Sperm glorious sperm! Insights from the world of marsupials

Abstract: Sexual conflict occurs in many species as males try to maximize paternity whilst females try to select males of superior quality to ensure offspring fitness. After mating, such conflict gives rise to both sperm competition and cryptic female choice. Eutherian and metatherian mammals diverged approximately 130 – 125 million years ago. While both classes of mammals are likely to have been subjected to similar post-copulatory selective forces, marsupials have responded with some unique reproductive adaptations in their ejaculates thought to further male-biased objectives in reproduction. These alternative strategies include semen barriers, efficient sperm transport and sperm co-operation. This paper examines the functional significance of these ejaculate adaptations using a number of promiscuous marsupial examples including the highly seasonal monovular tammar wallaby (Macropus eugenii) from Australia and the non-seasonal polyovular grey short-tailed opossum (Monodelphis domestica) from South America. Semen Barriers: Semen can act physically or chemically to disable rival sperm or manipulate female reproduction. The tammar, like other macropodids, has a highly elastic copulatory plug thought to act as a physical barrier to fertilization by rival males. We tested this hypothesis by examining seasonal changes in semen quality as well as paternity success following competitive mating in this species. We found that the copulatory plug was significantly more robust during the breeding season when sperm competition is most intense (P < 0.05), but no significant first male paternity advantage was observed (P = 0.112). Thus, the function of the copulatory plug remains unclear but preliminary evidence suggests it may prevent sperm loss from the female tract in this bipedal species. Efficient Sperm Transport and Sperm Cooperation: Ejaculation of great numbers of sperm by males is widely regarded as an adaptive strategy that confers success in sperm competition. To determine if promiscuous male marsupials also exhibit superfecundity, we conducted single-mating experiments and assessed sperm dynamics in the female reproductive tract in both the tammar and opossum. In contrast to the promiscuous rabbit, the tammar and opossum ejaculate 20 and 250-fold less sperm respectively. However, the opossum has extremely efficient sperm transport (1 in 300 ejaculated sperm reach the site of fertilization compared to 1 in 10,000 in the rabbit). This efficiency may be due to the formation of sperm pairs (a form of sperm cooperation) in ejaculates of this and other didelphid marsupials. Indeed, further examination revealed that paired sperm migrate almost exclusively to the isthmus of the oviduct shortly after mating. This is the first in vivo evidence of an adaptive advantage conferred by sperm cooperation in any mammal. In summary, the tammar, opossum and other marsupials provide important examples of how metatherians have evolved alternative reproductive adaptations in the face of selective pressures

Seasonal changes in the ejaculate of the male tammar wallaby, Macropus eugenii: implications for fertility and assisted breeding

The tammar wallaby, Macropus eugenii is a seasonally breeding macropodid marsupial. The seasonality observed in males is known to be driven by the reproductive state of the females. There is a significant increase in male prostate and Cowper's gland weights and testosterone concentration during the breeding season in January/February and again in October when the young females leave their i;nothers pouches and enter puberty. The dynamics of sperm production in the male tammar wallaby was assessed using changes in ejaculatory and sperm characteristics in and out of the breeding season in order to determine more accurately true seasonality in the male. Semen was collected from wild-caught adult males by electro-ejaculation at four times during the year (January, February, June/July and October). Ejaculates were assessed for semen volume, plug formation, sperm index, percentage and rating of motility, sperm and motile sperm concentration, and total sperm count. Increases were observed during the two breeding seasons in all traits assessed. Semen volume showed a steady increase from June/July (0.7 ml) to reach a peak in February (10.25 ml). In conclusion, we found a significant decrease in the size and coagulation properties of the ejaculate, and in sperm quality out of season. Implications for captive and assisted breeding programs are discussed

Appeasing pheromones for the management of stress and aggression during conservation of wild canids: could the solution be right under our nose?

Thirty-six species of canid exist globally, two are classified as critically endangered, three as endangered, and five as near threatened. Human expansion and the coinciding habitat fragmentation necessitate conservation interventions to mitigate concurrent population deterioration. The current conservation management of wild canids includes animal translocation and artificial pack formation. These actions often cause chronic stress, leading to increased aggression and the suppression of the immune and reproductive systems. Castration and pharmaceutical treatments are currently used to reduce stress and aggression in domestic and captive canids. The undesirable side effects make such treatments inadvisable during conservation management of wild canids. Pheromones are naturally occurring chemical messages that modulate behaviour between conspecifics; as such, they offer a natural alternative for behaviour modification. Animals are able to distinguish between pheromones of closely related species through small compositional differences but are more likely to have greater responses to pheromones from individuals of the same species. Appeasing pheromones have been found to reduce stress- and aggression-related behaviours in domestic species, including dogs. Preliminary evidence suggests that dog appeasing pheromones (DAP) may be effective in wild canids. However, the identification and testing of species-specific derivatives could produce more pronounced and beneficial behavioural and physiological changes in target species. In turn, this could provide a valuable tool to improve the conservation management of many endangered wild canids

Revisiting summer infertility in the pig: could heat stress-induced sperm DNA damage negatively affect early embryo development?

Temperature is a crucial factor in mammalian spermatogenesis. The scrotum, pampiniform plexus, and cremaster and dartos muscles in mammals are specific adaptations to ensure sperm production in a regulated environment 4-6°C below internal body temperature. However, the limited endogenous antioxidant systems inherent in mammalian spermatozoa compounded by the loss of cytosolic repair mechanisms during spermatogenesis, make the DNA in these cells particularly vulnerable to oxidative damage. Boar sperm is likely to be more susceptible to the effects of heat stress and thus oxidative damage due to the relatively high unsaturated fatty acids in the plasma membrane, low antioxidant capacity in boar seminal plasma, and the boar’s non-pendulous scrotum. Heat stress has a significant negative impact on reproductive performance in piggeries, which manifests as summer infertility and results in productivity losses that amount to millions of dollars. This problem is particularly prevalent in tropical and subtropical regions where ambient temperatures rise beyond the animal’s zone of thermal comfort. Based on preliminary studies in the pig and other species, this article discusses whether heat stress could induce sufficient DNA damage in boar sperm to significantly contribute to the high rates of embryo loss and pregnancy failure observed in the sow during summer infertility. Heat stress-induced damage to sperm DNA can lead to disrupted expression of key developmental genes essential for the differentiation of early cell lineages, such as the trophectoderm, and can distort the timely formation of the blastocyst; resulting in a failure of implantation and ultimately pregnancy loss. Confirming such a link would prompt greater emphasis on boar management and strategies to mitigate summer infertility during periods of heat stress

Recent developments in male fertility evaluation, sperm cryopreservation and artificial fertilisation, and their potential application to decapod crustacean aquaculture

To maximise productivity, a better understanding of the underlying causes of subfertility that lead to inferior offspring and high mortality is imperative. In decapod crustaceans, most research has focused on female reproductive performance, with little attention given to male fertility. Paternal genetic contribution is critical to both successful embryonic and post-embryonic development. Assessment of sperm quality can be a direct method to determine male subfertility in decapods. Sperm quality parameters such as sperm concentration and morphology have traditionally been used to determine male reproductive performance, but these procedures are time-consuming and can only assess a limited number of sperm cells and males. Alternative diagnostic biomarkers used widely in humans and other mammals could be adapted to decapod crustaceans and may be more indicative of sperm fertilisation competence and male reproductive performance. These predictive biomarkers use fluorescent cellular dyes and high-throughput flow cytometry or computer-assisted sperm microscopic analysis to evaluate sperm viability, mitochondrial function, acrosome reaction and DNA fragmentation. This review examines current and advanced biomarkers to evaluate sperm quality and further explores state-of-the-art procedures of sperm cryopreservation (conventional vs. vitrification techniques) and artificial fertilisation in decapod crustaceans. Sperm freezing coupled with artificial fertilisation in decapods permits the long-term storage, controlled timing and selection of individuals for reproduction. Collectively, these tools can be applied to commercial broodstock management to improve productivity and accelerate selective breeding in the crustacean aquaculture industry

Antioxidant therapy mitigates summer-induced DNA damage in boar spermatozoa

Introduction: While pork is the most widely eaten meat in the world, pig production is continually threatened by changing climate conditions resulting in poor reproductive performance, particularly in the tropics. The boar's inefficient capacity to sweat; non-pendulous scrotum and high susceptibility of boar sperm to temperature shock makes this species particularly vulnerable to the effects of heat stress. While DNA-damaged sperm may fertilize normally, key genes involved in early embryo development may be severely affected inducing early embryo loss as shown in the mice. Our study demonstrates that tropical summer significantly increases DNA damage in populations of sperm within the ejaculate without necessarily affecting sperm motility. Moreover, supplementing boars with antioxidants during summer could potentially reduce the negative impact of heat stress on sperm DNA integrity. Materials and Methods: Five mature Large White boars were housed individually in open gable pens at JCU in the dry tropics of Townsville, Queensland, Australia. Semen was collected by the gloved-hand technique using a dummy sow during spring (Oct 2014), summer (Feb 2015) & early winter (end May 2015) and diluted 1:3 in BTS media. Boar feed was supplemented with 100g/boar/day custom-made antioxidant formulation during summer (Jan - Apr 2016) and semen was collected after 42 & 84 days treatment. Sperm concentration was determined by haemocytometer and motility of 20 x 106 sperm/ml at 38°C was analyzed using CASA (Hamilton Thorne). Sperm DNA damage in 20,000 Percoll-purified sperm per boar per treatment was evaluated using TUNEL (Roche) & flow cytometry (Dako Cytomation; Fig. 1). Results: Sperm motility was similar in summer to winter & spring (P > 0.05) but total motility was lower in spring than winter (P ≤ 0.05). Antioxidant supplementation for 42 & 84 days during summer did not affect motility of spermatozoa (P > 0.05). Sperm DNA damage was more than 16-fold higher in summer than winter & nearly 9-fold higher than spring (P ≤ 0.05; Fig. 2). Antioxidant supplementation during summer reduced sperm DNA damage after 42 & 84 days antioxidant treatment (P ≤ 0.05; Fig. 3). Conclusions and discussion: Sperm DNA integrity is compromised in boars during summer, suggesting boar factors may contribute to embryo loss in sows. Moreover, such damage appears undetectable using traditional measures of sperm motility. Antioxidant therapy during summer appears to significantly alleviate heat stress-induced DNA damage in boar sperm, which may provide one solution to the problem of summer infertility in the pig

Tropical summer induces sperm DNA damage in boars which can be mitigated by antioxidant therapy

Summer infertility due to heat stress grossly affects reproductive performance in pigs, particularly in the tropics, and causes over $300 million in annual losses to the US swine industry. Boar's inefficient capacity to sweat; non-pendulous scrotum and the high susceptibility of boar sperm to temperature shock makes this species particularly vulnerable to heat stress. While traditionally considered a sow problem, recent studies demonstrate that heat stress-induced sperm DNA damage can result in early embryo loss in mice. Our study aimed to demonstrate higher sperm DNA damage during summer in boars and trial antioxidant therapy to alleviate the problem. Motility of sperm obtained from n=5 Large White boars housed in the dry tropics of Townsville, North Queensland, Australia was characterized by Computer-Assisted Sperm Analysis (CASA), but did not differ between spring, summer and winter (total motility: 70.8 ± 5.5% vs. 71.3 ± 8.1% vs. 90.2 ± 4.2%; progressive motility: 41.7 ± 2.8% vs. 35.4 ± 7.0% vs. 46.6 ± 4.0% respectively, both P ≥ 0.05). However, sperm DNA integrity in twenty-thousand spermatozoa per boar per treatment, evaluated using TUNEL staining and flow cytometry, revealed nearly 9-fold higher DNA damage in summer than spring and winter (16.1 ± 4.8% vs. 1.8 ± 0.5% vs 1.1 ± 0.2% respectively; P ≤ 0.05). However, boar feed supplemented with antioxidants during summer significantly reduced sperm DNA damage to 9.9 ± 4.5% and 7.2 ± 1.6% (P ≤ 0.05) after 42 and 84 days treatment respectively. Total and progressive motility were not altered by the supplement. Our results show sperm DNA integrity is compromised in boars during summer, suggesting boar factors may contribute to embryo loss in sows. Moreover, such damage may go undetected using traditional measures of sperm motility. Antioxidant supplementation during summer alleviates the negative impact of heat stress on sperm DNA integrity

Intrauterine expression of LIF and its receptors in the cycling and early pregnant mare

Leukaemia Inhibitory Factor (LIF) plays a critical role in blastocyst development and implantation, as clearly demonstrated by the failure of wild-type mouse embryos to implant in the uterus of LIF-knockout female mice unless the latter receive LIF supplementation. Expression of LIF and its receptors (LIF-R and gp130) in the early pregnant horse have not been described. However, the horse may be an interesting animal in which to study such ‘implantation factors’ because various aspects of implantation occur both unusually late and in a temporally distinct fashion in this species. The aim of this study was to examine LIF, LIF-R and gp130 gene expression in the endometrium of cycling and early pregnant mares, and in the early conceptus. Endometrial biopsies were recovered from 4 mares at each of late oestrus, days 7 and 14 of dioestrus (cycling mares), and days 7, 14 and 21 of gestation. Conceptuses were recovered by uterine lavage (day 7) or using a video-endoscopically guided net (days 14 and 21). For day 14 and 21 conceptuses, only the bilaminar trophoblast was used in gene expression studies. Expression of mRNA for LIF, LIF-R and gp130 was analysed by rtPCR, with relative expression calculated with respect to the 3 most stable housekeeping genes using GeNorm analysis. A dramatic increase in LIF mRNA expression (p<0.01) was observed in both the endometrium and the trophoblast on day 21 of pregnancy. Expression of LIF-R and gp 130 increased significantly in the trophoblast on day 21 (p<0.01), but did not vary in the endometrium at the various stages examined. We propose that LIF plays a role in the adhesion between trophoblast and endometrium which, in the mare, cannot begin until the blastocyst capsule is dissolved somewhere between days 18 and 22 of gestation

Boar sperm DNA damage induced by tropical heat stress can be alleviated using antioxidants

Seasonal heat stress is known to significantly diminish reproductive performance in pigs, particularly in the tropics, costing the industry millions in annual loses. The boar's reduced capacity to sweat, non-pendulous scrotum, and widespread use of European breeds in the tropics, makes this species particularly vulnerable to heat stress. While traditionally considered a sow problem, recent mouse studies demonstrate that heat stress-induced sperm DNA damage can result in arrested development and loss of early embryos. Our study investigated the impact of tropical summer heat stress on the quality and DNA integrity of boar sperm, and trialled antioxidant supplementation to alleviate the problem. Motility of sperm obtained from n = 5 Large White boars housed in the dry tropics of Townsville, North Queensland, Australia was characterized by Computer-Assisted Sperm Analysis but did not differ between summer, winter or spring (total motility: 71.3 ± 8.1 vs. 90.2 ± 4.2 vs. 70.8 ± 5.5% respectively, P > 0.05; progressive motility: 35.4 ± 7.0 vs. 46.6 ± 4.0 vs. 41.7 ± 2.8% respectively, P > 0.05). Sperm DNA integrity in 20,000 sperm/boar/season, evaluated using TUNEL and flow cytometry, revealed 16-fold more DNA damaged sperm in summer than winter, and nearly 9-fold more than spring (16.1 ± 4.8 vs. 1.0 ± 0.2 vs. 1.9 ± 0.5% respectively, P ≤ 0.05). However, boar feed supplemented with 100g/boar/day of proprietary custom-made antioxidants during summer significantly reduced sperm DNA damage to 9.9 ± 4.5% and 7.2 ± 1.6% (P ≤ 0.05) after 42 and 84 days treatment respectively. Total and progressive motility were not altered by the supplement. In summary, sperm DNA integrity is compromised in boars during summer, suggesting boar factors may contribute to seasonal embryo loss in sows. Moreover, such damage appears undetectable using traditional measures of sperm motility. Antioxidant supplementation during summer appears to mitigate the negative impact of heat stress on sperm DNA integrity