WWW.WAS.ORG • WORLD AQUACULTURE • JUNE 2025 71 (CONTINUED ON PAGE 72) FIGURE 2. Clarias hatchery tanks. motility of spermatozoa with an aim to establish the right dilution ratio so as to prevent over-dilution but did not study the effect of extended stay of C. gariepinus milt in saline water. Such information can be used to optimize the storage and transport of sperm for use in aquaculture and fish breeding programs. Thus, this study looked at the impact of duration of milt in saline water on its integrity and ability to induce fertilization in C. gariepinus by analyzing sperm motility, sperm count and density, sperm viability and fertilization, and hatchability of fertilized eggs. Methodology This research was carried out in the hatchery unit of the Department of Aquaculture and Fisheries Management, University of Benin, Benin City, Edo State, Nigeria. Eighteen broodstock C. gariepinus used were purchased from a reputable fish farm and selected using the guidelines stipulated by (Fregene et al., 2024). Three (3) male (average weight =2kg) and 15 female C. gariepinus (average weight 1.5kg), of the same age and nutritional background, were conditioned before use. The experiment was laid out in a Completely Randomized Design with five treatments and 8 weeks of culture period and the experiment was carried out three times. The treatments were derived with regards to the duration of milt after it had been retrieved from the sperm sac and preserved in saline solution of 0.9 ml/L concentration. The treatments included T0, the control where the milt was used immediately after extraction. In other treatments milt was placed in the refrigerator and used for fertilization after 3 hrs (T1), 6 hrs (T2), 9 hrs (T3) and 12 hrs (T4) respectively. Milt was extracted from a male in each trial and kept in a beaker containing 0.9ml/L of saline water in a ratio of 1:10. The milt-saline mixture was divided into five (5) equal volumes and coded T0, T1, T2, T3, and T4. T0 was used for fertilization immediately while the other four suspensions were kept at room temperature and used at the aforementioned intervals. The mature female fish were induced such that at the time of collection of the milt at 0, 3rd, 6th, 9th and 12th hour, a female fish was ready to ovulate. Hence five matured females of comparable age, size, nutritional and health status, and from the same source, were stimulated at different times thereby synchronising the time of ovulation with the treatment time (Figure 1). Before fertilization the hatchery tank, bowls and spawning mats were washed and disinfected and impounded with water (Figure 2). The eggs were significantly separated on the spawning mats after fertilization. About 5 mL from the total sperm sample in each treatment was evaluated for sperm motility just before using the remaining solution for fertilization, at 40× magnification after dilution of 20 µl distilled water as an activating solution. Sperm samples were assessed according to the technique outlined by Carina and Carles (2018). When positioning the sample on the microscope, the motility of the sperm cells was observed for no less than 5 minutes. Numbers of spermatozoa were determined using a cleaned small beaker to collect 5ml of sperm. Care was taken to prevent contamination during collection. The whole sperm cell (WSC) was counted (Canyurt and Akhan, 2008). A new Improved Neubauer Counting Chamber (Depth 0.0mm, 1/400mm2, B.S. 748 I.S. 10269, ROHEM INDIA) was used to count the number of sperm cells present after it had been diluted with a buffered isotonic solution. The motile sperm cell (MC) value was calculated using the formula: The sperm cells were counted on a microscope (with a 40x objective, with objective of 0.63 M.M. working distance and a 0.75 numerical aperture) connected to a camera and television monitor. A total of 30ml of stripped eggs was shed into each experimental bowl, and 5ml of the stripped eggs were then counted and multiplied by six to get the total number of eggs per bowl. Fertilization rate was determined 3 hours after fertilization (late morula to early gastrular stage) from sub samples kept (Egwenomhe and Obi, 2012). The fertilized eggs and unfertilized eggs were counted physically for each treatment. Fertilization rate was calculated as follows: The hatching rates were evaluated for 24-48 hours depending on the temperature after fertilization and determined using the same calculation as Egwenomhe et al., (2017). Water quality parameters were monitored throughout the culture period. Dissolved oxygen (DO) was measured using a digital dissolved oxygen meter, while pH and temperature were assessed with a digital pH meter and a thermometer, respectively. The collected data were analyzed using Analysis of Variance (ANOVA), and mean differences were determined using Duncan’s Multiple Range Test (DMRT) at a 0.05 significance level. Results and Discussion Significant differences (P<0.05) were observed among the treatments across all measured parameters (Table 1). The highest
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