In the present study, effects of storage duration on the quality and DNA integrity of Nili-Ravi buffalo bull spermatozoa frozen and stored in liquid nitrogen for up to three years were investigated. According to duration of storage, the semen samples were categorized into 10 groups, viz. i) 0 day, ii) one day, iii) two weeks, iv) one month, v) three months, vi) six months, vii) nine months, viii) one year, ix) two years and x) three years. A total of 50 straws, with 5 straws for each of the 10 groups were used. After thawing for 30 seconds at 37°C, these samples were evaluated for sperm motility, livability, livability index, plasma membrane integrity, acrosome damage and DNA damage. At 0 day, mean values for sperm motility (%), livability (hrs), livability index, plasma membrane integrity (%), acrosome damage (%) and DNA damage (%) were 53±1.22, 6.0±0.0, 130±5.2, 45.4±1.6, 3.8±0.6 and 2.2±0.4. After 3 years of storage, the corresponding values were 39±3.32, 4.4±0.2, 78.5±5.7, 28.2±2.2, 12.0±0.7 and 5.8±0.6. Sperm motility and livability remained unaffected up to 3 months of storage, followed by a decrease at 6^th month, while absolute index of livability and membrane integrity started decreasing at 1 month (P<0.05). Sperm with damaged acrosome increased at 6^th month (P<0.05). Similarly, sperm with damaged DNA increased after one year of storage (P<0.05). Thus, post-thaw motility, livability and acrosome integrity of spermatozoa of Nili-Ravi bulls started decreasing at 6^th month of storage, while sperm DNA integrity remained unaffected for one year of storage in liquid nitrogen.

Key words: DNA integrity, Frozen, Nili-Ravi bulls, Semen quality, Storage duration