Chilled storage of semen from Atlantic halibut, Hippoglossus hippoglossus L. II: effect of spermiation advancement, catheterization of semen, and production-scale application.

A method for in vitro storage of Atlantic halibut Hippoglossus hippoglossus L. semen can facilitate seed production. The study aimed at determining the effect of male spermiation advancement on viability of chilled stored spermatozoa. The use of catheterization of semen from the sperm duct was examined. Also, large volumes of semen were stored under sub-optimal production-like conditions in order to determine the suitability of the method into practical use. Semen was collected from two broodstocks: natural photoperiod males, being at the first phase of the reproductive season and 3-month advanced photoperiod males, being at the end of the reproductive season. Semen samples were diluted 1:5 (v/v) with modified Hanks' Balanced Salt Solution supplemented with antibiotics, and stored in Ziploc bags filled with air. Sperm motility parameters, assessed by computer-assisted sperm analysis (CASA), were assessed weekly. Experimental and production-scale fertilization trials were performed. Sperm samples from natural photoperiod males showed significantly longer viability under in vitro storage conditions than sperm from advanced photoperiod males. In the natural photoperiod group, the decrease in spermatozoa motility, curvilinear velocity and straight-line velocity occurred on day 50, 14, and 28 of storage, respectively. Spermatozoa from one of five males were still motile on day 80 of storage, and fertilization rates and embryo survival rates obtained using semen stored for 70 days did not differ from control values and they were significantly higher than values obtained with the use of fresh semen of the same male, but being at the end of reproductive season. Catheterization of semen showed no advantage to stripping the semen without a catheter, even for samples stored undiluted for 1 day of collection, before dilution. Under sub-optimal conditions, spermatozoa stored in large volumes (10-100mL of diluted semen) without any special treatments except for weekly swirling, remained viable for more than 1 month. Production-scale fertilizations with samples stored for 5-21 days resulted in high survivals of embryos and hatchlings. Because of its simplicity and efficiency, the method shows a high potential for use in commercial Atlantic halibut farming. It has already been applied to a halibut breeding programme for the next reproductive season at our research station.