Population decline: where demography, social science, and biology intersect.

In brief: Over the past half century, the world has witnessed an unprecedented decline in human fertility rates. This analysis reviews the various socioeconomic, cultural, and biological factors involved in driving this change and considers whether low fertility rates are a temporary or permanent feature of our future demographic profile. Abstract: Since the early 1960s, the world has witnessed the spectacular collapse of human fertility. As a result of this phenomenon, several countries are already seeing their population numbers fall and more will follow in the coming decades. The causes of this fertility decline involve a complex interplay of socio-economic, environmental, and biological factors that have converged to constrain fertility in posterity's wake. Since large numbers of offspring are no longer needed to compensate for high infant mortality in contemporary society, couples have opted to have small families in a quality-over-quantity investment in their progeny's future. Simultaneously, increases in female education, the enhanced participation of women in the paid workforce, and a resultant delay in childbearing has placed limits on achievable family size. Progressive urbanization, the improved availability of contraceptives, and the socio-economic pressures experienced by young adults in ageing societies are also contributing to fertility's demise. These factors, together with the individualism that pervades modern society and the increasing social acceptability of voluntary childlessness, have firmly established a low fertility ethos in most post-transition countries. Since none of these forces are about to relent, it looks as if extremely low fertility might be with us for some time to come. This may have long-term consequences. The lack of selection pressure on high fertility genotypes, the ability of ART to retain poor fertility genotypes within the population, and sustained exposure to reproductive toxicants in modern industrialized environments may all contrive to leave a permanent mark on the fecundity of our species.

Investigations into the role of platelet-activating factor in the peri-conception period of the mare.

In Brief: In many mammals, the lipid platelet-activating factor (PAF) has important functions in female reproduction and fertility. This study shows that PAF is present in the reproductive tissues of mares and is involved in processes related to ovulation and early pregnancy. Abstract: Platelet-activating factor (PAF) has been implicated in a number of reproductive processes ranging from ovulation to embryo motility but has not been widely explored in the mare. To identify the presence and examine the role of PAF in the equine periconception processes, targeted mass spectrometry coupled with chromatographic separation was performed on equine follicular fluid (FF), and PAF was quantitatively detected. Subsequently, untargeted high-resolution mass spectrometry-based lipidomic analysis was carried out to quantify PAF in different-sized pre-ovulatory follicles, whereby different molecular species of PAF, PAF (14:0) and PAF (16:1), were both seen to be increasing with follicle diameter. These findings suggest that PAF within FF is increasing as preovulatory follicles approach ovulation. Additionally, immunofluorescence staining identified the PAF receptor in the luminal pericellular, apical, and basal aspect of equine oviductal epithelial cells. Lastly, an equine oviductal epithelial organoid model was generated and showed that the addition of PAF significantly increased the ciliary beat frequency (CBF) (Hz), an action consistent with a role for PAF in embryo migration. It is proposed that the local action of PAF on the ciliated cells of the oviduct propels both the oocyte and the conceptus towards the uterus. In the mare, it appears that PAF is a contributor during the periconception period, potentially being a mediator in the mechanisms of ovulation and in the dialogue of very early pregnancy.

Establishment and characterization of oviductal organoids from farm and companion animals†.

Organoid technology has provided a unique opportunity to study early human development and decipher various steps involved in the pathogenesis of disease. The technology is already used in clinics to improve human patient outcomes. However, limited knowledge of the methodologies required to establish organoid culture systems in domestic animals has slowed the advancement and application of organoid technology in veterinary medicine. This is particularly true for the field of reproduction and the application of assisted reproductive technologies (ART). Here, we have developed a platform to grow oviductal organoids from five domestic species-bovine, porcine, equine, feline, and canine. The organoids were grown progressively from single cells derived from the enzymatic digestion of freshly collected infundibular/fimbrial samples. The addition of WNT, TGFß, BMP, ROCK, and Notch signaling pathway activators or inhibitors to the organoid culture medium suggested remarkable conservation of the molecular signals involved in oviductal epithelial development and differentiation across species. The gross morphology of organoids from all the domestic species was initially similar. However, some differences in size, complexity, and growth rate were subsequently observed and described. After 21 days, well-defined and synchronized motile ciliated cells were observed in organoids. Histopathologically, oviductal organoids mimicked their respective native tissue. In summary, we have carried out a detailed cross-species comparison of oviductal organoids, which would be valuable in advancing our knowledge of oviduct physiology and, potentially, help in increasing the success of ART.

Predicting the outcome of Thoroughbred stallion matings on the basis of dismount semen sample analyses.

In brief: A capacity to predict the likelihood of pregnancy following natural matings would be of considerable benefit to the Thoroughbred horse breeding industry. In this article, we describe a strategy for achieving this outcome through the analysis of dismount samples, that achieved an overall accuracy of 94.6%. Abstract: The purpose of this study was to determine whether the analysis of dismount semen samples from Thoroughbred stallions could be used to predict whether a given mating would result in a pregnancy. The analysis was based on 143 matings of 141 mares by a cohort of 7 Thoroughbred stallions over a 4-week period at an Australian Stud. The criteria of semen quality utilized in this analysis involved a preliminary assessment of the raw dismount sample in terms of semen volume, sperm number, and sperm movement characteristics using an iSperm® Equine portable device. Following this initial assessment, a subpopulation of progressively motile spermatozoa was isolated by virtue of the cells ability to migrate across a 5 µm polycarbonate filter in a Samson™ isolation chamber over a 15-minute period. These isolated cells were again evaluated for their number and quality of movement using the iSperm® system and, in addition, assessed for their ability to reduce WST-1, a membrane impermeant tetrazolium salt. These data were then combined with additional information describing the ages of the animals used in this study, their historical breeding records, and mating frequency during the breeding season. The total data set was then used to predict the occurrence of pregnancy, as confirmed by ultrasound at ~14 days post mating. The criteria used to predict fertility in the ensuing multivariate discriminant analysis were optimized for each individual stallion. Using this strategy, we were able to successfully predict the outcome of a cover with an overall accuracy of 94.6%.

Enhanced Recovery After Surgery (ERAS®) Society Consensus Guidelines for Emergency Laparotomy Part 3: Organizational Aspects and General Considerations for Management of the Emergency Laparotomy Patient.

BACKGROUND: This is Part 3 of the first consensus guidelines for optimal care of patients undergoing emergency laparotomy using an enhanced recovery after surgery (ERAS) approach. This paper addresses organizational aspects of care. METHODS: Experts in management of the high-risk and emergency general surgical patient were invited to contribute by the International ERAS® Society. PubMed, Cochrane, Embase, and MEDLINE database searches were performed for ERAS elements and relevant specific topics. Studies were selected with particular attention to randomized clinical trials, systematic reviews, meta-analyses and large cohort studies, and reviewed and graded using the Grading of Recommendations, Assessment, Development and Evaluation system. Recommendations were made on the best level of evidence, or extrapolation from studies on elective patients when appropriate. A modified Delphi method was used to validate final recommendations. RESULTS: Components of organizational aspects of care were considered. Consensus was reached after three rounds of a modified Delphi process. CONCLUSIONS: These guidelines are based on best current available evidence for organizational aspects of an ERAS® approach to patients undergoing emergency laparotomy and include discussion of less common aspects of care for the surgical patient, including end-of-life issues. These guidelines are not exhaustive but pull together evidence on important components of care for this high-risk patient population. As much of the evidence is extrapolated from elective surgery or emergency general surgery (not specifically laparotomy), many of the components need further evaluation in future studies.

Consensus Guidelines for Perioperative Care for Emergency Laparotomy Enhanced Recovery After Surgery (ERAS®) Society Recommendations Part 2-Emergency Laparotomy: Intra- and Postoperative Care.

BACKGROUND: This is Part 2 of the first consensus guidelines for optimal care of patients undergoing emergency laparotomy (EL) using an Enhanced Recovery After Surgery (ERAS) approach. This paper addresses intra- and postoperative aspects of care. METHODS: Experts in aspects of management of high-risk and emergency general surgical patients were invited to contribute by the International ERAS® Society. PubMed, Cochrane, Embase, and Medline database searches were performed for ERAS elements and relevant specific topics. Studies on each item were selected with particular attention to randomized clinical trials, systematic reviews, meta-analyses, and large cohort studies and reviewed and graded using the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) system. Recommendations were made on the best level of evidence, or extrapolation from studies on elective patients when appropriate. A modified Delphi method was used to validate final recommendations. Some ERAS® components covered in other guideline papers are outlined only briefly, with the bulk of the text focusing on key areas pertaining specifically to EL. RESULTS: Twenty-three components of intraoperative and postoperative care were defined. Consensus was reached after three rounds of a modified Delphi Process. CONCLUSIONS: These guidelines are based on best available evidence for an ERAS® approach to patients undergoing EL. These guidelines are not exhaustive but pull together evidence on important components of care for this high-risk patient population. As much of the evidence is extrapolated from elective surgery or emergency general surgery (not specifically laparotomy), many of the components need further evaluation in future studies.

Sperm cryopreservation: current status and future developments.

The cryopreservation of spermatozoa is an important reproductive technology for the preservation of fertility in man and animals. Since the serendipitous discovery of glycerol as an effective cryoprotectant in 1947, sperm cryopreservation has undergone many changes in terms of the freezing methods employed, the rates at which samples are frozen and thawed, and the media used to preserve sperm functionality and DNA integrity. An extensive literature survey has been conducted addressing the cryoprotectants employed for both animal and human semen and the freezing protocols utilised. The results indicate that glycerol remains the dominant cryoprotective agent, usually incorporated into a balanced salt solution containing energy substrates, buffers, osmolytes and protein in the form of human serum albumin (human) or skimmed milk (animal). Realisation that some of the damage observed in cryostored cells involves the generation of reactive oxygen species during the thawing process, has prompted many studies to assess the relative merits of incorporating antioxidants into the cryopreservation media. However, in the absence of systematic comparisons, there is currently no consensus as to which antioxidant combination might be the most effective. Utilising our fundamental understanding of cryodamage to optimise cryopreservation protocols for each species will be important in the future.

The future of assessing bull fertility: Can the 'omics fields identify usable biomarkers?†.

Breeding soundness examinations for bulls rely heavily on the subjective, visual assessment of sperm motility and morphology. Although these criteria have the potential to identify infertile males, they cannot be used to guarantee fertility or provide information about varying degrees of bull fertility. Male factor fertility is complex, and the success of the male gamete is not necessarily realized until well after the spermatozoon enters the oocyte. This paper reviews our existing knowledge of the bull's contribution from a standpoint of the sperm's cargo and the impact that this can have on fertilization and the development of the embryo. There has been a plethora of recent research characterizing the many molecular attributes that can affect the functional competence of a spermatozoon. A better understanding of the molecular factors influencing fertilization and embryo development in cattle will lead to the identification of biomarkers for the selection of bulls of superior fertility, which will have major implications for livestock production. To see this improvement in reproductive performance, we believe incorporation of modern technology into breeding soundness examinations will be necessary-although many of the discussed technologies are not ready for large-scale field application. Each of the 'omics fields discussed in this review have shown promise for the identification of biomarkers of fertility, with certain families of biomarkers appearing to be better suited to different evaluations throughout a bull's lifetime. Further research is needed for the proposed biomarkers to be of diagnostic or predictive value.

Proteomic analysis of spermatozoa reveals caseins play a pivotal role in preventing short-term periods of subfertility in stallions†.

Stallions experience transient fluctuations in fertility throughout the breeding season. Considering pregnancy diagnoses cannot be ascertained until ~14 days postbreeding, the timely detection of decreases in stallion fertility would enhance industry economic and welfare outcomes. Therefore, this study aimed to identify the proteomic signatures reflective of short-term fertility fluctuations and to determine the biological mechanisms governing such differences. Using liquid chromatography-mass spectrometry (LC-MS/MS), we compared the proteomic profile of semen samples collected from commercially "fertile" stallions, during high- and low-fertility periods. A total of 1702 proteins were identified, of which, 38 showed a significant change in abundance (P ≤ 0.05). Assessment of intra- and interstallion variability revealed that caseins (namely κ-, α-S1-, and α-S2-casein) were significantly more abundant during "high-fertility" periods, while several epididymal, and seminal plasma proteins (chiefly, epididymal sperm binding protein 1 [ELSPbP1], horse seminal plasma protein 1 [HSP-1], and clusterin), were significantly more abundant during "low-fertility" periods. We hypothesized that an increased abundance of caseins offers greater protection from potentially harmful seminal plasma proteins, thereby preserving cell functionality and fertility. In vitro exposure of spermatozoa to casein resulted in decreased levels of lipid scrambling (Merocyanine 540), higher abundance of sperm-bound caseins (α-S1-, α-S2-, and κ-casein), and lower abundance of sperm-bound HSP-1 (P ≤ 0.05). This study demonstrates key pathways governing short-term fertility fluctuations in the stallion, thereby providing a platform to develop robust, fertility assessment strategies into the future.

Standards in semen examination: publishing reproducible and reliable data based on high-quality methodology.

Biomedical science is rapidly developing in terms of more transparency, openness and reproducibility of scientific publications. This is even more important for all studies that are based on results from basic semen examination. Recently two concordant documents have been published: the 6th edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen, and the International Standard ISO 23162:2021. With these tools, we propose that authors should be instructed to follow these laboratory methods in order to publish studies in peer-reviewed journals, preferable by using a checklist as suggested in an Appendix to this article.

Causative mechanisms and functional correlates of MTT reduction in stallion spermatozoa.

MTT is a commonly used cell vitality probe, due to its ability to form insoluble formazan deposits at cellular locations of intense oxidoreductase activity. Although this response is considered a reflection of mitochondrial redox activity, extra-mitochondrial sites of MTT reduction have been recognized within the spermatozoa of several mammalian species. Therefore, the aim of this study was to determine the major sites and causative mechanisms of MTT reduction in stallion spermatozoa. Our results show that stallion spermatozoa displayed substantial mitochondrial formazan deposition, as well as a single extra-mitochondrial formazan deposit in various locations on the sperm head in approximately 20% of cells. The quality and capacitation status of stallion spermatozoa were positively correlated with the presence of an extra-mitochondrial formazan granule. Additionally, extra-mitochondrial formazan deposition was suppressed by the presence of an NADPH oxidase (NOX) inhibitor (VAS2870; active against NOX2, NOX4 and NOX5), MnTMPyP (SOD mimetic) and zinc (NOX5 inhibitor) suggesting that extra-mitochondrial MTT reduction may be facilitated by NOX-mediated ROS generating activity, conceivably NOX5 or NOX2. When comparing MTT to resazurin, another well-known probe used to detect metabolically active cells, MTT reduction had a higher correlation with sperm concentration and motility parameters (R2= 0.91), than resazurin reduction (R2 = 0.76). We conclude that MTT reduction in stallion spermatozoa follows a species-specific pattern due to a high dependence on oxidative phosphorylation and a degree of NOX activity. As such, MTT reduction is a useful diagnostic tool to assess extra-mitochondrial redox activity, and therefore, the functional qualities of stallion spermatozoa.

OXIDATIVE STRESS AND REPRODUCTIVE FUNCTION: Sperm telomeres, oxidative stress, and infertility.

In brief: Oxidative stress is recognized as an underlying driving factor of both telomere dysfunction and human subfertility/infertility. This review briefly reassesses telomere integrity as a fertility biomarker before proposing a novel, mechanistic rationale for the role of oxidative stress in the seemingly paradoxical lengthening of sperm telomeres with aging. Abstract: The maintenance of redox balance in the male reproductive tract is critical to sperm health and function. Physiological levels of reactive oxygen species (ROS) promote sperm capacitation, while excess ROS exposure, or depleted antioxidant defenses, yields a state of oxidative stress which disrupts their fertilizing capacity and DNA structural integrity. The guanine moiety is the most readily oxidized of the four DNA bases and gets converted to the mutagenic lesion 8-hydroxy-deoxyguanosine (8-OHdG). Numerous studies have also confirmed oxidative stress as a driving factor behind accelerated telomere shortening and dysfunction. Although a clear consensus has not been reached, clinical studies also appear to associate telomere integrity with fertility outcomes in the assisted reproductive technology setting. Intriguingly, while sperm cellular and molecular characteristics make them more susceptible to oxidative insult than any other cell type, they are also the only cell type in which telomere lengthening accompanies aging. This article focuses on the oxidative stress response pathways to propose a mechanism for the explanation of this apparent paradox.

Role of sperm DNA damage in creating de-novo mutations in human offspring: the 'post-meiotic oocyte collusion' hypothesis.

Spermatogonial stem cells exhibit a low level of spontaneous mutation that is heavily impacted by paternal age via mechanisms that appear to involve the aberrant repair of DNA damage. This background de-novo mutation frequency can be increased 1000-fold by mutations affecting a key signal transduction pathway that confers upon its descendants a selective advantage, leading to clonal expansion and nests of mutant germ cells in the testes of ageing males. This 'selfish selection' model effectively explains the origin of several dominant developmental disorders, such as achondroplasia and Apert syndrome, but cannot be generalized to account for the majority of de-novo mutations where no selective advantage is apparent. In this article, an additional germline mutation pathway is proposed that recognizes the unique susceptibility of spermatozoa to DNA damage and the importance of the oocyte in repairing these lesions prior to the S phase of the first mitotic division. Any deficiency or inaccuracy on the part of the oocyte in effecting this repair process has the potential to fix paternal DNA damage as a de-novo mutation in the embryo. Such a mechanism supports emerging data indicating that assisted conception procedures may enhance the mutational load carried by ART offspring.

Guidelines for Perioperative Care for Emergency Laparotomy Enhanced Recovery After Surgery (ERAS) Society Recommendations: Part 1-Preoperative: Diagnosis, Rapid Assessment and Optimization.

BACKGROUND: Enhanced Recovery After Surgery (ERAS) protocols reduce length of stay, complications and costs for a large number of elective surgical procedures. A similar, structured approach appears to improve outcomes, including mortality, for patients undergoing high-risk emergency general surgery, and specifically emergency laparotomy. These are the first consensus guidelines for optimal care of these patients using an ERAS approach. METHODS: Experts in aspects of management of the high-risk and emergency general surgical patient were invited to contribute by the International ERAS® Society. Pubmed, Cochrane, Embase, and MEDLINE database searches on English language publications were performed for ERAS elements and relevant specific topics. Studies on each item were selected with particular attention to randomized controlled trials, systematic reviews, meta-analyses and large cohort studies, and reviewed and graded using the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) system. Recommendations were made on the best level of evidence, or extrapolation from studies on non-emergency patients when appropriate. The Delphi method was used to validate final recommendations. The guideline has been divided into two parts: Part 1-Preoperative Care and Part 2-Intraoperative and Postoperative management. This paper provides guidelines for Part 1. RESULTS: Twelve components of preoperative care were considered. Consensus was reached after three rounds. CONCLUSIONS: These guidelines are based on the best available evidence for an ERAS approach to patients undergoing emergency laparotomy. Initial management is particularly important for patients with sepsis and physiological derangement. These guidelines should be used to improve outcomes for these high-risk patients.

Patterns of MTT reduction in mammalian spermatozoa.

MTT is widely used in biology as a probe for cell viability by virtue of its ability to generate deposits of insoluble formazan at sites of intense oxidoreductase activity. This response is generally held to reflect mitochondrial redox activity; however, extra-mitochondrial MTT reduction has also been recorded in certain cell types. Given this background, we set out to determine the major sites of formazan deposition in mammalian spermatozoa. In the mouse, most MTT reduction took place within the extensive mitochondrial gyres, with a single minor site of formazan deposition on the sperm head. By contrast, human spermatozoa generally displayed small disorganized midpieces exhibiting moderate MTT reduction activity accompanied by a major extra-mitochondrial formazan deposit on various locations in the sperm head from the neck to the anterior acrosome. Equine spermatozoa presented a combination of these two patterns, with major formazan deposition in the mitochondria accompanied by an extra-mitochondrial formazan deposit in around 20% of cells. The functionality of human spermatozoa was positively associated with the presence of an extra-mitochondrial formazan granule. Subsequent studies indicated that this extra-mitochondrial activity was suppressed by the presence of diphenylene iodonium, zinc, 2-deoxyglucose, co-enzyme Q, an SOD mimetic and NADPH oxidase inhibitors. We conclude that the pattern of MTT reduction to formazan by spermatozoa is species specific and conveys significant information about the relative importance of mitochondrial vs extra-mitochondrial redox activity that, in turn, defines the functional qualities of these cells.

Mass spectrometry reveals distinct proteomic profiles in high- and low-quality stallion spermatozoa.

The horse breeding industry relies upon optimal stallion fertility. Conventional sperm assessments provide limited information regarding ejaculate quality and are not individually predictive of fertilizing potential. The aim of this study was to harness mass spectrometry to compare the proteomic profiles of high- and low-quality stallion spermatozoa, with the ultimate goal of identifying fertility biomarker candidates. Extended stallion semen (n = 12) was fractionated using Percoll density gradients to isolate low-quality and high-quality sperm populations. Motility and morphological assessments were carried out, and proteomic analyses was conducted using UHPLC-MS/MS. High-quality spermatozoa recorded higher total (95.2 ± 0.52% vs 70.6 ± 4.20%; P ≤ 0.001) and progressive motilities (43.4 ± 3.42% vs 27.3 ± 4.32%; P ≤ 0.05), and a higher proportion of morphologically normal cells (50.2 ± 4.34% vs 38.8 ± 2.72%; P ≤ 0.05). In total, 1069 proteins were quantified by UHPLC-MS/MS, of which 22 proteins were significantly more abundant in the high-quality sperm population (P ≤ 0.05). A-kinase anchor protein 4 (AKAP4) and Hexokinase 1 (HK1) were considered possible biomarker candidates and their differential expression was confirmed by immunoblot. Protein expression was significantly correlated with total (AKAP4 R2 = 0.38, P ≤ 0.01; HK1 R2 = 0.46, P ≤ 0.001) and progressive motilities (AKAP4 R 2 = 0.51, P ≤ 0.001; HK1 R2 = 0.55, P ≤ 0.01), percentage rapid (AKAP4 R2 = 0.29, P ≤ 0.05; HK1 R2 = 0.58, P ≤ 0.001), straight-line velocity (HK1 R2 = 0.50, P ≤ 0.01) and straightness (HK1 R2 = 0.40, P ≤ 0.01). Furthermore, AKAP4 was highly susceptible to adduction by 4-hydroxynonenal (4HNE), which resulted in a global reduction in the phosphorylation profiles following capacitation. In conclusion, the proteomic profiles of high- and low-quality stallion spermatozoa differ substantially, and proteins such as AKAP4 and HK1 could serve as biomarkers of ejaculate quality.

Occupational Inhalation Exposures to Nanoparticles at Six Singapore Printing Centers.

Laser printers emit high levels of nanoparticles (PM0.1) during operation. Although it is well established that toners contain multiple engineered nanomaterials (ENMs), little is known about inhalation exposures to these nanoparticles and work practices in printing centers. In this report, we present a comprehensive inhalation exposure assessment of indoor microenvironments at six commercial printing centers in Singapore, the first such assessment outside of the United States, using real-time personal and stationary monitors, time-integrated instrumentation, and multiple analytical methods. Extensive presence of ENMs, including titanium dioxide, iron oxide, and silica, was detected in toners and in airborne particles collected from all six centers studied. We document high transient exposures to emitted nanoparticles (peaks of ∼500 000 particles/cm3, lung-deposited surface area of up to 220 µm2/cm3, and PM0.1 up to 16 µg/m3) with complex PM0.1 chemistry that included 40-60 wt % organic carbon, 10-15 wt % elemental carbon, and 14 wt % trace elements. We also record 271.6-474.9 pmol/mg of Environmental Protection Agency-priority polycyclic aromatic hydrocarbons. These findings highlight the potentially high occupational inhalation exposures to nanoparticles with complex compositions resulting from widespread usage of nano-enabled toners in the printing industry, as well as inadequate ENM-specific exposure control measures in these settings.

Depletion of thiols leads to redox deregulation, production of 4-hydroxinonenal and sperm senescence: a possible role for GSH regulation in spermatozoa†.

We hypothesized that thiols and particularly glutathione (GSH) are essential for the regulation of stallion sperm functionality. To test this hypothesis, we initially investigated the relationship between sperm function and GSH content, revealing highly significant correlations between GSH, sperm viability, motility, and velocity parameters (P < 0.001). Furthermore, the deleterious effects of GSH depletion using menadione and 1,3 dimethoxy 1,4, naphtoquinone (DMNQ) were able to be prevented by the addition of cysteine, but no other antioxidant. Pre-incubation with cysteine prevented menadione and DMNQ induced damage to sperm membranes after 1 h (P < 0.001; P < 0.05) and after 3 h of incubation (P < 0.001, P < 0.05). Pre-incubation with cysteine ameliorated both the menadione- and DMNQ-induced increase in 4-hydroxynonenal (P < 0.001). As cysteine is a precursor of GSH, we hypothesized that stallion spermatozoa are able to synthesize this tripeptide using exogenous cysteine. To test this hypothesis, we investigated the presence of two enzymes required to synthesize GSH (GSH and GCLC) and using western blotting and immunocytochemistry we detected both enzymes in stallion spermatozoa. The inhibition of GCLC reduced the recovery of GSH by addition of cysteine after depletion, suggesting that stallion spermatozoa may use exogenous cysteine to regulate GSH. Other findings supporting this hypothesis were changes in sperm functionality after BSO treatment and changes in GSH and GSSG validated using HPLC-MS, showing that BSO prevented the increase in GSH in the presence of cysteine, although important stallion to stallion variability occurred and suggested differences in expression of glutamate cysteine ligase. Mean concentration of GSH in stallion spermatozoa was 8.2 ± 2.1 µM/109 spermatozoa, well above the nanomolar ranges per billion spermatozoa reported for other mammals.

What makes a fertile sperm? Unique molecular attributes of stallion fertility.

Stallions experience lower per-cycle conception rates compared to other livestock species, largely because they are selected for breeding based on athletic prowess and not reproductive fitness. Mares are seasonal breeders, and pregnancies cannot be detected until 10-14 days post cover via transrectal ultrasonography. This means the detection of stallion fertility fluctuations is delayed by at least 2 weeks, which within the short breeding season employed by the thoroughbred horse breeding industry, can prove quite costly. For these reasons, there is increased demand for robust laboratory assays aimed at the accurate assessment of stallion fertility. This paper reviews our existing knowledge concerning the molecular mechanisms that underpin the functional competence of stallion spermatozoa, highlighting the relative importance of oxidative stress, DNA damage, sperm proteomics and RNA profile. We also consider the way in which fundamental improvements in our understanding of stallion sperm biology are informing the identification and development of possible biomarkers of fertility and thus avenues for the development of specific assays for fertility prediction.

CABYR is essential for fibrous sheath integrity and progressive motility in mouse spermatozoa.

Ca2+-binding tyrosine-phosphorylation-regulated protein (CABYR) has been implicated in sperm physiological function in several in vitro studies. It has also been implicated as a potential cause of and diagnostic tool in asthenozoospermic human males. CABYR is known to be localized to the fibrous sheath, an accessory structure in the flagellar principal piece. Utilizing the CRISPR-Cas9 technology, we have knocked out this gene in mice to understand its role in male fertility. Cabyr-knockout male mice showed severe subfertility with a defect in sperm motility as well as a significant disorganization in the fibrous sheath. Further, abnormal configuration of doublet microtubules was observed in the Cabyr-knockout spermatozoa, suggesting that the fibrous sheath is important for the correct organization of the axoneme. Our results show that it is the role of CABYR in the formation of the fibrous sheath that is essential for male fertility.