Gonazon, first registered GnRH spawning aid in EU/Norway

16 October 2003

In salmonid aquaculture, the normal spawning season extends over many weeks (typically 6 to 8), requiring repeated anaesthesia and handling of a large number of fish. As a consequence, it is difficult to optimise the use of labour and hatchery facilities. The season is even longer and ovulation efficiency is lower in Atlantic salmon when broodstock fish are kept in sea- or brackish water for sanitary and/or technical reasons. This means repeated handling of broodstock fish resulting in additional diseases and mortality, as well as increased labour. Thus, reducing both the duration of the spawning season and the number of fish manipulations will maximise egg production, 
limit broodstock mortality and optimise production and selection. It will also allow for better use of labour and facilities, and give an element of control to management that will optimise marketing efforts for egg suppliers. 

Ovulation is a complex physiological process wherein oocytes that are ready to be fertilised are shed. For fish farming purposes, the control of ovulation may be achieved by several hormonal means, such as pituitary extracts, 
human chorionic gonadotrophin (hCG; like Chorulon® see Intervet AAH Newsletter no. 4) and gonadotrophin-releasing hormone (GnRH, LHRH) analogues (GnRHa). Intervet has developed a GnRHa product, Gonazon™, to 
induce and synchronise ovulation of female salmonids and other finfish species. Due to the demand for full transparency in the food chain, a pre-requisite for the use of such products in aquaculture is the obtainment of a marketing authorisation (i.e., full registration). This was obtained for Gonazon in 2003 in the EMEA countries (EU/Norway) and the product will be available in certain markets early next year. 

Azagly-nafarelin, a long-acting GnRH agonist 
The active ingredient of Gonazon is azaglynafarelin, a synthetic GnRH analogue composed of 10 amino acids (pGlu-His-Trp-Ser-Tyr-[D-Nal(2)]-Leu-Arg-Pro-[aza-Gly]). The half-life of azagly-nafarelin in trout was prolonged (about 5 hr) compared with other GnRH analogues and other animals (typically minutes). This is presumably the result of its 
lower susceptibility to peptidases (that metabolise GnRH into smaller peptides and amino acids). 

The clinical trials with female broodstock salmonids were performed in commercial fish farms in France, Norway and Scotland between 1997 and 2000 and the studies involved about 900 rainbow trout, 500 Atlantic salmon and 360 
Arctic charr. This is the first time that such a large number of females were used to evaluate the efficiency of a specific GnRH in fish. All females were anaesthetised before manipulation to minimise stress. Gonazon was 
injected once intraperitoneally at the clinical dose of 32 µg/kg body weight (0.2-0.5 ml/kg body weight depending on the fish weight). 

Gonazon™, an effective inducer of ovulation 
One application of Gonazon is sufficient to advance and/or synchronise ovulation early in the spawning season in salmonids. This will decrease the cost of labour in the hatcheries and the number of anaesthesia sessions 
needed to strip females (currently, this is often 1-2 times per week for 6-10 weeks). In the trials, unless otherwise stated, treatment with Gonazon was performed when females had just begun their spawning season (0.1-6 % 
ovulation in the population). A tight synchronisation was observed with 90 to 95 % of ovulations being obtained in a narrow window of time (9-19 days depending on species and water temperature). Gonazon typically advanced mean spawning time by 2-3 weeks. 

A second very practical application of Gonazon is to decrease the cost of production and labour in the hatcheries in the last 3-4 weeks of the spawning season. At that time, fewer eggs are being produced in the broodstock facility due to the declining number of fish yet to spawn. Gonazon has been administered to rainbow trout and Atlantic salmon (see Figure below) when up to 70% of the total population had already spawned. In these trials, 94.2 and 
91.7% of these late-spawning fish ovulated by 9 days after treatment (vs. 27.6 and 33.3% in the controls, respectively). 

A third important application for Gonazon is to induce ovulation in sub-optimal environmental conditions when ovulation is delayed or blocked. This is the case in Atlantic salmon held in sea- or brackish water. Approx. 65% of 
Atlantic salmon held in brackish water (25-30 g NaCl/l) and receiving Gonazon were successfully stripped 9 days later (vs. 0 % in the controls); this had increased to 77% by day 16 (vs. 0.7% in the controls).

Gonazon™ does not significantly affect egg production and quality 
During all trials, there was no mortality, behavioural changes or changes in body weight associated with treatment. In all trials, there were also no statistical differences between fish treated with the recommended clinical dose (32 
µg/kg) and control fish in terms of relative fecundity, egg weight, ovarian fluid production and egg development to the eyed stage (see Figure below). Furthermore, no differences in hatchability, survival during yolk-sac resorption, 
mean weight of swimming fry, and survival and growth until 2 months after the first feeding were observed between treated and control rainbow trout. 

In one trout trial, a lower rate of development was observed but this can be easily explained by the fact that treatment was given 2 weeks before the beginning of the spawning season in rainbow trout. This is in keeping with the literature. It is concluded that the possible benefits of using Gonazon several weeks ahead of the initiation of the natural spawning season (to have eyed eggs earlier for sale and/or for juveniles to gain weight earlier) have to be 
balanced against the cost of the (limited) loss of embryos and larvae. 

Importance of using approved treatments in aquaculture 
Before Intervet obtained a marketing authorisation for Gonazon within the EU/Norway, the use of spawning inducers in fish was not legally allowed in these countries. Production of healthy food is the goal of all fish producers. Concurrently, consumers and national authorities are more and more focussed on the safety of the food consumed. 
The EU/Norway approval of Gonazon provides breeders with an efficient product that respects EU and national regulations for animal and human safety, and that is produced to a high pharmaceutical quality (according to Good 
Manufacturing Practices). In addition, Gonazon will be prescribed by veterinarians in these countries. This will ensure better traceability and will support the development of labelling, certification and HACCP procedures, more and 
more required by end-users (supermarket, consumers, etc.). 

Gonazon comprises a kit containing two sterile vials, one a concentrated solution of azaglynafarelin (2 ml) and the other an aqueous diluent (100 ml). These should be mixed in the sterile bottle(s) to give a concentration of active 
product to provide for a salmonid dose of 32 µg/kg body weight. This could be 320 µg/ml for use in large Atlantic salmon at 0.1 ml/kg body weight or could be 32 µg/ml for use in small Arctic charr at 1 ml/kg body weight, thus 
supplying lots of flexibility. 

Gonazon can be stored for 3 years at 4-8 °C and, when opened, for 4 weeks in the fridge. Once concentrate and diluent are mixed, the product should be injected immediately. The recommended delay between the injection and 
stripping is around 50-100 degree-days depending on water temperature and local farming conditions. When the water temperature is higher, a reduction in the delay between injection and stripping (to 50 degree days) is recommended as egg quality quickly decreases with increasing temperatures. 

Conclusion 
All field studies have underlined the ability of Gonazon to induce ovulation in salmonids and have clearly demonstrated that such a product can help farmers to optimise their production capacity, especially by simplifying the management of egg production. Synchronisation of ovulation, even under suboptimal environmental conditions, is also observed. Shortening of the late phase of the spawning season is another very useful application of this product. Gonazon could also advance the spawning season when administered around 2 weeks before its 
initiation. This may be, however, associated with a limited increase in mortality up to the fry stage (2-3 g). 

Other species 
Several Polish trials with Gonazon in common carp, even without the use of anti-dopaminergic compounds, have also proven the efficacy of azagly-nafarelin in this species (Mikolajczyk et al., 2003 [see 6th abstract below]). Gonazon has also been successfully used in Channel catfish. Trials to evaluate the specific effect of Gonazon on other species have not yet been performed, although it can be assumed that Gonazon will be at least as potent (on a µg to µg basis) as any GnRH product currently being used in aquaculture. The use of a single GnRHa injection regime has already been shown to be successful in species such as European sea bass, sea bream, turbot, various catfish 
species, etc. For many species that are synchronous or asynchronous multiple-batch spawners (flatfish, Sparids, etc.) or that present reproductive dysfunctions (e.g., sturgeons), control of ovulation by a single GnRHa injection 
may be a little more difficult but multiple injections should provide no problems. Further trials are also required to explore the ability of Gonazon to stimulate spermiation in male fish (both quality and quantity of milt).