Interrogating the seminal fluid proteome to resolve the molecular basis of fertility in poultry

With more than 50 billion chickens reared every year for both eggs (layers) and meat (broilers), poultry is one of the largest and fastest growing food systems worldwide. Poultry already outcompetes other meat markets and by 2025 more than half of all the meat produced globally is forecasted to be chicken. In the UK, growth in the poultry sector has soared in recent years, with an estimated contribution of £3.3bn to UK GDP in 2014. According to Defra, the UK poultry meat production increased to 1.8 million tonnes in 2017, with broilers accounting for around 85%. Currently, the UK is about 75% self-sufficient in poultry meat, and poultry meat represents the only UK livestock sector capable of quickly scaling-up production to support increased self-sufficiency of the UK. Ensuring consistently high fertility rates is key for meeting the demands of this expanding market. Fertility in these flocks is notoriously variable and tends to decline as birds get older, through reproductive ageing. Poor male fertilising efficiency requires increased female exposure to males, which has additional repercussions because males are often aggressive to females, which reduces female condition, health and overall fecundity. Even small improvements in the fertility of breeding stocks have vast financial consequences; e.g. a 1% variation in fertility in broiler flocks was estimated to be worth hundreds of millions of US$ in the US market.
It is becoming increasing clear that in several organisms, the proteins contained in the male seminal fluid can have a drastic influence on fertility by modulating sperm swimming velocity, sperm storage within the female reproductive tract, probability of fertilisation and female behaviour after mating including female receptivity to further matings. The advent of proteomics and the publication of a draft genome of the chicken present a unique opportunity to investigate the role of seminal fluid proteins in poultry fertility. Our recent work has characterised the seminal fluid proteome of natural ejaculates of a population of red junglefowl (the species that has given rise to the domestic chicken) and has shown that the seminal fluid proteome of these birds is complex with more than 1500 seminal fluid proteins (SFPs) identified so far, including proteins involved in various known biological functions e.g. immune responses and antibacterial defences, as well as sperm maturation and sperm motility. Our work has shown that some of these SFPs are associated with in vitro measures of sperm quality. Importantly, our work has further shown that the seminal fluid proteome undergoes rapid and marked compositional changes in response to socio-sexual factors such as the sexual familiarity of a female partner and the social dominance of a male, and longer term changes, as males age, with males that are able to retain high sperm quality in advanced age having a distinct seminal fluid proteome. In this project, we capitalise on this wealth of preliminary knowledge to develop a research programme, to identify seminal fluid proteins (SFPs) that are important in maintaining lifelong fertility in male poultry.
We use the red junglefowl as benchmark experimental system that has not been influenced by domestication and artificial selection, to characterise proteomic repertoires associated with rapid responses to socio-sexual conditions (objective 1) and reproductive ageing (obj. 2), and identify SFP signatures causally linked to fertility. We then confirm the role of seminal fluid in driving variation in fertility (obj. 3), and validate the commercial relevance and applicability of these findings by investigating patterns of intra- and inter-male variation in fertility-linked SFPs in commercial meat-production domestic chicken lines (broiler breeders, obj. 4). Collectively, these results will help us identify proteins linked to poultry fertility, which will inform new strategies to improve fertility in commercial stocks.

The poultry meat market is one of the fastest growing globally, and particularly in the UK, where it makes a substantial contribution to national GDP and is largely represented by broiler chickens. The efficiency and sustainability of broiler farming depends on fertility rates in broiler breeders, which is variable across individual birds and flocks, and longitudinally, due to reproductive ageing. Despite increasing evidence that seminal fluid proteins (SFPs) play fundamental roles in fertilising efficiency in different species, we know little about the role of SFPs in poultry fertility. We propose a novel experimental approach to advance our understanding of fertility in poultry, with a view to inform new approaches to better manage the fertility of commercial flocks. Based on recent research and building on our own work of red junglefowl (the main contributor of the domestic chicken), we postulate that an important source of fertility variation is determined by compositional changes in the seminal fluid (SF) proteome of natural ejaculates, through two mechanisms: rapid changes caused by differential SFP allocation in response to socio-sexual stimuli, and reproductive ageing processes. We first use the red junglefowl as benchmark reference. Obj.1 characterises compositional changes in the SF proteome experimentally induced by rapid responses to socio-sexual conditions and associated with sperm fertilising efficiency. Obj.2 investigates the way SF compositional changes covary with sperm fertilising efficiency as males age. Obj.3 validates the causal effect of SF on in vitro changes in sperm fertilising efficiency and in vivo probability of fertilisation, using ejaculate engineering and artificial insemination to disentangle sperm and SF effects. We then investigate the commercial relevance of these patterns in obj.4, where we study the SF proteome of natural ejaculates of broiler breeders, and variation linked to fertilising efficiency between and within males.

This project will deepen and qualitatively change our understanding of fertility and inform changes in livestock husbandry and selection regimes with key financial and societal implications.
Financial. The project has demonstrable scope to foster economic performance and the economic competitiveness of the British meat industry, by improving efficiency, sustainability and consistency of broiler breeder fertility. According to Defra, the UK poultry meat production increased to 1.8 million tonnes in 2017, with broilers accounting for around 85%, and generating £4.6 billion of gross value added contribution to national GDP, supporting directly and indirectly the employment of 84,500 (0.3% of the entire British workforce). In 2019, British production has continued to increase with a 10% year on year increment reported in October (Defra, 2019). The success and growth of this sector depends critically on male fertility, which can be poor, particularly in older birds. Even marginal fluctuations in hatchability cost the sector millions of pounds annually. According to USDA, in the first 5 months of 2017, US broiler breeder egg hatchability dropped to its lowest levels in the last 10 years, resulting in a 68% surge of wholesale prices and 3% consumer prices for boneless meat. At such costs, a drop of 1% in egg hatchability was estimated to result in $121M losses over 5 months. By identifying candidate SFPs associated with high and low male fertility, our project will enable the development of predictive diagnostic tools for earlier and more effective screening of broiler breeder males of consistently high fertility, e.g. genetic screening can occur within hours of hatching. Our results will also help devise artificial selection regimes taking into account SFP-coding genes linked to male fertility, and inform development of more effective semen extenders where artificial insemination is utilised (e.g. USA). By identifying the socio-sexual factors promoting high quality ejaculates, our project can also inspire more effective flock management strategies to maintain high lifelong fertility. Improved male fertility will reduce the need for 'spiking' flocks with new males and increase female productivity by limiting male harassment of females. Reduced male harassment of females can improve flock productivity by increasing female egg production by 2% in broiler breeder flocks (Leone & Estevez 2008 Poultry Science). While it is difficult to quantify precisely the financial benefits of these changes across a sector of such scale, all else being equal, few percentage point increases in egg production and hatching rate will translate into an annual profit increase in the millions of pounds for the UK poultry market and associated industries. For example, additional financial benefits can be accrued by precision livestock farming companies, which will be able to use our results to develop new technology for high-throughput fertility screening and flock management services tailored to the needs of different producers and farmers.
Societal. The project will also make demonstrable contribution to society through the benefits that more effective fertility management has for poultry welfare and sustainability. High standards of animal welfare in agriculture and farming are becoming increasingly important to the public, e.g. the majority of EU citizens feels that the welfare of farm animals is in need of improvement (EC Eurobarometer, 2016). Improving the efficiency of individual matings will enable farmers to increase female welfare by reducing female exposure to male aggression (e.g. by changing sex ratios and/or more prolonged sex segregation) and male-male aggression, by reducing the need for spiking flocks with new males. These improvements in production efficiency will also help significantly increase sustainability of the poultry industry and reduce its environmental impact.