EFFICACY OF COMMON CARP Cyprinus carpio TESTIS ENDUCING SEX REVERSAL OF NILE TILAPIA Oreochromis niloticus Rahul Ranjan, Narayan P. Pandit*, Nabin B. Khanal, Madhav K. Shrestha, and James S. Diana Department of Aquaculture and Fisheries Agriculture and Forestry University, Nepal

Introduction Sex reversal of Nile tilapia (Oreochromis niloticus) using 17α-methyl testosterone (MT) is the most commonly used method. There is concern about the residual effect of MT on human health and environment. It is not easily available and more costly in developing countries. There is a need of exploring alternative natural androgen sources. Use of animal testis as a source of natural androgen might be an alternative.

Testis is a good source of androgen. - Bull testis (Phelps et al., 1996), hog testis (Mayer et al., 2008), and ram testis (Haylor and Pascual, 1991) Fish testis is more suitable for sex reversal in tilapia Common carp (Cyprinus carpio) Early maturation Higher gonadosomatic index Two seasons of maturity Easily available Less costly than synthetic hormone Common carp testis (CCT)

Objectives To assess the effectiveness of common carp testis (CCT) feeding on sex reversal of Nile tilapia. To assess the optimum feeding dose of CCT for maximum sex reversal in Nile tilapia. To find optimum feeding duration of CCT for maximum rate of sex reversal in Nile tilapia.

Materials and Methods 8-dah (days after hatching) mixed-sex Nile tilapia (GIFT strain) were used

Experimental setup and design Six levels of CCT diets were prepared. 0 % CCT with fish meal 50 % CCT with fish meal 65 % CCT with fish meal 80 % CCT with fish meal 95 % CCT with fish meal 100% CCT with fish meal Fed for three different durations (25, 30 and 35 days) Experimental design- RCBD Treatment duration- block Feed types- factors

60 fry in each aquarium (1.5 ft x 1.0 ft x 1.5 ft) 90% water replaced in each 2 days Feeding (Twice a day; 10-11 am and 3-4 pm) 20% initial week Reduced 2.5% each week till 5% Quantity fixed on basis of sampled fish After treatment, fish were transferred to nylon hapa and reared until 160 dah with normal feed. Feed type allocated randomly to each aquarium Least survival- Aq 16- 24 fry I-1, 12, 17 II- 3, 8, 13 III- 2, 7, 15 IV- 4, 5, 11 V- 6, 9, 18 VI- 10, 14, 16

Feed preparation Collection of Common carp testes (CCT) Sun dried for 1-3 days Dried testes crushed into pieces and grinded Fine particles sieved Mixed in required amount with fish meal powder Testes stage- Mature and spermiating Fresh CCT for drying

Dried CCT CCT Powder

Feeding- 5% of body weight with 28% CP feed Fortnightly sampling Post treatment phase After treatment, fry were transferred to nylon happa (50 cm x 50 cm x 100 cm) and reared until 160 dah Feeding- 5% of body weight with 28% CP feed Fortnightly sampling 50% water changed weekly Feeding in hapa

Sex differentiation Gonad observation All fish taken out Dissected Gonad observed Determined with naked eyes Confirmed using staining material Acetocaramine (0.5 g caramine in 100 mL of 45% acetone)

Results and Discussion Sex reversal Sex reversal towards male was based on dose dependent manner Figure: Average male population with different doses of CCT

Treatment duration (days) Table: Effect of CCT dosage and treatment duration on proportion of male of Nile tilapia (Mean±SE). Feed type (CCT%) Treatment duration (days) Mean 25 30 35 0 (Control) 50.0±0.0 54.2±4.2 52.8±1.4c 50 62.5±7.2 75.4±4.0 66.8±4.3b 65 68.8±3.6 75.0±7.2 72.6±8.3 72.1±1.8b 80 69.6±10.9 79.2±4.2 70.4±4.8b 95 83.3±4.2 91.7±4.2 90.5±4.8 88.5±2.6a 100 87.5±0.0 95.8±4.2 93.1±2.8a Mean (Excluding Control) 72.9±5.3a 78.9±6.4a 82.7±4.5a Mean values with different superscript letters within column and within row are significantly different (p<0.05)

Gonadal structure Female Male Female gonad under 100x

Testis Ovary

Growth during treatment phase Figure: Growth trend of fish during sex reversal phase (35 days)

Table 7: Growth parameters of fry during treatment phase after 35 days Treatment (CCT %) Survival (%) DWG (mg/fish/day) SGR (%/day) 0 (Control) 90.00±4.41a 6.33±0.31c 8.55±0.14c 50 60.00±14.81b 11.93±0.78ab 10.29±0.19ab 65 57.78±10.18b 10.50±0.60b 9.94±0.16b 80 62.78±13.47b 10.98±1.55b 10.01±0.43b 95 65.00±13.64b 14.43±0.43a 10.83±0.08a 100 61.67±7.64b 11.57±1.14ab 10.19±0.27ab Mean values with different superscript letters within column and within row are significantly different (p<0.05)

Higher dietary protein Increased amount of hormone in diet Growth difference may be due to higher crude protein in CCT compared to fish meal. Higher dietary protein Increased amount of hormone in diet Lower density Asad et al. (2010)- protein level effect Davis et al. (2010)- hormone level effect (11KT)

Water quality during treatment phase Figure 13: Min- Max temperature and DO in aquarium during sex reversal phase

Figure 14: pH in aquarium during sex reversal phase

Conclusion 100% CCT fed for 30-35 days produce significantly higher (95.83±7.22%) male population. Male population increased with increased CCT dose Increased male population with increased treatment duration

Recommendations for further study Study on androgens present in CCT. Development of processing method. Histological, Immunohistological and steroid hormone profile of sex changed fish. Develop a methodology for extracting hormone from testis.

Funding for this research was provided by the The AquaFish Innovation Lab is supported in part by United States Agency for International Development (USAID) Cooperative Agreement No. EPP-A-00-06-00012-00 and by contributions from participating institutions. This presentation is made possible by the generous support of the American people through the United States Agency for International Development (USAID). The contents are the responsibility of the authors and do not necessarily reflect the views of USAID or the United States Government. Mention of trade names or commercial products in this presentation does not constitute endorsement or recommendation for use on the part of USAID or AquaFish. The accuracy, reliability, and originality of the work presented are the responsibility of the individual authors.

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