Appraisal of ATP1B1 and GSTM3 proteins as freezability factors in buffalo ejaculated spermatozoa

Abstract

Sperm cryopreservation intensifies the use of assisted reproductive technologies in food animals. However, variations in freezability confront frozen-thawed semen applications. Hence, this groundwork aimed to identify the fundamental differences between high (HF) and low freezability (LF) ejaculated spermatozoa in swamp buffaloes, which were limitedly described for this agricultural species. Twelve (12) different samples of bubaline semen were collected, cryopreserved and categorized into freezability phenotypes using post-thaw sperm motility as the basis. Sperm kinematics and functional plasma membrane integrity were evaluated in HF and LF samples using computer-assisted sperm analysis (CASA) and hypo-osmotic swelling test, respectively. The relative concentrations of some selected sperm proteins namely ATPase subunit beta 1 (ATP1B1) and glutathione S-transferase Mu 3 (GSTM3) were also comparatively quantified using dot blot and image processing analyses. The results revealed that all CASA parameters except wobble and straightness were significantly higher in HF than LF cluster (P<0.05). Moreover, HF spermatozoa also exhibited more intact plasma membranes under a hypo-osmotic state than the LF group (P<0.05). However, there was no significant difference in the abundance of ATP1B1 and GSTM3 proteins among freezability phenotypes, suggesting that other proteins related to energy production and oxidative stress protection influence bubaline ejaculated sperm cryoresilience. Nevertheless, these findings reaffirmed the existence of variabilities in sperm cryopreservation outcomes in buffaloes and established the key differences between HF and LF bubaline ejaculated spermatozoa in terms of motility, kinematics and plasma membrane integrity.