Bull Fertility Research Articles

An association weight matrix identified biological pathways associated with bull fertility traits in a multi-breed population.

Using seven indicator traits, we investigated the genetic basis of bull fertility and predicted gene interactions from SNP associations. We used percent normal sperm as the key phenotype for the association weight matrix-partial correlation information theory (AWM-PCIT) approach. Beyond a simple list of candidate genes, AWM-PCIT predicts significant gene interactions and associations for the selected traits. These interactions formed a network of 537 genes: 38 genes were transcription cofactors, and 41 genes were transcription factors. The network displayed two distinct clusters, one with 294 genes and another with 243 genes. The network is enriched in fertility-associated pathways: steroid biosynthesis, p53 signalling, and the pentose phosphate pathway. Enrichment analysis also highlighted gene ontology terms associated with 'regulation of neurotransmitter secretion' and 'chromatin formation'. Our network recapitulates some genes previously implicated in another network built with lower-density genotypes. Sequence-level data also highlights additional candidate genes relevant to bull fertility, such as FOXO4, FOXP3, GATA1, CYP27B1, and EBP. A trio of regulatory genes-KDM5C, LRRK2, and PME-was deemed core to the network because of their overarching connections. This trio probably influences bull fertility through their interaction with genes, both known and unknown as to their role in male fertility. Future studies may target the trio and their target genes to enrich our understanding of male fertility further.

Genomic prediction of male fertility in Brown Swiss cattle

Bull fertility has been recognized as an important factor affecting dairy herd fertility. The objective of this study was to assess the feasibility of predicting male fertility in Brown Swiss cattle using genomic data. The data set consisted of 1,102 Italian Brown Swiss bulls with sire conception rate (SCR) records and genotype data for roughly 480k SNP. The analyses included the use of linear kernel-based regression models fitting all SNPs or incorporating markers with large effect. Predictive performance was evaluated in 5-fold cross-validation using the correlation between observed and predicted SCR values and mean squared error of prediction. The entire SNP set exhibited predictive correlations around 0.19. Interestingly, the inclusion of 2 markers with large effect yielded predictive correlations around 0.32. Overall, using linear kernel-based models fitting markers with large effect is a promising approach. Our findings could help Brown Swiss breeders make enhanced genome-guided management and selection decisions on male fertility.

Effect of exogenous melatonin implant on post-thaw semen quality of buffalo bulls.

Melatonin is an intracellular antioxidant of sperm membrane that protects the cells from lipid peroxidation. Yet, its role as an antioxidant on semen quality of buffalo bulls is still obscure. The present study was undertaken to assess the effect of exogenous melatonin implant (18 mg/50 kg bodyweight) on post-thaw sperm characteristics, oxidative stress, endocrinological profiles and fertility of buffalo bulls. Six apparently healthy breeding Murrah buffalo bulls were randomly selected at bull farm, Guru Angad Dev Veterinary and Animal Sciences University for the present study and divided into two groups viz. control (n = 3) and melatonin implanted group (n = 3). A total of 120 ejaculates were collected from bulls of both groups (n = 60 each) throughout the study period. Most beneficial effects of melatonin implants were observed during post-implantation period. The percentages of post-thaw sperm total and progressive motility, viability and mitochondrial membrane potential were higher (p < .05) in melatonin implanted buffalo bulls compared to controls during post-implantation period. Following melatonin implantation, MDA production in post-thaw semen was lower (p < .05) in melatonin implanted group than in control group. Plasma melatonin and testosterone concentrations were higher (p < .05) in buffalo bulls implanted with melatonin as compared to their control counterparts. No differences (p > .05) in plasma LH concentrations were observed in both groups. First service pregnancy rate was 43.3% using semen of melatonin implanted bulls and 30.0% with semen of controls (p > .05). Thus, melatonin was able to protect sperm membrane against oxidative damage and improve post-thaw semen quality, thereby resulting in higher fertilizing potential of spermatozoa.

Seasonal variations in semen traits of exotic cattle bulls of different age groups in the subtropical region

Background: The age of the bull and season of semen collection may significantly impact semen characteristics, although the results of previously published studies vary somewhat. Unfortunately, breeding programs still do not account for male reproductive traits for a bull's fertility. The effects of season and age on the quality of exotic cattle breeds’ semen have been investigated in the current study. Methods: Two exotic cattle breeds, Jersey and Holstein Friesian, have been chosen from the subtropical region of India for study. The pooled dataset from the Information Network for Animal Productivity &amp; Health (INAPH, 2017–2022) containing data collected at the Frozen Semen Bank in Bassi, Jaipur, India, on three semen traits was used in the study. Three semen traits of exotic bulls – total semen volume, sperm concentration, and the number of sperms per ejaculation were examined in connection to the influences of age and season. Results: The age of the bulls was found to influence the concentration and sperm counts significantly. The semen volume was minimal in the older group, while the sperm concentration was lowest in the adult group. All the tested semen traits were highest in summer and lowest in winter. Conclusion: Therefore, it was concluded that the age and seasonal variations significantly influence the semen quality of exotic cattle breeds and suggested that the reproductive traits of the bulls should be considered for the breeding programs.

Survey of Southeast U.S. beef veterinarians regarding methods for bull breeding soundness evaluation

The purpose of this survey was to describe veterinarians’ methods for performing bull breeding soundness evaluations (BSEs). Veterinarians in Mississippi, Louisiana and Arkansas were emailed a survey regarding their experience, demo­graphics, opinions and perceptions of client pref­erences regarding BSEs. Logistic regression was used to test respondent characteristics for associations with dichotomous outcomes including evaluating semen morphology as part of every BSE and detecting the diadem defect. Of 3,757 veterinar­ians solicited, 204 (5%) responded and 83 (2%) qualified for analysis. Of these, 10/73 (14%) indicated they do not evaluate morphology during every BSE. When shown an image of the diadem defect, 18/73 (25%) indicated seeing it often or some­times, and 55/73 (75%) indicated rarely or never. The only fac­tor associated with evaluating morphology as part of every BSE, was indicating belief that morphology was most predic­tive of bull fertility vs. believing motility was most predictive, or being unsure (OR = 11.2, 95% C.I. = 1.3-94.1). Respondents who always evaluate morphology with bright field microscopy at 1,000X (OR = 4.1, 95% C.I. = 1.2-13.3) or with phase contrast microscopy at ≥ 400X (OR = 5.4, 95% C.I. = 1.1-27.7) had higher odds of indicat­ing they detect the diadem defect sometimes/often. There was an interaction between always using 1000X magnification, or not, and always using 400X, or not, on the reported detection of the diadem defect. Veterinarians’ perceptions of which as­pects of the BSE are most predictive of fertility influence their methods for performing BSEs, and influence the frequency of detecting diadem defects.

Estimation of genetic parameters for bull conception rate and its genetic correlations with semen production traits in Japanese Black bulls

The P of achieving pregnancy is an important trait of bull fertility in beef cattle and is defined as the bull conception rate (BCR). This study aimed to clarify and better understand the genetic architecture of the BCR calculated using artificial insemination and pregnancy diagnosis records from a progeny testing program in Japanese Black bulls. In this study, we estimated the genetic parameters of the BCR and their correlation with semen production traits. In addition, we assessed the correlated responses in BCR by considering the selection of semen production traits. Nine hundred and sixteen Japanese Black bulls were selected based on fertility, with 28 869 pregnancy diagnostic records from the progeny testing program. Our results showed that the heritability estimate was 0.04 in the BCR at the first service and 0.14 in BCR for the three services, and an increase in the inbreeding coefficient led to a significant decrease in BCR. The phenotypic trend of BCR remained almost constant over the years, whereas the genetic trend increased. In addition, the changes in the progeny testing year effect showed a similar tendency to the phenotypic trends, suggesting that the phenotypic trends could be mainly due to non-genetic effects, including progeny testing year effects. The estimated genetic correlation of BCR with sperm motility traits was favorably moderate to high (ranging from 0.49 to 0.97), and those with sperm quantity traits such as semen volume were favorably low to moderate (ranging from 0.23 to 0.51). In addition, the correlated responses in BCR at the first service by selection for sperm motility traits resulted in a higher genetic gain than direct selection. This study provides new insights into the genetic factors affecting BCR and the possibility of implementing genetic selection to improve BCR by selecting sperm motility traits in Japanese Black bulls.

MicroRNAs and Their Associated Genes Regulating the Acrosome Reaction in Sperm of High- versus Low-Fertility Holstein Bulls.

Bioinformatics envisage experimental data as illustrated biological networks, exploring roles of individual proteins and their interactions with other proteins in regulation of biological functions. The objective was to identify differentially expressed miRNAs and their associated genes regulating the acrosome reaction in capacitated sperm of high- compared to low-fertility dairy bulls and to elucidate biological functional pathways using a systems biology approach, featuring miRNA-mRNA cluster analysis. Categorized bovine-specific miRNAs (n = 84) were analyzed by RT-PCR; 19 were differentially expressed in high- compared to low-fertility sperm (p ≤ 0.05, fold regulation ≥ 2 magnitudes). Six miRNAs (bta-miR-129-5p, bta-miR-193a-3p, bta-miR-217, bta-mir-296-5p, bta-miR-27a, and bta-miR-320a) were highly upregulated (p < 0.05; fold regulation ≥ 5 magnitudes) in high- compared to low-fertility sperm. Highly scored integrated genes of differentially expressed miRNAs predicted associations with pathways regulating acrosome vesicle exocytosis, acrosome reaction, and sperm-oocyte binding. The mRNA expressions of genes associated with the acrosome reaction (including hub genes) were greater, ranging from 2.0 to 9.1-fold (p < 0.05) in high- compared to low-fertility capacitated bull sperm. In conclusion, differentially expressed miRNAs in high-fertility bovine sperm regulating acrosome functions have potential for predicting bull fertility.

Seminal cell-free DNA as a potential marker for in vitro fertility of Nellore bulls.

This study aimed to identify a marker for freezability and in vitro fertility of sperm samples before freezing. Semen was collected from nine Nelore bulls; half of the ejaculate was used for seminal plasma cell-free DNA (cfDNA) quantification, and the other half was cryopreserved. Evaluation of sperm movement using computer-assisted semen analysis and plasma membrane integrity and stability, acrosomal integrity, apoptosis, and mitochondrial potential using flow cytometry were performed on fresh and frozen/thawed semen at 0, 3, 6, and 12h after thawing. Frozen/thawed sperm was also used for in vitro embryo production. cfDNA was extracted from each bull, and the total DNA and number of cell-free mitochondrial DNA (cfmtDNA) copies were quantified. Semen from each animal was used for IVF, and cleavage, blastocyst formation, and cell counts were evaluated. Two groups were formed and compared based on the concentrations of cfDNA and cfmDNA present: low-cfDNA and high-cfDNA and low-cfmtDNA and high-cfmtDNA. Up to 12h post-thawing, there were no differences between the groups in the majority of the sperm parameters evaluated. Cleavage, day 6 and 7 blastocyst rates, and the number of cells were higher in the high cfDNA group than in the low cfDNA group. Similar results were observed for cfmtDNA, except for the number of cells, which was similar between the groups. The concentration of cfDNA and the relative number of copies of cfmtDNA in seminal plasma cannot predict the freezability of semen but can be used to predict in vitro embryo production.

Livestock Workshops: bull fertility testing

In this workshop Alice Miller discussed fertility testing in dairy and beef bulls, sharing practical tips and advice for practitioners. How to prepare the equipment to get the best results, and ensure you can rely on your findings by avoiding common artefacts was covered. Common morphological abnormalities and what they mean were discussed, helping the veterinarian with final assessment and diagnosis.

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.

Effects of Various Non-genetic Factors on Fertility Traits in Murrah Bulls in an Organized Herd

Background: In a herd going through improvement by selective breeding, bulls contribute more than half of the genetic gain due to higher selection intensity and wider coverage through AI. On the other side, meritorious sires might have a lower fertility due to various reasons which would render them less fit in their contribution to genetic improvement. Thus dairy buffalo breeders must find a delicate balance between production and reproduction superiority for selection of sires, for which an accurate assessment of genetic and non-genetic component of conception is essential. Methods: Present study was conducted on thirty five years data comprising of 5349 AI records on 1302 Murrah buffaloes using 86 bulls maintained and semen analysis of cryopreserved semen samples of 130 Murrah maintained at ICAR-National Dairy Research Institute Karnal were analyzed to study bull conception rate and sperm quality traits. Data were classified into four periods; four seasons of AI viz., Summer (April-June), rainy (July-September), autumn (October-November), winter (December-March); five parity (first, second, third, fourth, fifth and above) and five female AI number (1,2,3,4 and ³ 5 AI) to study the conception rate. Age at freezing (Years), period of freezing and season of freezing were analysed to observe their effects on semen quality traits. Mixed model analysis (SAS 9.3) and the analysis of variance were used to see the effects of various factors on conception rate as well as on semen quality parameters. Result: The analysis of variance revealed that period of AI and season of AI had significant (P less than 0.05) effect on conception rate while parity and female AI number were not found to affect the conception rate of bulls. For seminal parameters, acrosome integrity and HOST were significantly affected by age at freezing. Season affected all the semen quality traits while the effect of period of freezing was found to be non-significant for all the traits. Over all autumn season was found to be most suitable with respect to better seminal parameters. Considering season of AI, rainy season (July-September) had the lowest conception rate. The overall conception rate (%) in the study was 38.43±0.99; where Sires showed a wide variation for conception rate ranging from 20.00% to 60.46%. Concluding with the fact that there exists variation among bull fertility that can be utilized for not only herd conception rates but also be used as a criteria for bull selection and evaluation.

METABOLITES AND SEMEN CHARACTERISTICS IN DIFFERENT BULLS FERTILITY

This study was investigated the relationship between Holstein bulls' fertility with some biomarkers (amino، fatty، and organic acids) and semen characteristics. The study included exploring the relationship among the fertility of Holstein bulls، several biomarkers، and semen characteristics in straws (20-25 straws/bull) preserved for three years. The cows were artificially inseminated (9-10 cows/bull) from semen. Based on the fertility rate، the bulls were divided into three groups، the first bulls of medium fertility (˂65%)، the second of good fertility (65-79%)، and the third of high fertility (&gt;80%). The semen characteristics were evaluated، and the concentration of some amino acids and organic fatty acids was measured in the seminal plasma of bulls using gas chromatography and HPLC. The results revealed significant differences in the fertility rate among the different groups of bulls in favor of the highly fertile bulls. The results did not show any differences in most of the characteristics of the semen، except for a significant increase in the percentage of live sperm in good bulls compared to highly fertile ones. There were no significant differences for all the concentrations of carboxylic and most amino and fatty acids concentrations، except for an increase in the concentration of glycine acid (P˂0.05) and a decrease in the concentration of omega 9 (P˂0.05) in the highly fertile bulls compared to the medium fertile ones. In conclusion، glycine and omega-9 in the seminal plasma of bulls can be regarded as biomarkers of their fertility and the level of carboxylic acids (volatile fatty acids) can be adopted as an indicator of semen quality or energy level in the diet

Genetic parameters for yearling male reproduction traits in tropical composite cattle population.

Fertility is economically important but is hard to quantify and measure in breeding programs which has led extensive breeding programs to ignore fertility in their selection criteria. While female fertility traits have been extensively researched, male fertility traits have been largely ignored. It is estimated that 20% to 40% of bulls have sub-fertility, reducing the number of calves born and profits, highlighting the importance of investigating bull fertility. The most practical measure of male fertility is a bull breeding soundness evaluation (BBSE) which assesses structure as well as semen quality and quantity. Generally, traits recorded in a BBSE are neither genetically evaluated nor used for selection in breeding programs. All traits recorded during a BBSE were analyzed through a series of univariate and bivariate linear mixed models using a genomic relationship matrix to estimate genetic parameters. All genotype and phenotype data were obtained from a tropical composite commercial cattle population and imputed to 27,638 single-nucleotide polymorphisms (SNPs) with a total of 2,613 genotyped animals with BBSE records ranging from 616 to 826 animals depending on the trait. The heritabilities of the 27 traits recorded during a BBSE ranged from 0.02 to 0.49. Seven of the male fertility traits were recommended to be included in a breeding program based on their heritability and their phenotypic and genetic correlations. These traits are scrotal circumference, percent normal sperm, proximal droplets, distal midpiece reflex, knobbed acrosomes, vacuoles/teratoids, and sheath score. Using these seven traits in a breeding program would result in higher calving rates, increasing production and profitability.

Crossbred Bull Fertility Prediction Using a Transcriptome based SNP Methodology

Although various methods have been advanced and introduced to comprehend the most suitable approach for predicting fertility, extensive analysis has been undertaken. The utilization of high-throughput data sequencing has been instrumental in the generation of extensive datasets derived from diverse studies, with a primary focus on elements such as epigenetics, genomic variations, genome-wide associations, and differential gene expression in the context of bull fertility. However, the task of achieving effective prediction remains a formidable challenge. The current investigation aims to examine variations identified through RNA-Seq, with a particular emphasis on coding region alterations, and subsequently, map these variations to the region of genes associated with fertility functions and proximity. In this study, transcriptome analysis was carried over sperm samples obtained from 12 bulls, half of which exhibited high fertility (n = 6) and the other half low fertility (n = 6) based on their conception rates. Bioinformatics tools align the data, genetic variants were identified and mapped subsequently to known fertility quantitative trait loci (QTL). After filtering for fertility-specific single nucleotide polymorphisms (SNPs) with missense mutations, we identified a total of six significant biomarkers. This research demonstrates the efficacy of our innovative approach for screening genetic variants, ultimately leading to the identification of fertility markers within bovine spermatozoa.

Sperm HSP70: may not be an age-dependent gene but is associated with field fertility in Bali bulls (Bos sondaicus).

This study aimed to analyze the characteristics of the HSP70 gene and protein in spermatozoa of Bali bulls of different age groups and to examine its potential as a biomarker determining bull fertility. This study used frozen semen produced from six Bali bulls divided into two groups based on age (≤ 9 years and ≥ 12 years). Parameters of frozen semen quality analyzed included sperm motility and kinetics using computer-assisted semen analysis, sperm morphological defects using Diff-Quick staining, acrosome integrity using FITC-PNA staining, and DNA fragmentation using acridine orange staining. HSP70 gene expression characterization was analyzed using qRT-PCR, and HSP70 protein abundance was analyzed using enzyme immunoassays. Fertility field data were obtained by analyzing the percentage conception rate for each bull based on the artificial insemination service data contained in the Indonesian-integrated system of the National Animal Health Information System (iSIKHNAS). The results showed significant differences (P<0.05) in total and progressive motility, morphological defects of the neck and midpiece, and tail of sperm, and acrosome integrity between the age groups of Bali bulls. HSP70 gene expression and protein abundance showed no significant differences (P>0.05) in different age groups. HSP70 gene expression correlated with fertility rate (P<0.05). Age affected several semen quality parameters but did not affect HSP70 gene expression and protein abundance. The HSP70 gene molecule could be a biomarker that determines the fertility of Bali bulls.

Differential protein repertoires related to sperm function identified in extracellular vesicles (EVs) in seminal plasma of distinct fertility buffalo (Bubalus bubalis) bulls.

Buffalo bulls are backbone of Indian dairy industry, and the quality of semen donating bulls determine the overall production efficiency of dairy farms. Seminal plasma harbor millions of lipid bilayer nanovesicles known as extracellular vesicles (EVs). These EVs carry a heterogenous cargo of essential biomolecules including fertility-associated proteins which contribute to fertilizing potential of spermatozoa. In this study, we explored size, concentration, and complete proteome profiles of SP EVs from two distinct fertility groups to uncover proteins influencing bull fertility. Through Dynamic Light Scattering (DLS) it was found that purified EVs were present in 7-14 size exclusion chromatographic (SEC) fractions with sizes ranging from 146.5 to 258.7nm in high fertile (HF) and low fertile (LF) bulls. Nanoparticle Tracking Analysis (NTA) confirmed the size of seminal EVs up to 200nm, and concentrations varying from 2.84 to 6.82 × 1011 and 3.57 to 7.74 × 1011 particles per ml in HF and LF bulls, respectively. No significant difference was observed in size and concentration of seminal EVs between two groups. We identified a total of 1,862 and 1,807 proteins in seminal EVs of HF and LF bulls, respectively using high throughput LC-MS/MS approach. Out of these total proteins, 1,754 proteins were common in both groups and about 87 proteins were highly abundant in HF group while 1,292 were less abundant as compared to LF bulls. Gene ontology (GO) analysis, revealed that highly abundant proteins in HF group were mainly part of the nucleus and involved in nucleosome assembly along with DNA binding. Additionally, highly abundant proteins in EVs of HF group were found to be involved in spermatogenesis, motility, acrosome reaction, capacitation, gamete fusion, and cryotolerance. Two highly abundant proteins, protein disulfide-isomerase A4 and gelsolin, are associated with sperm-oocyte fusion and acrosome reaction, respectively, and their immunolocalization on spermatozoa may indicate that these proteins are transferred through EVs. Our evidences support that proteins in EVs and subsequently their presence on sperm, are strongly associated with sperm functions. Altogether, our investigation indicates that SPEVs possess crucial protein repertoires that are essential for enhancing sperm fertilizing capacity.

Correlation between in vitro sperm kinetic, oxidative stress assessments and field fertility of cryopreserved bull semen

This study assessed kinetic parameters and oxidative stress in bull sperm after post-thaw (PT) or after sperm selection by Percoll™ gradient, and thermo resistance test (SS + TRT) to identify useful indicators of field fertility. For the experiment, commercial doses of frozen semen were obtained from six Aberdeen Angus bulls. Three of the bulls were classified as high fertility and three as low fertility according to the IFert™ index provided by the international breeding company CRV Lagoa. Pooled semen samples were distributed between two treatment groups for analysis: post-thaw (PT) or sperm selection (SS) (Percoll™) and thermal resistance test (SS + TRT). The samples were evaluated using sperm kinetics (CASA) (motility %, progressive motility %, VCL µm/s, VSL µm/s, VAP µm/s, LIN %, STR % and WOB%), production of reactive oxygen species (ROS), lipid peroxidation, superoxide dismutase (SOD) enzyme activity and total antioxidant capacity. Data were analyzed using Two-Way ANOVA, considering the fertility index, the treatment used in the samples as effects, and the interaction between these factors. When a significant effect was observed, the values were compared using the Bonferroni test. A Pearson Correlation analysis was performed between the fertility indices and the sperm parameters analyzed in vitro, to evaluate the relationship between sperm quality and the fecundity rate obtained by the bulls. Sperm kinetic parameters, including total motility, progressive motile, and beat cross-frequency, were higher in low fertility compared to high fertility bulls (P &lt; 0.05). However, curvilinear velocity was greater in high fertility bulls followed by SS + TRT. Straight-line velocity, average path velocity, linearity, and beat cross-frequency beat were higher in high fertility bulls after SS + TRT. Reactive oxygen species was correlated with fertility after SS. In addition, there was a decrease in lipid peroxidation was observed only in high fertility bulls. However, lipid peroxidation and high fertility were correlated after PT and SS + TRT. The combination of in vitro sperm kinetic parameters predicted in vivo fertility more accurately than individual kinetic parameters. The lipid peroxidation of sperm is an important indicator of fertility in bulls. High fertility bulls appeared to be more susceptible to lipid peroxidation, which was only reduced in high fertility bulls, suggesting that their sperm can repair the damage induced by oxidative stress.

Sperm-borne tsRNAs and miRNAs analysis in relation to dairy cattle fertility

Sperm small non-coding RNAs (sncRNAs), such as microRNAs (miRNAs) and tRNA-derived small RNAs (tsRNAs), have been found to have implications for male fertility and play a role in the intergenerational transmission of specific phenotypes by influencing the early embryo's physiological processes in various animal species. This study postulates that there exists a correlation between sperm small non-coding RNAs (sncRNAs) and bull fertility, which in turn can influence the fertility of offspring through the modulation of early embryo development. To investigate this hypothesis, we generated comparative libraries of sperm sncRNAs from sires exhibiting high (n = 3) versus low bull fertility (n = 3), as well as high (n = 3) versus low daughter fertility (n = 3), as determined by the industry-standard Bull fertility index and Daughter fertility index. In total, 12 tsRNAs carried by sperm (11 down-regulated and 1 up-regulated) were found to be associated with bull fertility, while 19 tsRNAs (11 down-regulated and 8 up-regulated) were found to be associated with daughter fertility (q < 0.05, Log2foldchange>±1.5, base mean > 50). Notably, tRX-Glu–NNN–3811 exhibited potential as a biomarker for predicting fertility in both male and female dairy cattle. Moreover, a total of six miRNAs sperm-borne (two up-regulated and four down-regulated) and 35 miRNAs (27 up-regulated and eight down-regulated) exhibited a significant correlation with both bull fertility and daughter fertility individually (p < 0.05, base mean > 50, log2foldchange>±1.5), two microRNAs, namely miR-2385-5p (down-regulated) and miR-98 (up-regulated), exhibit a significant association (p < 0.05, base mean > 50, log2foldchange>±1.5) with the fertility of both bulls and daughter. The targets of these two microRNAs were subsequently identified and integrated with the transcriptomic database of the embryonic cells at the two-cell stage, which is known to be indicative of embryonic competence. The KEGG analysis revealed a potential correlation between these targets and choline metabolism, a crucial factor in embryonic epigenetic programming.In summary, the findings of this study indicate that sperm-borne small non-coding RNAs (sncRNAs) hold promise as biomarkers for predicting and enhancing fertility in dairy cattle. Furthermore, it is plausible that these sncRNAs may exert their effects on daughter fertility by targeting genes in the early embryo.

5 Age-dependent changes in DNA methylation levels of spermatozoa and the relationship to their motility and fertility in Japanese Black bulls

5 Age-dependent changes in DNA methylation levels of spermatozoa and the relationship to their motility and fertility in Japanese Black bulls

A proteomic approach to identifying spermatozoa proteins in Indonesian native Madura bulls.

Proteins assist sperm mature, transit the female reproductive tract, and recognise sperm oocytes. Indigenous Indonesian bulls, Madura bulls, have not been studied for reproductive proteomics. As local Indonesian beef livestock, Madura cattle assist in achieving food security; hence, their number must be improved. Thus, the identification of molecular proteomics-based bull fertility biomarkers is needed. This study aimed to characterise the sperm fertility function of the superior Madura bull (Bos indicus × Bos Javanicus) spermatozoa proteome. Frozen semen from eight Madura superior bulls (Bos indicus × Bos javanicus) aged 4-8 years was obtained from the artificial insemination centre (AIC) in Singosari and Lembang. Madura superior bulls are those that have passed the bull breeding soundness evaluation. Frozen sperm were thawed and centrifuged at 3000 × g for 30 min. Proteins in sperm were characterised through proteomic analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The resulting gene symbols for each protein were then subjected to bioinformatics tools, including UniProt, DAVID, and STRING databases. Regarding sperm fertility, the analysis revealed that 15 proteins were identified in the sperm of Madura bulls. Amongst the identified proteins, the superior Madura bull sperm contained several motilities, energy-related proteins, and chaperone proteins. A substantial portion of characterised proteins are linked to metabolic pathways and the tricarboxylic acid (TCA) cycle, contributing to sperm energy production. In conclusion, the first in-depth proteome identification of sperm related to sperm quality and bull fertility of a unique indigenous Madura breed of Indonesia was performed using the LC-MS/MS proteomic method. These findings may serve as a reference point for further studies related to the functions of bovine sperm and biomarkers of fertility and sperm quality.