Effect of Rosmarinic Acid on Dog Semen Quality after Freeze-drying by Applying Freeze-drying Machines for Vaccine Production

Freeze-drying (FD) is a sperm preservation method that eliminates the need for liquid nitrogen. However, the FD process can expose sperm to free radicals and temperature variations. This study aimed to determine the impact of rosmarinic acid (RA) as an antioxidant, initial sperm motility, and storage temperature on the DNA integrity of freeze-dried dog sperm. Forty-four ejaculates from 15 dogs of various breeds were divided into control (FD extender) and RA (FD extender with RA) groups, further categorized by motility (above or below 70%). After FD, samples were stored for three months at room temperature and 4 °C. Sperm quality analysis, including rehydrated freeze-dried samples, was conducted at intervals (immediately post FD, 7, 14, 30, and 90 days). Results indicated reduced viability and motility post-FD, with predominant abnormal sperm tail morphology. Initial motility had no impact on sperm quality. Storage temperature and the antioxidant RA improved DNA integrity but did not affect sperm morphology. DNA fragmentation occurred after 7 days at room temperature, slower at 4 °C, and further slowed with RA. The percentage DNA integrity after 3 months ranked from best to worst: RA group at 4 °C (1.41 ± 0.51), RA group at room temperature (4.59 ± 0.51), control group at 4 °C (4.76 ± 0.56), and control group at room temperature (38.80 ± 1.01). While sperm motility is crucial for frozen semen, freeze-dried semen presents an alternative for dogs with low fertility, given its easy storage, reduced shipping costs, and applicability in locations without liquid nitrogen. However, the FD extender and storage temperature choice significantly impacts DNA integrity in freeze-dried dog sperm.