Subfertile Bulls Research Articles

Multi-omics data integration for the identification of biomarkers for bull fertility.

Bull fertility is an important economic trait, and the use of subfertile semen for artificial insemination decreases the global efficiency of the breeding sector. Although the analysis of semen functional parameters can help to identify infertile bulls, no tools are currently available to enable precise predictions and prevent the commercialization of subfertile semen. Because male fertility is a multifactorial phenotype that is dependent on genetic, epigenetic, physiological and environmental factors, we hypothesized that an integrative analysis might help to refine our knowledge and understanding of bull fertility. We combined -omics data (genotypes, sperm DNA methylation at CpGs and sperm small non-coding RNAs) and semen parameters measured on a large cohort of 98 Montbéliarde bulls with contrasting fertility levels. Multiple Factor Analysis was conducted to study the links between the datasets and fertility. Four methodologies were then considered to identify the features linked to bull fertility variation: Logistic Lasso, Random Forest, Gradient Boosting and Neural Networks. Finally, the features selected by these methods were annotated in terms of genes, to conduct functional enrichment analyses. The less relevant features in -omics data were filtered out, and MFA was run on the remaining 12,006 features, including the 11 semen parameters and a balanced proportion of each type of-omics data. The results showed that unlike the semen parameters studied the-omics datasets were related to fertility. Biomarkers related to bull fertility were selected using the four methodologies mentioned above. The most contributory CpGs, SNPs and miRNAs targeted genes were all found to be involved in development. Interestingly, fragments derived from ribosomal RNAs were overrepresented among the selected features, suggesting roles in male fertility. These markers could be used in the future to identify subfertile bulls in order to increase the global efficiency of the breeding sector.

Abnormal centriolar biomarker ratios correlate with unexplained bull artificial insemination subfertility: a pilot study

The mechanisms underlying male infertility are poorly understood. Most mammalian spermatozoa have two centrioles: the typical barrel-shaped proximal centriole (PC) and the atypical fan-like distal centriole (DC) connected to the axoneme (Ax). These structures are essential for fertility. However, the relationship between centriole quality and subfertility (reduced fertility) is not well established. Here, we tested the hypothesis that assessing sperm centriole quality can identify cattle subfertility. By comparing sperm from 25 fertile and 6 subfertile bulls, all with normal semen analyses, we found that unexplained subfertility and lower sire conception rates (pregnancy rate from artificial insemination in cattle) correlate with abnormal centriolar biomarker distribution. Fluorescence-based Ratiometric Analysis of Sperm Centrioles (FRAC) found only four fertile bulls (4/25, 16%) had positive FRAC tests (having one or more mean FRAC ratios outside of the distribution range in a group’s high-quality sperm population), whereas all of the subfertile bulls (6/6, 100%) had positive FRAC tests (P = 0.00008). The most sensitive biomarker was acetylated tubulin, which had a novel labeling pattern between the DC and Ax. These data suggest that FRAC and acetylated tubulin labeling can identify bull subfertility that remains undetected by current methods and may provide insight into a novel mechanism of subfertility.

Adenylate kinase 9 is essential for sperm function and male fertility in mammals

Despite passing routine laboratory tests for semen quality, bulls used in artificial insemination exhibit significant variation in fertility. Routine analysis of fertility data identified a dairy bull with extreme subfertility (10% pregnancy rate). To characterize the subfertility phenotype, a range of in vitro, in vivo, and molecular assays were carried out. Sperm from the subfertile bull exhibited reduced motility and severely reduced caffeine-induced hyperactivation compared to controls. Ability to penetrate the zona pellucida, cleavage rate, cleavage kinetics, and blastocyst yield after IVF or AI were significantly lower than in control bulls. Whole-genome sequencing from semen and RNA sequencing of testis tissue revealed a critical mutation in adenylate kinase 9 (AK9) that impaired splicing, leading to a premature termination codon and a severely truncated protein. Mice deficient in AK9 were generated to further investigate the function of the gene; knockout males were phenotypically indistinguishable from their wild-type littermates but produced immotile sperm that were incapable of normal fertilization. These sperm exhibited numerous abnormalities, including a low ATP concentration and reduced motility. RNA-seq analysis of their testis revealed differential gene expression of components of the axoneme and sperm flagellum as well as steroid metabolic processes. Sperm ultrastructural analysis showed a high percentage of sperm with abnormal flagella. Combined bovine and murine data indicate the essential metabolic role of AK9 in sperm motility and/or hyperactivation, which in turn affects sperm binding and penetration of the zona pellucida. Thus, AK9 has been found to be directly implicated in impaired male fertility in mammals.

A potent allele marker related to low bull conception rate in Japanese Black bulls

Over the years, there has been considerable variation in the bull conception rate (BCR) of Japanese Black cattle; moreover, several Japanese Black bulls with a low BCR of ≤10% have been identified. However, the alleles responsible for the low BCR are not determined yet. Therefore, in this study, we aimed to identify single-nucleotide polymorphisms (SNPs) for predicting low BCR. To this end, the genome of Japanese Black bulls was comprehensively examined by a genome-wide association study with whole-exome sequencing (WES), and the effect of the identified marker regions on BCR was determined. The WES analysis of six sub-fertile bulls with a BCR of ≤10% and 73 normal bulls with a BCR of ≥40% identified a homozygous genotype for low BCR in Bos taurus autosome 5 in the region between 116.2 and 117.9 Mb. The g.116408653G > A SNP in this region had the most significant effect on the BCR (P-value = 1.0 × 10−23), and the GG (55.4 ± 11.2%) and AG (54.4 ± 9.4%) genotypes in the SNP had a higher phenotype than the AA (9.5 ± 6.1%) genotype for the BCR. The mixed model analysis revealed that g.116408653G > A was related to approximately 43% of the total genetic variance. In conclusion, the AA genotype of g.116408653G > A is a useful index for identifying sub-fertile Japanese Black bulls. Some positive and negative effects of SNP on the BCR were presumed to identify the causative mutations, which can help evaluate bull fertility.

Identification of genomic regions associated with total and progressive sperm motility in Italian Holstein bulls.

Sperm motility is directly related to the ability of sperm to move through the female reproductive tract to reach the ovum. Sperm motility is a complex trait that is influenced by environmental and genetic factors and is associated with male fertility, oocyte penetration rate, and reproductive success of cattle. In this study we carried out a GWAS in Italian Holstein bulls to identify candidate regions and genes associated with variations in progressive and total motility (PM and TM, respectively). After quality control, the final data set consisted of 5,960 records from 949 bulls having semen collected in 10 artificial insemination stations and genotyped at 412,737 SNPs (call rate >95%; minor allele frequency >5%). (Co)variance components were estimated using single trait mixed models, and associations between SNPs and phenotypes were assessed using a genomic BLUP approach. Ten windows that explained the greatest percentage of genetic variance were located on Bos taurus autosomes 1, 2, 4, 6, 7, 23, and 26 for TM and Bos taurus autosomes 1, 2, 4, 6, 8, 16, 23, and 26 for PM. A total of 150 genes for TM and 72 genes for PM were identified within these genomic regions. Gene Ontology enrichment analyses identified significant Gene Ontology terms involved with energy homeostasis, membrane functions, sperm-egg interactions, protection against oxidative stress, olfactory receptors, and immune system. There was significant enrichment of quantitative trait loci for fertility, calving ease, immune response, feed intake, and carcass weight within the candidate windows. These results contribute to understanding the architecture of the genetic control of sperm motility and may aid in the development of strategies to identify subfertile bulls and improve reproductive success.

Expression and Role of PIWI Proteins and piRNAs in Reproduction of Water Buffalo (Bubalus bubalis, Linn.)

High-fertile and productive dairy animals are important to satisfy the growing population's demand. Sire fertility is one of the essential factors that regulate the overall pregnancy rate of dairy herds. However, sire fertility varies from 10 to 90%, suggesting that male fertility largely accounts for varying fertility levels across the herd. Sub-fertile bulls and females should be identified and discarded promptly to improve the dairy herd's productivity. The most dominant factors implicated in culling are poor semen quality, poor semen freezability (<35% post-thaw motility), and poor libido for the bulls and hard breeders for females that cause huge economic loss to the raisers. Understanding the basic mechanism of male and female fertility has undergone tremendous change in recent times owing to the advancement of molecular tools judging the essential molecules responsible for fertility. Presently, a new molecular niche has surfaced in testes, strongly influencing the fertilization potential of spermatozoa. Over the last decade, there has arrived a conclusion that out of several factors, piRNA and PIWI proteins are largely implicated in regulating the vital aspects of fertility and embryogenesis. While this development is advancing in other animals, very limited information is available on PIWI protein and piRNAs in large animals. Except for a few sporadic information on PIWI protein in cattle, very limited information is available on piRNAs and PIWI protein in regulation with buffalo bull fertility and growth of embryos of buffaloes, posting a huge demand for research.

Bull breeding soundness and cryopreservation of semen

The Society for Theriogenology (SFT) adopted the current form for the bull breeding soundness evaluation (BSE) in 2018. The BSE consists of four parts: a physical exam, a minimal scrotal circumference by the age of the bull, minimum progressive motility of 30%, and minimum morphology of 70% normal cells. A bull deemed to be a satisfactory potential breeder should unequivocally meet or exceed these four requirements. These standards as set forth by the SFT, give the veterinar­ian an objective approach to evaluating a bull. It has been said that “rarely are bulls infertile, but there are a lot of sub-fertile bulls”. Identification of these sub-fertile bulls by veterinarians, allows cattle producers to negate a potential negative impact on the overall productivity of the herd due to poor reproductive efficiency.
 Each part of the exam should be of equal importance to the vet­erinarian and no part should be omitted if an accurate assess­ment of the bull is to be determined. Carson et al in review of BSE trends for the past 20 years, deemed that overwhelmingly the most common cause of unsatisfactory or deferred classi­fication of bulls was due to unacceptable semen morphology. The preparation and interpretation of the morphology slide must be proficient and consistent for the results of the exam to be valid.
 The challenges that food animal practitioners encounter re­garding remaining relevant in the livestock industry continue to surface, and the profession must find means to maintain their position in a changing world. Promoting ourselves as ex­perts and the benefits of a professional exam by a veterinarian are essential to suppressing emerging imitators that provide a mediocre version of this exam sometimes referred to as the “se­men check”.

394 Young Scholar Award Talk: Evaluation of new Markers to Predict Sperm Longevity and Bull Fertility

Abstract Bull fertility is currently evaluated through a breeding soundness exam (BSE) that can detect infertile/sterile bulls with high accuracy; however, subfertile bulls are often classified as satisfactory breeders. In the first study, Angus bulls (n=5) were used to evaluate the effect of different insemination doses (10, 20 and 40 million sperm/dose) on pregnancy/fixed-time AI (P/FTAI; cows n=4,866). The interaction between insemination dose and bull was not significant (P=0.53), and there was no effect of insemination dose on P/FTAI (P=0.31); however, there was an effect of bull on P/FTAI (P <0.01). Two bulls had high, one bull had average, and two bulls had low fertility. Post-thaw sperm analysis was performed by computer assisted sperm analysis (CASA) and flow cytometry; however, analysis performed could not explain field fertility differences. In a second study, Angus and Angus crosses bulls were used to investigate differences in sperm longevity between epididymal and ejaculated sperm. Epididymal and ejaculated semen was incubated at pH 5.8, 6.8, or 7.3. Proteins in epididymal fluid, seminal plasma, and proteins loosely attached to epididymal and ejaculated sperm were also investigated. It was identified that epididymal sperm maintained viability longer compared to ejaculated sperm regardless of media pH (P <0.05). Ejaculated sperm had increased longevity (total motility >20%) at pH 6.8 compared to pH 5.8 or 7.3. There were 458 unique proteins between all samples; 311 and 178 in epididymal fluid and seminal plasma, respectively, and 334 and 298 on epididymal and ejaculated sperm, respectively. Based on comparative proteomics, epididymal sperm energy metabolism is more glycolytic compared to ejaculated sperm. In addition, there were greater numbers of antioxidants available for epididymal sperm likely to maintain reactive oxygen species (ROS) at low concentrations to inhibit premature sperm activation. From this dataset, three proteins that are associated with cell-to-cell interaction were studied [plasma serine protease inhibitor (SERPINA5), dystroglycan (DAG1), and CD9]. Among dairy bulls, SERPINA5 and DAG1 were not associated with field fertility; thus, they are not potential fertility markers. The protein CD9 was evaluated to determine whether induction of sperm capacitation (in vitro) could be used to estimate/predict bulls fertility differences. Semen samples from first study were evaluated for total motility (CASA), and functional sperm analysis (flow cytometry) at pre-wash, post-wash, h 0, 3, 6, and 24 of incubation. Sperm analysis were not able to estimate fertility differences between high and average fertility bulls; however, low fertility bulls had decreased (P <0.05) viability, zinc signature 2, and zinc signature1 + 2, and had increased (P <0.05) dead CD9+ sperm. In summary, the use of an insemination dose of 10-40 million sperm can generate acceptable P/FTAI, and evaluation of CD9, viability and zinc signatures may increase the industry’s ability to estimate fertility of bulls that pass a BSE.

Sperm DNA methylation patterns at discrete CpGs and genes involved in embryonic development are related to bull fertility

BackgroundDespite a multifactorial approach being taken for the evaluation of bull semen quality in many animal breeding centres worldwide, reliable prediction of bull fertility is still a challenge. Recently, attention has turned to molecular mechanisms, which could uncover potential biomarkers of fertility. One of these mechanisms is DNA methylation, which together with other epigenetic mechanisms is essential for the fertilising sperm to drive normal embryo development and establish a viable pregnancy. In this study, we hypothesised that bull sperm DNA methylation patterns are related to bull fertility. We therefore investigated DNA methylation patterns from bulls used in artificial insemination with contrasting fertility scores.ResultsThe DNA methylation patterns were obtained by reduced representative bisulphite sequencing from 10 high-fertility bulls and 10 low-fertility bulls, having average fertility scores of − 6.6 and + 6.5%, respectively (mean of the population was zero). Hierarchical clustering analysis did not distinguish bulls based on fertility but did highlight individual differences. Despite this, using stringent criteria (DNA methylation difference ≥ 35% and a q-value < 0.001), we identified 661 differently methylated cytosines (DMCs). DMCs were preferentially located in intergenic regions, introns, gene downstream regions, repetitive elements, open sea, shores and shelves of CpG islands. We also identified 10 differently methylated regions, covered by 7 unique genes (SFRP1, STXBP4, BCR, PSMG4, ARSG, ATP11A, RXRA), which are involved in spermatogenesis and early embryonic development.ConclusionThis study demonstrated that at specific CpG sites, sperm DNA methylation status is related to bull fertility, and identified seven differently methylated genes in sperm of subfertile bulls that may lead to altered gene expression and potentially influence embryo development.

Predicting male fertility from the sperm methylome: application to 120 bulls with hundreds of artificial insemination records

BackgroundConflicting results regarding alterations to sperm DNA methylation in cases of spermatogenesis defects, male infertility and poor developmental outcomes have been reported in humans. Bulls used for artificial insemination represent a relevant model in this field, as the broad dissemination of bull semen considerably alleviates confounding factors and enables the precise assessment of male fertility. This study was therefore designed to assess the potential for sperm DNA methylation to predict bull fertility.ResultsA unique collection of 100 sperm samples was constituted by pooling 2–5 ejaculates per bull from 100 Montbéliarde bulls of comparable ages, assessed as fertile (n = 57) or subfertile (n = 43) based on non-return rates 56 days after insemination. The DNA methylation profiles of these samples were obtained using reduced representation bisulfite sequencing. After excluding putative sequence polymorphisms, 490 fertility-related differentially methylated cytosines (DMCs) were identified, most of which were hypermethylated in subfertile bulls. Interestingly, 46 genes targeted by DMCs are involved in embryonic and fetal development, sperm function and maturation, or have been related to fertility in genome-wide association studies; five of these were further analyzed by pyrosequencing. In order to evaluate the prognostic value of fertility-related DMCs, the sperm samples were split between training (n = 67) and testing (n = 33) sets. Using a Random Forest approach, a predictive model was built from the methylation values obtained on the training set. The predictive accuracy of this model was 72% on the testing set and 72% on individual ejaculates collected from an independent cohort of 20 bulls.ConclusionThis study, conducted on the largest set of bull sperm samples so far examined in epigenetic analyses, demonstrated that the sperm methylome is a valuable source of male fertility biomarkers. The next challenge is to combine these results with other data on the same sperm samples in order to improve the quality of the model and better understand the interplay between DNA methylation and other molecular features in the regulation of fertility. This research may have potential applications in human medicine, where infertility affects the interaction between a male and a female, thus making it difficult to isolate the male factor.

Use of insemination data for joint evaluation of male and female fertility in predominantly seasonal-calving dairy herds

Conception in dairy cattle is influenced by the fertility of the cow and the bull and their interaction. Despite genetic selection for female fertility in many countries, selection for male fertility is largely not practiced. The primary objective of this study was to quantify variation in male and female fertility using insemination data from predominantly seasonal-calving herds. Nonreturn rate (NRR) was derived by coding each insemination as successful (1) or failed (0) based on a minimum of at least 25 d. The NRR was treated as a trait of the bull with semen (male fertility) and the cow that is mated (female fertility). The data (805,463 cows that mated to 5,776 bulls) were used to estimate parameters using either models that only included bulls with mating data or models that fitted the genetic and permanent environmental (PE) effects of bulls and cows simultaneously. We also evaluated whether fitting genetic and PE effects of bulls as one term is better for ranking bulls based on NRR compared with a model that ignored genetic effect. The age of cows that were mated, age of the bulls with semen data, season of mating, breed of cow that mated, inbreeding of cows and bulls, and days from calving to mating date were found to have a significant effect on NRR. Only about 3% of the total variance was explained by the random effects in the model, despite fitting the genetic and PE effects of the bull and cow. The 2 components of fertility (male and fertility) were not correlated. The heritability of male fertility was low (0.001 to 0.008), and that of female fertility was also low (~0.016). The highest heritability estimate for male fertility was obtained from the model that fitted the additive genetic relationship matrix and PE component of the bull as one term. When this model was used to calculate bull solutions, the difference between bulls with at least 100 inseminations was up to 19.2% units (-9.6 to 9.6%). Bull solutions from this model were compared with bull solutions that were predicted fitting bull effects ignoring pedigree. Bull solutions that were obtained considering pedigree had (1) the highest accuracy of prediction when early insemination was used to predict yet-to-be observed insemination data of bulls, and (2) improved model stability (i.e., a higher correlation between bull solutions from 2 randomly split herds) compared with the model which fitted bull with no pedigree. For practical purposes, the model that fitted genetic and PE effect as one term can provide more accurate semen fertility values for bulls than the model without genetic effect. To conclude, insemination data from predominantly seasonal-calving herds can be used to quantify variability between bulls for male fertility, which makes their ranking on NRR feasible. Potentially this information can be used for monitoring bulls and can supplement efforts to improve herd fertility by avoiding or minimizing the use of semen from subfertile bulls.

Sperm Functional Genome Associated With Bull Fertility.

Bull fertility is an important economic trait in sustainable cattle production, as infertile or subfertile bulls give rise to large economic losses. Current methods to assess bull fertility are tedious and not totally accurate. The massive collection of functional data analyses, including genomics, proteomics, metabolomics, transcriptomics, and epigenomics, helps researchers generate extensive knowledge to better understand the unraveling physiological mechanisms underlying subpar male fertility. This review focuses on the sperm phenomes of the functional genome and epigenome that are associated with bull fertility. Findings from multiple sources were integrated to generate new knowledge that is transferable to applied andrology. Diverse methods encompassing analyses of molecular and cellular dynamics in the fertility-associated molecules and conventional sperm parameters can be considered an effective approach to determine bull fertility for efficient and sustainable cattle production. In addition to gene expression information, we also provide methodological information, which is important for the rigor and reliability of the studies. Fertility is a complex trait influenced by several factors and has low heritability, although heritability of scrotal circumference is high and that it is a known fertility maker. There is a need for new knowledge on the expression levels and functions of sperm RNA, proteins, and metabolites. The new knowledge can shed light on additional fertility markers that can be used in combination with scrotal circumference to predict the fertility of breeding bulls. This review provides a comprehensive review of sperm functional characteristics or phenotypes associated with bull fertility.

Targeted sequencing reveals candidate causal variants for dairy bull subfertility.

SummaryBull fertility is a key factor for successful reproductive performance in dairy cattle. Since the semen from a single bull can be used to inseminate hundreds of cows, one subfertile bull could have a major impact on herd reproductive efficiency. We have previously identified five genomic regions, located on BTA8 (72.2 Mb), BTA9 (43.7 Mb), BTA13 (60.2 Mb), BTA17 (63.3 Mb), and BTA27 (34.7 Mb), that show large dominance effects on bull fertility. Each of these regions explained about 5–8% of the observed differences in sire conception rate between Holstein bulls. Here, we aimed to identify candidate causal variants responsible for this variation using targeted sequencing (10 Mb per region). For each genomic region, two DNA pools were constructed from n≈20 high‐fertility and n≈20 low‐fertility Holstein bulls. The DNA‐sequencing analysis included reads quality control (using FastQC), genome alignment (using BWA and ARS‐UCD1.2), variant calling (using GATK) and variant annotation (using Ensembl). The sequencing depth per pool varied from 39× to 51×. We identified a set of nonsense mutations, missense mutations, and frameshift variants carried by low‐fertility bulls. Notably, some of these variants were classified as strong candidate causal variants, i.e., mutations with deleterious effects located on genes exclusively/highly expressed in testis. Genes affected by these candidate causal variants include AK9, TTLL9, TCHP, and FOXN4. These results could aid in the development of novel genomic tools that allow early detection and culling of subfertile bull calves.

Characterizing miRNA and mse-tsRNA in fertile and subfertile yak bull spermatozoa from Arunachal Pradesh

Male fertility in farm animals is considered as an important economic trait. The phenomenon of spermatogenesis plays a dynamic functional role in determining the viability of sperm and thereby can impact on fertility-driven complications. The process of spermatogenesis is controlled by numerous molecular factors and requires a precisely regulated pattern of gene expression. The role of small noncoding RNAs in altering gene expression has been extensively studied. However, limited information is available apropos their role in yak spermatogenesis. The present study aimed to evaluate the assessment of some significant microRNAs and their expression pattern in the body tissues and sperm of fertile and subfertile yak from Arunachal Pradesh besides identified a novel class of sperm enriched small RNA 'mature-sperm-enriched small RNA' (mse-tsRNA) in Yak spermatozoa. The RNAwas extracted from tissue and sperm using 27 gauge needles and subsequently reverse transcribed into small RNA cDNAs. The PCR positive sperm-predominant miRNAs were validated by quantitative reverse transcriptase PCR (qRT-PCR) for their expression in fertile and subfertile yak. Of the 22 microRNAs, the miRNA19a, miRNA142 and miRNA143 showed higher expression in the subfertile yak, whereas expression of miRNA7d, miRNA23a and miRNA23b were found elevated in the fertile animal. The presence of these small noncoding RNAs in yak sperm and testis indicated the legitimate involvement of their role in yak bull fertility.

Serum and seminal plasma IGF-1 associations with semen variables and effect of IGF-1 supplementation on semen freezing capacity in buffalo bulls

The objective of the study was to establish correlation of seminal and serum IGF-1 with seminal attributes, estimate antioxidant potential of IGF-1 by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assays and to study the effect of IGF-1 supplementation on semen cryopreservation. For this study, buffalo bulls were divided into sub-fertile (n = 2) and normal (n = 5) on the basis of sperm mass motility and individual motility. The serum IGF-1 concentration of normal bulls was greater than in sub-fertile bulls, but there was no difference in the seminal IGF-1 concentration among the groups. The values from correlation analyses indicated that serum IGF-1 concentration is positively correlated with semen mass motility and sperm concentration. In the second experiment, IGF-1 did not have antioxidant activities when assessed with DPPH and FRAP assays. In the third experiment, the ejaculates of normal and sub-fertile bulls were cryopreserved using semen extender in which there was IGF-1 supplementation at 0 (control), 50, 100, 150, 200, 250, 350 and 450 ng/mL of extender. Supplementation of IGF-1 at 250 ng/ml resulted in improved sperm motility, longevity and membrane intactness as compared to control after cryopreservation of semen from normal buffalo bulls, but not sub-fertile bulls. In summary, serum IGF-1 concentration was correlated with sperm mass motility and concentration in buffalo bulls and supplementation of IGF-1 protected sperm during the cryopreservation process but effects were not due to direct antioxidant activity.

Sperm chromatin alterations in fertile and subfertile bulls

Alterations in sperm chromatin have been related with subfertility in several mammals. In this study, chromatin alteration types (Base, Basal half, Central axis, Dispersed, and Whole) were assessed by toluidine blue (TB) staining, 6-diamidino-2-fenilindole (DAPI) and anti-protamine 1 antibody (anti-PR1) labeling in sperm samples of fertile and subfertile bulls. Semen samples were obtained from bulls kept in Artificial Insemination Center (fertile bulls) or from bulls subjected to scrotal insulation (subfertile bulls). The percentage of chromatin alterations identified by TB was similar (P > 0.05) in semen samples of fertile and subfertile bulls. In contrast, a greater (P < 0.01) chromatin decondensation and heterogeneity were recorded in semen samples of subfertile bulls. In DAPI and anti-PR1 methods, the subfertile bulls samples had a higher (P < 0.05) percentage of alteration in the base as well as overall chromatin alterations (P < 0.05). Moreover, the chromatin alterations recorded with TB, DAPI, and anti-PR1 were compared in semen samples of fertile and subfertile bulls. In fertile bulls, the overall chromatin alterations were similar (P > 0.05) among the methods In contrast, semen samples of subfertile bulls had a higher (P < 0.05) percentage of overall chromatin alterations when labeled with DAPI. In conclusion, our findings shown that all dye tested had specific sperm stainability and can be feasible to monitor subfertility condition in bulls. Also, different chromatin alteration types in sperm samples of fertile and suberftile bulls were recorded.

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

Can region-specific transcriptional profiling of the epididymis from fertile and sub-fertile bulls predict the etiology of fertility/sub-fertility in males? The highly regulated gene expression along the bovine epididymis is affected by the fertility status of bulls used for artificial insemination. 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. 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. 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. 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. The GEO number for public access of bovine epididymis microarray data is GSE96602. 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. 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. 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.

Estimation of endogenous levels of osteopontin, total antioxidant capacity and malondialdehyde in seminal plasma: Application for fertility assessment in buffalo (Bubalus bubalis) bulls.

This study was attempted to identify subfertile bulls by quantifying the endogenous levels of osteopontin (OPN), total antioxidant capacity (TAC) and malondialdehyde (MDA) in seminal plasma of buffalo bulls. On the basis of conception rate, buffalo bulls were classified into two groups: high-fertile (conception rate >50%) and subfertile bulls (conception rate <40%). A total of 100 ejaculates (10 ejaculates from each bull) were collected through artificial vagina method. The concentration of OPN, TAC and catalase (CAT) of high-fertile bulls was found to be higher (p<.05) than that of subfertile bulls. Further, MDA level in seminal plasma was found to be lower (p<.05) in high-fertile bulls compared with subfertile bulls. The fertility status had no effect on the superoxide dismutase (SOD) concentration in seminal plasma of both the groups. The levels of OPN (r=.678, p=0.013) and TAC (r=.648, p=.042) were found to be positively correlated with bull fertility and the level of MDA (r=-.718, p=.019) was found to be negatively correlated with bull fertility. However, the fertility of bulls was not found to be significantly correlated with SOD, CAT and sperm motility. In conclusion, seminal OPN, TAC and MDA tended to be more realistic in identification of subfertile bulls from breeding herds.

Functional insulin-like factor 3 (INSL3) hormone-receptor system in the testes and spermatozoa of domestic ruminants and its potential as a predictor of sire fertility.

Insulin-like factor 3 (INSL3) is essential for fetal testis descent, and has been implicated in the testicular and sperm functions in adult males; however, similar functions in domestic ruminants remain largely unknown. This study investigated the functional INSL3 hormone-receptor system in adult ruminant testes and spermatozoa, and explored its potential to diagnose the fertility of sires. Testes and spermatozoa were obtained from fertile bulls, rams and he-goats, whereas subfertile testes and spermatozoa were obtained only from bulls. As expected, INSL3 was visualized in Leydig cells, while we clearly demonstrated that the functional receptor, relaxin family peptide receptor 2 (RXFP2), enabling INSL3 to bind was identified in testicular germ cells and in the sperm equatorial segment of bulls, rams and he-goats. In comparison to fertile bulls, the percentage of INSL3- and RXFP2-expressing cells and their expression levels per cell were significantly reduced in the testes of subfertile bulls. In addition, the population of INSL3-binding spermatozoa was also significantly reduced in the semen of subfertile bulls. These results provide evidence for a functional INSL3 hormone-receptor system operating in ruminant testes and spermatozoa, and its potential to predict subfertility in sires.

Genomewide analysis of bull sperm quality and fertility traits.

Because the priority of AI industry is to identify subfertile bulls, a predictive model that allowed for the prediction of 91% bulls of low fertility was implemented based on seminological (motility) parameters and DNA status assessed both as DNA fragmentation index (DFI) and by TUNEL assay using sperm of 105 Holstein-Friesian bulls (four batches per bull) selected based on in vivo estimated relative conception rates (ERCR). Thereafter, sperm quality and male fertility traits of bulls were explored by GWAS using a high-density (777K) Illumina chip. After data editing, 85 bulls and 591,988 SNPs were retained for GWAS. Of 12 SNPs with false discovery rate <0.2, four SNPs located on BTA28 and BTA18 were significantly associated (LD-adjusted Bonferroni <0.05) with the non-compensatory sperm parameters DFI and TUNEL. Other SNPs of interest for potential association with TUNEL were found on BTA3, in the same chromosome where associations with non-compensatory in vivo bull fertility were already reported. Further suggestive SNPs for sperm membrane integrity were located on BTA28, the chromosome where QTL studies previously reported associations with sperm quality traits. Suggestive SNPs for ERCR were found on BTA18 in the vicinity of a site already associated with in vivo bull fertility. Additional SNPs associated with ERCR and sperm kinetic parameters were also identified. In contrast to other, but very few GWAS on fertility traits in bovine spermatozoa, which reported significant SNPs located on BTX, we have not identified SNPs of interest in this sexual chromosome.