WP 3 : REPRODUCTION & GENETICS Matura7on and spawning induc7on of Grey Mullet (Mugil cephalus)

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1 New species for EU aquaculture WP 3 : REPRODUCTION & GENETICS Matura7on and spawning induc7on of Grey Mullet (Mugil cephalus) Hanna Rosenfeld, Vered Zlatnikov, Chen Bracha, Eldad Toledano, Iris Meiri-Ashkenazi Israel Oceanographic & Limnological Research (IOLR), The National Center for Mariculture (NCM), Eilat, Israel Annual Coordina7on Mee7ng, January, Barcelona, Spain

Grey mullet (Mugil cephalus) Characteristics: A warm water

environmental value: An efficient aquaculture bioremediator High

value The problem: Mullet fingerling supply for aquaculture comes

2 Grey mullet (Mugil cephalus) Characteristics: A warm water euryhaline marine teleost with a worldwide distribution High environmental value: An efficient aquaculture bioremediator High economic value: Highly priced roe in addition to the fish flesh value The problem: Mullet fingerling supply for aquaculture comes almost exclusively from the wild, as techniques for artificial spawning remain incomplete

and fails to fertilize the eggs Dopaminergic inhibition appears to have a role in two different junctures of the reproductive

3 Mullets do not spawn spontaneously in captivity v Females often get stuck at early stages of vitellogenesis v Females do not undergo final matura*on & spawning v Spermiating males are rarely observed v In most cases the produced milt is highly viscous and fails to fertilize the eggs Dopaminergic inhibition appears to have a role in two different junctures of the reproductive cycle of grey mullet, i.e. early stages of gametogenesis & the stage of final gamete maturation (Aizen et al., 25) H DA (-) > GnRH (+) GtH P G

Major aims Ø To increase the abundance of spermia7ng males exhibi7ng high quality milt Ø To synchronize gonadal development in cap7ve grey mullet females & males Ø To induce spawning

4 Major aims Ø To increase the abundance of spermia7ng males exhibi7ng high quality milt Ø To synchronize gonadal development in cap7ve grey mullet females & males Ø To induce spawning Specific objec2ves: 1. Produce bio-potent recombinant gonadotropins (r- LH and r-fsh) 2. Evaluate their poten*al to act as therapeu*c agents allevia*ng reproduc*ve dysfunc*on in cap*ve mullet

5 Production and purification of grey mullet recombinant gonadotropins The lab-scale Pichia pastoris fermentation generated over 1 mg/l

In vitro functional characterization of r-fsh

11KT (pg/ml) 4 * 3 2 1 rfsh dose (ng/ml).

Both r-fsh and r-lh s7mulated 11-KT secre7on

6 In vitro functional characterization of r-fsh and r-lh A. B. 11KT (pg/ml) 4 * rfsh dose (ng/ml) rsblh Dose (ng/ml) rlh dose (ng/ml) Both r-fsh and r-lh s7mulated 11-KT secre7on 4-folds higher than the controls. Yet, r-fsh appears to be more potent than r-lh.

7 I n v i v o f u n c t i o n a l characterization of r-fsh and r-lh Experimental design During the onset of the reproductive season (early August, 214) grey mullet males received a single injection containing either: r-fsh, r-lh or saline (control). Saline r-lh r-fsh Three weeks later, fish were sampled to evaluate treatment effects on: 1) Body and gonad mass (BW and GSI values); 2) Testicular development; 3) Endogenous pituitary-gonad endocrine axis (i.e., gonadotropin β- subunit synthesis and sex steroid circulating levels).

In vivo effects of r-fsh and r-lh: I. Body weight (BW) and Gonadosomatic Index (GSI) Treatment Group BW (g) GSI (%) C 95 ± 49.45 ±.1 a r-fsh 863 ± 41.88 ±.

8 In vivo effects of r-fsh and r-lh: I. Body weight (BW) and Gonadosomatic Index (GSI) Treatment Group BW (g) GSI (%) C 95 ± ±.1 a r-fsh 863 ± ±.12 b r-lh 89 ± ±.14 ab No significant difference in BW across all treatment groups. The r-fsh- treated males exhibited significantly (P <.5) higher GSI values

9 In vivo effects of r-fsh and r-lh: r-fsh and to a lesser extent r-lh enhanced spermatogenic development among captive mullet males.

10 In vivo effects of r-fsh and r-lh: III. Plasma levels of 11-KT A 11 KT (pg/ml) 1 5 AB B Control rfsh rlh r-fsh treated males exhibited significantly (P <.5) higher plasma 11-KT levels as compared to those treated with r-lh.

11 In vivo effects of r-fsh and r-lh: IV. Pituitary expression of LH and FSH β-subunits Pituitary LH β mrna (Relative units) A 4 fold A B r-lh r-fsh Pituitary FSH β mrna (Relative units) A A A r-lh r-fsh

12 Interim conclusion The obtained results demonstrate posi7ve effects of r-fsh on pituitary LH synthesis and on 11-KT secre7on giving rise to enhanced gonadal growth and development.

13 rfsh vs. MT treatment First series of trials 1) 17α-methyltestosterone-EVAc (4 mg/kg BW) 2) rfsh (5 µg/kg rfsh) + MT-EVAc implantation (2-weeks apart) 3) Saline only (control group) Saline MT-EVAc r-fsh+ MT-EVAc % Control % MT % rfsh + MT Weeks from treatment Weeks from treatment Weeks from treatment The combined rfsh+mt treatment and to a lesser extent the MT- EVAc implanta*on increased the abundance of spermia*ng males and enhanced steady milt produc*on.

14 rfsh vs. MT treatment Second series of trials 1) rfsh (5 µg/kg rfsh)) 2) rfsh (5 µg/kg rfsh) + MT-EVAc implantation (2-weeks apart) 3) Saline only (control group) Treatment effect on sperm produc7on Saline r-fsh r-fsh+ MT-EVAc - no milt, 1- only traces 2- small amounts 3- fluid milt 4- flowing fluid, easily spread in the water

Hormonal treatment effects on synchronized ovarian development DA-Dopamine Antagonist (15 mg/kgbw) rfsh- recombinant FSH (5 µg/kg BW) GnRH- GnRH-EVAc implant (36 µg per implant) Rela7ve abundance of

15 Hormonal treatment effects on synchronized ovarian development DA-Dopamine Antagonist (15 mg/kgbw) rfsh- recombinant FSH (5 µg/kg BW) GnRH- GnRH-EVAc implant (36 µg per implant) Rela7ve abundance of post-vitellogenic females Saline DA DA+ GnRH--EVAc DA+ r-fsh+ Control DA DA+GnRH DA+rFSH Exp. I 27% 58% 71% Exp. II 29% 91% The DA+r-FSH was found to be the best performing treatment, giving rise to 91% post vitellogenic females

16 Hormonal treatment effects on spawning success Spawning induc*on protocol: Two injec*ons consis*ng of GnRH & DA given 22.5 h apart. Spawning successes has been improved in the pre-treated vs. control groups (42.9% and 29.4%, respec*vely).

17 Spawning season (216) Egg volume (ml) 5 4 Natural Shiaed Floating Sinking Spawning date Ø Ø Ø Ø Ø Ø Ø A rela*vely extended natural spawning season (~3 months). Shibed spawning season can be easily achieved via photo-thermal manipula*on. Improved spawning success (6%) among hormonally induced females. Improved synchroniza*on among breeding units increased fer*liza*on rate (7%). Fiby million eggs in total (average fecundity: 1.76 ±.52 million eggs/kg) Hatching rate: ± % Over two hundred thousand ﬁngerlings were produced

18 Thank you Zohar Ibarra (IRTA) Chen Bracha dano Eldad Tole Iris Meiri Ashkenazi This project has received funding from the European Union s Seventh Framework Programme for Research, technological development and demonstration (KBBE single stage, GA 63121, Vered Zlatnikov

larvi 2013 Pubertal development of Atlantic Bluefin tuna (Thunnus thynnus) in captivity Berkovich Nadia 6th fish & shellfish larviculture symposium

larvi 2013 Pubertal development of Atlantic Bluefin tuna (Thunnus thynnus) in captivity Berkovich Nadia 6th fish & shellfish larviculture symposium larvi 213 6th fish & shellfish larviculture symposium Pubertal development of Atlantic Bluefin tuna (Thunnus thynnus) in captivity Berkovich Nadia ghent university, belgium, 2- september 213 The key neuroendocrine