Proceedings of the Third World Fisheries Congress: Feeding the World with Fish in the Next Millenium—The Balance between Production and Environment
Effect of Gossypol on Survival of Spermatozoa of Nile Tilapia Oreochromis niloticus In Vitro
Huiguang Fu, Jidan Ye, Tongyan Lu, Liang Zhang
Cottonseed cake or meal is a cheap good source of plant protein. However, its toxicant component, gossypol, retards growth and especially inhibits male reproductive function (Xue 1983). Gossypol causes mortality in spermatozoa from rat, pig, mouse, monkey, dog, and humans in vitro (Li et al. 1986) and drastically inhibits the metabolism of interstitial cells in human testis (Paz and Homonnal 1984). The treatment of human spermatozoa with a fairly low concentration (100–300 µM) of gossypol affects the energetics and fertility of human spermatozoa in vitro (Kennedy et al. 1983; Paz and Homonnal 1984).
Although the effect of gossypol on male infertility is well established in mammals, it is uncertain whether this substance has a similar effect on fish. Limited reports have demonstrated that cottonseed meal and cake appear to be promising feed materials for Nile tilapia Oreochromis niloticus and catfish (El-Sayed 1990; Robinson et al. 1984), with the drawback of lacking lysine and methiomine as most other plant protein materials. Therefore, in this study, we report the effects of gossypol on survival of Nile tilapia spermatozoa.
Sexually mature male Nile tilapia weighing 400–510 g were supplied by a warmwater fish farm of Harbin Power Plant. The fish were acclimated in a 10-m2 concrete tank equipped with a recirculating filter, a thermostat (at 28°C), and fluorescent lights in a 12-h photoperiod. After acclimation for 1 month, individuals showing mating response were intramuscularly injected with luteinizing hormone–releasing hormone-A3 at a dose of 5 µg/kg body weight. Seven hours after the injection, the fish were stripped to collect sperm, which was then diluted in a ratio of 1:20 with Cosson’s trout spermatozoa reservation buffer (Cosson et al. 1985). Gossypol acetate (purity 99.9%, supplied by Pharmacy Institute of Chinese Medical Academy) were first dissolved in pure ethanol to make a 2.5 mM stock solution, then 1 mL of sperm reservation media was added to 1.5-mL eppendorf tubes to make final concentrations of 25, 50, and 100 µM. Corresponding amounts of ethanol were added in the control.
After incubation for 1 or 3 h at 28°C, the survival rate of the spermatozoa was calculated by counting live and dead spermatozoa with Trypan blue stain according to the method of Leung-Trujillo and Lawrence (1987), modified with examination by light microscopy at 4°C instead of stroboscopy. Because of the limited dissolvability of gossypol acetate in ethanol, dimethyl sulfoxide (DMSO) was used for 1, 5, and 10 mM gossypol treatments. The final concentrations of ethanol and DMSO in the spermatozoa reservation media was less than 1%.
For every treatment, milt from four to six fish was used with three replicates. The spermatozoa count was at least 400 cells in each of the three samples. The survival rate data were tested for significance first by analysis of variance and then Student’s t-test.
The survival rate of tilapia spermatozoa decreases with time. The survival rates of the spermatozoa in Cosson’s reservation buffer at 28°C for 0, 1, and 3 h are listed in Table 1. Incubation time of 1 h did not significantly influence survival rate, but a prolonged incubation of 3 h significantly decreased the survival rate.