Low Fertility Bulls Research Articles

Higher abundance of DLD protein in buffalo bull spermatozoa causes elevated ROS production leading to early sperm capacitation and reduction in fertilizing ability

BackgroudBefore fertilization, spermatozoa undergo a crucial maturation step called capacitation, which is a unique event regulates the sperm’s ability for successful fertilization. The capacitation process takes place as the spermatozoa pass through the female reproductive tract (FRT). Dihydrolipoamide dehydrogenase (DLD) protein is a post-pyruvate metabolic enzyme, exhibiting reactive oxygen species (ROS) production which causes capacitation. Additionally, other vital functions of DLD in buffalo spermatozoa are hyperactivation and acrosome reaction. DLD produces the optimum amount of ROS required to induce capacitation process in FRT. Depending on physiological or pathophysiological conditions, DLD can either enhance or attenuate the production of reactive oxygen species (ROS). Aim of this study was to investigate whether changes in the production of ROS in sperm cells can impact their ability to fertilize by triggering the capacitation and acrosome reaction.ResultsIn this study, abundance of DLD protein was quantified between high (n = 5) and low fertile bull (n = 5) spermatozoa. It was found that compared to high-fertile (HF) bulls, low-fertile (LF) bulls exhibited significantly (P < 0.05) higher DLD abundances. Herein, we optimised the MICA concentration to inhibit DLD function, spermatozoa were treated with MICA in time (0, 1, 2, 3, 4, and 5 h) and concentrations (1, 2.5, 5, and 10 mmol/L) dependent manner. Maximum DLD inhibition was found to be at 4 h in 10 mmol/L MICA concentration, which was used for further experimentation in HF and LF. Based on DLD inhibition it was seen that LF bull spermatozoa exhibited significantly (P < 0.05) higher ROS production and acrosome reaction in comparison to the HF bull spermatozoa. The kinematic parameters of the spermatozoa such as percent total motility, velocity parameters (VCL, VSL, and VAP) and other parameters (BCF, STR, and LIN) were also decreased in MICA treated spermatozoa in comparison to the control (capacitated) spermatozoa.ConclusionsThe present study provides an initial evidence explaining the buffalo bull spermatozoa with higher DLD abundance undergo early capacitation, which subsequently reduces their capacity to fertilize.

Prediction of Nili-Ravi buffalo bull fertility through Fourier harmonic analysis of sperm

Fourier harmonic analysis (FHA) is a robust method for identification of minute changes in sperm nuclear shape that are indicative of reduced fertility. The current study was designed to develop a fertility prediction model for Nili-Ravi buffalo bulls through FHA of sperm. In experiment I, FHA technique was standardized, average sperm nuclear perimeter was measured and sperm nuclear shape plot of buffalo bull was constructed. Sperm of buffalo bulls (n = 10) were stained with YOYO-1 and Hoechst-33342 to differentiate live and dead, and digital images were captured using phase contrast and fluorescent microscopy. The images were analyzed by ImageJ software and 100 sperm/bull were evaluated. The results are described as mean ± SEM values of mean harmonic amplitude (mharm), skewness harmonic amplitude (skharm), kurtosis harmonic amplitude (kurharm) and variance harmonic amplitude (varharm) at Fourier frequencies 0–5 along with the cartesian and polar coordinate plots of buffalo bull sperm. In experiment II, a fertility prediction model was developed based on FHA of buffalo bull sperm. Semen samples of low (n = 6), medium (n = 3) and high (n = 8) fertility bulls were investigated for FHA of sperm and harmonic amplitudes (HA) were generated. Firstly, to determine if live and dead sperm population have unique nuclear shape distribution; the mean, skewness, kurtosis and variance HA 0–5 of 1700 live and 1294 dead spermatozoa of 17 bulls were evaluated. T-test signified a difference in the mharm0 (2.363 ± 0.01 vs. 2.439 ± 0.02), skharm0 (−0.0002 ± 0.07 vs. −0.266 ± 0.09), kurharm0 (−0.156 ± 0.07 vs. 0.260 ± 0.18), kurharm2 (0.142 ± 0.11 vs. 1.031 ± 0.32) and varharm4 (0.109 ± 0.00 vs. 0.082 ± 0.00) of live vs. dead sperm population (p < 0.05). Therefore, 100 live sperm/bull were further evaluated for mean, skewness, kurtosis and variance HA 0–5 values among high (n = 6) and low-fertility (n = 6) groups. Results of T-test showed higher values of mharm2 (0.739 ± 0.01 vs. 0.686 ± 0.00), mharm4 (0.105 ± 0.001 vs. 0.007 ± 0.001), and skharm0 (0.214 ± 0.109 vs. −0.244 ± 0.097) in high vs. low-fertility group (p < 0.05). In next step, five significantly different combinations of discriminant measures between high and low-fertility groups were obtained by discriminant analysis. In conclusion, mharm4, skharm0 and varharm2 correctly identified 91.7 % of bulls into their respective fertility groups, and upon cross validation the value of the canonical correlation was 0.928.

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.

162 Differences in microRNA cargo of low-fertility bull spermatozoa before and after incorporation of extracellular vesicles isolated from proven fertilty bulls’ seminal plasma

162 Differences in microRNA cargo of low-fertility bull spermatozoa before and after incorporation of extracellular vesicles isolated from proven fertilty bulls’ seminal plasma

Identification of runs of homozygosity associated with male fertility in Italian Brown Swiss cattle.

Intensive selection for improved productivity has been accompanied by an increase in inbreeding rates and a reduction in genetic diversity. The increase in inbreeding tends to impact performance, especially fitness-related traits such as male fertility. Inbreeding can be monitored using runs of homozygosity (ROH), defined as contiguous lengths of homozygous genotypes observed in an individual's chromosome. The goal of this study was to evaluate the presence of ROH in Italian Brown Swiss cattle and assess its association with bull fertility. First, we evaluated the association between ROH and male fertility using 1,102 Italian Brown Swiss bulls with sire conception rate records and 572K SNPs spanning the entire genome. Second, we split the entire population into 100 high-fertility and 100 low-fertility bulls to investigate the potential enrichment of ROH segments in the low-fertility group. Finally, we mapped the significant ROH regions to the bovine genome to identify candidate genes associated with sperm biology and male fertility. Notably, there was a negative association between bull fertility and the amount of homozygosity. Four different ROH regions located in chromosomes 6, 10, 11, and 24 were significantly overrepresented in low-fertility bulls (Fisher's exact test, p-value <0.01). Remarkably, these four genomic regions harbor many genes such as WDR19, RPL9, LIAS, UBE2K, DPF3, 5S-rRNA, 7SK, U6, and WDR7 that are related to sperm biology and male fertility. Overall, our findings suggest that inbreeding and increased homozygosity have a negative impact on male fertility in Italian Brown Swiss cattle. The quantification of ROH can contribute to minimizing the inbreeding rate and avoid its negative effect on fitness-related traits, such as male fertility.

Bull field fertility differences can be estimated with in vitro sperm capacitation and flow cytometry

IntroductionThis study evaluated whether post in vitro capacitation changes in sperm could be used to estimate field fertility differences between bulls.MethodsFrozen-thawed semen from five bulls (two to four ejaculates per bull) previously identified as high (48.1% and 47.7%), intermediary (45.5%) or low (40.7% and 43.1%) fertility, based on pregnancy per AI (P/AI), were evaluated for total and progressive motility, sperm plasma membrane integrity (viability), acrosome integrity (viable sperm with an intact or disrupted acrosome), reactive oxygen species (ROS; viable sperm ROS+ or ROS-), mitochondrial membrane energy potential, zinc signatures (signatures 1-to-4) and CD9 protein populations at pre-wash and post-wash (only total and progressive motility), h0 (diluted with non-capacitation media), and at h0, h0 CM, h3, h6, and h24 after dilution with capacitation media (CM) and incubation at 37ºC. Data were analyzed using the GLIMMIX procedure as repeated measures in SAS with bull, time and the interaction as fixed effects.ResultsBull by time interaction was significant (P≤0.03) for total motility, viability, viable sperm with disrupted acrosome, and zinc signature 3. There tended (P=0.06) to be a bull by time interaction for zinc signatures 1+2 combined. Time was significant (P≤0.003) in all analyses, except viable ROS- (P=0.12). There was a significant effect of bull (P≤ 0.03) for viability, viable sperm with disrupted acrosome, zinc signatures 1, 2 and 1+2, viable CD9- and dead CD9+. High and intermediary fertility bulls had greater (P≤0.04) percentages of viable sperm, zinc signature 2 and zinc signature 1+2 compared to low fertility bulls. High and intermediary fertility bulls had decreased (P≤0.05) percentage of dead CD9+ compared to low fertility bulls. Viable CD9+ differed (P=0.02) and viable sperm with an intact acrosome and viable CD9+ tended to differ (P=0.06) amongst bulls; however, association with field fertility was not observed. There was a positive correlation between P/AI and zinc signature 2 (P=0.04), and there tended to be a positive correlation between P/AI and viability (P=0.10), and zinc signature 1+2 (P=0.10).DiscussionIn summary, incubation of sperm in CM and flow cytometry analyses for viability, zinc signatures 2 and 1+2, and dead CD9+ seems promising to estimate in vivo fertility differences amongst bulls.

The transcriptomic response of bovine uterine tissue is altered in response to sperm from high- and low-fertility bulls†.

Despite stringent quality control checks, some bulls with apparently normal semen quality yield lower than expected pregnancy rates. This study profiled the transcriptome and performed histological analysis of the bovine uterus in response to sperm from high-fertility (HF) and low-fertility (LF) bulls. Postmortem uterine biopsies and uterine explants were collected from heifers 12h after a fixed time artificial insemination (AI) to a synchronized estrus with frozen-thawed semen from five HF (fertility rate 4.01% ± 0.25) and five LF (fertility rate - 11.29% ± 1.11; mean ± SEM) bulls. Uterine biopsies were also collected from control (CTRL) heifers, which were not inseminated. RNA-sequencing and histological analysis were performed for differential gene expression and neutrophil quantification. In the HF treatment relative to CTRL heifers, there were 376 genes significantly differentially expressed in the endometrium with just one gene differentially expressed in the LF treatment relative to CTRL heifers. Comparing the HF and LF treatments directly, there were 40 significantly differentially expressed genes (P < 0.05). Transcriptomic analysis shows a predominant role for the inflammatory marker Interleukin-1 alpha, which was further confirmed by immunohistochemistry. Quantification of neutrophils in the endometrium showed a significant effect of sperm; however, there was no difference in neutrophil numbers between HF and LF groups. In conclusion, this novel study clearly shows a distinct inflammatory response to sperm in the endometrium and a divergent transcriptomic response to semen from HF and LF bulls.

Establishment of a repertoire of fertility associated sperm proteins and their differential abundance in buffalo bulls (Bubalus bubalis) with contrasting fertility

Sperm harbours a wide range of proteins regulating their functions and fertility. In the present study, we made an effort to characterize and quantify the proteome of buffalo bull spermatozoa, and to identify fertility associated sperm proteins through comparative proteomics. Using high-throughput mass spectrometry platform, we identified 1305 proteins from buffalo spermatozoa and found that these proteins were mostly enriched in glycolytic process, mitochondrial respiratory chain, tricarboxylic acid cycle, protein folding, spermatogenesis, sperm motility and sperm binding to zona pellucida (p < 7.74E−08) besides metabolic (p = 4.42E−31) and reactive oxygen species (p = 1.81E−30) pathways. Differential proteomic analysis revealed that 844 proteins were commonly expressed in spermatozoa from both the groups while 77 and 52 proteins were exclusively expressed in high- and low-fertile bulls, respectively. In low-fertile bulls, 75 proteins were significantly (p < 0.05) upregulated and 176 proteins were significantly (p < 0.05) downregulated; these proteins were highly enriched in mitochondrial respiratory chain complex I assembly (p = 2.63E−07) and flagellated sperm motility (p = 7.02E−05) processes besides oxidative phosphorylation pathway (p = 6.61E−15). The down regulated proteins in low-fertile bulls were involved in sperm motility, metabolism, sperm-egg recognition and fertilization. These variations in the sperm proteome could be used as potential markers for the selection of buffalo bulls for fertility.

Analysis of amplification and association polymorphisms in the bovine beta-defensin 129 (BBD129) gene revealed its function in bull fertility

β-defensins are adsorbable on the sperm surface in the male reproductive tract (MRT) and enhance sperm functional characteristics. The beta-defensin 129 (DEFB129) antimicrobial peptide is involved in sperm maturation, motility, and fertilization. However, its role in bovine fertility has not been well investigated. This study examines the relationship between the bovine BBD129 gene and Bos indicus x Bos taurus bull fertility. The complete coding sequence of BBD129 mRNA was identified by RNA Ligase Mediated-Rapid Amplification of cDNA End (RLM-RACE) and Sanger sequencing methodologies. It consisted of 582 nucleotides (nts) including 5' untranslated region (UTR) (46nts) and 3'UTR (23nts). It conserves all beta-defensin-like features. The expression level of BBD129 was checked by RT-qPCR and maximal expression was detected in the corpus—epididymis region compared to other parts of MRT. Polymorphism in BBD129 was also confirmed by Sanger sequencing of 254 clones from 5 high fertile (HF) and 6 low fertile (LF) bulls at two positions, 169 T > G and 329A > G, which change the S57A and N110S in the protein sequence respectively. These two mutations give rise to four types of BBD129 haplotypes. The non-mutated TA-BBD129 (169 T/329A) haplotype was substantially more prevalent among high-fertile bulls (P < 0.005), while the double-site mutated GG-BBD129 (169 T > G/329A > G) haplotype was significantly more prevalent among low-fertile bulls (P < 0.005). The in silico analysis confirmed that the polymorphism in BBD129 results in changes in mRNA secondary structure, protein conformations, protein stability, extracellular-surface availability, post-translational modifications (O-glycosylation and phosphorylation), and affects antibacterial and immunomodulatory capabilities. In conclusion, the mRNA expression of BBD129 in the MRT indicates its region-specific dynamics in sperm maturation. BBD129 polymorphisms were identified as the deciding elements accountable for the changed proteins with impaired functionality, contributing to cross-bred bulls' poor fertility.

Membrane remodulation and hyperactivation are impaired in frozen-thawed sperm of low-fertility bulls

Bulls used in artificial insemination programmes worldwide undergo quality control checks, which are typically based on the evaluation of sperm motility and morphology. Despite this, some bulls can have lower than expected field fertility and the reasons for this remain to be elucidated. Here we hypothesised that sperm from bulls of varying fertility will differ in their ability to undergo capacitation-related events including an increase in membrane fluidity, protein tyrosine phosphorylation, hyperactivation and the acrosome reaction. Firstly, we used frozen-thawed semen from 10 high-fertility (HF) and 10 low-fertility (LF) bulls, and subjected them to in vitro capacitating conditions, following which sperm viability, membrane fluidity, acrosome integrity and protein tyrosine phosphorylation were assessed using flow cytometry. We then assessed the ability of sperm to undergo hyperactivation (induced using caffeine) utilising computer-assisted sperm analysis, and the acrosome reaction (induced using calcium ionophore) using flow cytometry. When sperm were incubated in capacitating conditions, a higher percentage of viable sperm from HF bulls exhibited high membrane fluidity when compared to LF bulls (8.8 ± 0.8% and 5.8 ± 1.2%, respectively; mean ± standard error; P < 0.05). There was no difference between fertility groups in the percentage of acrosome-reacted sperm following the incubation in in vitro capacitating conditions or following the induction of the acrosome reaction using calcium ionophore. However, more sperm from HF bulls became hyperactive in response to caffeine stimulation than sperm from LF bulls (21.6 ± 2.5% versus 14.1 ± 2.4%, respectively; mean ± standard error; P < 0.05). Taken together, sperm from LF bulls had an impaired ability to undergo membrane remodulation and to hyperactivate when induced in vitro. These are key events in the journey of sperm along the female reproductive tract and in the interaction with the oocyte and thus could explain the lower field fertility exhibited by some bulls.

Identification of differentially expressed mRNAs and miRNAs in spermatozoa of bulls of varying fertility.

Bulls used in artificial insemination, with apparently normal semen quality, can vary significantly in their field fertility. This study aimed to characterize the transcriptome of spermatozoa from high (HF) and low (LF) fertility bulls at the mRNA and miRNA level in order to identify potential novel markers of fertility. Holstein-Friesian bulls were assigned to either the HF or LF group (n = 10 per group) based on an adjusted national fertility index from a minimum of 500 inseminations. Total RNA was extracted from a pool of frozen-thawed spermatozoa from three different ejaculates per bull, following which mRNA-seq and miRNA-seq were performed. Six mRNAs and 13 miRNAs were found differentially expressed (P < 0.05, FC > 1.5) between HF and LF bulls. Of particular interest, the gene pathways targeted by the 13 differentially expressed miRNAs were related to embryonic development and gene expression regulation. Previous studies reported that disruptions to protamine 1 mRNA (PRM1) had deleterious consequences for sperm chromatin structure and fertilizing ability. Notably, PRM1 exhibited a higher expression in spermatozoa from LF than HF bulls. In contrast, Western Blot analysis revealed a decrease in PRM1 protein abundance for spermatozoa from LF bulls; this was not associated with increased protamine deficiency (measured by the degree of chromatin compaction) or DNA fragmentation, as assessed by flow cytometry analyses. However, protamine deficiency was positively and moderately correlated with the percentage of spermatozoa with DNA fragmentation, irrespective of fertility group. This study has identified potential biomarkers that could be used for improving semen quality assessments of bull fertility.

Extracellular vesicles from seminal plasma to improve fertilizing capacity of bulls.

Seminal plasma contains extracellular vesicles (EVs) that vehicle RNA, proteins, and other molecules able to influence the biological function of sperm. The aim of this study was to improve the fertilizing capacity of male gametes of low-fertility bulls using EVs isolated by ultracentrifugation from the seminal plasma of a bull of proven fertility. After dose-response curve, 10×106 sperm of low-fertility bulls were co-incubated for an hour with 400×106 EVs/ml. In addition, it has been verified that the incorporation of EVs, which takes place in the sperm midpiece, is maintained for 5 hours and even after cryopreservation. Subsequently, the spermatozoa of low-fertility bulls, with EVs incorporated, were used for the in vitro production of embryos. The rate of blastocyst at seventh day yield in vitro, with the use of sperm with EVs incorporated, increased by about twice the yield obtained with the same sperm in the absence of EVs: bulls having an average embryonic yield of 6.41±1.48%, 10.32±4.34% and 10.92±0.95% improved their yield to 21.21±1.99%, 22.17±6.09% and 19.99±5.78%, respectively (P<0.05). These encouraging results suggest that it might be possible to keep breeding bulls with poor fertility. Further studies will be needed to evaluate the in vivo fertility of sperm treated with EVs and understand how the content of EVs is involve in the sperm-vesicle interaction and in the improved sperm performance.

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 &amp;lt;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 &amp;lt;0.05). Ejaculated sperm had increased longevity (total motility &amp;gt;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 &amp;lt;0.05) viability, zinc signature 2, and zinc signature1 + 2, and had increased (P &amp;lt;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.

Single nucleotide polymorphisms cumulating to genetic variation for fertility in crossbred (Bos taurus × Bos indicus) bull spermatozoa

Spermatozoa from high-fertile (HF) and low-fertile (LF) breeding bulls were subjected to high-throughput next-generation sequencing to identify important Single nucleotide polymorphisms (SNPs) and novel variants associated with fertility. A total of 77,038 genome-wide SNPs were identified, among which, 10,788 were novel variants. A total of 42,290 and 34,748 variants were recorded with 6115 and 4673 novel variants in in HF and LF bulls, respectively. Higher number of SNPs were identified in HF compared to LF bulls. GO analysis of filtered genes with significant variations in HF bulls indicated their involvement in oxidative phosphorylation and metabolic pathways. GO analysis of filtered genes with significant variation in LF bulls revealed their involvement in Ca2++ ion binding, structural constituent of ribosome, and biological processes like translation and ribosomal small subunit assembly. The study identified SNPs in candidate genes including TPT1, BOLA-DRA, CD74, RPS17, RPS28, RPS29, RPL14, RPL13, and RPS27A, which are linked to sperm functionality, survival, oxidative stress, and bull fertility. The identified SNPs could be used in selection of bulls for high fertility and the variation in these genes could be established as an explanation for the fertility differences in bulls upon validation in large number of bulls.

Integrated multi-omics analyses reveals molecules governing sperm metabolism potentially influence bull fertility

Bull fertility is of paramount importance in bovine industry because semen from a single bull is used to breed several thousands of cows; however, so far, no reliable test is available for bull fertility prediction. In the present study, spermatozoa from high- and low-fertility bulls were subjected to high-throughput transcriptomic, proteomic and metabolomic analysis. Using an integrated multi-omics approach the molecular differences between high- and low-fertility bulls were identified. We identified a total of 18,068 transcripts, 5041 proteins and 3704 metabolites in bull spermatozoa, of which the expression of 4766 transcripts, 785 proteins and 33 metabolites were dysregulated between high- and low-fertility bulls. At transcript level, several genes involved in oxidative phosphorylation pathway were found to be downregulated, while at protein level genes involved in metabolic pathways were significantly downregulated in low-fertility bulls. We found that metabolites involved in Taurine and hypotaurine metabolism were significantly downregulated in low-fertility bulls. Integrated multi-omics analysis revealed the interaction of dysregulated transcripts, proteins and metabolites in major metabolic pathways, including Butanoate metabolism, Glycolysis and gluconeogenesis, Methionine and cysteine metabolism, Phosphatidyl inositol phosphate, pyrimidine metabolism and saturated fatty acid beta oxidation. These findings collectively indicate that molecules governing sperm metabolism potentially influence bull fertility.

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.

Comparative high-throughput analysis of sperm membrane proteins from crossbred bulls with contrasting fertility.

The aim of the present study was to identify fertility associated sperm membrane proteins in crossbred bulls. Sperm membrane proteins from high- and low-fertile Holstein Friesian crossbred bulls (n=3 each) were subjected to high-throughput liquid chromatography-mass spectrometry (LC-MS/MS) for comparative proteomic analysis. Proteomic profiling identified a total of 456 proteins in crossbred bull spermatozoa; it was found that 108 proteins were up regulated while 26 proteins were down regulated (>1.5-folds) in spermatozoa from low- compared to high-fertile bulls. Gene ontology classification revealed that upregulated proteins in low-fertile bulls were involved in biological process such as oxidation-reduction process (p=3.14E-06), fusion of sperm to egg plasma membrane (p=7.51E-04), sperm motility (p=0.03), and capacitation (p=0.09), while down regulated proteins were associated with transport (p=6.94E-04), superoxide metabolic process (p=0.02), and tricarboxylic acid cycle (p=0.04). KEGG pathway analysis revealed that oxidative phosphorylation and tricarboxylic acid cycle pathways are the most significantly affected pathway in low-fertile bulls. It was concluded that expression of proteins associated with oxidative phosphorylation and tricarboxylic acid cycle pathways were altered in low-fertile crossbred bulls, and expression levels of SPATA19, ELSPBP1, ACRBP, CLU, SUCLA2, and SPATC1 could aid in assessing potential fertility of crossbred bulls.

PRM1 Gene Expression and Its Protein Abundance in Frozen-Thawed Spermatozoa as Potential Fertility Markers in Breeding Bulls.

Functional genes and proteins in sperm play an essential role in bulls’ reproductive processes. They are more accurate in determining bull fertility than conventional semen quality tests. Protamine-1 (PRM1) is a gene or protein crucial for packaging and protecting sperm DNA until fertilization affects normal sperm function. This study analyzes the genes and proteins potential from PRM1 as fertility markers for different breeds of bulls utilized in the artificial insemination programs, expected to be an accurate tool in interpreting bull fertility in Indonesia. This study used Limousin, Holstein, and Ongole Grade bulls divided into two groups based on fertility, high-fertility (HF) and low fertility (LF). The semen quality assessment included progressive motility (computer-assisted semen analysis), viability (eosin-nigrosine), and plasma membrane integrity (HOS test). Sperm DNA fragmentation (SDF) was assessed using the acridine orange staining and the Halomax test. Sperm PRM deficiency was evaluated with the chromomycin A3 method. Moreover, PRM1 gene expression was measured using qRT-PCR, and the PRM1 protein abundance was measured with the enzyme immunoassay method. Semen quality values, relative expression of PRM1 gene, and quantity of PRM1 protein were significantly higher (p < 0.05) in HF bulls than in LF bulls. The SDF and PRM deficiency values in LF bulls were significantly higher (p < 0.05) than HF bulls. Additionally, PRM1 at the gene and protein levels correlated significantly (p < 0.01) with fertility. Therefore, PRM1 is a potential candidate for fertility markers in bulls in Indonesia.

Gene Expression in Embryos From Norwegian Red Bulls With High or Low Non Return Rate: An RNA-Seq Study of in vivo-Produced Single Embryos.

During the last decade, paternal effects on embryo development have been found to have greater importance than previously believed. In domestic cattle, embryo mortality is an issue of concern, causing huge economical losses for the dairy cattle industry. In attempts to reveal the paternal influence on embryo death, recent approaches have used transcriptome profiling of the embryo to find genes and pathways affected by different phenotypes in the bull. For practical and economic reasons, most such studies have used in vitro produced embryos. The aim of the present study was to investigate the differences in the global transcriptome of in vivo produced embryos, derived from sires with either high or low field fertility measured as the non-return rate (NRR) on day 56 after first AI of the inseminated cows. Superovulated heifers (n = 14) in the age span of 12–15 months were artificially inseminated with semen from either high fertility (n = 6) or low fertility (n = 6) bulls. On day seven after insemination, embryos were retrieved through uterine flushing. Embryos with first grade quality and IETS stage 5 (early blastocyst), 6 (blastocyst) or 7 (expanded blastocyst) were selected for further processing. In total, RNA extracted from 24 embryos was sequenced using Illumina sequencing, followed by differential expression analysis and gene set enrichment analysis. We found 62 genes differentially expressed between the two groups (adj.p-value<0.05), of which several genes and their linked pathways could explain the different developmental capacity. Transcripts highly expressed in the embryos from low fertility bulls were related to sterol metabolism and terpenoid backbone synthesis, while transcripts highly expressed in the high fertility embryos were linked to anti-apoptosis and the regulation of cytokine signaling. The leukocyte transendothelial migration and insulin signaling pathways were associated with enrichments in both groups. We also found some highly expressed transcripts in both groups which can be considered as new candidates in the regulation of embryo development. The present study is an important step in defining the paternal influence in embryonic development. Our results suggest that the sire’s genetic contribution affects several important processes linked to pre-and peri implantation regulation in the developing embryo.

An ex-vivo assessment of differential sperm transport in the female reproductive tract between high and low fertility bulls

Despite passing all quality control checks at animal breeding centres, bulls with apparently normal semen quality can yield unacceptably low field fertility rates. This study took an ex-vivo approach to assess if bulls of divergent field fertility differ in the ability of their spermatozoa to interact with the female reproductive tract and its secretions. Six high and six low fertility Holstein Friesian bulls (+4.0 ± 0.2 and −15.7 ± 3.13, respectively; adjusted mean fertility ± s.e.m. mean of the bull population was 0) were selected from a population of 840 bulls with >500 field inseminations per bull. Thawed spermatozoa from each bull were analysed across a range of in vitro assays to assess their ability to transverse the female reproductive tract including; motility and kinematic parameters using computer-assisted sperm analysis, viability, membrane fluidity and acrosomal integrity using flow cytometry as well as mucus penetration tests, rheotactic behaviour and sperm binding ability to the oviductal epithelium. While there was no significant difference between high and low fertility bulls in most of the sperm motility, kinematic and sperm functional parameters (namely, motility, average path velocity, linearity, straightness, amplitude of lateral head movement), viability, membrane fluidity or acrosome intactness, high fertility bulls had higher curvilinear velocity compared to the low fertility group (P < 0.05) and a higher straight-line velocity was observed although it did not reach statistical significance (P = 0.08). There was no difference between treatment groups in the ability of spermatozoa to penetrate periovulatory cervical mucus or in their rheotactic response (P > 0.05). Interestingly, there was a positive correlation between the straight-line velocity of spermatozoa and their rheotactic response (r = 0.45, P < 0.001) and further linear regression analysis indicated 18.9% of the variance in sperm rheotaxis was accounted for by straight line velocity. A higher number of spermatozoa from the high fertility group compared to the low fertility group bound to oviductal explants (15.1 ± 0.98 and 12.5 ± 0.76, respectively; mean ± s.e.m; P < 0.05). In conclusion, the differences in the kinematics of sperm motility and ability to bind to oviductal explants between high and low fertility bulls were modest and are unlikely to explain the inherent differences in fertility between these cohorts of bulls.