Low sperm to egg ratio required for successful in vitro fertilisation in a pair-spawning teleost, Senegalese sole (Solea senegalensis)

Abstract

Cultured Senegalese sole (Solea senegalensis) breeders fail to spawn fertilised eggs and this bottleneck could be solved with the implementation of large-scale in vitro fertilisation protocols. However, low production of poor-quality sperm has frustrated the development of in vitro fertilisation protocols. Cultured females were induced to ovulate with a 5 µg kg-1 single injection of gonadotropin releasing hormone agonist (GnRHa) and good quality eggs (82.6 ± 9.2% fertilisation) were stripped 41:57 ± 1:46 h after the injection. Sperm was collected from cultured males, diluted in modified Leibovitz and used fresh to fertilise the eggs. A non-linear regression, an exponential rise to a maximum (R = 0.93, P < 0.0001) described the number of motile spermatozoa required to fertilise a viable egg and 1617 motile spermatozoa were sufficient to fertilise 99 ± 12% (± 95% CI) of viable eggs. Similar, spermatozoa egg-1 ratios of 592 ± 611 motile spermatozoa egg-1 were used in large-scale in vitro fertilisations with 190,512 ± 38,471 eggs. The sperm from a single male (145 ± 50 µL or 8.0 ± 6.8 × 108 spermatozoa) was used to fertilise the eggs. The mean hatching rate of the large-scale in vitro fertilisations was 70 ± 14 % to provide 131,540 ± 34,448 larvae per fertilisation. When unfertilised eggs were stored at room temperature the percentage of viable eggs decreased gradually and indicated the sooner eggs were fertilised after stripping the higher the viability of the eggs. The collection of sperm directly into a syringe containing modified Leibovitz significantly increased the percentage of motile spermatozoa (33.4 ± 12.2 %) compared to dilution in modified Leibovitz immediately after collection (6.6 ± 4.9 %). Senegalese sole have a pair-spawning reproductive behaviour characterised by external gamete fertilisation in close proximity with no sperm competition. The low spermatozoa egg-1 ratio required for maximum fertilisation was consistent with this reproductive behaviour and strategy. The provision of a large-scale in vitro fertilisation protocol (200 µL of sperm per 100 mL of eggs) will enable the industry to operate sustainably and implement breeding programs to improve production.