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Home > Archives > Volume 20, No 8 (2022) > Article

DOI: 10.14704/nq.2022.20.8.NQ22984

Early apoptotic changes and acrosomal integrity of cryopreserved Hariana bull spermatozoa on modified cryopreservation methods

Alok Kumar, Atul Saxena , Mukul Anand, Gyandev Singh and Sumit Singhal


Semen cryopreservation involves two major steps i.e. cooling and freezing. These steps play a significant role in post-thaw semen quality. The objective of the experiment was to compare different cooling and cooling-freezing combinations on survival, membrane integrity, and apoptotic changes in Hariana bull sperm. Forty ejaculates were collected from four bulls (ten collections/bull) using an artificial vagina at the biweekly interval. Extended semen was then split into three parts and subjected to different cooling regimens i.e. from 35oC to 4oC temperature drop at 2.21°C/min (rapid), 0.48°C/min (moderate), and 0.25°C/min (slow cooling). Each cooled part was again split into three parts and subsequently subjected to different freezing rates for each group i.e. rapid (from 4°C to -10°C @ 20°C/ min), moderate (from 4ºC to -10°C @ 10°C/ min) and slow (from 4°C to -10ºC @ 5°C/ min) using a programmable biological freezer. Samples were evaluated at pre-freeze and post-thaw stages for % viability, motility, membrane integrity, and apoptotic changes. The result showed that slow cooling and slow cooling-freezing combination were more effective as slow cooling resulted in 73.58±2.00% viability, 70.17±1.75% acrosomal integrity, 68.83 ± 1.79% plasma membrane integrity, and 61.96±2.17% non-apoptotic sperm. Similarly, the slow-cooling slow-freezing combination showed 62.50±1.61% viability, 61.01±1.71% acrosomal integrity, 59.25±1.68% plasma membrane integrity, and 51.46±1.51% non-apoptotic sperm. In conclusion, the slower rate of cooling followed by slower freezing rates were more efficient than rapid and moderate rates for cryopreservation of Hariana bull semen.


Annexin-V; Apoptosis; Cooling; Freezing; Hariana bull spermatozoa

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