Short-Term Storage of Japanese Koi (Cyprinus carpio var. koi) Sperm on the Egg Fertilization Performance

Abstract

Lack of mature male broodfish, insufficient sperm, and non-synchronized maturation times have always been a hindrance to the breeding program of Japanese koi (Cyprinus carpio var. koi) raised indoors. Therefore, it is believed that the preservation of Japanese koi sperm by short-term storage and cryopreservation could solve this problem. In this study, the appropriate diluent solution, sperm-to-diluent ratio, and storage temperature for short-term storage of Japanese koi sperm were determined, and the efficacy of the short-term stored sperm in fertilizing eggs was evaluated. Milt samples collected from sexually mature males were pooled and tested in modified calcium-free Hank's Balanced Salt Solution (CF-HBSS), modified Mahseer extender, and modified Kurokura extender at 1:1 and 1:5 ratios of sperm to diluent, respectively. Storage temperatures were tested at 4 °C and room temperature. Milt sample without diluent solution served as a control. The percentage of sperm motility was measured daily for one week. For the egg fertilization experiment, Japanese koi eggs were fertilized with sperm on the second day of short-term storage, while a freshly collected sperm sample served as a control. We found that sperm diluted 1:1 with a modified Kurokura extender and stored at 4 °C had a mean sperm motility of 76.00 ± 3.06% on the third day, compared with 54.67 ± 2.91% in the control treatment (P<0.05). Short-term stored spermatozoa showed equivalent egg fertilization ability compared to fresh spermatozoa (control) (P>0.05). In conclusion, the use of a modified Kurokura extender at a 1:1 ratio of sperm to diluent and storage at 4 °C was optimal for short-term storage of Japanese koi sperm, and these sperm still showed equivalent egg fertilization ability to freshly collected sperm after two days of storage. In addition, the current study also determined the appropriate extender solution for cryopreservation of Japanese koi sperm.