Many fish populations worldwide have experienced drastic reduction in number, largely due to the effects of the industry and habitat loss. One of the useful ways in which to replenish declining natural stocks is through captive breeding or hatchery program. Since 1997, population of snow trout Schizothorax zarudnyi has been decreasing. The indigenous fish, snow trout S. zarudnyi is an endangered species of fish endemic to Hamun Lake; Sistan, Iran shows much promise on the grounds of its wide popularity properties and hardiness towards environmental conditions.
Large areas of Sistan & Baluchistan can be used for aquaculture. Local fish species were considered for suitability in aquaculture and it was decided to select the snow trout S. zarudnyi as a possible candidate on account of its palatability and the high nutritional value of cyprinid in general.
Prerequisites for a successful aquaculture candidate are the ease in obtaining and raising fry or fingerlings, hardiness against disease and other environmental constraints, simple culture methods and acceptable marketing qualities (1).
Environmental and hormonal manipulation of ovulation in the fish have become of practical importance in the fish farming industry for two main reasons; to solve the problem of spawning asynchrony which necessitates frequent broodstock handling (2, 3) and for accelerating or delaying gametogenesis in captive broodstock, spawning may be scheduled to yield fry whenever needed (4).Use of exogenous hormones is an effective way to induced reproductive maturation and produce fertilized eggs (5). Originally, culturists utilized carp pituitary (CP) and this is still widely used particularly for the major Indian carps, Chinese carps and the common carp Cyprinus carpio (4-6). Human chorionic gonadotropin (hCG) has been used to induce final maturation of oocytes and also as a tool for utilization in commercial in aquaculture (5). The superactive luteinizing hormone-releasing hormone analogue (LHRHa) has been successfully used to induce final maturation and synchronize ovulation of many commercially cultured fish (7, 8). The use of different forms of gonadotropin releasing hormone agonist (GnRHa), which stimulate secretion of endogenous gonadotropin hormone (GtH) (9, 10) Ovaprim and Ovatide are a kind of analogue of salmon gonadotropin releasing hormone (sGnRHa) with a dopamine blocker (11). The use of sGnRHa resulted in successful stimulation of ovulation in some of cyprinids (12, 13) and catfishes (14).
The breeding performance is an important parameter to evaluate the breeding success in captive condition, which depends on the type of hormone used and its potency, dose of hormone and maturity status of the fish (15). The success of induced breeding also depends on latency period, which has been discussed for several species (16, 17). Appropriate combinations of the proper dose of inducing agent and stripping time always yield maximum egg output during induced breeding (18). Improper coordination between these two will lead to breeding failure (15). In view of the above, we investigated the latency period of S. zarudnyi under controlled condition by induced breeding with Ovaprim and Human chorionic gonadotropin (hCG).
Latency period is the time interval from the first injection to ovulation (19). The ‘‘latency period’’ varies greatly among species. For example, in salmonids this period extends to a few weeks (20), in Atlantic halibut Hippoglossus hippoglossus (Linnaeus, 1758) is 4–6 h (21), in groupers of the genus Epinephelus is 1–2 h (22), whereas in the white bass Morone chrysops (Rafinesque, 1820) it is only 30 minute (23).
Systematically, S. zarudnyi belongs to teleostei class, cypriniformes order, cyprinidae family and schizothorax genus (24). The world distribution of S. zarudnyi is in semi temporal freshwater of western Asia (25). This fish is endemic in Iran and found in Sistan region mainly (26).
Although, S. zarudnyi is believed to be "difficult to breed" in laboratory conditions, reports of success with the induced breeding of this species are available (27, 28). However, these studies are very restricted in analyzing the overall breeding performance.
The effect of different hormonal injections and latency time combinations on ovulation has not been evaluated properly in the induced breeding of S. zarudnyi. The objective of the study was to find the spawning latency period in the hormonal induced reproduction of Snow trout with two inducers (Ovaprim and hCG) separately and in combination.
3. Materials and Methods
The brood fishes for experimentation were obtained during October to December 2010 from Chahnimeh Reservoirs (Sistan province, Iran) using gillnets. Fish were transported to the Hatchery of the Department of Sistan Fisheries in plastic containers with well-oxygenated water for proper conditioning. After transport, the fish were treated with solution of formaline (40 ppm) for 2 hours and were then placed in earth pond (0.35 ha area) until March.
Females and males Snow trout used in the experiments were in mass 1328±45 and 632 ± 17.6 g respectively. Age of the fish was not determined. At the time of reproductive season (March-April), when the water temperature was increasing, 44 female fish and 53 male fish were selected. Females with soft, distended belly and pink-red genital papilla and males, which released milt when subjected to gentle pressure on the abdomen were selected. Males and females fish were placed in separate concrete tanks in running water of 14-18 ˚C for 10-12 hours. Prior to injections, fish were anaesthetized in a water bath containing 0.05-0.07 mg.L -1 clove solution.
Hormones used in this study were Human chronic gonadotropine –hCG (Pregnyl) were provided by Daroupakhsh Co. for Pharmaceuticals and Chemicals Industries, Tehran, I.R. of Iran, Under License of Organon, Oss Holland. Ovaprim contains the synthetic GnRH analog and domperidone dissolved in distilled water at 20 μg.mL -1 and 10 μg.mL -1, respectively obtained from Syndel Laboratories, Ltd., Vancouver, Canada.
After 24 hours of acclimation to water temperature of 15-17°C, the fish were treated with hormonal injections. Fish were divided into five groups Females in groups 1-4 received 1.5 ml Ovaprim.Kg -1 B.W., Ovaprim + high dose of hCG (1.2 ml.kg -1 + 5000 IU.Kg -1), Ovaprim + low dose of hCG (1.5 ml.Kg -1 + 1300 IU.Kg -1), 2000 hCG mg.Kg -1 respectively and the fifth treatment, NaCl 0.3 mg.kg -1 as control group. Males in groups 1-4 received hormones synchronized to the 2nd female’s injection as 0.3 mL Ovaprim.Kg -1 B.W., Ovaprim + hCG (0.3 mL.kg -1 + 1500 IU.Kg -1), Ovaprim + hCG (1.5 mL.Kg -1+ 200 IU.Kg -1), 500 hCG IU.Kg -1, respectively and the fifth NaCl 0.3 mg.Kg -1 as control group. All injections were intraperitoneal at the base of the pectoral fin. Time intervals between respective injections were 24 h, but between 3rd and 4th injections were 12 hours. Temperature during experiments was 15-17°C.
Fecundity rate estimated by using volumetric technique. This technique is relying on simple proportionality to estimate the total fecundity from a known number of eggs in a known volume of a subsample and a known value for the total volume of the sample, and then calculate the total number of eggs in the ovary.
3.1. Statistical Analysis
The differences in latency period, survival of embryos to the eyed stage and survival of embryos from eyed stage to the larvae data were analyzed by one way analysis of variance (ANOVA) at minimum significant of P
Results on the response to hormonal induction of ovulation, survival of embryos to the eyed stage, survival of embryos from eyed stage to the larvae, synchronization of ovulation and latency period for the different experiments are summarized in Table 1.
Successful ovulation was only obtained with Ovaprim. No female ovulated either in groups receiving hCG alone or control (groups 4 and 5).
The latency period or response time is time between the first hormonal injection and ovulation. This time is often related to water temperature and the period decreases with an increase in temperature (29), but in this study we investigate latency time at same temperature with different hormonal injections.
Important differences were observed in latency time after the application of different spawning media (30). Differences in the latency time of tench (Tinca tinca (Linnaeus, 1758)) were observed in the case of different spawning agents (31). In our study, the latency periods between the hormonal stimulation and the ovulation for Ovaprim, Ovaprim + hCG (low dose) and Ovaprim + hCG (high dose) were 60.15 ± 12.05 h, 59 ± 8.38 h, and 40.67 ± 6.35 h, respectively (Table 2). In order to perform statistical analysis, the hour:minute format was transformed in only minute format, thus the maximum latency time was for Ovaprim as 3609 ± 723.05, while 2440 ± 381.05 for Ovaprim + hCG (high dose). The longtime of latency was defined as lack of synchronization in achievement of readiness for spawning by the fish. The latency period of S. zarudnyi ranged from 34.5 - 71 hours at 16 - 17 °C after administration of Ovaprim.
In case of stimulation with Ovaprim, we suggest application of the preceding injection of Ovopel (preparation containing a mammal analogue of GnRH and dopamine antagonist – metoclopramide) allowed shortening the time of ovulation to 48 hours in case of the dace (Leuciscus leuciscus (Linnaeus, 1758)) and 36 hours in case of the ide (Leuciscus idus (Linnaeus, 1758)) and synchronize it significantly (32).The shortest time between injections and ovulation was noted when Ovaprim with high dose of hCG was used as a spawning agent, almost 30% smaller in contrast to the fish stimulated with Ovaprim singly or combination with low dose of hCG. In the case of ovaprim, females were responding in a longer period of time, but there was so much better ovulation (83.3%) comparison with females receiving Ovaprim and hCG, especially ovaprim and high dose of hCG (25%).
The differences in latency time in females treated with GnRHa, hCG and other hormonal preparations were reported in many species such as carp (Cyprinus carpio Linnaeus, 1758), Asian catfish Pangasius hypophthalmus (Sauvage, 1878). (33-35). It may be explained by the fact that GnRH release from the pituitary and the ovarian response to the released hormones is a sequential process while in fish injected with carp pituitary extract (C.P.E.) the ovarian response to the exogenous GtH was a single process. Probably hCG similar to C.P.E. acts on the gonads while GnRHa acts at a higher level of the reproductive axis. CPE usually involves a shorter latency time than Ovaprim, and this was noted in the case of cyprinids (35-37).
Another reason could be propylene glycol as a GnRHa + domperidone solvent cause lesser releasing of this compound in the blood circulation as compared to hCG solution, which cause higher levels of latency period in GnRHa + domperidone treated fish (10). In other words, it may be explained by the fact that GnRH release from the pituitary and the ovarian response to the released hormones is a sequential process while in fish injected with carp pituitary extract the ovarian response to the exogenous GtH was a single process (38). In snow trout, hCG caused shortening of latency time, although this advantage cannot cover other hCG disadvantages in snow trout induced spawning.
The statistical analysis shows a Pearson correlation coefficient of 41 between latency time and body weight of fish (group 1), meaning that there is moderate positive relationship between the two variables (figure 1). But, in groups 2 and 3, ovulation percent’s were very low in comparison with group 1 (Table 1), so it is not necessary to discuss about their relationships.
The weak intensity R square of 0.05 meaning that approximately no percent of the variation in the ovulation latency time can be explained by the body weight in group 1 and the equation of the linear regression being y=0.4112x+3046.9 + 3046.9 (Figure 1).
The statistical analysis shows a Pearson correlation coefficient of 0.02 between latency and ovulation percent of fish meaning that there is a moderate negative relationship between the two variables, and a weak intensity R square of 0.36, meaning that approximately forty percent of the variation in the ovulation latency time can be explained by the latency time, the equation of the linear regression being y = -0.0234x + 161.47 (Figure 2).
The study suggests that we cannot recommend a range of latency in S. zarudnyi by using Ovaprim; this has several reasons such as unknown age of wild broods, differences in broods weight, growth conditions, etc. It is necessary to obtain defined latency time for best breeding performance because the lower or higher doses reduced the egg output during breeding operation. This information is of value for a commercial hatchery to get maximum quantity of egg during induced spawning of this snow trout. It seems that further studies on ovulation stimulation with Ovaprim can be recommended. The results would allow optimizing reproduction effects in this interesting species.
There is no acknowledgment.
All authors have participated equally.
The study is self funded.
There is no conflict of interest.