Effect of Milk Type Subjected to Different Heat Treatments on Cryo-Survivability and In Vivo Fertility of Buffalo (Bubalus bubalis) Spermatozoa in a Milk-Based Extender

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Abstract
Oxidative stress is a major contributory factor to cellular damage during semen cryopreservation and results in a decreased fertilizing capacity of cryopreserved bull sperm. The inclusion of exogenous antioxidants sometimes exerts deleterious effects on sperm quality. Thus, enhancing the endogenous production of antioxidants is a requirement. This study aimed to investigate the effect of milk type heated at different temperatures on the antioxidant potential of extenders, and the subsequent post-thaw quality parameters and in vivo fertility of buffalo bull semen. Cow (C) and buffalo whole milk (B) were used separately for semen extender preparation, heated at five different temperatures (T1 = 90°C, T2 = 100°C, T3 = 110°C, T4 = 120°C, T5 = 130°C) for 10 minutes. Reactive sulfhydryl groups were measured in each subgroup by Ellman's reagents as CT1 = 143.2 μM, CT2 = 147.4 μM, CT3 = 151.5 μM, CT4 = 157.2 μM, CT5 = 161.8 μM, BT1 = 168.3 μM, BT2 = 172.5 μM, BT3 = 176.7 μM, BT4 = 196.3 μM, and BT5 = 205.7 μM. All semen samples were cryopreserved in milk-based extenders by using standard procedures. Post-thaw quality parameters including total and progressive motility, mitochondrial membrane potential, plasma membrane integrity, and acrosome integrity were found to be higher (p < 0.05) in the group (BT3) containing buffalo milk heated at 110°C, whereas in the same group, lipid peroxidation was found to be lower (p < 0.05) as compared with other treatment groups and control group. In vivo fertility of cryopreserved buffalo sperm was compared among BT3, CT1 (conventionally used milk extender), and a Tris egg yolk extender group. The fertility rates [47% (54/114), 30% (33/108), and 36% (37/103)] were higher (p < 0.05) in BT3 as compared with other groups. This study suggests that buffalo milk heated at 110°C has high antioxidant potential and improves post-thaw quality and in vivo fertility of cryopreserved buffalo bull semen.