When a retired racing stallion is selected for breeding, their reproductive fitness is generally unknown. Where a severely sub-fertile or infertile stallion commences an ill-fated breeding career, it can generate a host of unnecessary economic and welfare costs, stemming from futile, repeated breedings. To date there are no effectual processes in place to tackle this problem. As such, this study aimed to compare the proteomic profiles of spermatozoa collected from stallions of variable fertility status, to identify robust biomarkers of fertility that could be used as pre-purchase diagnostic tools, thereby identifying unsuitable sires in the future.
Semen samples were collected from two infertile, four sub-fertile (conception rates 0–33%) and 15 commercially ‘fertile’ stallions (conception rates 61.5–100%), and assessed using LC-MS/MS. This analysis identified a complex, proteomic signature comprising 1,076 proteins; of which 37 proteins were more abundant in samples collected from sub-fertile and infertile stallions. These included α-aminoadipic semialdehyde dehydrogenase (ALDH7A1) and voltage-dependent anion-selective channel protein 3 (VDAC3;FC≥-1.5;P≤0.0001). When comparing the proteomic conservation between the fertile and infertile stallion ejaculates, a notable 79 proteins were absent from infertile samples. These included isocitrate dehydrogenase 3 (NAD+)-α (IDH3A); stress-70 protein, mitochondrial (HSPA9); arylsulfatase A (ARSA); and phospholipase C zeta 1 (PLCZ1), which are individually associated with asthenospermia, fertilisation and oocyte activation. Conversely, a comparison of fertile and severely sub-fertile stallion ejaculates revealed 113 proteins were exclusively conserved to fertile ejaculates. These included testisin (PRSS21); α-enolase (ENO1); apolipoprotein E (APOA1); and tektin-3 (TEKT3), which are associated with zona-pellucida binding, fertility, and varicocele.
We have identified a suite of putative protein biomarkers that have substantial potential for assessing stallion reproductive fitness. These findings will serve to inform the development of novel diagnostics to improve pregnancy success rates, thereby dramatically reducing the economic wastage and welfare implications associated with futile breeding practices.