Evaluation of In Vitro Capacitation of Buffalo Frozen/Thawed Sperm by Different Techniques

Abstract

This study aimed to determine the most reliable method to evaluate capacitation of buffalo frozen/thawed sperm. Frozen/thawed sperm cells were incubated in Tyrode albumin lactate pyruvate medium (TALP) in absence of capacitating agents (control) and in presence of 10 g/ml heparin for 2 and 4 h. Capacitation was assessed by Trypan blue/Giemsa after lysophosphatidylcholine (LPC) exposure, chlortetracycline (CTC) fluorescence assay and immunelocalization of tyrosine phosphorylated protein. Furthermore, we evaluated the effect of heparin on penetration, cleavage rates and kinetics of embryo development after heterologous IVF. The percentage of LPC-induced acrosome reacted (AR)-sperm increased (P<0.05) with heparin compared to the control after 2 h (28.2 vs 24.4%, respectively) and 4 h (35.1 vs 32.0 %, respectively). No differences in CTC pattern B (capacitated sperm) were found between groups and incubation times (on average 63%). On the contrary, heparin decreased (P<0.01) the percentage of tyrosine phosphorylation pattern A after 2 and 4 h (34.3 and 35.3%, respectively) compared to the control (54.5 and 51.8%, respectively) and increased (P<0.01) that of pattern EA after 2 and 4 h (59.2 and 54.2 %, respectively) compared to the control group (44.7 and 45.2 %, respectively). Both cleavage and penetration rates, as well as the percentage of fast developing embryos, were higher (P<0.01) in the heparin-treated group (77.2, 80.4 and 74.0 %, respectively) compared to the control (56.6, 58.0 and 55.2 %, respectively). In conclusion, Trypan blue/Giemsa staining to evaluate LPC-induced AR and tyrosine protein phosphorylation assay can be successfully used to evaluate capacitation of buffalo frozen/thawed semen.

Exogenous cholesterol prevents cryocapacitation‐like changes, membrane fluidity, and enhances in vitro fertility in bubaline spermatozoa

“…The most interesting results arising from this study was that there was a strong reduction in sperm cryocapacitation when the buffalo sperm was processed with CLC. This is particularly important because the percentage of sperm undergoing cryocapacitation in buffalo sperm is much higher than in other domestic species (Elkhawagah et al, 2014) because the buffalo spermatozoon contains poor C: P ratio (0.34, 0.24 and 0.17 at fresh, pre‐freeze and post‐thaw, respectively, Rajoriya et al, 2014; Rajoriya et al, 2016) and easily susceptible to cold shock. These results clearly indicated that the Gr III (3 mg CLC) followed by Gr II (2 mg CLC) has protected the sperm cells from cryocapacitation and premature acrosomal reaction in buffalo semen.…”

“…Nevertheless, as suggested by Moce et al (2010), the CLC technology could be useful to reduce the higher cryocapacitation damages especially in high merit bulls with sperm that do not fulfil the threshold semen quality parameters after cryopreservation. In bubaline species, due to both the high individual variability in fertilizing ability (Presicce, Révay, Nagy, Dinnyés, & Kovács, 2003) and the alarming proportion of cryocapacitated sperm at thawing (Elkhawagah et al, 2014), this approach (CLC technology) may allow to increase the number of bulls to enrol as the semen donors in frozen semen banks. Therefore, it would be interested in future studies to evaluate the effect of the CLC treatment on sperm from males that do not freeze well or so‐called poor freezability.…”