Effects of chilled storage and cryopreservation of sperm, and sperm to egg ratio on fertilization success in lumpfish (Cyclopterus lumpus)

Abstract

Lumpfish males usually release small amounts of milt and are therefore often sacrificed for surgical harvesting of gonads. To optimize the utilization of sperm obtained by dissection of the gonads, a series of small-scale fertilization experiments was carried out in petri dishes. Sperm extracted from homogenized gonads were diluted 1:1 with a commercial extender solution before use (AquaBoost®SpermCoat). Fertilization success was evaluated as percentage eyed eggs ∼120 day-degrees post fertilization. Firstly, the effect of chilled storage (3 °C) of diluted milt were evaluated by fertilizing eggs (2.27 × 105 sperm/egg) from two freshly stripped females every 2nd or 3rd day over a 14-day period. Storage time did not affect the fertilization rates which were high throughout the 14-days trial period (89.1–99.8%). Secondly, the effect of sperm to egg ratio of cryopreserved and chilled stored sperm (3 °C for 3 days) on fertilization rate was investigated and compared by fertilizing eggs from three separate females with six different sperm to egg ratios (1.25 × 105 to 2 × 109). For both storage methods, only the lowest sperm to egg ratio (1.25 × 105) resulted in a significant lower but still high fertilization rate (80.7%). Using freshly extracted sperm at lower sperm to egg ratios (1.25 × 105 to 3.1 × 104) to fertilize eggs from three females revealed, however, no relationship between sperm to egg ratio and fertilization rates as they were high for all ratios (91.2–93.4%). Two additional experiments were undertaken at a real scale to validate the petri dish results at higher egg numbers and volumes. A fixed volume of 40 ml eggs (∼4800 eggs) was fertilized with four sperm to egg ratios ranging from 3.1 × 104 to 5 × 105. All sperm to egg ratios resulted in high fertilization rates ranging from 93.7 to 99%. In the second real scale experiment increasing volumes of eggs (1 up to 160 ml) were fertilized with a fixed sperm to egg ratio (2.5 × 105). The fertilization success (95.9–97.5%) was not affected by egg volume. In conclusion, the current study showed that lumpfish sperm can be chilled stored at 3–4 °C for 14 days and can be cryopreserved and thawed without any significant reduction in fertilization success under the conditions used in this study. Compared to other marine teleosts, lumpfish require a low sperm to egg ratio for successful fertilization, but the critical low ratio to obtain high fertilization was not revealed in this study.