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Background Reproductive development may be advanced in bull calves through enhanced dietary intake during the early life period. This effect between enhanced nutrition with subsequent earlier reproductive development is orchestrated through signalling within the hypothalamic-pituitary-testicular axis. Within the hypothalamus, the arcuate nucleus (ARC) is crucial for the integration of peripheral metabolic status with subsequent gonadotropin releasing hormone (GnRH) signalling; however, the precise molecular control regulating this effect is not fully known. The aim of this study was to evaluate the global transcriptomic and proteomic responses to varied plane of nutrition during early calf-hood in young dairy bull calves. Additionally, we sought to integrate these ‘omics’ datasets to determine key genes and proteins contributing to earlier reproductive development. Between 2–12 weeks of age, 30 Holstein-Friesian bull calves (mean age: 17.5 days; mean bodyweight 48.8 kg), were offered either a high or moderate plane of nutrition with 15 calves in each group. At 12 weeks of age, all calves were euthanised and the ARC tissue isolated from each calf. The ARC tissue was then used for global transcriptomic (miRNAseq and mRNAseq) and proteomic analyses. Results Bioinformatic analyses were undertaken to determine differentially expressed transcripts (FDR < 0.1; fold change > 1.5) between the dietary treatment groups, resulting in the identification of 1 differentially expressed miRNA (miR-2419-3p) and 83 differentially expressed mRNA in the ARC region. mRNA target gene prediction identified Complement 3 ( C3 ) as a target of miR-2419-3p, suggesting a relationship between the two transcripts. Furthermore, through a co-regulatory network analysis conducted on the proteomics dataset, C3 was revealed as a hub protein. Additionally, through the proteomic network analysis, C3 was interacting with proteins involved in both insulin and GnRH signalling, highlighting a potential role for C3 in mediated the effect of enhanced nutritional status with earlier reproductive development within the ARC. Conclusion This study highlights an effect of altered plane of nutrition in early life on the molecular control of the hypothalamic ARC. Additionally, results generated suggest a potential role for the C3 gene in mediating the interaction between enhanced metabolic status with reproductive development within the ARC, regulated by miR-2419-3p.

Enhanced early life nutrition accelerates sexual development in the bull calf through neuroendocrine-signalling mediated via the hypothalamic–pituitary–testicular axis. Our aim was to assess the impact of contrasting feeding regimes in bull calves during the first 12 weeks of life on the testes transcriptome and proteome. Holstein–Friesian bull calves were offered either a high (HI) or moderate (MOD) plane of nutrition, designed to support target growth rates of 1.0 and 0.5 kg/day, respectively. At 12 weeks of age all calves were euthanized, testicular parenchyma sampled, and global transcriptome (miRNAseq and mRNAseq) and proteome analyses undertaken. Bioinformatic analyses revealed 7 differentially expressed (DE) miRNA and 20 DE mRNA. There were no differentially abundant proteins between the two dietary groups. Integration of omics results highlighted a potential role for the cadherin gene, CDH13 , in earlier reproductive development. Furthermore, co-regulatory network analysis of the proteomic data revealed CDH13 as a hub protein within a network enriched for processes related to insulin, IGF-1, androgen and Sertoli cell junction signalling pathways as well as cholesterol biosynthesis. Overall, results highlight a potential role for CDH13 in mediating earlier reproductive development as a consequence of enhanced early life nutrition in the bull calf.

Pregnancy rates for elite bulls used in artificial insemination (AI) can vary significantly and therefore the identification of molecular markers for fertility and targets to improve bull selection is important. β-defensin peptides have diverse regulatory roles in sperm function across multiple species but the role of copy number variation (CNV) on fertility parameters has not been previously evaluated. In this study, Holstein-Friesian bulls were screened based on reliable field fertility data to identify two groups (High and Low fertility (HF and LF, respectively)) of n = 10 bulls/group which were genotyped for β-defensin 103 ( DEFB103 ) gene CNV by droplet digital PCR. Overall, low DEFB103 copy number (CN) was associated with increased sperm motility across all bulls (n = 20, p < 0.05). As genetic diversity of DEFB103 CN was only apparent in the LF group, three bulls per CNV class (low, intermediate and high CN) were chosen for more detailed comparative functional analysis. Sperm from low CN bulls exhibited higher binding to the oviductal epithelium, while high CN increased sperm membrane fluidity in vitro (p < 0.05). To investigate the functional effect of DEFB103 CNV on the uterine response in vivo , 18 heifers were inseminated with sperm from bulls with low, intermediate and high CN. Transcriptomic analysis on uterine tissue harvested 12 h post-insemination showed differential expression of 58 genes (FDR < 0.1) involved in sperm migration, immune signalling and chemotaxis. Although field fertility results from a complex number of interactive factors, these novel results suggest a contributory role for DEFB103 CN in both sperm function and the uterine response to bull sperm, thereby potentially contributing to pregnancy outcomes in cattle. Further analysis of the role of CNV in additional β-defensin genes in bull fertility is now warranted.

The use of artificial insemination (AI) with frozen-thawed semen in sheep is limited internationally due to low pregnancy rates. An exception is Norway, where high success rates routinely occur following vaginal deposition of frozen-thawed semen during natural estrus. Previous research suggests that breed-specific differences in pregnancy rates may result from impaired cervical sperm transport. This study compared cervical microbiomes among sheep breeds with known differences in pregnancy rates after AI. Cervical samples were collected from Suffolk (low fertility) and Belclare (medium fertility) breeds in Ireland, and Norwegian white sheep (NWS) and Fur breeds (both high fertility) in Norway, during the follicular phase of both natural and synchronized estruses, and the luteal phase of synchronized estrus. Amplicon sequencing revealed significantly higher bacterial abundance during the follicular phase in the low-fertility Suffolk breed compared to high-fertility breeds. Alpha diversity was higher in Suffolk and Belclare breeds, especially during the natural follicular phase, coinciding with pronounced beta diversity differences among breeds. Genus Histophilus was the top feature leading to microbial differences between ewe breeds and types of cycle. Ewe breed was the main driver of cervical microbial composition; increased microbial load in lower-fertility breeds may negatively impact sperm survival/transport, hampering AI success.

Background Despite 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. Results The 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. Conclusion This 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.

Leydig cell functional capacity reflects the numbers and differentiation status of the steroidogenic Leydig cells in the testes and becomes more or less fixed in early adulthood with the final establishment of the hypothalamo-pituitary-gonadal (HPG) axis after puberty. Factors influencing Leydig cell functional capacity and its role in puberty are poorly understood. Using a bovine model of dairy bulls fed four different nutritional regimes from 1 month to 12 months, and applying circulating Insulin-like peptide 3 (INSL3) as an accurate biomarker of Leydig cell functional capacity, showed that a high plane of nutrition in the first 6 months of life, but not later, significantly increased INSL3 in young adulthood. Moreover, INSL3 concentration at 4 months indicated a marked differential in early feeding regime and correlated well (negatively) with the timing of puberty, as reflected by the age in days for the first production of an ejaculate with >50 million sperm and >10% forward motility, as well as with testis size at 18 months. Reversing the diet at 6 months was unable to rectify the trend in either parameter, unlike for other parameters such as testosterone, body weight, and scrotal circumference. This study has shown that early prepubertal nutrition is a key factor in the development of Leydig cell functional capacity in early adulthood and appears to be a key driver in the dynamic progression of puberty.

Background The outcome of cervical artificial insemination (AI) with frozen-thawed semen in sheep is limited by the inability of sperm to traverse the cervix of some ewe breeds. Previous research has demonstrated that cervical sperm transport is dependent on ewe breed, as sperm can traverse the cervix in greater numbers in some higher fertility ewe breeds. However, the molecular mechanisms underlying ewe breed differences in sperm transport through the cervix remain unknown. In this study, we aimed to characterise the cervical transcriptome of four European ewe breeds with known differences in pregnancy rates following cervical AI using frozen-thawed semen at the follicular phase of a natural oestrous cycle. Cervical post mortem tissue samples were collected from two Irish ewe breeds (Belclare and Suffolk; medium and low fertility, respectively) and from two Norwegian ewe breeds (Norwegian White Sheep (NWS) and Fur; high fertility compared to both Irish breeds) at the follicular phase of a natural oestrous cycle ( n  = 8 to 10 ewes per breed). Results High-quality RNA extracted from biopsies of the mid-region of the cervix was analysed by RNA-sequencing and Gene Ontology (GO). After stringent filtering ( P  <  0.05 and FC > 1.5), a total of 11, 1539 and 748 differentially expressed genes (DEGs) were identified in Belclare, Fur and NWS compared to the low fertility Suffolk breed, respectively. Gene ontology analysis identified significantly enriched biological processes involved in muscle contraction, extracellular matrix (ECM) development and the immune response. Gene co-expression analysis revealed similar patterns in muscle contraction and ECM development modules in both Norwegian ewe breeds, which differed to the Irish ewe breeds. Conclusions These breed-specific biological processes may account for impaired cervical sperm transport through the cervix in sheep during the follicular phase of the reproductive cycle. This novel and comprehensive dataset provides a rich foundation for future targeted initiatives to improve cervical AI in sheep.

Cation channels of sperm (CatSper) are sperm-specific calcium channels with identified roles in the regulation of sperm function in humans, mice, and horses. We sought to employ a comparative genomics approach to identify conserved CATSPER genes in the bovine genome, and profile their expression in reproductive tissue. We hypothesized that CATSPER proteins expressed in bull testicular tissue mediates sperm hyperactivation and their rheotactic response in the reproductive tract of the cow. Bioinformatic analysis identified all four known CATSPER genes (CATSPER 1–4) in the bovine genome, and profiling by quantitative real-time polymerase chain reaction identified site-specific variation in messenger ribonucleic acid (mRNA) expression for all four genes along the reproductive tract of the bull. Using a novel antibody against CATSPER 1, protein expression was confirmed and localized to the principal piece of bull sperm, in agreement with what has been reported in other species. Subsequent treatment of bull sperm with either the calcium chelator ethylene glycol tetraacetic acid;mibefradil, a specific blocker of CatSper channels in human sperm; or CATSPER1 antibody all significantly inhibited caffeine-induced hyperactivation and the rheotactic response, supporting the concept that the calcium influx occurs via CatSper channels. Taken together, the work here provides novel insights into expression and function of CatSper channels in bull testicular tissue and in the function of ejaculated sperm. Summary Sentence The effect of blocking calcium channels on hyperactivation and rheotactic response.

Background Cervical artificial insemination (AI) with frozen-thawed semen results in unacceptably low pregnancy rates internationally. The exception is in Norway, where vaginal deposition of frozen-thawed semen to a natural oestrous routinely yields pregnancy rates in excess of 70%. Previous studies by our group has demonstrated that this is due to differences in cervical sperm transport. However, a potentially important contributory factor is that ewes are inseminated to a natural oestrous in Norway but to a synchronised oestrous across most of the rest of the world. In this study, we interrogated the gene expression of the sheep cervix of four ewe breeds with known differences in pregnancy rates following cervical AI using frozen-thawed semen under the effect of exogenous hormones to synchronise the oestrous cycle. These four ewe breeds ( n  = 8 to 11 ewes per breed) are from two countries: Ireland (Belclare and Suffolk; medium and low fertility, respectively) and Norway (Norwegian White Sheep (NWS) and Fur; both with high fertility compared to the Irish ewe breeds). Results RNA extracted from cervical biopsies collected from these breeds was analysed by RNA-sequencing and differential gene expression analysis. Using the low-fertility Suffolk breed as a reference level; 27, 1827 and 2641 genes were differentially expressed in Belclare, Fur and NWS ewes, respectively ( P  <  0.05 and FC > 1.5). Gene ontology (GO) analysis revealed that Fur and NWS had an up-regulation of enriched pathways involved in muscle contraction and development compared to Suffolk. However, there was a down-regulation of the immune response pathway in NWS compared to Suffolk. In addition, GO analysis showed similar expression patterns involved in muscle contraction, extracellular matrix (ECM) development and cell-cell junction in both Norwegian ewe breeds, which differed to the Irish ewe breeds. Conclusions This novel study has identified a number of conserved and breed-specific biological processes under the effect of oestrous synchronisation that may impact cervical sperm transport during the follicular phase of the reproductive cycle.

β-Defensins are peptides with antimicrobial roles, characterized by a conserved tertiary structure. Beyond antimicrobial functions, they exhibit diverse roles in both the immune response and fertility, including involvement in sperm maturation and function. Copy number variation (CNV) of β-defensin genes is extensive across mammals, including cattle, with possible implications for reproductive traits and disease resistance. In this study, we comprehensively catalogue 55 β-defensin genes in cattle. By constructing a phylogenetic tree to identify human orthologues and lineage-specific expansions, we identify 1 : 1 human orthologues for 35 bovine β-defensins. We also discover extensive β-defensin gene CNV across breeds, with DEFB103, in particular, showing extensive multi-allelic CNV. By comparing β-defensin expression levels in testis from calves and adult bulls, we find that 14 β-defensins, including DEFB103 , increase in expression during sexual maturation. Analysis of β-defensin gene expression levels in the caput of adult bull epididymis, and β-defensin gene copy number, in 94 matched samples shows expression levels of four β-defensins are correlated with genomic copy numbers, including DEFB103 . We therefore demonstrate extensive CNV in bovine β-defensin genes, in particular DEFB103 , with potential functional consequences for fertility.