EFFECT OF HCG IMPLANTS ON CHANGE IN TESTOSTERONE AND ESTRADIOL LEVEL IN BLOOD SERUM OF MURREL, CHANNA STRIATUS

Transcription

1 EFFECT OF HCG IMPLANTS ON CHANGE IN TESTOSTERONE AND ESTRADIOL LEVEL IN BLOOD SERUM OF MURREL, CHANNA STRIATUS Selvaraj, S 1., T. Francis 2, M. Venkatasamy and S. Santhoshkumar Department of Fisheries Biology and Resource Management Fisheries College and Research Institute Thoothukudi ABSTRACT The breeding period of Murrels in India is variable. It is well known that environmental cues play an important role in regulating reproduction in teleost fish. Choosing a successful hormone therapy involves the selection of effective hormone formulations, proper duration of hormonal treatment and timing of the hormone administration. Recent advances in chronic hormone delivery vehicle have produced a variety of pellets and capsules which slowly release hormones when implanted into the musculature. Hence, in the present study, an attempt has been made to study the effect of hcg implants or change in hormone profile of both male and female striped Murrel, Channa striatus. An increase in the testosterone level was observed in hcg implanted male fishes during different sampling periods. The testosterone level was highest during the month of May (24.5pg/ml) in implanted fish. Like male fish, highest level of estradiol 17 ß level (62 pg/ml) was observed during the month of May in female fish. A highly significant differences (P<0.01) were observed between control and HCG implanted fish. The levels of estradiol 17 ß in the control and hcg implanted female fish were highly significant (P<0.01). Keywords: hcg implants, murrels, testosterone, estradiol 17 ß and ELISA. INTRODUCTION Murrels belonging to the family Channidae form a unique group of food fish in India. High market price, delicious taste, boneless flesh, tolerance to a variety of habitats and carnivorous food habit make it a desirable candidate species for fish farming presently, most of the commercial Murrel culture relies on wild fry, which are then practiced to accept formulated feed. Their availability in the market is also declining every year, even though they fetch good market value. The major reason is lack of a well established Murrel hatchery and lack in promotion of Murrel culture to fish farmers. The Striped Murrel, C. striatus is a native freshwater fish of tropical Africa and, Asia (Ng and Lim, 1990). It is the most widely cultured species of Murrel in many South East Asian countries (Ling, 1977). The breeding period of Murrels in India is variable. Alikunhi (1953) reported that C. striatus breeds during November to January in Southern *Corresponding author:1 - selvarajsethu1@gmail.com; 2 - t_franciz2000@gmail.com 290

2 Selvaraj et.al., India. It is well known that environmental cues play an important role in regulating reproduction in teleost fish. Environmental cues mediate secretion of hormones by the brain and pituitary which synchronize the activities of various organs involved in reproduction. Under culture conditions, the required environmental stimuli may be lacking and this may pose a physiological barrier to the commencement of reproductive processes. Under such conditions, hormonal therapy needs to be applied to bypass this block (Lam, 1982). Choosing a successful hormone therapy involves the selection of effective hormone formulations, proper duration of hormonal treatment and timing of the hormone administration according to appropriate physiological stages of receptivity. Third generation technologies utilize Gonadotropin Releasing Hormone analogs (GnRHa) with or without dopamine antagonists such as domperidone, pimozide and metaclopramide. It is the third generation techniques that have attracted significant interest in recent years especially the administration of hormones through oral and controlled release intramuscular implants (Donaldson, 1996). Recent advances in chronic hormone delivery Vehicles have produced a variety of pellets and capsules which slowly release hormones when implanted into the musculature or abdominal cavity. Since release is directly into the body fluid, any hormone can be administered using this method and because there is no loss of hormone, the treatment dosage can be significantly reduced. The technique of sustained hormone implantation serves as an alternative to the liquid vehicle administration of hormone by injection, where a short term treatment is usually the objective Hormone implantation has been found to be successful in inducing maturation and spawning of Goldfish, Carassius auratus (Sokolowska et al., 1984), Sea bass, Lates calcarifer (Harvey et al., 1985), Rabbitfish, Siganus guttatus (Harvey et al., 1985), Atlantic salmon, Salmo salar (Crim et al., 1986), Milkfish, Chanos chanos (Lee et al., 1986), Grey mullet, Mugil cephalus (Lee et al., 1996), Winter flounder, Pseudopleuronecte asmericanus (Harmin and Crim, 1992). Common tench, Tinca tinca (Linhart et ai., 1995), Striped trumpeter, Latris lineata (Morehead et al., 1998), Asian catfish, Clarias batrachlls (Sahu and Sahoo, 2000), Starry flounder, Platichthys stellatus (Lim et al., 2002) and Dusky grouper, Epinephelus marginalis (Marino et al., 2003). These hormone implantation techniques have not been evaluated in Striped Murrel, C. striatus for inducing gonadal maturation in captivity. The effectiveness of chronic release hormone implants for stimulating testicular and ovarian maturation in Striped Mullet, Mugil cephalus was studied by Lee et al. (1996). Mylonas et al. (1997) observed the changes in plasma gonadotropin II level of sex steroid hormones and sperm production of Striped bass, (M. sexatilis) after treatment with controlled release GnRHa delivery systems (50µg GnRHa/fish) The influence of pellets of 100 mg LHRHa/ fish and 150 IU hcg/fish was investigated by Sahu et al. (2000) in Asian catfish. Clarias batrachus and found that the above treatments brought about early maturation in the fish well advance of monsoon. Lim et al. (2004) experimented the effects of slow release of GnRHa implants on milt characteristics and plasma levels of gonadal steroids in Greenback flounder, Rhombosolea tapirina. Kumakurna et al. (2004) studied the effects of GnRHa implant on pituitary ovarian axis of one year old pre-pubertal red Seabream, Pagrus major. Hence, in the present study, an attempt has been made to study the effect of human Chorionic Gonadotropin implants on change in reproductive hormone profile of Striped Murrel, Channa striatus. 291

3 Effect of... MATERIALS AND METHODS Implantation of hcg implants The Striped Murrel, C. striatus used in the present study was collected from Pazhayakayal area of Thoothukudi District. The specimens were transported to the field laboratory in plastic crates of 50 liters capacity. The fish were acclimatized for 5days in FRP tanks of 500 liters capacity. The average initial length and body weight of the fish were to 64.40cm and 659± g respectively. During the period of acclimatization for 5 days, cooked chicken intestine at 5 percent of the body weight of fish was given as daily feed ration. After acclimatization, both male and female fish were segregated based on the opening (Dehadrai et al., 1973), which is circular in female and much elongated in male After segregation of sexes, fishes were transferred to a round cement tanks of size 3m diameter and 1m height having 0.5m water depth. Preparation of hcg implants Human Chorionic Gonadotropin (Trade name, Profassi) of 20,000 IU was mixed with 0.5g of cholesterol until a gel like consistency was attained and the resultant paste was incubated in incubator at 35 C till it dried up (Huat, 1980; Lee et al., 1986b). After drying of hcg-cholesterol mixture, it was powdered well for packing in silastic capsule. Specfications of silastic capsule Length of the empty silastic capsule cm Diameter of the empty silastic capsule- 0.5cm Weight of the empty silastic capsule g Based on the individual weight of fish, hcgcholesterol mixture was packed in the silastic capsule at the concentration of 1000 IU of hcg/kg of body weight (Francis et al., 2000). The average weight of hcg implant were 0.082g. For control, cholesterol mixture without hcg hormone was packed in the silastic capsule. The fish were anaesthetized in 0.1 percentage benzocaine solution. Following anaesthetizing, a small incision was made near the dorsal musculature and the implants were implanted intramuscularly. After implantation, the wound was swabbed with oxytetracycline ointment. The wound got healed within 5 days. Experimental design The fishes were divided into two groups. Each group consisted of male and female fish stocked in separate tanks. One group was implanted with hcg implants while the control group was implanted without hcg hormone. After the start of experiment, the fishes were fed alternately with cooked chicken intestine and clam meat, twice a day at ad libitum during 6 a.m. and 6 p.m. till the end of experiment. Monthly recording of various water quality parameters were carried out in the experimental tanks using photometer, a water quality kit. About 80 percentage water exchange was carried out fortnightly. Hormone implantation was carried out two times in the present study with an interval period of five months. During the fifth month, second implantation was carried out in the remaining fishes. Steroid hormone analysis Blood samples were collected from the caudal peduncle of both hcg implanted and control fish during the monthly sampling. The collected blood samples were allowed to clot at 4ºC and the serum was removed for storage at -20 C until the hormone analysis was undertaken. The steroid hormones namely testosterone in males and estradiol-17ß in females was measured in blood serum using Enzyme Linked lmmunosorbent Assay (ELISA) kit.

4 Statistical analysis Monthly mean values of testosterone and estradiol-17ß levels in serum of control and hcg implanted fish were compared by Analysis of variance (ANOVA). RESULTS & DISCUSSION Testosterone and estradiol-17ß levels in blood serum of control and hcg implanted fish were measured using Enzyme Linked immunosorbent Assay kit to study the influence of hcg implants on serum steroid hormone profiles. The initial values of testosterone and estradiol-17ß in the blood serum of fish were O.2ng/ ml and 8pg/ml respectively. The levels of testosterone in male fish and estradiol-17ß in female fish analysed during various sampling periods (August 2004 to June 2005) are presented in Tables 1 and 2. Testosterone levels in blood serum of hcg implanted male fish were found to increase from 3.5ng/ml (September) to 18ng/ml (December). A sudden increase in the levels of testosterone was observed after two months of hcg implantation. Likewise, a decrease in the testosterone level was noticed for two months after it attained a peak in December. The testosterone level again increased to 14ng/ml (March) to 24.5ng/ml (May) followed by a fall in the testosterone level (18.2ng/ml) during the month of June. In hcg implanted male fishes, testosterone level was highest during the month of May (24.5ng/ml). An increase in the testosterone levels were observed in control male fishes for the first three months, followed by a steady fall till the month of March (0.1ng/ml). The testosterone level was highest during the month of June (8.2ng/ml) in control male fish. The changes in the levels of testosterone in control and hcg implanted male fishes during different sampling period are depicted in Fig. 1. A highly significant differences (P<O.Ol) were Selvaraj et.al., observed between control and hcg implanted male fish (Table 3). Estradiol-17ß levels in hcg implanted female fish were found to increase from 34pg/ml (September) to 61.5pg/ml (November). A sudden fall in the level of estradiol followed by steady increase and reaching its peak (62pg/ml) during the month of May. Again, a reduction in estradiol- 17ß levels were observed in June (23.5pg/ml). The estradiol-17 ß level was highest during the month of May (28.6pg/ ml) in control female fish. The levels of estradiol- 17ß in the control and hcg implanted female fish were highly significant (P<O.OI) (Table 4). Changes in testosterone and estradiol-17ß levels in blood serum of control and hcg implanted fish (Tables 1 and 2) were analysed to understand the endocrine control of maturation in fish. After first hcg implantation, testosterone and estradiol- 17ß levels were high during the month of November and December (Fig. 1 and Fig. 2). When the animals were implanted with hcg implants for second time, testosterone and estradiol -17ß levels again increased and attained a peak during the month of May. In hcg implanted fish, testosterone and estradiol-17ß levels were always higher than the control fish. The present study is in agreement with the findings of Zairin et al. (1992a). Zairin et al. (1992b) showed that the levels of testosterone and estradiol-17ß were always higher in hcg treated group than the control group in Clarias batrachus. Thus, it is evident that the higher levels of testosterone and estradiol-17ß in blood serum were present in hcg implanted male and female fish. In the present experiment, higher testosterone levels in hcg implanted group than in the control group may be due to the activation of enzymes involved in testosterone synthesis as reported by Zairin et al. (1993) in C. batrachus. Similarly, higher estradiol- 17ß levels in the hcg implanted group than in the control group may be due to the action of hcg on the vitellogenic oocytes as observed by Zairin et al. 293

5 Effect of... (1992a) in C. batrachus. The data was statistically analyzed to compare the testosterone (male) and estradiol-17ß (female) levels in hcg implanted and control fish (Tables 3 and 4). Highly significant difference (P<O.O1) was observed in the testosterone and estradiol-17ß levels of control and hcg implanted fish. The steroid profiles during maturation and induced spawning of the Striped Mullet; M. cephalus was studied by Tamaru et al. (1991) and found that both serum testosterone and estradiol- 17ß are highly correlated with oocyte growth. Zairin et al. (1992a) analysed the plasma steroid hormone profiles during hcg induced ovulation in female Walking catfish, C. batrachus and found that the plasma estradiol-17 ß levels in hcg treated fish remained constant throughout the experiment, whereas levels in the control group decreased. Similarly, changes in the plasma steroid hormone profiles of hcg injected male Walking catfish, C. batrachus was studied by Zairin et al. (1993) and found that the hcg induced an increase in plasma testosterone and ll-ketotestosterone (11-KT) Levels. Plasma steroid levels and oogenesis in female specimens of Trichogaster trichopterus were measured by radioimmunoassay and high performance liquid chromatography after injections of the LHRHa, with and without Pimozide (PIM) (Degani et al., 1995). The serum steroid hormone profiles in hatchery bred female catfish, C. macrocephalus during an annual reproductive cycle was observed by Fermin et al. (1997). Likewise, Fermin et al. (1997) also observed serum steroid hormone profiles in hatchery bred male catfish, C. macrocephalus during an annual reproductive cycle and found that serum testosterone levels ranged between l5-25ng/ml. Like her observation, increased levels of plasma steroid hormones were observed in HCG implanted male and female Murrel, Channa striatus. It showed that, HCG implantation have an influence on the increase in steroid hormone levels. Nayak et al. (2000) studied the plasma steroid profiles during oocyte maturation and LHRHa pimozide induced ovulation in the Asian catfish, C. batrachus and reported that the levels of estradiol-17ß and estrone rapidly increased reaching a peak during the vitellogenic phase, while a decline was observed during the spawning phase. The sex steroid profiles in female and male Sturgeon (Acipenser stellatus) during final maturation induced by hormonal treatment was studied by Semenkova et al. (2002) and found that following hormonal treatment, ll-ketotestosterone (11-KT) levels decreased slightly in females, but increased significantly in males 24hr after LHRHa injection. The plasma sex steroid and vitellogenin profiles during vitellogenesis in Tasmanian female Atlantic salmon (S. salar) was studied by King and Pankhurst (2003) and reported that the plasma levels of estradiol-17ß and testosterone increased from 3 to 20ng/ml and plasma levels of vitellogenin increased from 2.5 to 35mg/ml. The same increase in trend of hormone levels were observed in the present experiment induced with hormone implants. In the light of the above observation it is evident that hcg implants could play a major role in increasing level of steroid hormones. Thus increase the maturation of Murrel for off seasonal breeding. ACKNOWLEDGMENTS The authors are grateful to the Dean, Fisheries College and Research Institute, Thoothukudi and the authorities of Tamil Nadu Veterinary and Animal Sciences University, Chennai for providing facilities to carry out the study 294

6 Selvaraj et.al., REFERENCES Alikunhi, K. H. (1953). Notes on the bionomics, breeding and growth of the murrel, Ophiocephalus striatus (Bloch). Proc. Indian Acad. Sci., 38(1): Crim, L.W., Glebe, B. D. and Scott, A P. (1986). The influence of LHRH analog on oocyte development and spawning in female Atlantic salmon, Salmo salar. Aquaculture. 56: Degani, G., Jackson, K and Marmelstein, G. (1995). The effect of LHRH analog on sex steroid profiles and oogenesis in female Trichogaster trichopterus. J. Aqua. Trop., 10(4): Dehadrai, P. V., Banerji, S. R., Nirmal, K. T and Das, N. K. (1973). Sexual dimorphism in certain air breathing teleosts. J. Inland Fish. Soc. India, 5: Donaldson, E. M. (1996). Manipulation of reproduction in farmed fish..anim. Reprod. Sci., 42: Fermin, ld. T., Miura, T., Veda, H., Adachi, S and Yamauchi, K. (1997). Testicular histology and serum steroid hormone profiles in hatchery bred catfish Clarias macrocephalus during an annual reproductive cycle. Fish. Sci., 63(5): Francis, T., Ramanathan, N. Athithan, S. and Cheryl, H. F. (2000). Induced breeding of murrel, Channa striatus using various inducing agents. Fishing Chimes, 19 (10-11): Harmin, S. A. and Crim, L.W. (1992). Gonadotropic Hormone Releasing Hormone analog induced ovulation and spawning in female Winter flounder, Pseudopleuronectes americanus. Aquaculture, 104: Harvey, B., Nacario, J.., Crim, L.W., Juario, J. V and Marte, C.L. (1985). Induced spawning of sea bass, Lates calcarifer, and rabbitfish, Siganus guttatus, after implantation of pelleted LHRH analogue. Aquaculture, 47(1): Huat, K. K. (1980). Stimulation of ovarian maturation in fish by sustained hormone preparations. Aquaculture, 20: King, H. R. and Pankhurst, N. W. (2003). Ovarian growth and plasma sex steroid and vitellogenin profiles during vitellogenesis in Tasmanian female Atlantic salmon (Salmo salar). Aquaculture, 219: Kumakura. N., Okuzawa, K., Gen, K., Yamaguchi, S., Lim, B. S and Kagawa, H. (2004). Effects of gonadotropin-releasing hormone on pituitaryovarian axis of one-year old pre-pubertal red seabream. Gen Comp Endocrinol. 138(2): Lam, T. J. (1982). Applications of endocrinology to fish culture. Can. J. Fish. Aquat. Sci., 39: Lee, C. S., Kelly, C. D and Tamaru, E. S. (1996). Hormonal induction of maturation in Striped Mullet,Mugil cephalus. Asian Fish. Sci., 9: Lee, C. S., Tamaru, C. S and Kelley, C. D. (1986b). Technique for making chronic release LHRHa and 17a-methyltestosterone pellets for intramuscular implantation in fishes. Aquaculture, 59: Lee, C. S., Tamaru, C. S., Banno, le., Kelley, C. D., Bocek, A and Wyban, la. (1986a). Induced maturation and spawning of Milkfish, Chanos chanos Forsskal, by hormone implantation.aquaculture, 52: Lim, H. K., Han, H. S and Chang, Y. J. (2002). Effects of Gonadotropin Releasing Hormone analog on milt production enhancement in Starry flounder Platichthys stellatus. Fish. Sci., 68: Lim, H. K., Pankhurst, N. W and Patrick Fitzgibbon, Q. (2004). Effects of slow release Gonadotropin 295

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8 SI. No SI. No Table 1 Testosterone level in blood serum of control and hcg implanted male fishes Sampling period Testosterone level (ng/ml) (Monthly /year) Control Treatment August 2004 (Initial) September 2004 October 2004 November 2004 December 2004 January 2005 February 2005 March 2005 April 2005 May 2005 June 2005 Table Estradiol-17ß level in blood serum of control and hcg implanted female fishes Sampling period Estradiol-17ß level (pg/ml) (Monthly /year) Control Treatment August 2004 (Initial) September 2004 October 2004 November 2004 December 2004 January 2005 February 2005 March 2005 April 2005 May 2005 June 2005 Selvaraj et.al., Table ANOVA table showing the effect of hcg implants on changes in testosterone level in blood serum of control and hcg implanted male fishes during different months Sources of Degrees Sum of Mean sum Table F value F ratio variation of freedom squares Treatment Block Error Total

9 Effect of... Table 4 ANOVA table showing the effect of hcg implants on changes in estradiol-17ß level in blood serum of control and hcg implanted female fishes during different months Sources of variation Degrees of freedom Sum of squares Mean sum of squares F ratio Table Treatment Block Error Total