Fertility in Adverse Environments Group: who we are and what we do

GAME Lab: About Us Research Interests: - Assisted reproduction in wilflife (marsupials, African wild dogs & barramundi) - Impact of adverse conditions (IVF, heat-stress, social-stress, disease & maternal-age)on fertility (various animal models) Techniques Used: - highly sensitive molecular tools as 'barometer' to detect compromised viabilityin sperm & embryos: o CASA, TUNEL, immunof luorescent microscopy, qRT-PCR, semen collection, sperm cryopreservation, artificial insemination & IV

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

Antioxidant supplementation alleviates DNA damage in boar spermatozoa induced by tropical heat stress

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. Data, expressed as mean ± SEM, were analysed by one-way repeated measures ANOVA with pairwise Bonferroni tests. 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

Summer induces DNA damage in boar sperm: implications for the management of seasonal infertility

At 40% share, pork is the most widely eaten meat globally. As such, research efforts must improve production and efficiency in the pig industry to meet growing demand. However, summer heat stress has a significant negative impact on pig fertility; causing embryonic death and decreased litter size that cost the industry millions in productivity losses. This problem is particularly prevalent in the tropics where ambient temperatures rise beyond the animal’s zone of thermal comfort. Boars are particularly vulnerable to the effects of heat stress due to their inefficient capacity to sweat; non-pendulous scrotum; and the high susceptibility of boar sperm to temperature shock. Moreover, due to limited endogenous antioxidant systems inherent in mammalian spermatozoa and the loss of cytosolic repair mechanisms during spermatogenesis, the DNA in these cells are particularly susceptible to oxidative damage. While a seemingly healthy looking sperm may swim and fertilize an oocyte normally, studies in mice demonstrate that heat stress-induced DNA damage can disrupt expression of key developmental genes in early embryos after fertilization and distort the formation of the blastocyst; resulting in implantation failure and pregnancy loss. The aim or our study is to determine whether heat stress induces DNA damage to boar sperm that could significantly contribute to the high rates of embryo loss and pregnancy failure observed in sows during summer infertility. The quality of sperm obtained from n=6 Large White boars housed in the dry tropics of Townsville, North Queensland, Australia was evaluated across different seasons (summer, winter and spring) during 2014 - 2015. Sperm motility was characterised by Computer-Assisted Sperm Analysis (CASA; IVOS version 10: Hamilton Thorne, USA), and sperm DNA integrity evaluated by Terminal deoxynucleotidyl transferase dUTP Nick-End Labelling (TUNEL; In situ cell death detection kit, fluorescein: Roche, Germany). Twenty-thousand spermatozoa per boar per treatment were analysed using flow cytometry (CyAn ADP analyser: Beckman Coulter, USA). Sperm had equal motility across all seasons (total motility: 70.8 ± 5.5% vs. 71.3 ± 8.1% vs. 90.2 ± 4.2%, P ≥ 0.05; progressive motility: 41.7 ± 2.8% vs. 35.4 ± 7.0% vs. 46.6 ± 4.0%, P ≥ 0.05 for spring, summer and winter respectively). However, sperm in summer exhibited ~9-fold higher DNA damage than that in winter and spring (16.1 ± 4.8% vs. 1.1 ± 0.2% and 1.8 ± 0.4% respectively; P ≤ 0.05). These results demonstrate that summer negatively affects sperm DNA integrity in boars without depressing sperm motility. This means traditional methods of evaluating semen quality may not detect inherently compromised spermatozoa. We are currently evaluating the effect of this DNA-damaged sperm on rates of fertilization, development and survival in pig embryos. Our study emphasizes the need for improved management practices and development of strategies to mitigate heat stress in boars during summer

Antioxidant therapy improves sperm DNA integrity in boars during summer

Background: Summer infertility due to heat stress significantly affects the reproductive potential of pigs causing over $300 million per year in lost productivity to the US pig industry and billions worldwide. The boar's inefficient capacity to sweat; non-pendulous scrotum, and the high susceptibility of boar sperm to temperature shock appears to correlate with higher sperm DNA damage during summer. Heat stress-induced sperm DNA damage can result in early embryo loss, as demonstrated in mice. This study investigated whether supplementing boars with antioxidants during summer could improve boar sperm DNA integrity. Method: Motility of sperm obtained from n=5 Large White boars housed in the dry tropics of Townsville, Queensland, Australia were analysed using Computer-Assisted Sperm Analysis. Sperm DNA integrity during summer was compared without or after 42 and 84 days antioxidant supplementation, by Terminal deoxynucleotidyl transferase dUTP Nick-End Labelling and flow cytometry. Paired T-tests were used to determine significant differences between treatments (P ≤ 0.05). Results and Discussion: Total and progressive motility of sperm did not differ between treatments (P ≥ 0.05). However, antioxidant supplementation of boars during summer resulted in a 38% and 55% reduction of DNA-damaged spermatozoa after 42 and 84 days treatment respectively (16.1 ± 4.8% untreated vs 9.9 ± 4.5% vs 7.2 ± 1.6%; P ≤ 0.05). Conclusions: Supplementing boars with antioxidants during summer improves boar sperm DNA integrity which could potentially mitigate the negative impact of heat stress on male fertility. Such improvements may also increase downstream fertilisation rates and early embryo survival in the sow

Cryoprotective effect of glycerol against sperm DNA damage in frozen-thawed boar spermatozoa.

The use of frozen-thawed boar sperm still lags behind chilled boar semen in artificial insemination (AI) operations despite its advantages [1]. Freezing however, may damage the structure and function of sperm, leading to reduced fertilization success. Moreover, glycerol, which is a critical cryoprotectant in most sperm freezing protocols, can be toxic to cells in high concentrations [2]; affecting sperm motility and acrosomal integrity. This study was conducted to determine the cryoprotective effect of glycerol on sperm DNA integrity and motility in frozen-thawed boar sperm

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

Forest loss during 2000–2019 in pygmy hippopotamus (Choeropsis liberiensis) habitats was driven by shifting agriculture

The Upper Guinea Forest (UGF; West Africa), a global biodiversity hotspot, has lost more than 90% of its original area since 1900, threatening endemic species such as the endangered pygmy hippopotamus (Choeropsis liberiensis). However, little is known about the proximate causes of this deforestation. We classified Sentinel-2 data using the random forest algorithm to differentiate between three main human processes (shifting agriculture, intensive agriculture or urban expansion) driving deforestation between 2000 and 2019 across the pygmy hippopotamus distribution area. Out of c. 89 600 km2 in the year 2000, 15 900 km2 (17%) of forest were lost, primarily to shifting agriculture (14 900 km2). Côte d’Ivoire and Liberia accounted for 14 900 km2 (94%) of the net area of forest lost, c. 15 times greater than deforestation in Sierra Leone and Guinea combined (953 km2). Forest loss inside protected areas is pervasive, and it is essential to prioritize conservation efforts in areas where deforestation is still low (e.g., Taï, Sapo and Gola Rainforest national parks). We suggest that the preservation of the UGF will face challenges associated with people’s demand for food and income. Continued landscape-scale planning and action to reduce deforestation are urgently needed to limit the impact of shifting agriculture on pygmy hippopotamus habitat

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