Comparison of donkey and stallion semen preservation: effects of egg yolk plasma, seminal plasma and extenders on sperm motility, MALDI profiles and fertility

Abstract The conservation of donkey breeds at risk of extinction requires the development and optimization of semen preservation and artificial insemination strategies aimed at improving reproductive efficiency, increasing genetic diversity, and supporting sustainable population management. The effects of commercial extenders (INRA96, BotuSemen Gold, Beyond), seminal plasma (SP) removal, and egg yolk plasma (EYP; 0–10%) supplementation on jack semen stored at 4 °C for 24–48 h were evaluated, using stallion semen as a reference. Sperm motility was assessed by computer-assisted sperm analysis (CASA), while intact-cell MALDI-TOF mass spectrometry (ICM-MS) was used to characterise peptidoform and proteoform profiles. Fertility was evaluated following low-volume deep intrauterine insemination. Among the preservation strategies tested, the milk protein-based extender INRA96, SP removal, and supplementation with 5% EYP were the most effective at preserving sperm motility during storage. After 24 h at 4 °C, EYP-supplemented semen exhibited higher total and progressive motility and achieved a conception rate of 35%, compared with 5% for semen stored without EYP (fresh semen: 45%). These functional improvements were accompanied by dynamic remodelling of the sperm proteome, with EYP primarily mitigating cooling-induced molecular alterations rather than the basal sperm proteome in both jacks and stallions. The birth of four Grand Noir du Berry (GNB) foals from semen stored for 24 h provided in vivo validation of this preservation strategy and highlighted its potential to support assisted reproduction and the management of genetic diversity in donkey populations. Lay Summary The conservation of endangered donkey breeds relies on maintaining genetic diversity and improving reproductive success, particularly in populations with a limited number of breeding animals. This study aimed to optimise the preservation of donkey semen by evaluating different commercial extenders, as well as the effects of seminal plasma removal and egg yolk plasma supplementation during cooling, building on previous findings in the stallion. The results showed that supplementation of a milk-based extender with 5% egg yolk plasma improved sperm motility and viability after 24 hours of storage at 4 °C and partially limited alterations in protein profiles associated with cellular ageing. This improvement translated into a conception rate of 35%, compared with only 5% without egg yolk plasma, approaching that achieved with fresh semen (45%). The birth of four Grand Noir du Berry foals from semen stored for 24 hours provides in vivo validation of this approach and open new opportunities for assisted reproduction programmes.