The kiss system is widely known to control puberty and to be involved in the onset of reproduction, acting upstream of the Brain-Pituitary-Gonad (BPG) axis in gonadotrophin releasing hormone (GnRH) neurons and stimulating secretion of GnRH. Kisspeptin has been proven to be a novel, very promising therapeutic for fertility disorders in humans. In fish, also positive results have shown the potential of this hormone to induce gamete development, acting in a more physiological way than classical treatments. Such evidences promoted the idea that kisspeptin could be used to solve reproductive disorders of Senegalese sole G1 breeders, which represents a major bottleneck for the industry of this species. In this sense, this study aimed at testing the effectiveness of the kisspeptin hormonal treatment (using Kiss2) as an innovative therapeutic to induce reproduction in Senegalese sole G1 breeders, in terms of sperm motility, sex steroids and gonadotrophins concentrations.
Senegalese sole G1 broodstocks were maintained at CCMAR facilities in 4 tanks of 3m3 (n=12/per tank, sex ratio 1:1) under natural photoperiod and temperature conditions . During breeding season, the animals from each 2 of the tanks were treated either with SSkiss2_v2 decapeptide (GeneBank HM116743) in a single intramuscular injection at a dose of 250 µg/Kg body weight, or with a PBS injection used as a placebo control.
Before (T0) and after (2h, 4h, 2 days and 4 days) the treatment, all fish were anesthetized (2-phenoxyethanol, 300 ppm) and sampled for blood plasma to determine concentrations of sex steroids (Testosterone, T; Estradiol, E2; 11Ketotestosterone, 11KT) and gonadotrophins (F ollicle s timulating hormone, FSH; L uteinizing hormone, LH) by ELISA. Sperm was collected from males to check motility with CASA software and cell viability was evaluated by flow cytometry.
Hormonal levels revealed a positive effect of kisspeptin treatment in FSH and LH levels at 4h and 2 days after treatment, both in males and females. For sex steroids, the same effect was observed for testosterone, 2 and 4 days after treatment, respectively for females and males. Sperm analysis showed a transient elevation of sperm motility after the treatment, however statistical analysis did no confirm differences between groups. Cell viability was similar among the treated and the control groups .
Kiss2 seems to play an important role inside the hormonal cascade of Senegalese sole BPG axis, in both sexes. This treatment appears to be a very promising therapeutic to ind uce Senegalese sole reproduction, although more research is still needed, namely testing repeated injections or slow-release implants.
Acknowledgements: Research funded by ReproF1 Project (Programa Operacional Mar2020, MAR-16-02-01-FMP-0059) and Portuguese national funds (FCT-Foundation for Science and Technology) through project UID/Multi/04326/2019 and contract DL 57/2016/CP1361/CT0007.