Jessica Paige Rickard

Context: Motility longevity offers valuable insights into sperm function and metabolic resilience; however, the extent of inter-individual variation and its mechanistic underpinnings remain poorly defined. Aims: This study aimed to characterise variation in mass motility duration in rams and determine whether differences in motility longevity are associated with distinct metabolic profiles. Methods: Successive ejaculates (n = 314) were collected and assessed from 23 Merino rams. Seminal plasma from ejaculates exhibiting the shortest and longest motility duration was analysed immediately post-collection and at motility cessation using targeted mass spectrometry to characterise changes in metabolite abundance over time. Key results: Mass motility duration varied markedly between rams, ranging from 11 min to over 20 h. Although mass motility duration, ejaculate volume, and sperm concentration were influenced by sequential ejaculate collection, mass motility duration was not strongly correlated with ejaculate volume or sperm concentration, indicating that variation in motility longevity is not simply explained by these traits. Rather, distinct metabolic strategies appear to drive variation among phenotypes. Short-lasting ejaculates showed rapid depletion of fructose and marked lactate accumulation, consistent with heavy reliance on glycolysis and limited mitochondrial engagement. In contrast, long-lasting ejaculates maintained stable fructose levels while exhibiting increased hexose phosphate abundance and substantial pyruvate utilisation, suggesting more controlled glycolytic flux and sustained mitochondrial oxidation. Accumulation of α-ketoglutarate and 2-hydroxyglutarate was exclusive to short-lasting ejaculates, indicating mitochondrial bottlenecks and redox imbalance. Conclusions: These findings highlight metabolic regulation, rather than substrate availability alone, as a key determinant of motility longevity in ram spermatozoa. Implications: Understanding how long-lasting ejaculates maintain coordinated glycolytic and mitochondrial activity provides a framework for developing metabolically informed strategies to prolong sperm survival during processing and storage.

Following semen deposition, the female reproductive tract must balance immune tolerance towards spermatozoa with preservation of antimicrobial defence. To date, much of our understanding of female immune response following insemination has been derived from models of pathogen defence and uterine receptivity in humans, with comparatively little on how these same immune pathways shape successful sperm transport. Here, we summarise the relationship between early sperm transport immediately following semen deposition and acute innate immune activation, characterised by complement engagement and rapid leukocyte recruitment. Polymorphonuclear neutrophils, traditionally viewed as terminal effectors of sperm clearance, are reframed as active mediators of innate surveillance and sperm immune adaptation. Spermatozoa enter the female environment bearing a molecular identity shaped by testicular development, epididymal maturation and exposure to seminal plasma, yet the link between specific mediators, immunity and fertility has yet to be explored in livestock. Seminal plasma extracellular vesicles stand out as a promising mediator of complement activity and cytokine signalling. Disruption of this male-derived immune conditioning through processing for cryopreservation is hypothesised to explain impaired sperm transport in sheep. Orchestrating male and female contributions to the maternal immune response reframes reproductive success as coordinated sperm–immune communication. This review identifies knowledge gaps whose resolution will advance our understanding of immune tolerance to spermatozoa, informing strategies to enhance the success of assisted reproductive technologies in livestock. Lay Summary: Successful reproduction requires a highly functional immune response to defend against pathogens while facilitating sperm transport. This review explores how spermatozoa and seminal plasma actively communicate with the female immune system immediately following semen deposition. We highlight how immune cells and complement proteins shape sperm survival, and how alterations to the sperm immune profile during in vitro processing may disrupt this delicate balance of communication within the female.

Context: Mass motility assessment is widely used to evaluate semen quality in the sheep breeding industry, yet the biological factors influencing motility duration remain poorly characterised. At ejaculation, spermatozoa are combined with seminal plasma (SP), which initially supports motility and function but may compromise long-term metabolic endurance. Aims: This study aimed to investigate the effects of SP on mass motility duration and pH regulation of epididymal ram spermatozoa. Methods: Epididymal sperm were incubated with whole SP, saline, or left undiluted to assess motility duration. Subsequently, sperm were incubated with varying SP concentrations and molecular weight-separated fractions (protein-free 3 kDa) and assessed for motility and pH over 24 h. Metabolomic and lipidomic profiling via targeted mass spectrometry characterised the biochemical composition of each SP fraction. Key results: SP exposure significantly reduced mass motility duration (195 ± 11 min) compared with undiluted sperm (1797 ± 185 min; P = 0.0123) and saline dilution (2667 ± 134 min; P < 0.0001). All SP-treated groups exhibited a progressive pH decline, consistent with increased glycolytic flux, whereas SP-naïve sperm showed stable or increasing pH, indicating a more regulated metabolic state and greater metabolic flexibility. The protein-free SP fraction was most detrimental to motility, which was likely to be the result of the depletion of key proteins, lipids, and metabolites associated with energy production. Conclusions: Prolonged exposure to SP impairs ram sperm motility longevity by promoting a glycolysis-biased metabolic state and reducing metabolic endurance. Implications: Understanding how seminal plasma composition influences sperm metabolic regulation may inform strategies to better preserve sperm motility during semen handling and storage.

The complex anatomical, biochemical and immunological architecture of the ovine cervix presents a substantial barrier to frozen-thawed (FT) ram spermatozoa migration following cervical artificial insemination (AI). A key component of the immunological barriers involves interactions between FT sperm and polymorphonuclear neutrophils (PMN), which are known to infiltrate the female reproductive tract (FRT) following semen deposition and engage in effector mechanisms, specifically degranulation, release of neutrophil extracellular traps (NETs) and phagocytosis. Seminal plasma (SP) has been shown to play a critical immunomodulatory role in regulating sperm–PMN interactions across several species, including sheep, reducing sperm–PMN binding in vitro. During in vitro processing and cryopreservation, SP is inherently diluted, potentially exacerbating immune-mediated clearance of FT sperm. Identifying the specific components of SP responsible for this immunoprotective effect may inform targeted supplementation strategies. Extracellular vesicles (EVs), a heterogenous class of SP-containing proteins, lipids, metabolites and nucleic acids, are also emerging as key mediators of immunological communication. Their potential role in modulating sperm–PMN interactions warrants further investigation to help understand sperm–PMN interactions and implications for improving survival of FT ram sperm in the cervix following AI.

Artificial reproductive technologies (ARTs) are central to genetic improvement strategies in livestock, yet adoption remains inconsistent across species. In sheep, uptake has lagged behind that of cattle due to species‑specific reproductive biology, variable oocyte and embryo competence following use of exogenous hormones, economic feasibility and inconsistent success rates. This review synthesises the principles, methodological development, and contemporary performance of semen collection and cryopreservation, artificial insemination (AI), multiple ovulation embryo transfer (MOET), and in vitro fertilization and embryo production (IVF) in sheep, and integrates insights from a global survey of commercial sheep artificial breeding providers (68% response rate; N = 41) on the current size and scale of the industry. It also collates data on optimisation studies working to improve success rates in these technologies. Survey data reveal marked heterogeneity in semen processing practices, cryopreservation method, extender use, insemination dose preparation, and oestrus synchronisation protocols. Laparoscopic intrauterine AI remains the predominant technique, yielding a global weighted pregnancy rate of 71%, despite substantial site, sire, and operator‑dependent variation. MOET is widely implemented, producing an average of 6–8 transferable embryos per donor, with fresh embryo transfer outperforming frozen–thawed embryos across global regions. In contrast, IVF in sheep continues to face technical and commercial constraints related to oocyte recovery, in vitro maturation, and blastocyst development. Collectively, this review highlights critical opportunities for standardised semen assessment, optimised hormone protocols, advanced predictive analytics, and targeted commercialisation of ovine IVP systems to enhance reproducibility, efficiency, and genetic gain within the global sheep artificial breeding industry.

In brief: Cervical explants alter gene expression and cytokine secretion in response to seminal plasma compared with spermatozoa alone. Results validate the model to study mechanisms behind sperm-immune tolerance in the ovine cervix ex vivo. The ovine cervix presents a unique barrier to frozen–thawed ram spermatozoa, reducing pregnancy following cervical artificial insemination (AI). This is hypothesised to be related to a modified sperm molecular profile following seminal plasma (SP) exposure and cryopreservation, altering communication with the female environment. However, the impact of different sperm types on the cervix itself has never been explored, in part due to the difficulty of profiling these in vivo interactions. Here, an ex vivo cell culture model was used to explore changes in the transcriptome of cervical explants following exposure to spermatozoa and SP. Explants were collected from Merino ewes (n = 6) post-mortem and cultured with epididymal, epididymal + SP, frozen and frozen + SP ram spermatozoa, SP only, or a media control for 6 h. Gene expression was profiled by RNA sequencing. Multiplex ELISAs quantified the abundance of cytokines in culture supernatant. Cytokeratin and H&E staining confirmed the presence of cervical epithelial cells in cultured explants. The presence of epididymal sperm caused the differential expression of 18 genes compared with the media-only control, while the addition of SP to epididymal sperm altered 781 genes. Differentially expressed genes were involved in pathways related to inflammation, signalling, and ATP synthesis. SP exposure to frozen sperm led to significantly increased IL1A and reduced TGFB1 production compared with other treatments. Results show an altered cervical immune response in the presence of ram SP and sperm. Further research is required to determine molecular candidates which facilitate immune tolerance and sperm transit through the cervix.

In brief: Frozen-thawed ram spermatozoa show reduced fertility following cervical artificial insemination, likely attributed to an elevated immune response in the ewe. This paper identifies the immunoprotective effect of ram seminal plasma (SP) against polymorphonuclear neutrophil binding, highlighting potential for fertility improvement. Following cervical artificial insemination, frozen-thawed (FT) ram spermatozoa display reduced fertility compared to fresh spermatozoa, irrespective of sperm motility and viability, likely attributed to an elevated immune response in the ovine cervix. SP has previously been shown to be beneficial to sperm transport in the ovine cervix, yet the components responsible for this protective effect remain unknown. As such, the present study examined the immune dynamics of frozen-thawed ram spermatozoa with polymorphonuclear neutrophils (PMNs), utilising a neutrophil binding assay. The effect of 25% v/v SP supplementation on binding susceptibility was also investigated. A portion of SP was heat-treated before incubation (HTSP) to isolate the impact of SP proteins. The presence of SP significantly reduced sperm-PMN binding (37.15 ± 0.02%, 46.59 ± 0.02%, 38.83 ± 0.02%) compared to FT alone (62.83 ± 0.02%, 75.74 ± 0.02%, 56.0 ± 0.03%) across all serum groups (no serum, serum and heat-treated serum, respectively). HTSP showed comparable binding susceptibility to the FT treatments, indicating that the protective effect of SP is attributed to a heat-labile factor. Serum significantly increased sperm-neutrophil binding irrespective of SP treatment. However, this was reduced to serum-free levels following heat-treatment, suggesting sperm-neutrophil dynamics are further mediated by protein components within ewe serum, likely complement proteins. The viability of sperm or the presence of freezing medium did not influence PMN binding. Furthermore, PMN viability and therefore binding were not influenced by the presence of serum or SP. Together, the current study presents new evidence for the immunoprotective properties of SP in sheep, which could be leveraged to aid transit through the cervix.

Sperm DNA integrity is increasingly considered a useful measure of semen quality in mammalian reproduction. However, the definition of DNA integrity, the ideal means by which it should be measured, and its predictive value for fertility remain a topic of much discussion. With an emphasis on livestock species, this review discusses the assays that have been developed to measure DNA integrity as well as their correlation with in vitro and in vivo fertility.

The assessment of semen quality is used to identify factors that influence sperm performance and diagnose male infertility