Evaluation of the function of fresh and frozen - thawed sex-sorted and non-sorted stallion spermatozoa using a heterologous oocyte binding assay

Abstract

The aim of the present study was to evaluate the potential oocyte binding ability and functional integrity of fresh or frozen-thawed, sex-sorted or non-sorted stallion spermatozoa. In the absence of effective IVF procedures in the horse, a heterologous sperm-binding assay was used as an indicator of fertilising capacity to assess differences in the ability of stallion spermatozoa to bind to bovine oocytes. The functional integrity of four treatment groups was assessed: (1) fresh non-sorted spermatozoa; (2) fresh sex-sorted spermatozoa; (3) frozen-thawed non-sorted spermatozoa; and (4) frozen-thawed sex-sorted spermatozoa. Spermatozoa found in association with the zona pellucida of the bovine oocytes were deemed 'attached' or 'bound' depending on their characterisation as either acrosome intact or acrosome reacted, respectively. Significantly less frozen-thawed spermatozoa were found attached to the oocytes compared with fresh spermatozoa. No significant differences were identified between the number of attached sex-sorted and non-sorted frozen-thawed spermatozoa. However, significantly more sex-sorted than non-sorted fresh spermatozoa were found attached to the oocytes after 1 h coincubation, although after 3 h coincubation this difference was no longer apparent. In conclusion, sex-sorted fresh and frozen-thawed stallion spermatozoa are functionally capable of attaching and binding to bovine oocytes in vitro. Furthermore, fresh sex-sorted spermatozoa attach better than non-sorted spermatozoa, suggesting that they have a more advanced capacitation-like status.

Heterologous murine and bovine IVF using bottlenose dolphin (Tursiops truncatus) spermatozoa

“…This result agrees the only study that described heterologous IVF using cryopreserved dolphin spermatozoa, in which maximum attachment and binding were achieved at 2 h of co-incubation with zona-free hamster oocytes [9]. Similar times are used in co-incubation studies of other species [46][47][48][49].…”

Capacitation in the presence of methyl-β-cyclodextrin results in enhanced zona pellucida-binding ability of stallion spermatozoa

“…4D). This assay has recently been developed to address difficulties associated with the acquisition of sufficient horse oocytes and, importantly, has been used to assess stallion sperm capacitation status (Clulow et al 2010, Coutinho da Silva et al 2012. The use of this assay in our study demonstrated that media containing PVA and mbcd supported the highest levels of ZP binding with an average of 119.4 sperm bound to the ZP of each bovine oocyte, a result that significantly exceeded the number of sperm bound after incubation in NC conditions (11.6 cells/oocyte).…”

“…The use of this assay in our study demonstrated that media containing PVA and mbcd supported the highest levels of ZP binding with an average of 119.4 sperm bound to the ZP of each bovine oocyte, a result that significantly exceeded the number of sperm bound after incubation in NC conditions (11.6 cells/oocyte). The gamete co-incubation method used in these studies was adapted from that established by Clulow et al (2010) in studies of fresh and frozen, sex-sorted and non-sorted stallion spermatozoa. These authors demonstrated that after incubation in TALP and co-incubation with heterologous bovine oocytes, an average of fewer than 30 sperm were bound to each oocyte (Clulow et al 2010).…”

“…The gamete co-incubation method used in these studies was adapted from that established by Clulow et al (2010) in studies of fresh and frozen, sex-sorted and non-sorted stallion spermatozoa. These authors demonstrated that after incubation in TALP and co-incubation with heterologous bovine oocytes, an average of fewer than 30 sperm were bound to each oocyte (Clulow et al 2010). Similar results were obtained through binding assays incorporating equine sperm and bovine and equine oocytes, with averages of 29 and 36 sperm/egg being obtained respectively (Coutinho da Silva et al 2012).…”

“…Heparin and TALP-HEPES medium or modifications of Krebs-Ringer bicarbonate have often been used to capacitate equine spermatozoa for IVF as they can promote reproducible responses in these cells (Dell' Aquila et al 1996, Choi et al 2003, Clulow et al 2010. Notwithstanding their reproducibility, these capacitation media are not able to promote levels of acrosomal exocytosis or protein tyrosine phosphorylation that are comparable to those achieved in the spermatozoa of other species such as cattle (Galantino-Homer et al 2004, Purdy & Graham 2004 or humans (Lu et al 2006).…”