ABSTRACT: Assisted reproductive technologies, such as in vitro fertilization, remain inefficient in camelids, largely due to gaps in understanding the molecular interactions that regulate sperm capacitation. Fertilization requires not only viable spermatozoa but also precise modulation of capacitation by the peri-ovulatory microenvironment, including follicular fluid (FF) and oviductal fluid (OF). In this study, spermatozoa were incubated in Fert-TALP medium supplemented with FF or OF, and both functional outcomes and proteomic remodeling were assessed. FF was collected from preovulatory follicles (7–9 mm) and OF from the corresponding ipsilateral oviducts, thereby reflecting the in vivo environment encountered by sperm in the female reproductive tract following mating. Incubation with FF enhanced motility, viability, and acrosome responsiveness, and was associated with a proteomic shift involving ~12% of proteins. These included factors implicated in zona pellucida binding (LYPD4, PGK1, ANXA2, TCP1 complex members) and galactose metabolism (MAOA, AKR1B1, GLA, HK1). The enriched processes included glycolysis/gluconeogenesis, cytoskeletal reorganization, and protein maturation, all consistent with sperm capacitation. By contrast, sperm incubated with OF showed underrepresentation of capacitation-related pathways, including the proteasome complex, fibrous sheath, and TCA cycle. Moreover, OF proteome revealed decapacitation-associated factors such as PEBP1 and PAFAH1B3, which likely stabilize membranes and delay premature capacitation. Together, these findings demonstrate complementary yet contrasting roles of FF and OF in modulating sperm physiology: FF acting as a capacitating medium, and OF providing a stabilizing environment. This work delivers the first partial proteome of capacitated alpaca sperm together with matched reproductive fluids, providing mechanistic insights with direct implications for improving assisted reproduction in camelids.
Martin Argañaraz 