Freezing dog semen using -80 °C ultra-freezer: Sperm function and in vivo fertility

Long term storage of canine frozen semen is conventionally performed in liquid nitrogen (LN₂). However, previous works in freezing canine semen using a -80 °C ultra-freezer (-80°C-UF) showed no differences on sperm quality after thawing. The main objective of this study was to compare the effects of the freezing techniques using LN₂ or -80°C-UF on sperm function and in vivo fertility of frozen-thawed dog semen. The sperm-rich fraction of the ejaculate was collected separately from five Chihuahua breed, and each one divided into two aliquots, and frozen and stored in LN₂ or -80°C-UF. Sperm function was analyzed for motility and viability, acrosome integrity, mitochondrial function and phosphatidylserine translocation by flow cytometry before and after cryopreservation. A total of 10 bitches were intravaginal inseminated (IVAI; LN₂ frozen-thawed semen = 5 and -80°C-UF frozen-thawed semen = 5). Pregnancy status was confirmed 30 d after IVAI by transabdominal ultrasonography and live born puppies at term were recorded. Sperm function parameters were affected for both freezing protocols. Differences (P < 0.05) were found between freezing and storage methods in most of the parameters of sperm function analyzed, except in the phosphatidylserine translocation. The percentages of pregnancies were not different between the two freezing and storage protocols used. Semen freezing and storage using -80 °C UF is an effective technique for long-term preservation of canine spermatozoa.