Enhancing zona penetration by spermatozoa from a teratospermic species, the cheetah (Acinonyx jubatus)

Abstract

Cheetahs (Acinonyx jubatus) produce poor quality ejaculates that can limit the efficiency of standard assisted reproduction including artificial insemination (AI) and in vitro fertilization (IVF). The purpose of this study was to: (1) further study sperm‐oocyte interaction in this teratospermic species by examining the ability of malformed sperm to interact with various oocyte barriers; and (2) assess the potential of zona piercing for assisting IVF in a teratospermic felid. Zonae of salt‐stored (SS), domestic cat oocytes were mechanically pierced (ZnPd) three times each. Semen was collected by electroejaculation from six male cheetahs and ejaculates were processed for IVF. Sperm aliquots from each ejaculate were assessed for a sperm motility index (SMI) over time. Zona‐intact (ZnIn‐SS) oocytes (n=78) and ZnPd‐SS oocytes (n=74) were coincubated with spermatozoa in vitro for 6 h. The proportion of morphologically abnormal spermatozoa per ejaculate was high for all males (range 81.5% to 95.9% ). SMI values at 0 and 6 h were variable, ranging from 50 to 75 and 0 to 40, respectively. Spermatozoa from all ejaculates bound to and penetrated the outer zona pellucida of ZnIn‐SS and ZnPd‐SS oocytes similarly (P > 0.05). The proportion of oocytes containing spermatozoa within the inner zona layer and the average number of spermatozoa per oocyte in this region were greater (P < 0.05) for the ZnPd‐SS than ZnIn‐SS oocytes (39.2% and 1.0 versus 12.8% and 0.2, respectively). Although zona piercing enhanced sperm penetration, there was no increase (P > 0.05) in pleiomorphic spermatozoa penetrating the inner zona pellucid or PVS. Penetration of both ZnIn‐SS and ZnPd‐SS oocytes was positively correlated (P < 0.05; r =0.91) with SMI at 6 h but not to the proportion of the pleiomorphic spermatozoa ejaculated (P > 0.05). In summary, altering the integrity of the zona pellucida by creating artificial channels increases the number of cheetah spermatozoa entering the inner zona region, but not the PVS. This phenomenon occurs without increasing the number of pleiomorphic sperm entering the zona/oocyte interface, reinforcing the role of the zona pellucida, especially the inner region, as a powerful filter for malformed sperm.