozoa (presumably X-chromosome-bearing spermatozoa) could fertilize cattle oocytes in vitro. However, embryos generated from non-agglutinated (enriched in X-chromosome-bearing spermatozoa) had a lower (p < 0.05) ability to cleave (66.4 ± 2.5% vs. 75.1 ± 3.3%) than those of non-treated control sperm. Nevertheless, the percentage of blastocysts developed from cleaved embryos did not differ (p > 0.05) between the groups (34.8 ± 3.7% vs. 35.8 ± 3.4%). Duplex PCR of blastocysts, using a bovine-specific universal primer pair and a Y-chromosome-specific primer pair, showed a sex ratio of 95.8% females from sex-sorted spermatozoa, which was higher than those of non-treated control spermatozoa (46.4%). In conclusion, the results of the present study suggest that monoclonal antibody-based enrichment of X- chromosome-bearing spermatozoa can be applied to fresh bull semen without compromising their post-fertilization early embryonic development to the blastocyst stage. Future studies should investigate the term development and sex ratio of calves from antibody-treated spermatozoa.

Comparative Evaluation of Developmental Competence of Immature Cattle Oocytes in Three Different Culture Media

Comparative Evaluation of Developmental Competence of Immature Cattle Oocytes in Three Different Culture Media

Identification of unique transcriptomic signatures through integrated multispecies comparative analysis and WGCNA in bovine oocyte development

BackgroundCattle (Bos taurus) are a major large livestock, however, compared with other species, the transcriptional specificity of bovine oocyte development has not been emphasised.ResultsTo reveal the unique transcriptional signatures of bovine oocyte development, we used integrated multispecies comparative analysis and weighted gene co-expression network analysis (WGCNA) to perform bioinformatic analysis of the germinal follicle (GV) and second meiosis (MII) gene expression profile from cattle, sheep, pigs and mice. We found that the expression levels of most genes were down-regulated from GV to MII in all species. Next, the multispecies comparative analysis showed more genes involved in the regulation of cAMP signalling during bovine oocyte development. Moreover, the green module identified by WGCNA was closely related to bovine oocyte development. Finally, integrated multispecies comparative analysis and WGCNA picked up 61 bovine-specific signature genes that participate in metabolic regulation and steroid hormone biosynthesis.ConclusionIn a short, this study provides new insights into the regulation of cattle oocyte development from a cross-species comparison.

Effect of Temperature Change and Estradiol Levels on Cattle Oocytes Developmental Competency: Comparing X-Sexed and Non-Sexed Sperm Following in vitro Fertilization

Effect of Temperature Change and Estradiol Levels on Cattle Oocytes Developmental Competency: Comparing X-Sexed and Non-Sexed Sperm Following <i>in vitro</i> Fertilization

Enhancement of Developmental Competence of Immature Cattle Oocytes with Leukemia inhibitory Factor as a Culture Media Supplement

Background: Leukemia Inhibitory Factor (LIF) plays an essential role in oocyte maturation and during early embryonic development. The present study aims to improve the in vitro cattle embryo production by supplementing culture media with LIF. Methods: Fresh ovaries and oviducts were collected from an abattoir in 0.9% saline (30-35°C) supplemented with antibiotics. Total 542 cumulus-oocyte complexes were aspirated from ovaries and cultured in maturation media in 5% CO2 incubator at 38.5°C with maximum humidity after 5-6 times washing. After 24 h matured oocytes were co-incubated with in vitro capacitated sperms in FBO medium. After 15-18 h cumulus cells were stripped off and presumptive zygotes were cultured in mCR2aa medium. After 40 to 42 h, cleavage was observed and embryos were cultured for 7-9 days. Culture media used to replace with fresh media after every 24 h. LIF was supplemented with 15, 30, 45 ng/ml. Result: Supplementation of LIF in culture media increased maturation rate, cleavage rate significantly (P less than 0.05). LIF Supplementation @ 30 ng/ml during culture increased blastocyst development (in control group 7.0±1.3 and 7.1±1.2, 12.9±0.4, 10.2±1.5 in 15 ng/ml, 30 ng/ml and 45 ng/ml respectively) significantly.

How I overcame problems in in vitro fertilisation of livestock animals

In my research life of 35 years, growing with IETS as a researcher of in vitro maturation and fertilisation (IVM/IVF) of porcine and cattle oocytes, I suffered from hard times related to solving problems that prevented the progress of my research and conferment of my degrees. Many researchers may have similar problems. Thus, I would like to address a few examples of how I overcame these problems related to IVF and help young researchers with similar troubles. There were four main problems to be solved in my experiments. Problem 1: Establishment of IVF using only defined medium in pigs. Problem 2: Establishment of successful in vitro culture (IVC) of IVM/IVF bovine oocytes in defined medium. Problem 3: Low rate of male pronucleus formation in IVM porcine oocytes after IVF. Problem 4: Sedimentation of Ca2+ in the sperm capacitation solution for IVF in pigs. Problem 1 was solved by a lucky accident, in which a sperm suspension that would have otherwise been discarded happened to be successfully used for IVF in pigs. Problems 2, 3 and 4 were solved by communication with scientists whose fields were different from mine, where similar problems had been solved already. Young researchers are encouraged to transcend the boundaries of their research fields and solve problems by interacting with researchers in different fields. There are many good connections or answers around us that may be effective in resolving the problems that are hindering the progress of pending research.

Comparison of in vitro Maturation Media on Cattle Oocytes after in vitro Embryo Production

The study was conducted to compare differences in vitro maturation media and their effect on subsequent in vitro embryo production. The study procedures were approved by the agricultural research council animal ethics committee (Ref no: APAEC 2020/05) and the Tshwane university of technology animal research ethics committee (Ref no: AREC 2021/08/004). Follicular fluid containing a suspension of oocytes was retrieved from ovaries through aspiration and slicing techniques. The retrieved oocytes were graded and randomly matured into four media (TCM199, Vitromat Protect, and BO IVM®) groups for 22 h, evaluated for polar body extrusion, fertilized through their respective in vitro fertilization media, and evaluated for pronucleus status. Presumptive zygotes were further cultured in vitro in their respective media, incubated for either 18 or 96 h, and evaluated for cleavage rates. The data was subjected to an appropriate analysis of variance. Student's Least Significant Differences (t-LSD) was calculated at a 5% significance level to compare means of significant treatment effects. The BO-IVM® (57.4%), TCM 199 (55.6%), and VitroMat Protect® (54.0%) recorded higher results than EGF (26.9%) for polar body formation on oocytes (P<0.05). Vitrofert® medium recorded a higher percentage (43.3%) of total fertilization rate when compared to the other media (P<0.05). VitroCleave Plus® medium recorded a higher total cleavage rate (43.3%) as compared to the other media (P<0.05). In conclusion, TCM 199, BO-IVM®, and VitroMat Protect® media rendered higher results of oocyte polar body extrusion rates compared to the EGF medium. Vitrofert® medium recorded a higher percentage (43.3%) of total fertilization rate when compared to the other media. Higher embryo development (4-8 cells and total cleavage) rates were observed in VitroCleave Plus® medium. Overall, the introduced media (VitroMat Protect®) and its media suite successfully adapted to the laboratory environment in South Africa (SA) and can therefore be adopted for optimizing the in vitro Embryo Production (IVEP) of cattle oocytes.

136 Influence of sex-sorted semen on developmental competency of cattle oocytes

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

Genetic evaluation of oocyte and embryo production in dairy Gir cattle using repeatability and random regression models

The objective of this work is to estimate genetic parameters and breeding values to improve embryo and oocyte production, using repeatability and random regression models (RRM) for Gir dairy cattle. We used 11,398 records of ovum pick-up from 1,747 dairy Gir donors and evaluated sixteen different models: the traditional repeatability model and fifteen RRM, each of which considered a different combination of Legendre polynomial regressors to describe the additive genetic and permanent environment effects. The 4G1P model (four regressors [...]

CONDITIONS FOR PREPARATION OF CATTLE OOCYTES FOR IN VITRO MICROMANIPULATION

When cattle oocytes are fertilized through intracytoplasmic sperm injection, the sperm is injected directly into the cytoplasm of the egg cell, allowing three natural barriers to be overcome outside the body. If the loss of highly valuable genetic material&#x0D; in a male or female parent is threatened, this may be the only way to preserve valuable hereditary traits with a high cost recovery, and it may also serve as the basis for further development of genetic engineering. In this regard, the Laboratory for Molecular Biotechnology and DNA Testing of the RUE “Scientific and Practical Center of the National Academy of Sciences of Belarus for Animal Breeding” has developed and studied conditions for preparation of cattle oocytes for in vitro micromanipulation. It has been established that it is preferable to use the ovaries in the luteal and follicular phases.

Augmentation of Developmental Competence of Immature Cattle Oocytes Supplemented with Growth Factors in Culture Media

Augmentation of Developmental Competence of Immature Cattle Oocytes Supplemented with Growth Factors in Culture Media

Tight gene co-expression in BCB positive cattle oocytes and their surrounding cumulus cells

BackgroundCytoplasmic and nuclear maturation of oocytes, as well as interaction with the surrounding cumulus cells, are important features relevant to the acquisition of developmental competence.MethodsHere, we utilized Brilliant cresyl blue (BCB) to distinguish cattle oocytes with low activity of the enzyme Glucose-6-Phosphate Dehydrogenase, and thus separated fully grown (BCB positive) oocytes from those in the growing phase (BCB negative). We then analyzed the developmental potential of these oocytes, mitochondrial DNA (mtDNA) copy number in single oocytes, and investigated the transcriptome of single oocytes and their surrounding cumulus cells of BCB positive versus BCB negative oocytes.ResultsThe BCB positive oocytes were twice as likely to produce a blastocyst in vitro compared to BCB- oocytes (P < 0.01). We determined that BCB negative oocytes have 1.3-fold more mtDNA copies than BCB positive oocytes (P = 0.004). There was no differential transcript abundance of genes expressed in oocytes, however, 172 genes were identified in cumulus cells with differential transcript abundance (FDR < 0.05) based on the BCB staining of their oocyte. Co-expression analysis between oocytes and their surrounding cumulus cells revealed a subset of genes whose co-expression in BCB positive oocytes (n = 75) and their surrounding cumulus cells (n = 108) compose a unique profile of the cumulus-oocyte complex.ConclusionsIf oocytes transition from BCB negative to BCB positive, there is a greater likelihood of producing a blastocyst, and a reduction of mtDNA copies, but there is no systematic variation of transcript abundance. Cumulus cells present changes in transcript abundance, which reflects in a dynamic co-expression between the oocyte and cumulus cells.

Effect of three schemes of ovum pick-up on the follicular dynamics, gene expression, and in-vitro developmental competence of oocytes in Sahiwal cattle.

This study aimed to compare the effect of three schemes of ovum pick-up (OPU) on follicular dynamics, oocytes recovery, oocytes quality, gene expression, nuclear maturation and in-vitro developmental competence of oocytes in Sahiwal cattle. Considering the follicle population, all the cows were divided equally in a 3× 3 cross over design, and each cow received one of the three treatments: (a) twice weekly (TW; n=6), (b) once weekly (OW; n=6) and (c) bi-weekly OPU (BW; n=6) in three periods, with the first OPU conducted on 4, 7 and 14 days after second dominant follicle puncture (DFP) in the TW, OW and BW OPU interval groups, respectively. The collected cumulus oocytes complexes (COCs) were graded into A, B, C and D grades depending on the number of layers of cumulus cells and homogeneous nature of cytoplasm. Nuclear maturation was assessed by staining the oocytes with Hoechst 33342. The growth rate (mm/day) of dominant follicle (DF) (F1) (0.49 ± 0.21 vs. 0.71 ± 0.26 vs. 1.30 ± 0.27) and first subordinate follicle (F2) (0.85 ± 0.27 vs. 0.71 ± 0.25 vs. 1.06 ± 0.29) did not differ (p > .05) among all the three groups. The proportion of animals bearing a corpus luteum (CL) in the BW OPU interval group (53.3%) was significantly higher (p < .05) as compared to TW (13.3%) and OW (18.3%) OPU interval groups. The number of medium-sized follicles and oocyte with grade A and B were significantly higher (p < .05) in the TW (1.16 ± 0.21 and 33.88 ± 0.03) OPU interval group as compared to the OW (0.88 ± 0.22 and 21.54 ± 0.03) and BW (0.55 ± 0.21 and 21.89 ± 0.02) OPU interval groups. However, the number of degenerated oocytes in BW (0.85 ± 0.16) OPU interval group was significantly higher (p < .05) as compared to the TW (0.16 ± 0.15) and OW (0.44 ± 0.16) OPU interval groups. Expression level of growth differentiation factor 9 in TW OPU interval group was significantly higher (p < .05) as compared to the OW and BW OPU interval groups. Likewise, expression level of bone morphogenetic protein 15 (BMP15) in the TW and BW OPU interval groups was significantly higher (p < .05) as compared to the OW OPU interval group. The nuclear maturation rate was significantly higher in the TW (63.64 ± 0.07) and BW (59.26 ± 0.08) OPU groups as compared to OW (51.43 ± 0.06) OPU interval group. However, the cleavage rate (59.30 ± 0.06 vs. 44.29 ± 0.06 vs. 56.67 ± 0.06) did not differ (p > .05) among the three groups. Whereas, the blastocyst rate tended to be higher (p=.06) in the TW (29.07 ± 0.05) and BW (28.33 ± 0.04) OPU interval groups as compared to OW (18.57 ± 0.05) OPU interval group. Taken together, it can be concluded that TW OPU interval scheme enhances the medium-sized follicles resulting in good quality oocytes, regulates the oocyte-derived paracrine factors, leading to higher nuclear maturation rates and improved embryonic development in-vitro.

Influence of endometritis on the follicular dynamics, recovery, quality, gene expression, nuclear maturation and in-vitro developmental competence of oocytes in Sahiwal cattle.

Uterine infections often lead to culling of valuable animals from a herd, resulting in genetic drain. The genetic potential of problematic females could be harvested by in-vitro embryo production. The objective of this study was to evaluate the effect of clinical endometritis on follicular dynamics, recovery, quality, gene expression, nuclear maturation and in-vitro developmental competence of oocytes in Sahiwal cattle. The B-mode ultrasonography was performed to examine the uterus for the presence of pus. Based on the history and reproductive examination of cows, a total of twelve (n=12) Sahiwal cattle were selected for the experiment: (1) healthy group (n=6) and (2) clinical endometritis group (n=6). The 1st ovum pick-up (OPU) was conducted on day 165 postpartum. The collected cumulus oocytes complexes (COCs) were graded into A, B, C and D grades depending on the number of layers of cumulus cells and homogeneous nature of cytoplasm. Nuclear maturation was assessed by staining the oocytes with Hoechst 33,342. The results revealed that the number of medium-sized follicle (1.3±0.1 versus 0.6±0.1) and total number of follicles (9.1±0.7 versus 6.6±0.7) were higher (p<.05) in the healthy group as compared to clinical endometritis group, respectively. Similarly, the number of oocytes recovered (5.0±0.4 versus 2.8±0.4), oocytes with grade A, B and C (2.9±0.3 versus 1.5±0.3), proportion of oocytes with grade A or B (33±0.0 versus 20±0.1) and nuclear maturation (68±0.1 versus 55±0.1) were also higher (p<.05) in the healthy group as compared to clinical endometritis group, respectively. Perhaps, cleavage rate (55.1±0.1 versus 46.2±0.1) and blastocyst rate (29.7±0.0 versus 26.3±0.1) did not differ (p>.05) between the groups. Likewise, the expression level of growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) in immature oocytes did not differ (p>.05) between both the groups. In conclusion, clinical endometritis has a negative effect on follicular dynamics, oocyte recovery, oocyte quality and nuclear maturation; furthermore, the developmental competence of COCs is not compromised by it.