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The study aimed to validate the double versus single freezing protocol for Beetal buck (Capra hircus) spermatozoa in tris‐citric acid (TCA) based extender both in terms of quality and fertilization potential. Computer‐assisted sperm motion and kinematic (CASA) variables, i.e. total (%), and progressive motilities (TM and PM, %) and rapid velocity (RV, %), average path (VAP, μm/s), straight line (VSL, μm/s) and curved line velocities (VCL, μm/s), straightness, (VSL/VAP, %) and linearity, (VSL/VCL, %) as well as supra‐vital plasma membrane integrity (SV‐PMI, %), mitochondrial membrane potential (MMP, %), viable/intact acrosome (V–IACR, %) and DNA integrity (DNA–I, %) had significantly greater values (p < .05) during single freeze–thawing as compared with the double freeze–thawing at 0, 30, 90, 150 and 210 days, respectively. All CASA and other assays alone did not show significant differences (p > .05) between both freeze–thaw cycles at all treatment durations, respectively. No statistical significance (p > .05) was observed for the in vivo fertility between single (n = 84/141 = 59.72%) and double freeze–thawing (n = 72/136 = 52.9%) cycles, respectively. In conclusion, sperm motion, kinematics, plasma membrane, acrosome, mitochondria and DNA integrities and in vivo fertility are acceptable after the double freezing protocol despite being lower than after one freeze cycle in Beetal buck.

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Effects of kisspeptin‐10 as antioxidant in cryodiluent were evaluated on post‐thaw quality of buffalo spermatozoa. Qualified semen samples from five bulls were pooled, divided into five aliquots and extended in Tris‐citric acid cryodiluent containing differential doses of kisspeptin‐10 (5, 10, 15, and 20 μmol L−1 and negative control. Extended sperm suspension was cooled to 4°C, packaged in 0.54 ml straws and cryopreserved. At post‐thawing, catalase (unit mg−1), peroxidase (unit mg−1) and reduced glutathione (μmol L−1) levels were highest (p < 0.05) with 20 μmol L−1 of kisspeptin‐10 as compared to negative control. Moreover, lipid peroxidation (nmol L−1 min−1 mg protein−1) level was lowest (p < 0.05) with 20 μmol L−1 of kisspeptin‐10. Sperm progressive motility (%), rapid velocity (%) and kinematics were higher (p < 0.05) with 15 and 20 μmol L−1 of kisspeptin‐10 as compared to negative control. Supra‐vital plasma membrane integrity (%), viable sperm with intact acrosome (%) and DNA integrity (%) were improved (p < 0.05) with all doses of kisspeptin‐10 as compared to negative control. It was concluded that the addition of 15 and 20 μmol L−1 kisspeptin‐10 in cryodiluent ameliorated the overall frozen‐thawed quality parameters of Nili‐Ravi buffalo spermatozoa.