The greater amberjack (Seriola dumerili) in the Mediterranean is a species with high potential for the diversification of the aquaculture production, however, reproductive dysfunctions in captivity have been observed in wild-caught breeders. Milt production was decreased compared to the wild, and lower fertilization success has been recorded in greater amberjack compared to other farmed species. Agonists of gonadotropin-releasing hormone (GnRHa) and human chorionic gonadotropin (hCG) have been used to overcome reproductive problems in many fishes, and in the present study we i) compared these hormonal spermiation enhancement methods in terms of plasma sex steroid production and sperm quality, and ii) applied the best spermiation enhancement method in an industrial egg production scenario.
Materials and methods
Four-year-old hatchery-produced (First generation – F1) male breeders were maintained in a sea cage (Salamina, Greece) and were fed with a broodstock diet (Skretting, Vitalis Cal, 22mm). On May 18th 2021, after blood and sperm collection, the fish (7.4±0.7 kg, mean±SD) were PIT tagged and were treated either with a GnRHa implant (n=6) with an effective dose of 139±17 µg GnRHa kg-1 or GnRHa injection (n=6) with an effective dose of 24±7 µg GnRHa kg-1 or hCG injection (n=6) with an effective dose of 972±120 IU kg-1 or left untreated (n=6)(day 0). Fish were sampled again for blood and sperm on day 7 and 13 after the hormonal treatments.
For egg production, and based on the result of the above experiment, wild-captured breeders were maintained similarly in a sea cage (Salamina, Greece). On June 7th 2022, after reproductive stage evaluation through ovarian biopsies and sperm collection, the males were treated either with a hCG injection (n=5) with an effective dose of 1083±93 IU kg-1 (day 0) or GnRHa implants (n=5) with an effective dose of 118±10 µg GnRHa kg-1. Six females were implanted with GnRHa at 57±8 µg GnRHa kg-1, while two females at the oocyte maturation stage were left untreated; all females were equally divided in two 70-m3tanks (OUT 1: with hCG treated males, OUT 2: with GnRHa implanted males). For the following days, egg production and quality were evaluated and on day 13 males were sampled again for blood and sperm, and females for oocyte diameter through ovarian biopsies. Plasma testosterone, 11-ketotestosterone, 17-β estradiol, 17α,20β-dihydroxy-4-pregnen-3-one and cortisol were quantified with the use of liquid chromatography tandem mass spectrometry (LC-MS/MS). Sperm quantity was evaluated according to their spermiation condition –which is a measure of the available milt in the testes- after gentle abdominal pressure was applied, determined by a subjective scale from 0 to 3, as follows: Spermiation index S0 = no milt released, S1= only a drop of milt released after multiple stripping attempts, S2= milt was released easily after the first stripping attempt and S3= copious amounts of milt released with very little pressure. The sperm quality parameters that were evaluated using computer assisted sperm analysis (CASA) included: (a) sperm density (number of spermatozoa ml-1 of milt), (b) survival of spermatozoa under cold storage at 4°C (days), (c) motile spermatozoa immediately after activation (%), (d) progressive motile spermatozoa (%), (e) rapid motile spermatozoa (%), (f) straight-line velocity (VSL, μm sec-1), (g) curvilinear velocity (VCL, μm sec-1), (h) average-path velocity (VAP, μm sec-1) and (i) straightness (STR, %).
Results and discussion
In 2021, neither plasma sex steroids (ANOVA, P>0.05) nor sperm quality parameters were statistically different among the four groups (Control, GnRHa implants, GnRHa injections, hCG), while plasma cortisol was elevated in all fish after handling. However, all the hCG treated males were spermiating on day 13 compared to only 33% of those treated with GnRHa implants or injections, while no spermiating male was found in the Control group. In 2022, no statistical differences were observed in egg production and quality-even though 11 spawns were recorded in OUT 1 compared to the 7 spawns in OUT 2 tank- and plasma sex steroids on day 13 between the two groups. However, the hCG treated males had significantly higher percentage of motile, progressive and rapid spermatozoa, compared to those treated with GnRHa implants (ANOVA, P<0.05) (Fig.1). The study demonstrated that hCG was better compared to GnRHa in enhancing sperm quality parameters, but this enhancement was not translated to improved spawning performance (egg fecundity and fertilization) in greater amberjack in the Mediterranean.
The project BestBrood has received funding from the EPAnEK Operational Programme, ERA-NETS 2019b (MIS 5075029), cofund BlueBio.