Saint-Hyacinthe, Canada
Saint-Hyacinthe, Canada

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D'Amours O.,Laval University | Bordeleau L.-J.,Laval University | Frenette G.,Laval University | Blondin P.,LAlliance Boviteq Inc. | And 2 more authors.
Reproduction | Year: 2012

Previously, we showed that binder of sperm 1 (BSP1) and epididymal sperm binding protein 1 (ELSPBP1) proteins are more abundant in the immotile bovine sperm subpopulation following cryopreservation. In this study, we investigated the association of BSP1 and ELSPBP1 with sperm in relation to their ability to survive the cryopreservation process. Fresh and cryopreserved semen samples from the same ejaculate collected from nine Holstein bulls were incubated with a fixable viability probe, fixed and permeabilised and then immunolabelled with rabbit anti-BSP1, rabbit anti-ELSPBP1 or rabbit IgG as negative control. Spermatozoa were then incubated with Alexa 488-conjugated secondary antibody and Hoechst 33342. For each sample, 10 000 'Hoechst positive' events were analysed by flow cytometry. Alternatively, sperm populations were obtained by fluorescence-activated cell sorting. In freshly ejaculated live sperm, two distinct BSP1 detection patterns were revealed: a first population where BSP1 is present along the flagellar region (P1 subpopulation) and a second population where BSP1 is localised on both the flagellar and the acrosomal regions (P3 subpopulation). The dead population presented a BSP1 distribution similar to P3 but with a more intense fluorescence signal (P4 subpopulation). In the corresponding cryopreserved samples, all sperm in the P3 subpopulation were dead while only a small proportion of the P1 subpopulation was dead (P2 subpopulation). ELSPBP1 was detected only in dead spermatozoa and in comparable proportions in both freshly ejaculated and cryopreserved semen. These results show that the presence of BSP1 over the acrosomal region characterises spermatozoa sensitive to cryopreservation and that ELSPBP1 characterises spermatozoa that are already dead at ejaculation. © 2012 Society for Reproduction and Fertility.


Plourde D.,Laval University | Vigneault C.,LAlliance Boviteq Inc. | Laflamme I.,Laval University | Blondin P.,LAlliance Boviteq Inc. | Robert C.,Laval University
Theriogenology | Year: 2012

One of the main objectives related to performing comparative analysis of embryonic transcriptomes is to share information with other reproductive biologists or commercial service providers. Biological extracts influence performance of in vitro production systems and affect the reproducibility of results between production sites; these sources of variation could impede the potential for knowledge transfer. The objective of the present study was to assess the impact of the production site when sharing a common in vitro embryo production protocol. Biological extracts and semen were shared between production sites and thus removed as potential sources of variation. To remove the impact of blastocyst staging, all comparisons used expanded blastocysts. Although blastocyst yields and the number of Tunel positive cells per embryo differed between production sites, blastocysts were morphologically very similar in regards to cell number, their allocation to either the trophoblast or inner cell mass, or their gender ratio. These observations were also confirmed at the gene expression level, as indicated by highly similar transcript abundances. Only 36 genes out of the 16,121 expressed during bovine prehatching development were statistically differentially expressed, of which a large proportion were associated with the apoptotic process. These results highlighted the impact of laboratory set up, including personnel experience, when replicating an in vitro production system. Although inherent differences may arise, given the similarity of results between production sites, we concluded that embryo production protocols have the potential to be transferred and shared. © 2012 Elsevier Inc.


Plourde D.,Laval University | Vigneault C.,LAlliance Boviteq Inc. | Lemay A.,Laval University | Breton L.,Laval University | And 4 more authors.
Theriogenology | Year: 2012

Bovine embryo production is practiced worldwide for commercial purposes. A major concern of embryo suppliers is the impact of in vitro production systems on embryo quality. In the present study, we compared Buffalo Rat Liver cell coculture with semidefined, medium-based culture, oocytes recovered postmortem with those obtained from live animals, and in vitro with in vivo embryo development. Gene expression levels in expanded blastocysts were measured using microarray and quantitative RT-PCR. The systems were similar in terms of blastocyst yield and rate of development, whereas embryo productivity was greater for immature oocytes collected in vivo. Although immature oocytes collected in vivo had greater developmental competence, they yielded blastocysts that were indistinguishable (in terms of level of gene expression) from embryos derived from immature oocytes recovered postmortem. Culture conditions had a significant impact on gene expression, particularly among genes involved in lipid metabolism. Numerous uncharacterized novel transcript regions were also influenced by in vitro treatments. In conclusion, ovum pick-up combined with in vitro culture in semidefined medium provided a high blastocyst yield, without the deleterious effects associated with coculture. © 2012 Elsevier Inc.


Baldoceda-Baldeon L.M.,Laval University | Gagne D.,Laval University | Vigneault C.,LAlliance Boviteq Inc | Blondin P.,LAlliance Boviteq Inc | Robert C.,Laval University
Reproduction | Year: 2014

Mitochondria play an important role during early development in mammalian embryos. It has been shown that properly controlled follicular preparation increases the likelihood of in-vitro-produced bovine embryos reaching the blastocyst stage and that competent embryos exhibit heightened expression of genes associated with mitochondrial function.We hypothesized that apparently incompetent embryos could be rescued by restoring mitochondrial function. It has been shown that vitamin K2. (a membrane-bound electron carrier similar to ubiquinone) can restore mitochondrial dysfunction in eukaryotic cells. The aim of this study was therefore to investigate the effects of vitamin K2. on bovine embryonic development in vitro. The vitaminwas found most effectivewhen added 72 h after fertilization. It produced a significant (P!0.05) increase in the percentage of blastocysts (C8.6%), more expanded blastocysts (C7.8%), and embryos of better morphological quality. It improved the mitochondrial activity significantly and had a measurable impact on gene expression. This is the first demonstration that current standard conditions of in vitro production of bovine embryos may be inadequate due to the lack of support for mitochondrial function and may be improved significantly by supplementing the culture medium with vitamin K2. © 2014 Society for Reproduction and Fertility .


de Montera B.,Laval University | Fournier E.,Laval University | Shojaei Saadi H.A.,Laval University | Gagne D.,Laval University | And 4 more authors.
BMC Genomics | Year: 2013

Background: It was recently established that changes in methylation during development are dynamic and involve both methylation and demethylation processes. Yet, which genomic sites are changing and what are the contributions of methylation (5mC) and hydroxymethylation (5hmC) to this epigenetic remodeling is still unknown. When studying early development, options for methylation profiling are limited by the unavailability of sufficient DNA material from these scarce samples and limitations are aggravated in non-model species due to the lack of technological platforms. We therefore sought to obtain a representation of differentially 5mC or 5hmC loci during bovine early embryo stages through the use of three complementary methods, based on selective methyl-sensitive restriction and enrichment by ligation-mediated PCR or on subtractive hybridization. Using these strategies, libraries of putative methylation and hydroxymethylated sites were generated from Day-7 and Day-12 bovine embryos.Results: Over 1.2 million sequencing reads were analyzed, resulting in 151,501 contigs, of which 69,136 were uniquely positioned on the genome. A total of 101,461 putative methylated sites were identified. The output of the three methods differed in genomic coverage as well as in the nature of the identified sites. The classical MspI/HpaII combination of restriction enzymes targeted CpG islands whereas the other methods covered 5mC and 5hmC sites outside of these regions. Data analysis suggests a transition of these methylation marks between Day-7 and Day-12 embryos in specific classes of repeat-containing elements.Conclusions: Our combined strategy offers a genomic map of the distribution of cytosine methylation/hydroxymethylation during early bovine embryo development. These results support the hypothesis of a regulatory phase of hypomethylation in repeat sequences during early embryogenesis. © 2013 de Montera et al.; licensee BioMed Central Ltd.


PubMed | LAlliance Boviteq Incorporated and Laval University
Type: Journal Article | Journal: Physiological genomics | Year: 2016

Oocyte developmental competence in superstimulated cows is dependent in part on the duration of the FSH coasting. FSH coasting refers to superstimulation with FSH (2 days of endogenous FSH following follicle ablation and 3 days of FSH injections) followed by no FSH for a specific duration. The optimal duration varies among individuals. FSH coasting appears to modulate the transcriptome of different follicular compartments, which cooperate as a single functional unit. However, the integrative effects of FSH coasting on different follicular compartments remain ambiguous. Meta-analysis of three independent transcriptome studies, each focused on a single cell type (granulosa, cumulus, and oocyte) during FSH coasting, allowed the identification of 12 gene clusters with similar time-course expression patterns in all three compartments. Network analysis identified HNF4A (involved in metabolic functions) and ELAVL1 (an RNA-binding protein) as hub genes regulated respectively upward and downward in the clusters enriched at the optimal coasting time, and APP (involved in mitochondrial functions) and COPS5 (a member of the COP9 signalosome) as hub genes regulated respectively upwards and downwards in the clusters enriched progressively throughout the coasting period. We confirmed the effects on HNF4A downstream targets (TTR, PPL) and other hub genes (ELAVL1, APP, MYC, and PGR) in 30 cows with RT-quantitative PCR. The correlation of hub gene expression levels with FSH coasting indicated that a combination of these genes could predict oocyte competence with 83% sensitivity, suggesting that they are potential biomarkers of follicle differentiation. These findings could be used to optimize FSH coasting on an individual basis.


PubMed | LAlliance Boviteq Inc and Laval University
Type: Journal Article | Journal: Theriogenology | Year: 2016

The use of oocytes obtained from younger donors for IVF followed by embryo transfer represents an opportunity to accelerate genetic gain by reducing generation time. In this study, we investigated the relationship between donor age and the invitro developmental competence of oocytes obtained from Holstein females (aged 5-18months) after FSH stimulation and coasting. The follicle size patterns showed a significantly higher total number of small follicles (5-6mm) from donors aged 5 to 10months and a higher total number of medium-sized follicles (7-10mm) in donors aged 6 to 7months. Our analysis also revealed that the total number of follicles was significantly higher (P<0.05) in donors aged 5 to 8months and tended to be higher (P=0.053) in nine-month-old donors. However, oocytes obtained from donors aged 5 to 10months yielded fewer embryos reaching the morula and blastocyst stages. In summary, our results demonstrate that a higher number of oocytes can be obtained from younger animals but lower developmental competence negates this gain.


Chen L.,University of Alberta | Li C.,University of Alberta | Li C.,Agriculture and Agri Food Canada | Sargolzaei M.,University of Guelph | Schenkel F.,LAlliance Boviteq Inc.
PLoS ONE | Year: 2014

The aim of this study was to evaluate the impact of genotype imputation on the performance of the GBLUP and Bayesian methods for genomic prediction. A total of 10,309 Holstein bulls were genotyped on the BovineSNP50 BeadChip (50 k). Five low density single nucleotide polymorphism (SNP) panels, containing 6,177, 2,480, 1,536, 768 and 384 SNPs, were simulated from the 50 k panel. A fraction of 0%, 33% and 66% of the animals were randomly selected from the training sets to have low density genotypes which were then imputed into 50 k genotypes. A GBLUP and a Bayesian method were used to predict direct genomic values (DGV) for validation animals using imputed or their actual 50 k genotypes. Traits studied included milk yield, fat percentage, protein percentage and somatic cell score (SCS). Results showed that performance of both GBLUP and Bayesian methods was influenced by imputation errors. For traits affected by a few large QTL, the Bayesian method resulted in greater reductions of accuracy due to imputation errors than GBLUP. Including SNPs with largest effects in the low density panel substantially improved the accuracy of genomic prediction for the Bayesian method. Including genotypes imputed from the 6 k panel achieved almost the same accuracy of genomic prediction as that of using the 50 k panel even when 66% of the training population was genotyped on the 6 k panel. These results justified the application of the 6 k panel for genomic prediction. Imputations from lower density panels were more prone to errors and resulted in lower accuracy of genomic prediction. But for animals that have close relationship to the reference set, genotype imputation may still achieve a relatively high accuracy. © 2014 Chen et al.


Hamilton C.K.,University of Guelph | Verduzco-Gmez A.R.,LAlliance Boviteq Incorporated | Favetta L.A.,University of Guelph | Blondin P.,LAlliance Boviteq Incorporated | King W.A.,University of Guelph
Sexual Development | Year: 2012

Testis-specific protein Y-encoded (TSPY) is present in varying copy number in both human (20-76 copies) and cattle (37-200 copies), and some studies have linked this variation to semen quality in men. The purpose of this study was to determine if TSPY copy number is associated with fertility in bulls by using adjusted non-return rates, a commonly used measure of field fertility in Canada. In addition, we investigated the associations between TSPY copy number and its expression as well as specific semen parameters, such as average sperm concentration, sperm count, ejaculate volume, and motility. In 2 independent trials, TSPY copy number was shown to be positively correlated to adjusted non-return rates (trial #1: Spearman r = 0.34, p < 0.05; trial #2: Spearman r = 0.77, p < 0.01). Furthermore, TSPY copy number was inversely correlated to TSPY mRNA expression in the testis (Pearson r = -0.71, p < 0.0001). There were no correlations of TSPY copy number or expression with the semen parameters measured. Therefore, TSPY copy number might represent a potential marker of bull fertility, but its mechanism does not appear to be directly related to the semen characteristics analyzed as part of this study. Copyright © 2012 S. Karger AG, Basel.


PubMed | University of Calgary and LAlliance Boviteq Inc.
Type: Journal Article | Journal: Theriogenology | Year: 2016

Enhanced early-life nutrition (130% of required energy and protein) increased testes size and weight (20-25%) and reduced age at puberty (1month) in beef and dairy bulls, compared with those fed 70% of dietary requirements. The objective was to determine effects of early-life (2-31weeks) nutritional modulation on feed costs, predicted number of harvestable sperm and doses of semen, and semen quality. Calves (1week old) were randomly allocated into three groups that were fed 4, 6, or 8L/day of milk (low [n=8], medium [n=9], and high groups [n=9], respectively) from ages 2 to 8weeks. Thereafter, they were weaned, transitioned onto barley silage-based diets, to receive 70, 100, or 130% of recommended amounts of energy and protein (feed costs were CDN$280 more per bull to feed high versus low diets from 2 to 31weeks). After 31weeks, all bulls were fed a medium diet. Semen was collected, by electroejaculation, from 51 to 73weeks, extended, chilled, and cryopreserved. Bulls fed high nutrition were numerically younger (P=0.45) at sexual maturity (sperm with 30% progressive motility, 70% morphologically normal, and 20% abnormal heads), first acceptable post-chill sperm motility (>50%; P=0.66) and first acceptable post-thaw motility (>25% progressive; P=0.25) than bulls in the low-nutrition group. Semen from three bulls per group was used for invitro fertilization (total of 1249 bovine oocytes); there were no significant differences among groups in fertilization percentage (meanSEM of 68.08.7, 77.13.5, and 68.74.5% for low, medium, and high, respectively) or blastocyst yield (31.55.6, 41.44.9, and 33.74.6%). On the basis of analysis of 2D gels of sperm proteins, 380 spots were identified on the fused master gel, but no spots were differentially expressed across groups. Overall, there were no significant differences in semen quality or sperm function among bulls fed three levels of nutrition from ages 2 to 31weeks. However, bulls fed high-nutrition early in life had potential to produce more sperm that could be harvested and sold, which would increase profitability, thereby supporting enhanced early-life nutrition as a management tool to improve reproductive potential of dairy bulls.

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