For the first objective testosterone levels of regular adult female, adult male and sex inversed tilapia (O. niloticus) were determined. For getting regular male and female tilapia, a stock was identified in a homestead earthen pond in Hatgodagari area of Pobaupazilla of Rajshahi district where natural stock of tilapia was maintained at least for last 5 years and no tilapia fingerling was released during that period. Therefore, it was considered as free from any kinds of hormonal feeding. Fish were caught with cast net; the blood sample were taken from the caudal vain of live fish, and then by dissecting the fish, sex was determined by observing (often under microscope for confirmation) the primary sexual organs (testis/ovary) to label the blood sample as regular male or female tilapia. At the same time the condition of the sex organs was documented. Similarly, live adult sex inversed (confirmed by the fish farmer) tilapia (treated group) were bought from the wholesale market located in Naodapara of Rajshahi city in several batches in between May to August-2018 and blood samples were taken. After taking the blood sample, the fishes were dissected for confirmation of their sex (often by observing under microscope) and condition of the sex organ was documented. For normal population, 53 blood samples were collected from 53 different fish. For sex inversed population 50 blood samples were collected from 50 different fish as well. The size of the fish ranged from 200 g to 500 g each. For each fish 2 to 3 ml blood sample was taken from caudal blood vessel using 5 ml disposable syringe.
After taking the blood sample the needle was removed from the syringe. Then the blood from the syringe was transferred to the sterile red top blood tube (glass tube) without anticoagulant. Then the blood tubes with blood sample were left for half an hour without shaking in room temperature. The serum was separated following the standard serum separation protocol (Texas Department of State health services, 2018). After half an hour the blood tubes were put in a centrifuge machine to centrifuge for segregating the blood serum from the blood cells. The blood samples were centrifuge for 10 minutes @ 2000 rmp.
After completion of the centrifuge, the blood serum from the top were taken using vacuum dropper and put it in sterile Eppendorf tubes and put in freezer immediately. Because the collection of blood samples took several months, the frozen blood serum samples were stored in freezer at -800 C in Biochemistry Department of Rajshahi Medical College until used for testing the testosterone level. Due to the distance of the collection site from Rajshahi University, to avoid the transfer of live fish frequently during the experimental period, a mobile laboratory (consisting of a compound microscope, a small centrifuge machine, blood tubes, syringe, Eppendorf tubes, dissecting box, ice box etc.) for fish blood collection was set near the sample collection site.
2.1. Feed Preparation For feeding ‘AIT’ pellet carp feed was used (proximate composition was: 27.26% protein, 7.20% lipid, 11.54% ash, 13.70% moisture, 5.90% fiber and 34.40% carbohydrate). To prepare the 15 kg feed with hormone, first 150 mg (at the rate of 10 mg/kg feed) Methyl testosterone (marketed by ‘Argenta’ in Bangladesh) were diluted in 15 ml of lab grade ethanol. Then it was made 250 ml by adding tap water. Then the 250 ml solution was sprayed over the 15 kg feed and mixed up well so that the feed soaked up all the liquid containing 150 mg methyl testosterone. Then the moist feed was air dried in the shed. Despite being air dried the feed contained extra moisture, therefore, to avoid the fungal growth, the feed was preserved in the refrigerator, in a plastic bag.
2.3. Collection of Blood Samples Blood samples were collected from 2 to 3 male and female on every 5 days following the process mentioned above to monitor the serum testosterone level. After collection of the blood sample, the fishes were released in the hapa again. Because there were limited number of fish, in this case fish were not dissected after the collection of blood sample, rather sex was identified by observing their genital opening. Fish from different hapas were collected on rotational basis for sampling to allow the fish recover from the physical injury that incurred in the process of blood collection. Thus, over the period of one month the fish were fed with methyl testosterone with feed and blood samples were collected to monitor the change in serum testosterone level in O. niloticus. Blood samples were kept collecting till another month after feeding of testosterone was stopped.
2.4. Determination of Testosterone Level Serum testosterone level were determined by ‘Das Plate Reader’- manufactured in Italy (http://www. dasitaly. com/ en/prodotti/Plate% 20 Reader), marketed by Bio-Trade International in Bangladesh, using ‘Accu Bind ELISA Microwells’ for Testosterone from ‘Monobind Inc.’, USA, following the prescribed systems/procedure of the ELISA kit using the reagents supplied with the kit (Monobind Inc., 2018) in the laboratory of Royal Hospital (pvt.) Ltd located in Laxmipur area of Rajshahi city. Since the ELISA kit is customized for human use it can determine the maximum concentration of 12 ng/ml of testosterone. After testing the blood serum, for those samples where values were over 12 ng/ml, were diluted with the diluent and tested again for determining the actual testosterone concentration.
2.5. Statistical Analysis Comparison of means test (t test for independent samples) was used to determine whether there is any difference between the testosterone level of regular tilapia and sex inversed tilapia of marketable size.