In this study annual fluctuations of DNA fragmentation and quality of cold-stored equine sperm were evaluated. Ejaculates were collected weekly during one year from 15 stallions. Ejaculate volume, sperm concentration and total sperm count were determined and semen was then extended and cold-stored for 48 h. Sperm motility was evaluated by CASA before and after 24 as well as 48 h of cold storage. In addition, the percentages of sperm with intact plasma membrane and acrosome (PMAI %) and with low intracellular Ca level were determined in cold-stored semen (24 h, 48 h). SCSA™ was performed to assess mean DFI, SD of DFI and % DFI in raw frozen-thawed as well as in extended sperm after 24 and 48 h of storage. The month of semen collection affected (P < 0.05) all parameters evaluated in raw semen and all criteria except progressive motility as well as rapid cells in semen stored for 24 and 48 h, respectively. Ejaculate volume was higher and sperm concentration lower in summer compared to winter and motility lower in July than in any other month of the year (P < 0.05). In semen processed in April and stored for 24 h the percentage of rapid cells was improved compared to January and after 48 h of storage progressive motility (%) was higher in January and October than in July (P < 0.05). After 24 h of cold storage PMAI % was higher in October than in January and after 48 h values were higher in September compared to January and February as well as from April to July (P < 0.05). Regarding sperm with low intracellular Ca level (%) after storage for 24 and 48 h, higher values were measured in winter and in October compared to April, June and July (P < 0.01). Seasonal changes in DNA fragmentation were most evident with respect to mean DFI. In raw frozen-thawed semen mean DFI was lower from August to November than in June and July (P < 0.001). Values were lower during winter compared to spring and early summer (P < 0.05) and lower in December than from April to September (P < 0.001). After 24 h of cold storage mean DFI was lower in September and October when compared to January, February, May, July and November (P < 0.05) and after 48 h storage mean DFI was reduced in spring and autumn compared to February, June and July (P < 0.05). In conclusion, a seasonal effect was evident on semen characteristics of raw and cold-stored sperm. Semen quality was impaired in midsummer when low sperm motility and viability were combined with an elevated DNA fragmentation and Ca level of sperm.

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