K.J. Rana

The viability of cryopreserved tilapia spermatozoa

Abstract Tilapia ( Oreochromis spp.) spermatozoa protected with 12.5% methanol (MeOH) in fish Ringers, stored in 1.5-ml cryotubes and held in a vapour phase liquid nitrogen refrigerator remained viable for at least 13 months. The results, however, showed a high degree of variation; of the 16 samples tested only 66% produced viable cells on thawing. Mean fertilization rates of eggs were independent of storage time and ranged from 38.7 to 93.4%. To minimize variability a protocol using 0.5-ml plastic straws, which were held under liquid nitrogen, was developed and tested on the ability of post-thaw spermatozoa to fertilize eggs in comparison with an unfrozen control. Choice of cryoprotectant and its concentration, cooling and dilution rates and the fertilizing capacity of 0.5 ml of extended (ca. 25 μl milt) milt were investigated. The best pre-freezing activation of spermatozoa was obtained with MeOH and maximum cell protection was achieved using 10% MeOH. Cooling spermatozoa at rates of between 5 and 50°C/min and diluting milt at rates of between 1:2 and 1:20 had no significant ( P >0.05) influence on the rates of fertilization of eggs. Mean embryo yields ranged from 86.2 to 98.6%. Milt diluted at 1:20 could satisfactorily fertilize up to 500 eggs (140×10 3 spermatozoa/egg). The growth and survival of 30-day-old fry produced from milt stored for up to 12 months were not significantly ( P >0.05) different from normally produced fry.

Influence of egg size on the growth, onset of feeding, point-of-no-return, and survival of unfed Oreochromis mossambicus fry

Abstract The variation in egg size within and between egg clutches of O. mossambicus , and the subsequent influence of mean egg size on growth, feeding incidence and survival of unfed fry were investigated. The individual egg sizes within 90% of the egg clutches examined were normally distributed and the coefficient of variation of egg size within clutches ranged from 7.4% to 15.4%. Fry emerging from the small, medium and large egg size groups reached their maximum length and weight at 9, 9 and 12 days after hatching, respectively. Maximum attainable fry length and weight were significantly correlated with egg size ( r = 0.885 and 0.947, respectively, with P Egg size had little influence on the onset of feeding. Fry were capable of exogenous feeding within 6 to 7 days of hatching. Fry originating from small and medium egg size classes reached the point-of-no-return at 15–16 days after hatching compared to 21 days for fry from the large egg size class. The mean survival time (ST 50 ) of fry on their yolk reserves was significantly correlated ( r = 0.923; P

Induction of diploid androgenetic and mitotic gynogenetic Nile tilapia (Oreochromis niloticus L.)

Androgenesis is a potentially valuable technique for recovering fish from gene banks composed of cryopreserved sperm, developing inbred lines, and analyzing patterns of inheritance. The procedure for producing diploid organisms whose nuclear DNA is wholly of paternal origin is dependent on: (1) the denucleation of “host” eggs, and (2) the inhibition of the first mitotic division in order to double the haploid sperm chromosome complement following fertilization of host eggs. Denucleation of tilapia (Oreochromis niloticus L.) eggs was carried out using UV irradiation. Treatment durations of 5–8 min (total dose of 450–720 J/m2) produced acceptable yields of viable denucleated eggs [22.9±1.6% (±SE) of controls] as estimated by the survival of haploid androgenetic tilapia to 48 h post-fertilization. Successful mitotic inhibition was accomplished using a heat-shock of 42.5 °C for 3–4 min, applied at 2.5-min intervals from 22.5 to 30 min post-fertilization (mpf). The mean survival of androgenetic diploid fish to yolk-sac absorption for treatment groups varied from 0.4% to 5.3%, relative to the controls. Differences in the suceptibility of eggs from different females to UV irradiation were a significant factor in the overall yield of androgenetic diploids. Paternal effects did not significantly influence the androgenetic yield, suggesting that individual males would not be selected against. For comparative purposes mitotic gynogenetic “mitogyne” diploids were produced from UV-irradiated sperm. Mean survival to yolk-sac absorption varied from 0.5% to 10.64%, relative to controls. Similar optima for androgenetic and gynogenetic induction were found in the period 25–27.5 mpf (minutes post-fertilization). Induction treatments would appear to be operating on the same developmental events in both these techniques, and the results suggest that the UV irradiations used do relatively little damage to the eggs beyond nuclear inactivation. The results indicate that the production of androgenetic O. niloticus is possible on a consistent basis and that the application of this technique may be useful in quantitative and conservation genetics.

Influence of incubation temperature on Oreochromis niloticus (L.) eggs and fry. I, Gross embryology, temperature tolerance and rates of embryonic development

Abstract The influence of constant rearing temperatures on the thermal tolerance of O. niloticus embryos was evaluated using two acclimation conditions: Group A eggs were fertilized at 28°C and then subjected to test temperatures; Group B eggs were fertilized and reared to the retinal pigmentation stage (48 h) at 28°C before subjection to test temperatures. Eggs from both groups were incubated at 11, 17, 20, 24, 28, 30, 34.5 and 39.5°C. Incubation temperature and acclimation conditions affected embryonic survival and hatching success. At 17°C and below or at 39.5°C cleavage in Group A eggs failed. At each constant rearing temperature between 20 and 34.5°C the proportion of embryos advancing from the cleavage stage to hatching decreased. Optimal development to hatching (>90%) for Group A eggs occurred at 25–30°C compared with 23.5–32°C for Group B eggs. The upper and lower median tolerance limits (TL 50 -with 95% confidence limits) for Group A eggs were 34.0°C (33.3–34.7) and 21.8°C (21.1–22.5) compared with 35.1°C (34.3–35.8) and 14.8°C (13.9–15.8) for Group B eggs. The time to hatch (>50%) for Group A and B eggs was inversely related to their incubation temperature. For Group A and B eggs hatching times ranged from 2.3 days at 34.5°C to 6 days at 20°C. The rate of development to hatching ( 1 t days ), however, was best described by a curvilinear relationship of the second order.

Influence of incubation temperature on Oreochromis niloticus (L.) eggs and fry: II. Survival, growth and feeding of fry developing solely on their yolk reserves

Abstract The consequence of constant water temperature of 17, 20, 24, 28, 30 and 34.5°C on the survival, growth and first feeding capabilities of O. niloticus hatchlings developing solely on their yolk reserves were investigated. The survival times (ST 50 ) of fry were temperature-dependent and ranged from 2.5 days post-hatching at 34.5 to 18.5 days at 20°C. With the exception of 20 and 34.5°C, mass mortality of fry coincided with the onset of yolk exhaustion. The mean upper and lower median tolerance limits (with 95% confidence limits) from hatching to first feeding stage were 32.1°C (31.9–32.2°C) and 21.8°C (21.4–22.3°C), respectively. Optimal survival to first feeding (>90%) during this period occurred at 28–30°C. Incubation temperature influenced the weight of the body and yolk reserves of emergent fry; fry hatching with a lower mean dry body weight contained greater yolk reserves. Maximum mean body weight and minimum mean yolk weight of emergent fry occurred at 30°C. The growth rate (SGR, %/day) of fry was temperature-dependent. Maximum growth rate occurred between days 3 and 6 at 28 and 30°C and maximum body weights were attained by 18, 9 and 6 days at 24, 28 and 30°C, respectively. The gross yolk utilization efficiency between hatching and maximum dry body weight was temperature-dependent between 24 and 30°C and ranged between 55.4% and 61.7%. At higher rearing temperatures feeding commenced earlier. Onset of feeding and point-of-no-return occurred at 8, 5 and 4 days and 23, 20 and 18 days, respectively, at 24, 28 and 30°C.

Ontogenetic changes in location and morphology of chloride cells during early life stages of the Nile tilapia Oreochromis niloticus adapted to fresh and brackish water.

Ontogenetic changes in the location, size, density and morphology of chloride cells in the Nile tilapia Oreochromis niloticus adapted to fresh and brackish water are described using Na(+) /K(+) -ATPase immunohistochemistry, light microscopy (LM), scanning electron microscopy (SEM) and confocal scanning laser microscopy (CSLM). The pattern of chloride cell distribution changed during development under both treatments, with chloride cell density decreasing significantly from hatch to 7 days post-hatch, but appearing on the inner opercular area at 3 days post-hatch and increasing significantly thereafter (P < 0·05). Chloride cells were always denser in fresh- than in brackish-water larvae. In both treatments, chloride cells located on the outer operculum and tail showed a marked increase in size with age, but cells located on the abdominal epithelium of the yolk sac and the inner operculum showed a significant decrease in size (P < 0·05). Chloride cells from brackish-water adapted larvae from 1 day post-hatch onwards were always significantly larger (P < 0·05) than those from freshwater-adapted larvae. SEM revealed structural differences in chloride cell apical morphology according to environmental conditions. There appears to be clearly defined temporal staging of the appearance of adaptive mechanisms that confer an ability to cope with varying environmental conditions during early development.

The influence of maternal age and delayed initial feeding on the survival and growth of previously unfed O. niloticus (L.) and O. mossambicus (Peters) fry

Abstract Naturally fertilized eggs from four 0+, 1+ and 2+ O. niloticus and O. mossambicus females were artificially incubated at 28°C. All growth and survival trials were conducted at 28°C and to 20 days post-hatch in a recirculatory system containing 48 2-l compartments. Five samples, each of twenty 5-day-old fry representing each female were transferred to separate containers and the age of initial feeding of fry in each compartment progressively delayed. Fry were fed to excess at either 6, 9, 12, 15 or 18 days post-hatching four times a day. In both species feeding at 6 days (10 days post-spawn) resulted in maximum sized 20-day-old fry. Within each species and age-class and further delay in feeding resulted in a significant ( P P O. niloticus females only realized 15, 25 and 36% of their maximum growth potential when compared with 38, 67 and 86% in O. mossambicus . Similar trends were noted for their specific growth rates and body condition. The longest delay in initial feeding to obtain a 50% probability of survival of fry from 0+, 1+ and 2+ broodstock at 20 days was 11, 5, 15 and 17 days for O. niloticus and 10, 11 and 16 days for O. mossambicus , respectively. The implications of the above findings are discussed with respect to the natural and artificial rearing of mouth-brooding tilapias.