Optimization of a fertilization protocol for spotted wolffish (Anarhichas minor)

Spotted wolffish, Anarhichas minor, is considered a potential species for marine cold water aquaculture diversification. Some biological bottlenecks, including broodstock rearing procedures, hamper the full commercial production of this species. Peculiarities of their reproduction, as the putative internal fertilization and the sperm that is slow moving and motile on stripping, make this species different from most other cultivated species. In this work, we describe practical ways to improve the A. minor fertilization protocols through ameliorate usage of the limiting amount of sperm frequently reported in this species. This was achieved by 1) establishing a methodology to accurately measure the sperm concentration; 2) determining the optimal sperm to egg ratio, and; 3) adjusting the gametes contact time accordingly to the used sperm to egg ratio. In the first trial, sperm concentration was measured by direct counting in a haemocytometer; by spectrophotometry and; with the spermatocrit. The absorbance values in a spectrophotometer set at 300 nm after pre-diluting the sperm sample in an extender 100 times had the strongest correlation (R2 = 0.9185) with the haemocytometer cell-counting. In the second trial, the eggs were fertilized, using the following six egg to sperm ratios: 5 × 103, 1 × 104, 5 × 104, 1 × 105, 5 × 105 and 1 × 106. There were higher fertilization rates, above 90%, when 5 × 105 and 1 × 106 sperm per egg were used with a gametes contact time of 2 h. Finally, in the third trial, eight combinations of contact times (1 h, 2 h, 4 h and 6 h) and sperm to egg ratio (5 × 103, 5 × 104 and 5 × 105) were tested. Increased gametes contact time, 6 h vs 2 h, partially compensated for the lower sperm volumes when 5 × 103 and 5 × 104 sperm to egg ratios were used. In addition to the standardization of the fertilization protocols for the A. minor, the information in this work is also valuable for other aspects of broodstocks management, and contributes to the understanding of the reproductive biology of this species.