Effects of ultra-violet irradiation on sperm motility and diploid gynogenesis induction in large yellow croaker (<Emphasis Type="Italic">Pseudosciaena crocea</Emphasis>) undergoing cold shock

Large yellow croaker, Pseudosciaena crocea, exhibit sexually dimorphic growth, with females growing faster and reaching larger adult sizes than males. Thus, development of techniques for preferentially producing females is necessary to optimize production of these species. We have established a protocol to produce all-female croaker P. crocea through induction of meiotic gynogenesis with homologous sperm. The first set of experiments investigated the ultra-violet (UV) irradiation on sperm motility and duration of sperm activity to determine the optimal UV dosage for genetic inactivation of sperm, yet retaining adequate motility for activation of eggs. Milt from several males was diluted 1:100 with Ringer’s solution and UV irradiated with doses ranging from 0–150 J cm⁻². The results indicated that motility and duration of activity generally decreased with increased UV doses. At UV doses greater than 105 J cm⁻², after fertilization, motility was <10% and fertilization rates were significantly lower. Highest hatching rate was obtained at 75 J cm⁻². A second set of experiments was carried out to determine appropriate conditions of cold shock for retention of the 2nd polar body in P. crocea eggs after fertilization with UV-inactivated sperm by altering the timing, temperature and duration of shock. At 20°C, shock applied at 3 min after fertilization resulted in higher survival rate of larvae at 6 h after hatching. Results of different combinations of three shock temperatures (2°C, 3°C or 4°C) and five shock durations (4 min, 8 min, 12 min, 16 min or 20 min) at 3 min after fertilization demonstrated that shocks of 12 min gave highest production of diploid gynogens. Statistical analysis revealed that maximum production of diploid gynogens (44.55 ± 2.99%) were obtained at 3°C. The results of this study indicate that the use of UV-irradiated homologous sperm for activation of P. crocea eggs and cold shock for polar body retention is an effective method for producing gynogenetic offspring.     

Induction of Diploid Androgenesis in the Stinging Catfish,Heteropneustes fossilis

This study reports the results on induced diploid androgenesis in the Indian catfish, Heteropneustes fossilis. Ultraviolet (UV) irradiation was used to inactivate maternal genome of H. fossilis. Complete inactivation of maternal genome was recorded at 12,500 ergs/mm². These genome‐inactivated eggs of H. fossilis were inseminated with conspecific sperm. The sperm suspension was diluted to 1 × 10⁷ sperm ml/L in Hank's Balanced Salt Solution. Egg viability was assessed for different exposure durations at fertilization, hatching, haploidy, and diploidization. Majority of the larvae derived from irradiated eggs had abnormal appearance. Complete inactivation of maternal genome was detected by haploid syndrome and confirmed by chromosome counting (n = 29). These eggs activated with sperm were subjected to heat shock at 40 and 41 C for different postactivation times and durations. Diploid androgens had a normal appearance as controls and confirmed by chromosome counting (n = 58). A maximum of 21 and 14% of diploidization was recorded at 30 min after activation, at 40 and 41 C, which corresponds to the first cleavage suppression time.     

紫外辐射灭活大黄鱼和黄姑鱼精子及其激活大黄鱼卵子发育为胚胎的效果

为探讨大黄鱼(Larimichthys crocea)和黄姑鱼(Nibea albiflora)精子的紫外辐射灭活适宜剂量及其激活大黄鱼卵子发育为胚胎的效果,以Ringer氏液为稀释液,按1:30稀释大黄鱼和黄姑鱼精子,采用自制紫外灭活装置[紫外辐射强度2200μW/(cm~2·s),紫外波长254 nm]对这2种鱼的精子进行紫外照射处理及活力测定,然后与正常大黄鱼卵子进行人工授精,授精后一部分卵未作冷休克处理,另一部分卵进行了冷休克处理(受精2 min 30 s,3℃海水,冷休克10 min),并进行了早期胚胎成活率和仔鱼孵化率的测定与比较。结果显示:1)大黄鱼和黄姑鱼精子的激活率与紫外照射处理时间呈负相关,精子的快速运动时间变化呈典型的Hertwig效应。2)未冷休克组中大黄鱼和黄姑鱼诱导的早期胚胎成活率与精子的紫外照射时间总体呈负相关,而仔鱼孵化率呈Hertwig效应。3)冷休克组中大黄鱼和黄姑鱼精子诱导的早期胚胎成活率和仔鱼孵化率随紫外照射时间的增加呈Hertwig效应,分别于2 min 20 s和1min 30 s时达相对峰值,此时,大黄鱼早期胚胎成活率和仔鱼孵化率分别为(38.3±4.3)%和(66.5±5.1)%,黄姑鱼早期胚胎成活率和仔鱼孵化率分别为(43.3±3.3)%和(67.7±6.3)%。分析认为,大黄鱼和黄姑鱼精子遗传失活的紫外辐射剂量分别以308 m J/cm~2和198 m J/cm~2为佳。本研究旨在为大黄鱼雌核发育技术的改进提供依据。     

低渗溶液浓度对黄颡鱼精子活力和受精率的影响

为提高黄颡鱼(Pelteobagrus fulvidraco)人工授精的稳定性,分别用卵子和精子同步激活法以及预激活精子法,在6个梯度浓度(0%～0.5%)范围的低渗溶液中进行人工授精试验,比较精子活力、受精率和孵化率。结果显示:低渗溶液浓度与精子活力、精子激活率和受精率密切相关,在相同浓度的低渗溶液组中采用预激活精子法的受精率显著高于卵子和精子同步激活法(P<0.05),受精率相对应提高了7.7%～14.5%;低渗溶液的浓度和人工授精方式对孵化率无显著影响。结果表明在浓度0.3%的低渗溶液中采用预激活精子法的效果最好。     

Ultraviolet-induced androgenesis in Nile tilapia, Oreochromis niloticus (L.), and hybrid Nile × blue tilapia, O. aureus (Steindachner)

Fertilization of ultraviolet (UV) irradiated oocytes of Nile tilapia, Oreochromis niloticus (L.), with sperm from O, niloticus or blue tilapia, O, aureus (Steindachner), and subsequent suppression of the first cleavage of fertilized eggs successfully induced androgenesis in Nile and blue tilapia. The optimal doses of UV irradiation to denucleate a female genome of Nile tilapia prior to androgenesis ranged from 5940 to 6930 erg mm^?2 for 54-63 s at a fixed intensity of 110 erg mm^?2 s^?1. Putative androgenetic fish were created from eggs which were irradiated at various times and several durations of heat-shock. Eggs which were treated for 5 min at 41.6 ^oC at 2 7.5 min after fertilization were the most successful at suppressing the first cleavage and producing viable androgenetic diploids in Nile or hybrid Nile X blue tilapia. The maximal survivals of putative androgenetic diploids in relation to the control were 1.60% and 0.90% in Nile and hybrid Nile X blue tilapia, respectively. The androgenetic offspring established exhibited active feeding and normal growth.     

Growth and survival of olive barb,Puntius sarana (Hamilton 1822) larvae produced with cryopreserved versus fresh sperm

Despite the success in fertilization and hatching of fish eggs with cryopreserved sperm, report on growth and survival of larvae produced from frozen‐thawed sperm is inadequate. The study evaluates the applicability of cryopreserved sperm for mass seed production by comparing the growth and survival of a popular food‐fish olive barb, Puntius sarana (Hamilton 1822) larvae produced from cryopreserved and fresh sperm. The eggs were artificially fertilized with cryopreserved and freshly collected sperm, and the growth and survival of produced larvae from both group recorded up to 12 weeks. The independent sample t‐test statistic showed the difference in lengths, t(718) = 0.241; P = 0.810 and weights, t(718) = 0.412; P = 0.680 were insignificant between two groups. There was also no significant difference, t(718) = ?0.758, P = 0.448 in survival of larvae produced from cryopreserved and freshly collected sperm. The study indicates that larvae of olive barb produced from cryopreserved sperm are equally compatible in growth and survival as the larvae produced from fresh sperm. Therefore, cryopreserved sperm can be applied for artificial fertilization of P. sarana to supply quality seed for aquaculture.     

Artificial fertilization in turbot, Scophthalmus maximus (L.): different methods and determination of the optimal sperm–egg ratio

With the aim of improving artificial fertilization (AF) in turbot, Scophthalmus maximus (L.), a series of fertilization experiments was carried out under dry conditions and different wet conditions (eggs/sea water: 2V/V and V/V). Another series of fertilization experiments was carried out with different quantities of sperm pool to determine the optimal ratio of spermatozoa to eggs for each AF method. Sperm pool from two males and eggs from spawns with a viability rate of > 70% were used. The sperm pool’s density (0.4–5.18 × 10⁹ sperm mL^–1) and motility (1–5) had been assessed previously. Significantly different fertilization rates were found when comparing 2V/V and V/V wet conditions. Significantly higher fertilization rates were found in dry fertilization when the sperm–egg ratio was > 9000 spermatozoa per egg and, under wet condition V/V, at 3000–4000 spermatozoa per egg.     

Phenotypic trait of Crassostrea hongkongensis♀× C. angulata♂ hybrids in southern China

The Hong Kong oyster (Crassostrea hongkongensis) and the Portuguese oyster (Crassostrea angulata) are important aquaculture species in southern China. To understand the potential feasibility of hybridization between the two species, two‐by‐two factorial cross‐experiments were conducted in Shenzhen (Guangdong province). An asymmetry in fertilization was revealed: C. hongkongensis eggs could be fertilized by C. angulata sperm, but the reciprocal cross was not possible. The fertilization and hatching success in the C. hongkongensis females × C. angulata males (HA) cross was lower than the two intraspecific crosses. Moreover, hybrid larvae had a slower growth rate than those from parental progenies due to genome incompatibility. However, the size of the spat and adult growth were greater than those of the two parental progeny, with obvious heterosis during the grow‐out stage. The survival advantage of hybrids was examined at all times. The surprising finding was that all hybrids were completely fertile, meaning they could produce normal and functional gametes. Genetic analysis by molecular markers was applied to confirm that the HA spat were true hybrids. Our results revealed that the interspecific hybridization between C. hongkongensis and C. angulata is completely successful in one direction. Furthermore, hybrids are viable, fast growing and completely fertile, which provides a promising method for the genetic improvement of oysters as a new aquaculture stock in southern China.     

Captive breeding of climbing perch Anabas testudineus (Bloch, 1792) with Wova-FH for conservation and aquaculture

Induced breeding of climbing perch, Anabas testudineus was conducted by synthetic hormone Wova‐FH in the intensity level of 0.1, 0.2 and 0.3 mL kg^?1 of body weight respectively. The brooders were injected one time and left to spawn in the spawning hapa in the sex ratio between male and female as 2:1. It was found that at all the intensity level hormone Wova‐FH could enhance the fishes to breed and lay eggs whereas no breeding was observed in control set. The spawning time, quantity of the brooder spawn, fertilization rate, hatching rate and survival rate were quantified in each set of experiment. The egg output/female was significantly higher in 0.3 mL in comparison with 0.1 and 0.2 mL kg^?1 of body weight. The statistical analysis showed significant (P≤0.05) effect between hormone dose on fertilization rate, egg output and hatching rate. The present experiment suggests that Wova‐FH at the dose of 0.3 mL kg^?1 body weight of fish is more effective which might be considered for raising captive population.     

The Influence of Ovarian Fluid pH of Stripped Unfertilized Channel Catfish,Ictalurus punctatus,Eggs on the Hatching Success of Channel Catfish ♀ x Blue Catfish,Ictalurus furcatus♂, Hybrid Catfish Eggs

This study was designed to determine the effect of ovarian fluid pH of stripped unfertilized channel catfish, Ictalurus punctatus, eggs on fertilization and hatch rate of channel catfish ♀ x blue catfish, Ictalurus furcatus♂, hybrid catfish eggs. A significant correlation was established between ovarian fluid pH of stripped channel catfish eggs and hybrid embryo hatch rate (R² = 0.75, P = 0.01), suggesting ovarian fluid pH of stripped catfish eggs prior to fertilization can be predictive of the hatching success of hybrid catfish embryos. These data were used to categorize pH of stripped eggs: pH <7.0 as “low pH eggs,” pH 7.0–7.4 as “medium pH eggs,” or pH >7.4 as “high pH eggs” quality eggs. The range in percent hatch rate for these pH categories was <15%, 15–30%, and >30%, respectively. Higher calcium concentrations during incubation do not appear to improve hatching success of “low pH eggs.” The predictive model presented herein describes a quick method for evaluating stripped catfish eggs for hybrid fry production in catfish hatcheries.     

Induction of diploid gynogenesis in turbot <Emphasis Type="Italic">Scophthalmus maximus</Emphasis> with left-eyed flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis> sperm

Turbot Scophthalmus maximus exhibits sexually dimorphic growth, with females growing faster and reaching larger adult sizes than males. Thus, development of techniques for preferentially producing females is necessary to optimize production of these species. In this paper, gynogenetic diploids of turbot were induced by activating egg development with ultraviolet (UV)-irradiated left-eyed flounder Paralichthys olivaceus sperm combined with cold shock to prevent extrusion of the second polar body. The results of UV irradiation experiments showed that survival, motility, and duration of activity of P. olivaceus sperm generally decreased with increase in UV dose. The typical Hertwig’s effect was observed after fertilized turbot eggs with UV-irradiated P. olivaceus sperm and the optimal UV dose for gynogenetic haploid production was 36,000 erg mm⁻². At 15°C, appropriate timing of cold shock for retention of the second polar body in turbot eggs was at 6 min after fertilization. Results of different combinations of two shock temperatures (1 or 3°C) and four shock durations (15, 25, 35 or 45 min) at 6 min after fertilization demonstrated that shock of 25 min at 1°C gave the highest production of diploid gynogens (39.58% relative to its diploid control). The results of this study reveal that the use of UV-irradiated P. olivaceus sperm for activation of turbot eggs and cold shock for polar body retention is an effective method to produce gynogenetic offspring.     

Evaluation of different procedures for fertilization and larvae production in Hediste diversicolor (O.F. Müller, 1776) (Nereididae,Polychaeta)

Different experimental trials were performed to clarify some aspects of the biology of the polychaete Hediste diversicolor (O.F. Müller, 1776) as a further step towards the development of appropriate breeding protocols for indoor farming systems. In particular, the trials were addressed to evaluate the effectiveness of two fertilization conditions (in vitro and “natural‐like”); induce gamete spawning by exposing mature individuals to thermal shock or to tissue homogenates; estimate the density effects on larval growth and survival; and evaluate the most suitable parameters to be used as proxy for biomass assessment. The highest percentages of fertilized eggs and larvae were obtained by the in vitro fertilization condition. Mature organisms were induced to spawn by exposure to thermal shock although the spawned eggs revealed low rates of fertilization and hatching. The treatment with male tissue homogenates induced females to successful spawning, and the resulting eggs showed high fertilization and hatching rates. The density of larvae in the rearing phase had no effect on growth or on survival rates of juveniles. Finally, allometric evaluations showed that fresh weight and L3 length are the most reliable parameters to be used as proxy for biomass assessment of this species.     

Chemical surface disinfection of eggs of Baltic cod,Gadus morhua L.

The effect of two disinfectants on eggs and larvae of Baltic cod, Gadus morhua, was investigated. The eggs were disinfected for 10 min using various concentrations of either glutaraldehyde (100, 200, 400, 600 and 800 mg L^?1) or iodophor (10, 50, 100 and 150 mg L^?1), 1–4‐days post‐fertilization. Bactericidal effect of disinfection, survival to hatching, hatching success and larval abnormalities were assessed. Larval survival was recorded at 5‐, 10‐ and 15‐days post‐hatch (dph). Although Baltic cod eggs have an unusually thin chorion, they could tolerate surface disinfection. A reduction in bacterial growth was observed with increased concentrations of disinfectant (3.0 × 10⁷–1.6 × 10¹ CFU mL^?1). Abnormalities in newly hatched larvae were not related to disinfection. Survival of the yolk sac larvae was significantly better for eggs treated with 400 mg L^?1 glutaraldehyde for 10 min at 10 and 15 dph. Effective disinfection was also recorded using 100 mg L^?1 Actomar K30. Egg batch effect rather than initial bacterial concentration, disinfectant type or incubation method determined the survival of the eggs to hatching and survival of larvae. Because of the carcinogenic effect of glutaraldehyde, iodophor is recommended for routine disinfection of cod eggs.     

Larval growth of turbot, Scophthalmus maximus (L.) produced with fresh and cryopreserved sperm

The present paper assesses the fertilization and hatching rates, as well as the growth, of larvae obtained from four artificial fertilizations (AF) using fresh and cryopreserved sperm of the turbot Scophthalmus maximus (L.). Larvae growth in both sperm groups, measured in terms of length and weight at culture days 0, 7, 14 and 31, are compared, as well as their growth rates. The two groups' fertilization and hatching rates were not significantly different. Likewise, no significant differences in length and wet weight of 7‐ and 14‐day‐old larvae were found using fresh and cryopreserved sperm; however, significant differences were found in 31‐day‐old larvae, which were more attributable to the variability inherent in larval turbot culture, and to variability in the reproductive specimens used in our study, than to the type of sperm employed. These results indicate that the type of sperm used in artificial fertilization, i.e. fresh or cryopreserved, is not a determining factor, either for fertilization and hatching, or for subsequent larval development. Our results also confirm once again the high quality of cryopreserved turbot sperm, and its usefulness in commercial hatcheries.     

Changes in fertility of rainbow trout eggs retained in coelom

ABSTRACT:    Effects of prolonged retention time of ovulated eggs in the parental coelom on fertilization success were studied in rainbow trout Oncorhynchus mykiss using cryopreserved sperm with a uniform fertilizing ability. Proportions of successful fertilization, eyed eggs and hatched alevins were examined at different time periods up to a retention time of 14 days beyond the ordinary stripping time, and were compared with eggs incubated in artificial coelomic fluids (ACF). Eggs that were retained longer in the coelom showed gradual decreases in all the proportions of successful fertilization, eyed eggs and hatched alevins. The progress of cleavage after fertilization slowed with prolonged retention times. Eggs incubated in ACF lost their fertilizing ability much sooner than those retained in the coelom. The hatching rate of eggs retained for 2 weeks in coelom was 36%, while it was 1% in those eggs incubated for 4 days in ACF. Thus, eggs retained in the coelom showed higher fertilization success than those incubated in ACF.     

Development of cryopreservation for maintaining yellow catfish Pelteobagrus fulvidraco sperm

Yellow catfish (Pelteobagrus fulvidraco) is a candidate freshwater fish for aquaculture in China with its high consumer demand. The aim of this study was to examine the possibility of storage of the sperm of yellow catfish by cryopreservation in liquid nitrogen. Experiments were designed to investigate the effects of the different combinations of three extenders (Ringer extender, Kurokura-1 extender and D-15 extender) and three cryoprotectants (DMSO, Glycerol and Methanol) on the cryopreservation of yellow catfish sperm. Post-thaw sperm motility, fertilization and hatching rate were detected to evaluate the reliability of sperm cryopreservation. The results demonstrated that Ringer extender and 10% methanol was the best combination for protecting the sperm during freezing in liquid nitrogen by a three-step method and thawing in a water bath at 37 °C for 60 s. In this combination for cryopreservation, sperm maintained the highest post-thaw motility (65 ± 5%), fertilization (90.47 ± 3.67%) and hatching rate (88 ± 4%). And more interestingly, the fertilization and hatching rate were similar to those of fresh sperm (97.55 ± 2.74% and 92 ± 5%). Successful sperm cryopreservation techniques for yellow catfish have been developed for hatchery purpose.     

Storage of common carp, Cyprinus carpio L., eggs for short durations

In this study, a short-term storage of pre-activated eggs of the Japanese ornamental (koi) carp, Cyprinus carpio L., at three different temperature regimes is reported. Koi eggs were stored at low temperatures (6-9^oC), at variable or high temperatures (12-31C) and at moderate-stable temperatures (20-24.5^oC). Survival of the developing embryos was examined at the first and the second day post-activation, and in hatch-out larvae. Survival was calculated for each treatment by linear regression Y = a-bX (P± 0.01), except for eggs incubated at temperature of about 20 C(P > 0.1).The specific mortality index (b/a 100), as a ratio between the mortality rate (b) and the fertilization rate (a), indicated that the highest mortality is associated with the high and unstable storage temperatures, while the lowest mortality is with the moderate and stable storage temperatures. Eggs can be stored at moderate and stable temperatures for a maximal duration of 6h, yielding survival of hatch-out larvae higher than 50%. In two trials, fertilization potency of sperm stored in a domestic refrigerator (5-9^oC) for 5h, was compared with freshly stripped sperm and no differences in fertilization rates were found between the two treatments.     

Larval Development and Rearing of Longtooth Grouper Epinephelus bruneus in Jeju Island, Korea

Eggs and sperm were obtained from a female (6.3 kg/BW) and a male (8.4 kg/BW) longtooth grouper Epinephelus bruneus following HCG injection in July 2003. The eggs were fertilized artMcially with the sperm and incubated in one of two 50-m³ tanks after washing the fertilized eggs. The fertilized eggs were 830–950 pn (average 900 ± 2 μm) in diameter and the respective fertilization and hatching rates were 97.7 ± 0.6% and 96.8 ± 0.5% at a water temperature of 25.0 ± 0.5 C. With this regime, the survival rate by day 93 was 7.5% in the 50-m³ tank. The elapsed time from hatching to opening the mouth was 3 d at 25 C. The initial mouth size (z) of the larvae was 0.22–0.23 mm. The newly hatched larvae were 2.02 ± 0.02 mm TL; this increased to 4.12 ± 0.09 mm TL by day 11. By day 54, the larvae had metamorphosed into juveniles and reached 41.12 ± 1.20 mm TL, and by day 93 the juveniles reached 93.78 ± 1.98 mm TL. In all, 49.5% of the larvae were malformed and the type of malformation was diverse.     

Fertilization by refrigerator stored sperm of the Indian major carp, Labeo calbasu (Hamilton, 1822)

This study reports on the spermatological properties, and on the development of a protocol for refrigerator storage (4°C) of Labeo calbasu (Hamilton, 1822) sperm for artificial breeding. Volume, motility, concentration and pH of the freshly collected sperm were 2.21 ± 0.53 (μL g^?1 of fish weight) (mean ± SD), 95 ± 1 (%), 1.93 ± 0.44 × 10⁹ (cells mL^?1) and 7.56 ± 0.17 respectively. Sperm activation was evaluated at different osmolalities of NaCl solution, and motility ceased completely when osmolality of the extender was ≥287 mOsmol kg^?1. Sperm retained motility for 24, 72 and 108 h, after refrigerator storage when sperm were undiluted, suspended in Alsever's solution and suspended in Alsever's solution containing 5% methanol respectively. Fertilization rate of fresh eggs with sperm stored at 4°C in Alsever's solution and Alsever's solution containing 5% methanol was 77% and 60% with a hatching rate of 60% and 43% respectively. The fertilization and hatching success of the stored sperm suggests potential to use refrigeration for transporting genetic material to hatcheries for artificial breeding of L. calbasu in Bangladesh.     

Long-term effects of the cryopreservation of turbot (Psetta maxima) spermatozoa

The survival of turbot eggs and the rearing capacities of larvae stemmed from artificial fertilization practices using frozen-thawed spermatozoa were evaluated. Furthermore, the viability of sperm samples stored during a 9 month period in liquid nitrogen was assessed. No significant difference in the fertilization rate, hatching rate, survival and wet weight of 10-day old larvae were observed using fresh or frozen-thawed spermatozoa. The motility recorded at 10 s and 60 s post-activation and the fertilization capacity of frozen-thawed spermatozoa were not significantly decreased during a 9 month storage period in liquid nitrogen. These results confirm the high quality of the turbot spermatozoa stemmed from the cryopreservation process, allowing their use for routine aquaculture practices.