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.

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 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.

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.

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.

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.

Cyclic nucleotide phosphodiesterases in human spermatozoa and seminal fluid: Presence of an active PDE10A in human spermatozoa.

BACKGROUND: Cyclic adenosine monophosphate (cAMP) plays a crucial role as a signaling molecule for sperm functions such as capacitation, motility and acrosome reaction. It is well known that cAMP degradation by phosphodiesterase (PDE) enzyme has a major impact on sperm functions. The present study was undertaken to characterize cAMP-PDE activity in human semen. METHODS: cAMP-PDE activity was measured in human sperm and seminal plasma using family specific PDE inhibitors. Three sperm fractionation methods were applied to assess cAMP-PDE activity in spermatozoa. Western blots were used to validate the presence of specific family in sperm and seminal plasma. RESULTS: Using three sperm fractionation methods, we demonstrated that in human sperm, the major cAMP-PDE activity is papaverine-sensitive and thus ascribed to PDE10. In seminal plasma, total cAMP-PDE activity was 1.14±0.39fmol of cAMP hydrolyzed per minute per µg of protein. Using specific inhibitors, we showed that the major cAMP-PDE activity found in human seminal plasma is ascribed to PDE4 and PDE11. Western blot analysis, immunoprecipitation with a specific monoclonal antibody, and mass spectrometry confirmed the presence of PDE10 in human spermatozoa. CONCLUSION: This study provides the first demonstration of the presence of functional PDE10 in human spermatozoa and functional PDE4 and PDE11 in human seminal plasma. GENERAL SIGNIFICANCE: Since the contribution of cyclic nucleotides in several sperm functions is well known, the finding that PDE10 is an active enzyme in human spermatozoa is novel and may lead to new insight into fertility.

Impact of whole-genome amplification on the reliability of pre-transfer cattle embryo breeding value estimates.

BACKGROUND: Genome-wide profiling of single-nucleotide polymorphisms is receiving increasing attention as a method of pre-implantation genetic diagnosis in humans and of commercial genotyping of pre-transfer embryos in cattle. However, the very small quantity of genomic DNA in biopsy material from early embryos poses daunting technical challenges. A reliable whole-genome amplification (WGA) procedure would greatly facilitate the procedure. RESULTS: Several PCR-based and non-PCR based WGA technologies, namely multiple displacement amplification, quasi-random primed library synthesis followed by PCR, ligation-mediated PCR, and single-primer isothermal amplification were tested in combination with different DNA extractions protocols for various quantities of genomic DNA inputs. The efficiency of each method was evaluated by comparing the genotypes obtained from 15 cultured cells (representative of an embryonic biopsy) to unamplified reference gDNA. The gDNA input, gDNA extraction method and amplification technology were all found to be critical for successful genome-wide genotyping. The selected WGA platform was then tested on embryo biopsies (n = 226), comparing their results to that of biopsies collected after birth. Although WGA inevitably leads to a random loss of information and to the introduction of erroneous genotypes, following genomic imputation the resulting genetic index of both sources of DNA were highly correlated (r = 0.99, P<0.001). CONCLUSION: It is possible to generate high-quality DNA in sufficient quantities for successful genome-wide genotyping starting from an early embryo biopsy. However, imputation from parental and population genotypes is a requirement for completing and correcting genotypic data. Judicious selection of the WGA platform, careful handling of the samples and genomic imputation together, make it possible to perform extremely reliable genomic evaluations for pre-transfer embryos.

Gene expression analysis of bovine oocytes at optimal coasting time combined with GnRH antagonist during the no-FSH period.

Ovarian stimulation with FSH combined with an appropriate period of FSH withdrawal (coasting) before ovum pick-up now appears to be a successful way to obtain oocytes with high developmental competence in bovine. Recent results showed that extending follicular growth by only 24 hours has a detrimental effect on oocyte quality as shown by the reduced blastocyst formation rate. Although these treatments are initiated during the luteal phase with low LH level, the small LH pulsatility present at that time could potentially impact follicular development as well as oocyte quality. In this study, a GnRH antagonist (Cetrotide) was used to suppress LH secretion during follicular differentiation to get a better insight into the physiological importance of the LH support during that period. Oocytes were collected by ovum pick-up, and quality was assessed by measuring the blastocyst formation rate obtained after IVM-IVF. The oocyte transcriptome from GnRH antagonist-treated animals was also compared with that from a control group (coasting duration of 68 hours) to detect possible alterations at the messenger RNA (mRNA) level. The oocyte quality was not statistically affected by the treatment as shown by the blastocyst formation rate obtained. However, microarray analysis showed that a total of 226 genes had a significant difference (fold change > 2; P < 0.05) at the mRNA level, with the majority being in overabundance in the treated group. Many genes related to RNA posttranscriptional modifications presented different abundance at the mRNA level significant differences in the control group (68 hours), whereas translation function appeared to be affected, with many genes related to structural constituents of the ribosome presenting a overabundance in the GnRH antagonist-treated group. Specific mRNAs with crucial roles in chromosome segregation control also showed significant difference at the mRNA level after Cetrotide treatment. The results presented here indicated that the suppression of the LH secretion in an optimal stimulated context would have an impact on the oocyte, with the possible alteration of critical functions related to translation capacity and chromosome segregation control.