Comparison of cattle derived from in vitro fertilization, multiple ovulation embryo transfer, and artificial insemination for milk production and fertility traits.

The use of assisted-reproduction technologies such as in vitro fertilization (IVF) is increasing, particularly in dairy cattle. The question of consequences in later life has not yet been directly addressed by studies on large animal populations. Studies on rodents and early data from humans and cattle suggest that in vitro manipulation of gametes and embryos could result in long-term alteration of metabolism, growth, and fertility. Our goal was to better describe these presumed consequences in the population of dairy cows produced by IVF in Québec (Canada) and to compare them to animals conceived by artificial insemination (AI) or multiple ovulation embryo transfer (MOET). To do so, we leveraged a large phenotypic database (2.5 million animals and 4.5 million lactations) from milk records in Québec aggregated by Lactanet (Sainte-Anne-de-Bellevue, QC, Canada) and spanning 2012 to 2019. We identified 304,163, 12,993, and 732 cows conceived by AI, MOET, and IVF, respectively, for a total of 317,888 Holstein animals from which we retrieved information for 576,448, 24,192, and 1,299 lactations (total = 601,939), respectively. Genetic energy-corrected milk yield (GECM) and Lifetime Performance Index (LPI) of the parents of cows were used to normalize for genetic potential across animals. When compared with the general Holstein population, MOET and IVF cows outperformed AI cows. However, when comparing those same MOET and IVF cows with only herdmates and accounting for their higher GECM in the models, we found no statistical difference between the conception methods for milk production across the first 3 lactations. We also found that the rate of Lifetime Performance Index improvement of the IVF population during the 2012 to 2019 period was less than the rate observed in the AI population. Fertility analysis revealed that MOET and IVF cows also scored 1 point lower than their parents on the daughter fertility index and had a longer interval from first service to conception, with an average of 35.52 d compared with 32.45 for MOET and 31.87 for AI animals. These results highlight the challenges of elite genetic improvement while attesting to the progress the industry has made in minimizing epigenetic disturbance during embryo production. Nonetheless, additional work is required to ensure that IVF animals can maintain their performance and fertility potential.

Machine learning and hypothesis driven optimization of bull semen cryopreservation media.

Cryopreservation provides a critical tool for dairy herd genetics management. Due to widely varying inter- and within-bull post thaw fertility, recent research on cryoprotectant extender medium has not dramatically improved suboptimal post-thaw recovery in industry. This progress is stymied by the interactions between samples and the many components of extender media and is often compounded by industry irrelevant sample sizes. To address these challenges, here we demonstrate blank-slate optimization of bull sperm cryopreservation media by supervised machine learning. We considered two supervised learning models: artificial neural networks and Gaussian process regression (GPR). Eleven media components and initial concentrations were identified from publications in bull semen cryopreservation, and an initial 200 extender-post-thaw motility pairs were used to train and 32 extender-post-thaw motility pairs to test the machine learning algorithms. The median post-thaw motility after coupling differential evolution with GPR the increased from 52.6 ± 6.9% to 68.3 ± 6.0% at generations 7 and 17 respectively, with several media performing dramatically better than control media counterparts. This is the first study in which machine learning was used to determine the best combination of constituents to optimize bull sperm cryopreservation media, and provides a template for optimization in other cell types.

Hydroxytyrosol and resveratrol improves kinetic and flow cytometric parameters of cryopreserved bull semen with low cryotolerance.

There is considerable interest in breeding programs to "rescue" semen with poor post-thaw fertility from bulls known as "bad freezers". We hypothesized that there may be an interaction between the post-thaw recovery of sperm and the efficacy of antioxidant addition to extenders. The current study assesses the effects of antioxidant additives hydroxytyrosol (HT) and resveratrol (RSV) on the post-thaw semen parameters in two groups of bulls classified as either high or low cryotolerant (i.e., "good" and "bad" freezers). Semen samples were collected from 40 bulls and processed in the extenders containing different concentrations of HT (experiment 1; 0, 25 and 50 µM) and RSV (experiment 2; 0.0, 0.01, 0.1 and 1 mM). In experiment 1, bulls in the low cryotolerance group had a significant improvement in post-thaw recovery at 25 µM and 50 µM (P < 0.05). These improvements were observed in motility and several cellular parameters. However, post-thaw semen quality in the high cryotolerance group was not significantly affected by the HT addition. In experiment 2, although RSV did not have any positive impact in high cryotolerance group, post-thaw recovery in the low cryotolerance bulls was significantly improved in 0.1 mM RSV. Oxidative stress markers in either high or low cryotolerance groups were not significantly changed by RSV addition (P > 0.05). It can be concluded that addition of optimized concentrations of HT and RSV to the extender could be a strategy for improving the post-thaw semen, especially in bulls with high genetic merit but low initial cryotolerance.

Preimplantation Genetic Testing for Aneuploidy Improves Live Birth Rates with In Vitro Produced Bovine Embryos: A Blind Retrospective Study.

Approximately one million in vitro produced (IVP) cattle embryos are transferred worldwide each year as a way to improve the rates of genetic gain. The most advanced programmes also apply genomic selection at the embryonic stage by SNP genotyping and the calculation of genomic estimated breeding values (GEBVs). However, a high proportion of cattle embryos fail to establish a pregnancy. Here, we demonstrate that further interrogation of the SNP data collected for GEBVs can effectively remove aneuploid embryos from the pool, improving live births per embryo transfer (ET). Using three preimplantation genetic testing for aneuploidy (PGT-A) approaches, we assessed 1713 cattle blastocysts in a blind, retrospective analysis. Our findings indicate aneuploid embryos have a 5.8% chance of establishing a pregnancy and a 5.0% chance of given rise to a live birth. This compares to 59.6% and 46.7% for euploid embryos (p < 0.0001). PGT-A improved overall pregnancy and live birth rates by 7.5% and 5.8%, respectively (p < 0.0001). More detailed analyses revealed donor, chromosome, stage, grade, and sex-specific rates of error. Notably, we discovered a significantly higher incidence of aneuploidy in XY embryos and, as in humans, detected a preponderance of maternal meiosis I errors. Our data strongly support the use of PGT-A in cattle IVP programmes.

Sperm miRNAs- potential mediators of bull age and early embryo development.

BACKGROUND: Sperm miRNAs were reported to regulate spermatogenesis and early embryonic development in some mammals including bovine. The dairy cattle breeding industry now tends to collect semen from younger bulls under high selection pressure at a time when semen quality may be suboptimal compared to adult bulls. Whether the patterns of spermatic miRNAs are affected by paternal age and/or impact early embryogenesis is not clear. Hence, we generated small non-coding RNA libraries of sperm collected from same bulls at 10, 12, and 16 months of age, using 16 months as control for differential expression and functional analysis. RESULTS: We firstly excluded all miRNAs present in measurable quantity in oocytes according to the literature. Of the remaining miRNAs, ten sperm-borne miRNAs were significantly differentially expressed in younger bulls (four in the 10 vs 16 months contrast and six in the 12 vs 16 months contrast). Targets of miRNAs were identified and compared to the transcriptomic database of two-cell embryos, to genes related to two-cell competence, and to the transcriptomic database of blastocysts. Ingenuity pathway analysis of the targets of these miRNAs suggested potential influence on the developmental competence of two-cell embryos and on metabolism and protein synthesis in blastocysts. CONCLUSIONS: The results showed that miRNA patterns in sperm are affected by the age of the bull and may mediate the effects of paternal age on early embryonic development.

Specific imprinted genes demethylation in association with oocyte donor's age and culture conditions in bovine embryos assessed at day 7 and 12 post insemination.

The production of bovine embryos through in vitro maturation and fertilization is an important tool of the genomic revolution in dairy cattle. Gene expression analysis of these embryos revealed differences according to the culture conditions or oocyte donor's pubertal status compared to in vivo derived embryos. We hypothesized that some of the methylation patterns in oocytes are acquired in the last step of folliculogenesis and could be influenced by the environment created in the follicles containing these oocytes. These altered patterns may not be erased during the first week of embryonic development in culture or may be sensitive to the conditions during that time. To quantify the changes related to culture conditions, an in vivo control group consisting of embryos (Day 12 post fertilization for all groups) obtained from superovulated and artificially inseminated cows was compared to in vitro produced (IVP) embryos cultured with or without Fetal Bovine Serum (FBS). To measure the effect of the oocytes donor's age, we also compared a fourth group consisting of IVP embryos produced with oocytes collected following ovarian stimulation of pre-pubertal animals. Embryonic disk and trophoblast cells were processed separately and the methylation status of ten imprinted genes (H19, MEST, KCNQ1, SNRPN, PEG3, NNAT, GNASXL, IGF2R, PEG10, and PLAGL1) was assessed by pyrosequencing. Next, ten Day 7 blastocysts were produced following the same methodology as for the D12 embryos (four groups) to observe the most interesting genes (KCNQ1, SNRPN, IGF2R and PLAGL1) at an earlier developmental stage. For all samples, we observed overall lower methylation levels and greater variability in the three in vitro groups compared to the in vivo group. The individual embryo analysis indicated that some embryos were deviant from the others and some were not affected. We concluded that IGF2R, SNRPN, and PEG10 were particularly sensitive to culture conditions and the presence of FBS, while KCNQ1 and PLAGL1 were more affected in embryos derived from pre-pubertal donors. This work provides markers at the single imprinted control region (ICR) resolution to assess the culture environment required to minimize epigenetic perturbations in bovine embryos generated by assisted reproduction techniques, thus laying the groundwork for a better comprehension of the complex interplay between in vitro conditions and imprinted genes.

Effects of follicular ablation and GnRH on synchronization of ovulation and conception rates in embryo recipient heifers.

Two experiments were performed to determine effects of follicular ablation (FA) and GnRH treatment on conception rate and synchronization in timing of ovulation among Holstein heifers. In Experiment 1, heifers were randomly allocated to four groups: Control (n = 84): prostaglandin F2α (PGF) IM on Day 0; FA-5/GnRH (n = 43): FA 5 days before PGF and GnRH on Day 2; FA-4/GnRH (n = 48):FA 4 days before PGF and GnRH on Day 2; andFA-3/GnRH (n = 21): FA 3 days before PGF and GnRH on Day 2. Ultrasonography was performed to determine follicular size, ovulation occurrence, and size of CL. In Experiment 2, heifers were assigned to three groups: Control (n = 264), FA-5/GnRH, and FA-4/GnRH. Pregnancy diagnosis was performed at Days 30 and 60. In Experiment 1, size of largest follicle at time of PGF was less variable (P ≤ 0.05) in all FA groups compared to the Control group. With the FA-5/GnRH and FA-4/GnRH treatments, there were greater (P ≤ 0.05) proportions of timing of ovulation synchronization (86 % and 85 %, respectively) compared to the Control (61 %) and FA-3/GnRH (62 %) groups. In Experiment 2, conception rates did not differ among groups, however, there were more pregnancies per cow when timing-of-ovulation treatments were imposed. In conclusion, follicular ablation combined with GnRH treatment resulted in an increased proportion of heifers having synchronized ovulation and, therefore, number of recipient heifers available for embryo transfer. Additionally, there was no effect on conception rate when there was greater synchronization in timing of ovulation among heifers.

Low concentrations of 3-O-methylglucose improve post thaw recovery in cryopreserved bovine spermatozoa.

A number of studies have explored the use of membrane permeable (usually metabolizable) and membrane impermeable saccharides to protect cells in general, and sperm in particular during cryopreservation. Critical concentrations for protective levels of sugars frequently range between 50 mmol/L and 500 mmol/L, where efficacy is attributed to the sugar's membrane stabilizing and glass forming attributes and colligative effects that reduce intra- and extracellular salt concentrations during freezing. Many studies on bull sperm have demonstrated that both permeating and non-permeating sugars have negligible positive effects on post-thaw viability. Recently, however, a non-metabolizable sugar, 3-O-Methylglucose (3-OMG), was shown to protect hepatocytes during liver cryopreservation at 0.1-0.3 mol/L. Because glucose is readily transported into sperm, we hypothesized that 3-OMG could be a new class of cryoprotectant to explore in bull sperm. Here we present positive results demonstrating that 3-OMG improves post thaw viability in bull sperm, and that this effect is not likely due to improved glass forming capabilities. In particular, in experiment 1, 3-OMG was added to the Tris-egg yolk-glycerol base media at levels from 0 mmol/L to 200 mmol/L. Semen from four bulls was collected and diluted with one of the cryopreservation media, cooled, and frozen following industry standard practices. Motility and mitochondrial activity were negatively impacted when concentration of 3-OMG was more than 25 mmol/L. Therefore, we explored lower concentrations in experiment 2, where semen from eight bulls was used to evaluate concentrations 5 mmol/L, 15 mmol/L and 25 mmol/L of 3-OMG compared with control. Motility and progressive motility in 5 mmol/L 3-OMG and in the control were significantly higher than 15 mmol/L and 25 mmol/L 3-OMG, whereas mitochondrial activity and acrosome integrity in 5 mmol/L 3-OMG were significantly better than the control freezing medium. In experiment 3, to evaluate whether the improved effects of 3-OMG are due to its non-metabolizing property, or due to colligative effects, we compared post-thaw viability in semen from four bulls cryopreserved with 5 mmol/L glucose, sucrose, or 3-OMG. Motility and progressive motility was significantly improved in 3-OMG compared to glucose or sucrose groups which were comparable to the EY control. In conclusion, 3-OMG at a concentration of 5 mmol/L in Tris-egg yolk-glycerol medium improves the post thaw motility, progressive motility and viability of bull sperm. The mechanism of action is not understood but because the efficacy is maximal at low concentrations, it is not likely due to improved intra- or extracellular glass forming abilities and may demonstrate a different protective mechanism than was shown in hepatocytes.

DNA methylation status of bovine blastocysts obtained from peripubertal oocyte donors.

In the dairy industry, the high selection pressure combined with the increased efficiency of assisted reproduction technologies (ART) are leading toward the use of younger females for reproduction purposes, with the aim to reduce the interval between generations. This situation could impair embryo quality, decreasing the success rate of the ART procedures and the values of resulting offspring. Young Holstein heifers (n = 10) were subjected to ovarian stimulation and oocyte collection at 8, 11, and 14 months of age. All the oocytes were fertilized in vitro with semen from one adult bull, generating three pools of embryos per animal. Each animal was its own control for the evaluation of the effects of age. The EmbryoGENE platform was used to compare the DNA methylation status of blastocysts obtained from oocytes collected at 8 versus 14 and 11 versus 14 months of age. Age-related contrast analysis identified 5,787 and 3,658 differentially methylated regions (DMRs) in blastocysts from heifers at 8 versus 14 and 11 versus 14 months of age, respectively. For both contrasts, the DMRs were distributed nonrandomly in the different DNA regions. The DNA from embryos from 8-month-old donors was more hypermethylated, while the DNA from embryos from 11-month-old donors displayed an intermediate phenotype. According to Ingenuity Pathway Analysis, the upstream regulator genes cellular tumor antigen p53, transforming growth factor ß1, tumor necrosis factor, and hepatocyte nuclear factor 4α are particularly associated with methylation sensitive targets, which were more hypermethylated in embryos from younger donors.

The age of the bull influences the transcriptome and epigenome of blastocysts produced by IVF.

Genetic selection for the best suited offspring drives the dairy industry to use young genitors and assisted reproductive technologies (ART) to reduce generation intervals. However, sperm samples collected from peri-pubertal bulls have lower counts and quality compared to samples from adult bulls. Moreover, our previous study identified differentially methylated regions (DMRs) in sperms from early-, peri- and post-pubertal bulls. The aim of this study was to further investigate the impacts of paternal age on early embryos. To achieve this, we evaluated the transcriptome and the epigenome of bovine blastocysts generated from spermatozoa of bulls at 10, 12, and 16 months of age and used in vitro fertilization (IVF) of oocytes recovered from the same adult cows. A total of 259 probes were differentially expressed and 6953 probes were differentially methylated in the 10- vs 16-month and the 12- vs 16-month groups. Ingenuity Pathway Analysis (IPA) of transcriptomic data demonstrated that energy-related pathways such as oxidative phosphorylation, EIF2 signaling, and mitochondrial dysfunction were affected the most by the age of the bull. Meanwhile, IPA analysis of the epigenome revealed that protein kinase A signaling, RAR activation, and other pathways were influenced by paternal age. Overall, we showed that the bull's age mainly influenced metabolism-related pathways in blastocysts, and this could therefore impact subsequent development.

The use of adenosine to inhibit oocyte meiotic resumption in Bos taurus during pre-IVM and its potential to improve oocyte competence.

One of the major challenges of artificial reproductive technologies is to develop new methods for producing greater numbers of embryos. An oocyte fosters the ability to develop into an embryo before oocyte meiotic resumption. The aim of the present study was to assess the effect of adenosine (ADO), a purine nucleoside found in follicular fluid, on the inhibition of oocyte meiotic resumption and the production of blastocysts. The results showed the efficacy of ADO to inhibit oocyte meiotic resumption. The use of ADO (3 mM) during a pre-in vitro maturation (pre-IVM) culture period of 6 h resulted in a significant increase (p < 0.05) of blastocysts compared to control conditions with no pre-IVM culture period. No effect on the percentage of cleavage was observed. The effect of adenosine on blastocyst yield was time- and concentration-dependent with an optimum effect at 3 mM for 6 h. Supplementing the ADO pre-IVM culture medium with estradiol, follicle-stimulating hormone, progesterone, epidermal growth factor, insulin-like growth factor-2 or reelin did not improve the blastocyst yield. Transcriptional analyses of ADO-treated cumulus cells revealed that NRP1, RELN, MAN1A1, THRA and GATM were up-regulated. Finally, bioinformatic analysis identified mitochondrial function as the top canonical pathway affected by ADO. This opens up new opportunities for further investigations.

Proteomic markers of low and high fertility bovine spermatozoa separated by Percoll gradient.

In the context of artificial insemination, male fertility is defined as the ability to produce functional spermatozoa able to withstand cryopreservation. We hypothesized that interindividual variations in fertility depend on the proportion of the fully functional sperm population contained in the insemination dose. The objective of this study was to identify protein markers of the fully functional sperm subpopulation. Insemination doses from four high-fertility (HF) and four low-fertility (LF) bulls with comparable post-thaw quality parameters were selected for proteomic analysis using iTRAQ technology. Thawed semen was centrifuged through a Percoll gradient to segregate the motile (high density [HD]) from the immotile (low density [LD]) sperm populations. Sperm proteins were extracted with sodium deoxycholate and four groups were compared: LD and HD spermatozoa from LF and HF bulls. A total of 498 unique proteins were identified and quantified. Comparison of HD spermatozoa from HF and LF bulls revealed that five proteins were significantly more abundant in the HF group (AK8, TPI1, TSPAN8, OAT, and DBIL5) whereas five proteins were more abundant in the LF group (RGS22, ATP5J, CLU, LOC616319, and CCT5). Comparison of LD spermatozoa from HF and LF bulls revealed that four proteins were significantly more abundant in the HF group (IL4I1, CYLC2, OAT, and ARMC3) whereas 15 proteins were significantly more abundant in the LF group (HADHA, HSP90AA1, DNASE1L3, SLC25A20, GPX5, TCP1, HIP1, CLU, G5E622, LOC616319, HSPA2, NUP155, DPY19L2, SPERT, and SERPINE2). DBIL5, TSPAN8, and TPI1 showed potential as putative markers of the fully functional sperm subpopulation.

Effect of heifer age on the granulosa cell transcriptome after ovarian stimulation.

Genomic selection is accelerating genetic gain in dairy cattle. Decreasing generation time by using younger gamete donors would further accelerate breed improvement programs. Although ovarian stimulation of peripubertal animals is possible and embryos produced in vitro from the resulting oocytes are viable, developmental competence is lower than when sexually mature cows are used. The aim of the present study was to shed light on how oocyte developmental competence is acquired as a heifer ages. Ten peripubertal Bos taurus Holstein heifers underwent ovarian stimulation cycles at the ages of 8, 11 (mean 10.8) and 14 (mean 13.7) months. Collected oocytes were fertilised in vitro with spermatozoa from the same adult male. Each heifer served as its own control. The transcriptomes of granulosa cells recovered with the oocytes were analysed using microarrays. Differential expression of certain genes was measured using polymerase chain reaction. Principal component analysis of microarray data revealed that the younger the animal, the more distinctive the gene expression pattern. Using ingenuity pathway analysis (IPA) and NetworkAnalyst (www.networkanalyst.ca), the main biological functions affected in younger donors were identified. The results suggest that cell differentiation, inflammation and apoptosis signalling are less apparent in peripubertal donors. Such physiological traits have been associated with a lower basal concentration of LH.

Interval of gonadotropin administration for in vitro embryo production from oocytes collected from Holstein calves between 2 and 6 months of age by repeated laparoscopy.

Laparoscopic Ovum Pick-Up (LOPU) in calves followed by in vitro embryo production (IVEP) and transfer (ET) into adult recipients has great potential for accelerated genetic gain through shortening of the generation interval. In this study, 11 Holstein calves were subjected to up to six LOPU procedures between the ages of 2-6 months at 2-3 weeks interval. In all cases, the animals received a CIDR 5 days prior to LOPU and were gonadotropin-stimulated starting at 72 h before LOPU using one of three protocols that were rotated twice among the animals during the study. Calves were injected with FSH every 12 h (FSH12h), or every 8 h (FSH8h) or every 8 h until -36 h from LOPU at which point the FSH was replaced with a single dose of 400 IU eCG (FSH8h-eCG). No statistical differences were observed among the 3 treatments in terms of mean follicles available for aspiration (35.7 ±â€¯16 vs. 38.5 ±â€¯25 vs. 31.1 ±â€¯22), mean oocytes recovered (26.5 ±â€¯14 vs. 21.6 ±â€¯10 vs. 19.4 ±â€¯14) and cleavage rate (66.0 ±â€¯14 vs. 61.1 ±â€¯11 vs. 72.2 ±â€¯8), for FSH12h, FSH8h and FSH8h-eCG, respectively. However, FSH8h-eCG resulted in a significantly higher rate of transferable embryos (17.5 ±â€¯8%) compared with FSH12h (8.9 ±â€¯5%, P < 0.05). Oocytes from follicles of ≥5 mm in diameter yielded a higher rate (P < 0.05) of development to the blastocyst stage (13.8%) than those collected from <5 mm follicles (6.8%). Animal age, by comparing animals at <100, 101 to 130 and > 130 days of age, did not affect the mean number of follicles (34.2 ±â€¯15 vs. 39.3 ±â€¯26 vs. 31.6 ±â€¯25), the mean number of oocytes recovered (21.2 ±â€¯10 vs. 24.5 ±â€¯15 vs. 22.6 ±â€¯17), and the cleavage rate (68.6 ±â€¯11 vs. 61.7 ±â€¯12 vs. 70.7 ±â€¯10%), respectively. However, animals in the older age range had significantly higher development to the blastocyst stage (19.9 ±â€¯6 vs. 9.5 ±â€¯8%, P < 0.01) and better embryo quality, as evidenced by higher average cell numbers (119.1 ±â€¯47 vs. 91.5 ±â€¯25, P < 0.05) compared with those in the lower age. Finally, we tested the benefits of relieving endoplasmic reticulum stress by supplementing the culture medium with 50 µM tauroursodeoxycholic acid (TUDCA) and found a numerically higher rate of development to the blastocyst stage (21.1 ±â€¯8 vs. 18.6 ±â€¯4%), but not statistically different, compared with control culture. Overall, our findings indicate that a significant number of transferable embryos (range 10-30) can be produced from Holstein calves before they reach 6 months of age.

ASAS-SSR Triennial Reproduction Symposium: The use of natural cycle's follicular dynamic to improve oocyte quality in dairy cows and heifers.

The selection of the best dairy heifers is mainly driven by the genetic value of their parents. The phenotype analysis of cows and of the daughters of bulls has been used to identify the best genetic value for decades before being replaced by genomic selection of individuals that are not yet parents. Because it is possible to predict the future value of an individual by its genetic makeup, it becomes feasible to do it as early as the blastocyst stage and to decide which should be transferred or not. Because we know the genotype of an animal at birth, or even before, it is becoming desirable to reproduce this animal as soon as possible to reduce generation interval and improve selection speed. Nature provides constraints that can be overcome: a single oocyte per cycle and age at puberty. Indeed, it is now possible to super-stimulate the ovary at any age and to start collecting oocytes at 6 mo by trans-vaginal ultrasonography. The challenge becomes the production of good eggs and embryos capable of implanting and developing into healthy calves. Our understanding of ovarian follicular physiology has been instrumental in designing stimulation protocols that may be adjusted to any physiological context including age, and even the individual animal, to obtain a good response. Therefore, the combination of procedures developed in cows to optimize oocyte quality, for example, FSH coasting, in association with in vitro fertilization and optimal culture conditions can now result in the production of several female embryos twice a month from animals 6 to12 mo of age. The transcriptomic and epigenetic analyses of embryos produced from the same females at different ages were compared and few differences were noted in particular in relation to embryo metabolism. These embryos are as good as the ones obtained from adult animals and can be produced with sexed sperm of bulls 12 mo of age. This combination of these technical optimizations with blastocyst genotyping allows the selection of a second generation within a year.

Influence of luteinizing hormone support on granulosa cells transcriptome in cattle.

In cows, the use of follicle-stimulating hormone (FSH) to stimulate follicular growth followed by a short period of FSH withdrawal has been shown to be beneficial for oocyte developmental competence. Although this treatment represents a useful optimization to generate highly competent oocytes, the underlying physiological process is not completely understood. The goal of this study was to investigate the role of luteinizing hormone (LH) action during FSH withdrawal before ovulation. To accomplish this, LH release was pharmacologically inhibited during the coasting period with gonadotropin-releasing hormone (GnRH) antagonists. Granulosa cells samples were obtained from cows stimulated with FSH during 3 days followed by a coasting period of 68 h and treated with a GnRH antagonist (cetrorelix group) or not (control). A significant reduction in the number of follicles at >10 mm diameter was observed with the cetrorelix group and gene expression of granulosa cells reveals that 747 transcripts are potentially regulated by LH. Further analysis indicates how the absence of LH may trigger early atresia, the upregulation of atretic agent as tumor protein P53 and transforming growth factor ß1 and the inhibition of growth support. This work allows identification of genes that are associated with maintained follicular growth and conversely the ones leading to atresia in dominant pre-ovulatory follicles.

Spermatozoa DNA methylation patterns differ due to peripubertal age in bulls.

In the dairy industry, using semen as soon as the bull is mature enough to produce it is advantageous for breeding purposes. Mammalian spermatogenesis is a hormone-dependent developmental program in which a complex cascade of events must take place to ensure that germ cells reach the proper stage of development at the proper time. Conventional indicators of semen quality such as sperm cell motility and viability usually improve as bulls mature, meeting quality criteria satisfactorily at around 16 months. Using semen before that age may affect embryo viability, but other changes occurring during the peripubertal period should be considered. Although it is known that establishment of these patterns begins during foetal life, the extent to which sperm cell DNA methylation changes during puberty has not been studied. The aim of this study is to correlate the age of a young bull with the overall DNA methylation pattern of its spermatozoa. Spermatozoa were collected from bulls at the ages of 10 months (early pubertal), 12 months (late pubertal) and 16 months (pubertal). Each animal (n = 4) was compared to itself with 16 months as control. Genome-wide DNA methylation was analyzed by microarray using the EmbryoGENE DNA Methylation Analysis platform. Using a fold change over 1.5 and a 5% FDR p-value correction, a total of 2602 differently methylated regions were found in common between 10 months of age and 16 months of age. No differently methylated regions between 12 months and 16 months of age were found at the same level of statistical significance. We conclude that spermatozoa from bulls aged 10 months have a different epigenetic profile, which could compromise their value.

Proteomic Markers of Functional Sperm Population in Bovines: Comparison of Low- and High-Density Spermatozoa Following Cryopreservation.

Mammalian semen contains a heterogeneous population of sperm cells. This heterogeneity results from variability in the complex processes of cell differentiation in the testis, biochemical modifications undergone by spermatozoa during transit along the male reproductive tract, interactions with secretions from accessory sex glands at ejaculation, and, in the context of reproductive technologies, in the ability of ejaculated spermatozoa to resist damage associated with freeze-thaw procedures. When submitted to density gradient centrifugation, ejaculated spermatozoa distribute themselves into two distinct populations: a low-density population characterized by low motility parameters, and a high-density population with high motility characteristics. To understand the origin of ejaculated spermatozoa heterogeneity, cryopreserved semen samples from bulls used by the artificial insemination (A.I.) industry were submitted to Percoll gradient centrifugation. Proteins from low and high density spermatozoa were then extracted with sodium deoxycholate and submitted to proteomic analysis using iTRAQ (isobaric tag for relative and absolute quantitation) methodologies. Quantification of selected sperm proteins was confirmed by multiple reaction monitoring (MRM). Overall, 31 different proteins were more abundant in low-density spermatozoa, while 80 different proteins were more abundant in the high-density subpopulation. Proteins enriched in high-density spermatozoa were markers of sperm functionality such as the glycolytic process, binding to the egg zona pellucida, and motility. Low-density spermatozoa were not solely characterized by loss of proteins and their associated functions. Chaperonin-containing TCP1s and chaperones are hallmarks of the low-density subpopulation. iTRAQ analysis revealed that other proteins such as binder of sperm proteins, histone, GPX5, ELSPBP1, and clusterin are overexpressed in low-density spermatozoa suggesting that these proteins represent defects occurring at different steps during the sperm journey. These differences contribute to the sperm cell heterogeneity present in mammalian semen.

The effect of age and length of gonadotropin stimulation on the in vitro embryo development of Holstein calf oocytes.

The use of oocytes recovered from prepubertal donors for in vitro embryo production has great potential for accelerating the rate of genetic gain in the dairy industry. However, these oocytes are known to be less developmentally competent than those from adult donors. In this study, we investigated the effect of age and gonadotropin stimulation in Holstein heifers subjected to oocyte collection every two weeks between 2 and 6 months of age. In order to assess the effect of gonadotropin stimulation, animals were subjected to one of three treatments, namely Short (ST; 36-42 h), Long (LT; ≥72 h) and No Treatment (NT) prior to laparoscopic ovum pick-up (LOPU). Our results show that the LT significantly improved the proportion of large follicles (>5 mm diameter) present in the ovary (LT 34.0% vs. ST 11.2% vs. NT 2.4%, P < 0.05), as well as the percentage of good-quality cumulus oocyte complexes (COCs) recovered (LT 95.3 ± 18% vs. ST 85.4 ± 22% vs. NT 82.2 ± 14%, P < 0.05) and blastocyst rate (LT 36.7 ± 26% vs. ST 18.3 ± 15% vs. NT 16.7 ± 9%, P < 0.05). Recovery rate was affected by treatment (LT 70.4 ± 25 vs. ST 85.4 ± 29 vs. NT 72.7 ± 23, P < 0.05). To assess the impact of age, data was grouped into <100 days (A), 100-130 days (B) and >130 days (C) of age at LOPU. We found that as animals got older, although the average number of COCs per donor per LOPU declined (A: 17.5 ± 11 vs. B: 14.7 ± 7 vs. C: 11.9 ± 8), the blastocyst rate increased (A: 12.8 ± 20% vs. B: 17.1 ± 21% vs. C: 21.8 ± 25%, P < 0.05). We also evaluated the incidence of polyspermy and confirmed it is a critical limitation for IVF in calf oocytes. The incidence of polyspermy was unaffected by gonadotropin treatment, but significantly decreased with age. The capacity for full development to term of in vitro produced embryos from calf oocytes was tested by embryo transfer into 21 synchronized adult recipients, which resulted in 13 pregnancies (62%), full development to term and healthy calves born. Finally, the study allowed evaluating the safety of the procedure since, on average, each animal was subjected to 8 LOPU procedures over a period of 4 months. Our results showed that the procedure is safe (no incidents during laparoscopy), and was not harmful for the reproductive future of the animals, as those that were bred became pregnant after reaching sexual maturity.

Impact of male fertility status on the transcriptome of the bovine epididymis.

STUDY QUESTION: Can region-specific transcriptional profiling of the epididymis from fertile and sub-fertile bulls predict the etiology of fertility/sub-fertility in males? SUMMARY ANSWER: The highly regulated gene expression along the bovine epididymis is affected by the fertility status of bulls used for artificial insemination. WHAT IS KNOWN ALREADY: In mammals, sperm maturation and storage occur in the epididymis. Each epididymal segment has his own transcriptomic signature that modulates the intraluminal composition and consequently governs sequential modifications of the maturing male gamete. STUDY DESIGN, SIZE, DURATION: Epididymides from six Holstein bulls with documented fertility were used. These bulls were divided into two groups: high fertility (n = 3), and medium-low fertility (n = 3) and their epididymal transcriptomic profiles were analyzed. PARTICIPANTS/MATERIALS, SETTING, METHODS: Bovine cDNA microarray probing and bioinformatic tools were used to identify genes that are differentially expressed in caput, corpus and cauda epididymidal tissues of bulls with the documented fertility index. MAIN RESULTS AND THE ROLE OF CHANCE: Hierarchical clustering and principal component analysis revealed a clear separation between caput, corpus and cauda epididymides. Some transcripts characterize a particular anatomical segment, whereas others are expressed in two out of three epididymal segments. Gene ontology analysis allowed deduction of specific functions played by each epididymal segment. The transcriptional profiles between fertile versus sub-fertile conditions clustered most closely in the corpus and cauda segments, whereas the profiles in the caput segment were distinct between fertile and sub-fertile bulls. Of the differently expressed genes, 10 (AKAP4, SMCP, SPATA3, TCP11, ODF1, CTCFL, SPATA18, ADAM28, SORD and FAM161A) were found to exert functions related to reproductive systems and 5 genes (DEAD, CYST11, DEFB119, DEFB124 and MX1) were found to be associated with the defense response. LARGE SCALE DATA: The GEO number for public access of bovine epididymis microarray data is GSE96602. LIMITATIONS, REASONS FOR CAUTION: Further work is required to link these modulations of epididymal functions with sperm fertilizing ability in order to understand the etiology of certain cases of idiopathic infertility in livestock and men. WIDER IMPLICATIONS OF THE FINDINGS: As fertility can be quantified in bulls used for artificial insemination, this species is a unique model to aid in the understanding of male fertility/sub-fertility in man. Our data provide a molecular characterization that will facilitate advances in understanding the involvement of epididymal physiology in sub/infertility etiology. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by a grant to R.S. from the Natural Sciences and Engineering Research Council (NSERC) of Canada. C.L., A.A., E.C. and R.S. have no conflict of interest to declare. P.B. is R&D director at Alliance Boviteq Inc., a bovine artificial insemination company.