Ectoine, a compatible solute produced by extremophilic bacteria, is widely used for mucosal protection due to its cytoprotective and anti‑inflammatory properties. However, its effects within the female reproductive tract and its interaction with sperm‑associated immune responses remain unknown. This study provides the first assessment of ectoine's immunomodulatory activity in bovine cervical and uterine tissues and its influence on sperm-polymorphonuclear neutrophil (PMN) interactions. Cervical and uterine explants from follicular‑phase heifers (n = 5) were pre‑incubated with 0, 0.5, 5, or 50 mM ectoine prior to stimulation with lipopolysaccharide (LPS) or sperm. Cytokine concentrations (IL‑8, IL‑6, and IL‑1β) were measured by ELISA, and sperm-PMN binding and sperm motility following PMN co‑incubation were evaluated. Baseline cytokine secretion was higher in uterine versus cervical explants (P < 0.05), indicating distinct tissue‑specific immune profiles. Ectoine alone did not alter basal cytokine secretion (P > 0.05). LPS increased IL‑8, IL‑6, and IL‑1β secretion from cervical explants and IL‑8 and IL‑1β in uterine explants (P < 0.05), and ectoine did not modify these responses (P > 0.05). Sperm elevated IL‑1β release from uterine explants (P < 0.01), while IL‑6 and IL‑8 remained unchanged; this response was also unaffected by ectoine (P > 0.05). Ectoine did not influence sperm-PMN binding or sperm motility in the presence of PMNs (P > 0.05). Collectively, this study provides the first reproductive‑tract safety evaluation of ectoine, demonstrating that it does not disrupt constitutive or stimulus‑induced inflammatory signaling nor interfere with sperm-immune interactions. These findings support ectoine's safe incorporation into semen preservation and assisted reproductive technologies.

Heterospermic (HS) bull semen involves mixing the semen from two or more sires in the same semen straw. It has the potential to protect against sub-fertile bulls which pass semen quality control checks, due to the presence of sperm from other males in the same straw. Previous studies suggest that its use could increase conception rates due to a more heterogeneous population of sperm in the female reproductive tract. Despite increasing use of HS semen in the recent years, driven mainly by the introduction of sex-sorted semen and increased usage of HS beef semen on dairy cows, few studies have focused on the physiological mechanisms underpinning its fertility. This review aims to summarize the existing studies on HS bull semen, present hypotheses for its potential role in sperm interaction with the female reproductive tract, introduce the topic of HS sex-sorted semen and, finally, discuss the challenges for the large-scale production and implementation of this product in breeding programs.

Ectoine is a small, amino acid-derived osmolyte produced by extremophilic bacteria that acts as a compatible solute, protecting cellular macromolecules and structures from extreme environmental stress without disrupting essential cellular functions. The aim of this study was to evaluate the biocompatibility of ectoine with bull sperm and to assess the potential of ectoine to enhance the resilience of sperm under varying stress conditions. Thawed bovine sperm in the presence (0.5, 5, and 50 mM) or absence (control; 0 mM) of ectoine were subjected to a biocompatibility test (37 °C for 6 h; n = 8 bulls), heat stress (39 or 42 °C for 6 h; n = 8 bulls) or osmotic stress (150 or 400 mOsm for 15 min; n = 12 bulls), whereby motility and kinematic parameters, as well as viability, acrosome integrity, and membrane fluidity by flow cytometry were assessed. Sperm motility in cervicovaginal mucus (37 °C for 3 h; n = 6 bulls) was also assessed. All results are reported as mean ± SEM. Ectoine displayed a non-toxic effect across all motility and functional parameters (viability, acrosome integrity, and membrane fluidity). Nonetheless, a reduction in kinematic parameters, including straight line velocity (VSL), average path velocity (VAP) and straightness (STR) was observed at 50 mM ectoine (P < 0.05). Under heat stress at 39 and 42 °C, ectoine concentrations of 0.5 and 5 mM maintained motility and viability, comparable to controls across all time points (P > 0.05). In hypoosmotic conditions (150 mOsm), individual bulls displayed different degrees of osmotic resistance. In those bulls with poor osmotic resistance (n = 4), ectoine (0.5 and 5 mM) maintained sperm viability similar to the 0 mM control (P > 0.05). However, the viability of sperm incubated with 50 mM solute was 2-fold higher relative to the control (P < 0.001). In hyperosmotic conditions, addition of ectoine to sperm prior to exposure did not affect the total motility or viability compared to the no ectoine treatment (P > 0.05). When sperm were incubated in cervicovaginal mucus, there was an effect of ectoine treatment. Sperm treated with 50 mM ectoine exhibited higher motility throughout incubation compared to the control (0 mM) (P < 0.05). In conclusion, these findings establish ectoine as a promising candidate for improving sperm resilience and warrants further studies to assess additional protective effects of ectoine.

ABSTRACT:Bull field fertility varies greatly and is largely influenced by differences in early embryo survival rather than fertilization failure alone. We, therefore, investigated whether sire fertility status is reflected in the metabolomic profile of embryo-conditioned culture media. Six Holstein-Friesian bulls were selected from a population of 840 sires and classified using an adjusted sire fertility index. Mean (±SD) values were 4.27 ± 0.61% for highly fertile bulls (HF, n = 3) and -13.1 ± 1.85% for sub-fertile bulls (SF, n = 3). Cumulus-oocyte complexes were fertilized using frozen-thawed semen from individual bulls. On day 7 post-fertilization, grade 1 blastocysts (pools of n = 15 per bull; n = 3 replicates) were transferred to fresh culture medium for 24 h. On day 8, embryo-conditioned media from both fertility treatments and unconditioned media were collected, snap-frozen, and subjected to high-throughput untargeted metabolomic profiling. A total of 149 metabolites were detected. Unsupervised analysis revealed substantial overlap between HF and SF sire-derived embryo-conditioned media, while both differed from unconditioned media. The relative abundance of 30 metabolites was altered by embryo presence, irrespective of sire fertility. Direct comparison of HF vs SF sire-derived embryo-conditioned media identified only three differentially abundant metabolites. However, multivariate machine learning analysis identified a panel of eight N-acetylated amino acids, which discriminated embryos derived using semen from bulls of divergent fertility status with high accuracy (AUROC = 0.903). In conclusion, sire fertility status imparts a subtle but detectable metabolic imprint on the early embryo, which can be exploited to non-invasively discriminate embryos according to potential developmental competence. LAY SUMMARY:Reproductive efficiency is fundamental to productive and profitable cattle farming. Even bulls that pass routine fertility assessments can achieve very different pregnancy rates. Understanding the biological basis of this variation has important practical implications. We studied whether differences in early embryo metabolism might help explain why some bulls consistently outperform others in fertility. We created embryos in the laboratory using sperm from highly fertile and sub-fertile bulls. Then, we analyzed hundreds of small molecules that reflect metabolic activity in the fluid surrounding the embryo, and we detected very few differences between the two groups. However, using a machine learning approach, we identified a panel of eight molecules that distinguished embryos by sire fertility status with high accuracy. These findings suggest that a bull's fertility leaves a subtle but detectable metabolic imprint on early embryos, with potential future applications in non-invasive embryo quality assessment.

Early and accurate assessment of bull fertility is critical for the success of artificial insemination (AI) programs in cattle production. However, current selection tools, including genomic predictions and standard semen evaluations, offer limited reliability in forecasting field fertility outcomes. To address this limitation, we explored the sperm metabolome as a potential source of novel fertility-associated biomarkers. Using high-throughput untargeted metabolomics, we profiled frozen-thawed sperm from Holstein-Friesian bulls with high (n = 12) and low (n = 12) adjusted fertility scores, each with a minimum of 500 AI service records (range from 519 to 99,953 per bull). Raw peak intensities for 615 metabolites were normalized to the total protein concentration of each sample, and, following data filtration, 547 metabolites were retained for downstream analyses. Unpaired t-tests combined with fold-change thresholding identified 18 differentially abundant metabolites between high fertility and low fertility groups (P < 0.1, absolute fold change >1.5), with significant enrichment in pathways relating to lipid and energy metabolism. Further interrogation of these differentially abundant metabolites in the literature revealed possible metabolic differences associated with calcium channel inhibition and reactive oxygen species production in the low fertility bulls. Machine learning-based biomarker discovery further identified a subset of five metabolites-3-phosphoglycerate, phenylalanine, ceramide, citrate, and citrulline-capable of distinguishing fertility status with high predictive accuracy (AUROC = 0.877; P = 0.02). Overall, these data support sperm metabolomics as a promising omics-based approach to enhance bull fertility evaluation and improve selection strategies in AI programs.

The variability observed in bull field fertility cannot be fully explained by conventional semen analysis. Work by our group and others has shown that ejaculates are highly heterogeneous and contain distinct sperm subpopulations (SSPs) that differ in motility, functionality, and susceptibility to processes such as cryopreservation, with recent evidence indicating differential interactions within the female reproductive tract (FRT). Therefore, these SSPs have the potential to influence sperm survival and the uterine and oviductal environments, which not only influence fertilization but also early embryo development success. This review discusses key sperm-related processes within the FRT, including molecular changes, penetration into the uterine glands, sperm reservoir formation, and, in particular, the role of the immune response and endometrial priming in fertility. Comparative studies between high- and low-fertility bulls offer valuable models for the investigation of the mechanisms behind fertility variation, providing indications of the relevance of some SSPs. By summarizing the existing knowledge on SSPs, this review aims to provide a framework for understanding sperm behavior within the FRT and highlights the need to better understand sperm heterogeneity and its role in successful pregnancy establishment.

The influence of dead sperm on their healthy counterparts in bovine semen is not well established. This is particularly relevant to artificial insemination (AI), since semen handling and biotechnological procedures can increase the percentage of dead sperm. This study aimed to evaluate the impact of acrosome-disrupted (sonicated) spermatozoa on the quality of neighbouring untreated viable cells after cryopreservation. Semen samples from 12 healthy Holstein bulls were diluted (80 × 106 sperm/mL) in pre-warmed OptiXcell extender at 38°C. A 6 mL portion of diluted semen underwent sonication, and both sonicated and untreated semen samples were mixed to produce treatment groups (TG) as: TG25%, TG50% and TG75% sonicated sperm. Control (CTRL) was not mixed with sonicated sperm. Progressive sperm motility was assessed during a thermo-resistance test after 30 (on-test) and 120 min (off-test) of incubation at 38°C. Results of delta and relative variation of progressive sperm motility showed a significant decline in the TG75% compared to the CTRL (p = 0.013 and 0.034, respectively). Flow cytometry revealed a gradual decline in percentage of viable acrosome-intact sperm with low membrane fluidity and low intracellular calcium (p < 0.001). A comparable decrease was observed for percentage of viable acrosome-intact sperm with high mitochondrial membrane potential (p < 0.001). Considering these findings, this study suggests that post-sonication leakage of acrosomal/cellular content could compromise the functionality of untreated spermatozoa, highlighting the necessity to conduct further mechanistic investigation to evaluate possible damaging pathways.

In sheep, cervical artificial insemination (AI) of frozen-thawed semen is not practiced worldwide due to the unacceptably low pregnancy rates achieved (typically <30%). The exception to this is in Norway where farmers perform shot-in-the-dark vaginal AI with frozen-thawed semen and achieve pregnancy rates over 60%. Research in Ireland has shown that this is due to the breed of the ewe used in Norway and specifically the ability of sperm to traverse the cervix in some ewe breeds (such as those in Norway) but not others. This review summarizes the published studies performed over the last two decades, which have focused on characterizing the biological differences in the cervix of ewe breeds of divergent fertility. It not only discusses how this unique ewe breed effect can be used to develop our understanding of how frozen-thawed semen can cross the ovine cervix but also provides new in vivo opportunities through the addition of specific biomolecules that will protect sperm, thus improving the success of cervical AI using frozen-thawed semen in sheep.

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.

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. 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. 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.

In vaginal depositors, semen is deposited at the opening of the cervix, and sperm quickly enter the cervical canal, which is filled with cervical mucus in the lead up to ovulation. The outward flow of cervical mucus in the cervical lumen is a major challenge to sperm progression, and only a small percentage of the deposited sperm make their way into the uterus. Sperm-cervical mucus penetration tests are useful to assess the ability of both sperm and mucus to interact when in varying physiological states of the oestrous cycle. As sheep are vaginal depositors of semen, they are a useful model to study cervical sperm transport and thus ex vivo sperm-mucus penetration tests are often used by researchers developing products to manipulate cervical mucus to either block sperm or promote sperm transit across the cervix. Here, we describe a method to collect ovine cervical mucus, assess its quality, and perform a mucus penetration test using fluorescently tagged sperm which simplifies sperm counting.

The aim of this experiment was to assess the effect of media viscosity on ram sperm motility, kinematics and rheotaxis in vitro by using methylcellulose as a media thickener. Frozen-thawed semen of three rams was thawed and diluted in Tyrode's albumin lactate pyruvate (TALP) media supplemented with 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6% and 0.7% w/v of methylcellulose. Sperm motility and kinematic characteristics were analysed using computer-assisted sperm analysis (CASA). The rheotactic behaviour was assessed in a microfluidic channel, and the number of spermatozoa that passed the 10 mm point of a microfluidic channel over a 2min period against a flow rate of 30 mu m/sec was assessed. The use of media with higher viscosity (higher levels of methylcellulose) resulted in significantly lower (p < .05) sperm motility and kinematic parameters. Moreover, higher levels of methylcellulose reduced (p < .05) the number of spermatozoa that exhibited positive rheotaxis. In conclusion, viscosity affected the kinematic properties and rheotactic behaviour of ram sperm.

Internationally, cervical artificial insemination (AI) in sheep yields low pregnancy rates when frozen-thawed semen is used. An exception to this is in Norway where vaginal AI of frozen-thawed semen to a natural oestrus yields non-return rates in excess of 60%, which has been attributed to the ewe breed used in Norway. This study used both metabolomics and a RNA-sequencing approach to assess the lipid production and composition from cervical mucus and tissue of four European ewe breeds (n=28 to 30 ewes per breed) with previously reported differences in pregnancy rates following cervical AI with frozen-thawed semen. These breeds included Suffolk (exhibiting low fertility), Belclare (medium fertility) as well as Norwegian White Sheep (NWS) and Fur (both with high fertility and pregnancy rates > 60%) at both a synchronised and natural oestrous cycle. The aim was to explore the differences between ewe breeds in the lipidomic profile and to identify candidate biomarkers associated with an optimal environment for cervical sperm transport. The results revealed the identification of 255 lipids, of which 170, 102 and 83 were different between ewe breeds, types of cycle and affected by their interaction, respectively (P < 0.05). Reduced levels of lipids involved in the resolution of inflammation (i.e. 14-HDoHE,17-HDoHE, 15-HETE) were identified in the low fertility Suffolk breed compared to high fertility ewe breeds. However, there was an up-regulation of the COX pathway accompanied by increased levels of prostaglandins in the Suffolk breed. These findings indicated a sub-optimal and pro-inflammatory environment that could have a negative effect on cervical sperm transport.

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-defensins are peptides which have diverse regulatory roles in sperm function across multiple species. In this study, Holstein-Friesian bulls were screened based on field fertility data to identify two groups (High and Low fertility (HF and LF, respectively)) of n=10 bulls per group which were genotyped for copy number variation (CNV) in the DEFB103 gene. 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 functional analysis. Sperm from LF bulls with low CN exhibited higher binding to the oviduct epithelium in vitro, while high CN affected sperm membrane fluidity in non-capacitating conditions 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 significant differential expression of 58 genes (FDR<0.1) involved in sperm migration, immune signalling and chemotaxis. These novel results confirm an important role for DEFB103 CN in both sperm function and the uterine response to bull sperm, thereby potentially influencing pregnancy outcomes in cattle. ### Competing Interest Statement The authors have declared no competing interest.

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.

In the female reproductive tract, mucin proteins are the main component of mucus secreted by cervical goblet cells and play an essential role in many biological functions. They act as a medium for lubrication and mainting a cervical mucosal barrier against ascending pathogens from the vagina while also allowing sperm migration. The expression of mucin genes as well as the levels of O-glycosylation changes across the oestrous cycle. Detection and characterization of mucins and their glycans is important to understand the interface between the external and the internal environment, as the cervical epithelium represents the first line of defense against infections of the upper reproductive tract. Advances in the field of molecular biology have made possible to study differences in mucin and glycan gene expression which can help to understand impeded sperm transport as well as variation in the susceptibility to infection. This chapter discusses procedures relevant for both animals and humans on how to recover cervical tissue and perform a gene expression analysis of genes corresponding to mucins and their glycans using RNA sequencing.

Abstract 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 12 h 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. Summary Sentence The concept that sperm from high fertility bulls are priming the endometrium for implantation and a subsequent pregnancy merits further investigation. Graphical Abstract

Introduction Cervical artificial insemination (AI) with frozen-thawed semen in sheep has yielded unacceptably low pregnancy rates. The exception is in Norway where vaginal AI yields non-return rates in excess of 60%, which has been attributed to the ewe breed used. Objectives and methods This study aimed to characterise, for the first time, the ovine follicular phase cervical mucus metabolome, with a focus on the amino acid profile. Cervical mucus was collected from four European ewe breeds with known differences in pregnancy rates following cervical AI with frozen-thawed semen. These were Suffolk (low fertility), Belclare (medium fertility), Norwegian White Sheep (NWS) and Fur (both high fertility). Results A total of 689 metabolites were identified in the cervical mucus of all the four ewe breeds. Of these, 458 metabolites were altered by ewe breed, which had the greatest effect in the dataset ( P < 0.05). We detected 194 metabolites involved in the amino acid pathway, of which 133, 56 and 63 were affected by ewe breed, type of cycle and their interaction, respectively ( P < 0.05). N-methylhydantoin and N-carbamoylsarcosine (degradation products of creatinine pathway) exhibited the greatest fold change decrease in the Suffolk breed compared to Fur and NWS ( P < 0.001). Oxidized metabolites were also decreased in Suffolk compared to high fertility breeds ( P < 0.05). In contrast, other metabolites such as 3-indoxyl-sulfate, putrescine, cadaverine were significantly increased in Suffolk at the synchronised cycle. Conclusion The suboptimal amino acid profile in the cervical mucus of the low fertility Suffolk breed may have negative consequences for sperm transport.

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