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Abstract: Tetraploidy is induced in rainbow trout by applying a pressure shock at a specific time point between insemination and first cleavage, or the first cleavage interval (FCI). Previous studies suggested that variation in the FCI among individuals and populations of fish prevents the identification of a single time point that can be used for all trout. In this study we confirmed the optimal time to apply pressure is 65±5% of the FCI. In addition, we found that variation in FCI of fish from a common environment can be within limits that allow a single time point to be established for that group of fish, if ova post ovulatory aging is taken into account. Aging of ova, either in vivo or in vitro, increased FCI to a degree that is a concern for tetraploid induction. The FCI was about 12min longer at 7days post ovulation, and 30min at 10–14days, than at 1day. The FCI for a group of fish was consistent throughout the spawning season. Survival to hatching and frequency of spinal abnormalities were similar for progeny of first and second generation tetraploid males, but survival was doubled and abnormalities reduced by approximately 90% in second generation tetraploid females compared with first generation females. All progeny of tetraploid by tetraploid crosses were determined to be tetraploids based on flow cytometry of embryonic cells. In summary, attention to ova aging and use of second generation female tetraploids allows efficient production of a tetraploid rainbow trout broodstock. [Copyright &y& Elsevier]

Abstract: Discriminating the male influence on egg fertilization in Penaeus monodon is needed so that accurate measures of male fertility can be developed and subsequently used to evaluate methods aimed at improving fertility. In this study, we employed a novel, pairwise comparison approach to discriminating the fertilization influence of males: female broodstock were artificially inseminated with one spermatophore each from a pair of males. We achieved 22 successful ‘spawnings’ involving selected pairs from 33 males, and estimated the proportion of embryos fertilized by each paired male by individual genotyping of embryos. The non-inseminated, ‘twin’ spermatophore was used to estimate measures of male fertility; sperm number and the number of normal sperm of each inseminated spermatophore. Relationships between measures of male fertility and egg fertilization were then evaluated. Using this approach we were able to show that the egg fertilization potential of males could not be determined simply by total sperm number or the number of normal sperm (as determined by gross morphology under light microscopy). We also found extremely high variability in fertilization between paired males. The results highlighted male infertility issues (e.g. sperm viability) that are not simply overcome by greater sperm numbers. Our novel approach provides a method that can be used to determine whether more complex measures of male fertility can reliably predict egg fertilization potential. [Copyright &y& Elsevier]

Abstract: A simple technique is described for isolating DNA from individual embryos or nauplii of the Black Tiger shrimp, Penaeus monodon. The protocol used a commercial prepGEM™ insect DNA extraction kit in combination with water bath ultrasonic disruption. DNA quality and yields were sufficient for multiplex PCR discrimination of up to six DNA microsatellites in a single test. The utility of the method was demonstrated in defining the parentage of 1279 embryos (7h old) spawned from 21 females and 684 newly-hatched N1 to N2 nauplii spawned from 12 females each inseminated artificially with the spermatophores of two different males. Overall, parentage assignments based on the six microsatellite markers were deduced to be 89.5±6.7% and 93.0±4.4% effective using DNA isolated from individual embryos and nauplii, respectively. The technique is simple, cost-effective and should provide a robust means of isolating DNA from individual embryos and nauplii of aquatic invertebrates suitable for PCR-based genotyping. [ABSTRACT FROM AUTHOR]

Abstract: The feasibility of cross-breeding of Pacific white shrimp, P. (Litopenaeus) vannamei (PV) and blue shrimp, P. (Litopenaeus) stylirostris (PS), by means of artificial insemination, was evaluated. Intra-specific (pure species) crosses were used as control treatments: 1) ♂PV×♀PV and 2) ♂PS×♀PS. Reciprocal, inter-specific (hybrid) crosses 3) ♂PV×♀PS and 4) ♂PS×♀PV, were performed employing a variety of artificial insemination techniques. Twelve intra-specific inseminations of white shrimp (♂PV×♀PV) were performed. All inseminated females spawned eggs, ranging from 70,111 to 139,940 eggs per spawn. Five out of the 12 spawns produced nauplii (14,241 nauplii per spawn), with a mean hatch rate of 11.7%. For intra-specific blue shrimp crosses (♂PS×♀PS), 13 out of 14 inseminated females spawned eggs ranging from 112,349 to 282,141 eggs per spawn, and obtaining three fertile spawns with a mean hatch rate of 12.8% and 24,588 nauplii per spawn. For the inter-specific crosses, a total of 49 hybridization attempts were made, 27 for the cross ♂PV×♀PS and 22 for the reciprocal cross ♂PS×♀PV. In spite of trying artificial insemination in various ways, none of the hybridization attempts was successful; indicating that cross-breeding of white and blue shrimp is not possible by means of traditional artificial insemination techniques. [Copyright &y& Elsevier]

This paper reports on cryopreservation of sperm from turbot (Scophthalmus maximus). The effects of various extenders, cryoprotectants and sperm–egg insemination ratios on motility score and/or fertilization capacity of post-thaw turbot spermatozoa were examined to optimize cryopreservation procedures. Post-thaw motility of frozen sperm obtained with extender TS-2 was higher than those achieved with extenders D-15 and modified plaice Ringer solution (MPRS). The most effective cryoprotectant was determined to be 10% dimethyl sulfoxide (DMSO). Fertilization of small egg batches (2 ml eggs) with frozen sperm resulted in average fertilization rate (FR) (70.1±8.9%) and hatching rates (HR) (46.8±5.2%) similar to the fertilization rates (74.7±8.0%) and hatching rates (47.5±6.8%) of fresh sperm. The minimal density of frozen sperm required to obtain satisfactory fertilization rate was determined to be 2000:1 (sperm/egg). Fertilization of larger egg batches (40 ml eggs) with sperm frozen in 1.8-ml cryovials provided similar fertilization rates (71.6±7.3%) to that of the fresh sperm (76.2±5.7%), whereas the hatching rates (34.7±3.6%) of eggs fertilized with frozen sperm were slightly lower than those of fresh sperm (41.1±3.7%). Our results demonstrated that cryopreservation technique for turbot sperm in 1.8-ml cryovial could be used for hatchery purposes. [Copyright &y& Elsevier]

The present work optimizes procedures for artificial insemination of European catfish, Silurus glanis L., with goals to increase collection of sperm and hatching rate. We address technical aspects of immobilizing solution (IS), activation solution (AS), process of insemination, activation of gametes and elimination of eggs stickiness with a practical solution for artificial propagation in hatcheries. Spermiation was stimulated and could be sustained over a period of 1 month after weekly carp pituitary (CP) injection of 5 mg kg-1 b.w. Males produced significantly the largest quantity of sperm, 0.12–0.13·109 spermatozoa kg-1 b.w., after third and fourth injections. The best IS for sperm was determined as solution containing 170–200 mM NaCl, 30 mM Tris–HCl, pH 7 and activation solution for activation of gametes containing 17 mM NaCl, 5 mM Tris–HCl, pH 7. The ratio of 1:2 rather than ratio of 1:1 in the control batch (volume of activation solution in milliliters/grams of eggs) increased the hatchability up to 68%. The highest hatching success of 82–88% from four individual females were found for sperm number of 800–80,000 spermatozoa egg-1, in comparison to 26% hatching at 80 spermatozoa egg-1. The best time for application of alcalase enzyme for elimination of egg stickiness was 3 min after gamete activation at concentration of 20 ml enzyme l-1 with exposure of 2 min. [Copyright &y& Elsevier]

Viable F1 hybrids were obtained from crosses of female Macrobrachium nipponense and male Macrobrachium hainanense involving spermatophore transfer and artificial insemination. This represents the first successful known case of hybridization of two Macrobrachium species by means of artificial insemination. The hatching rate was over 90%. About 20–60% of newly hatched larvae metamorphosed to postlarvae. The morphological characteristics of the hybrids resembled a combination of features of both parents. Malate dehydrogenase (MDH) and esterase (EST) isozyme electrophoresis indicated parents and F1 hybrids showed co-dominant expression of the paternal and maternal alleles controlling the isozymes and confirmed the hybridization. [Copyright &y& Elsevier]

We developed a simple, easy method with a microplate to artificially incubate Japanese crayfish (Cambaroides japonicus) eggs for their cultivation. We prepared 6-, 12-, 24-, 48-, and 96-well microplates containing sterile water heated to 5, 10, 15, and 20 °C. Fourteen experimental groups for each water temperature were prepared for each of different water volumes (0.125–10 ml) in each well. One embryonic egg was placed in each well. Experiments were also conducted with water collected from the lake where the eggs were harvested from and held at 15 °C. In the microplates with sterile water, high proportions of eggs hatched (60–100%) at 15 °C in all volumes of water, although the proportions of hatching were low (0–20%) at 5, 10, and 20 °C. All eggs died in the experiments that used lake water. We conclude that the 96-well would be the most effective size to hatch crayfish eggs in, because of its convenience. This method using a microplate is simpler and easier compared with methods of previous studies to artificially incubate crayfish eggs, and therefore it might be useful to incubate eggs of other freshwater crayfish species. [Copyright &y& Elsevier]

The application of artificial insemination techniques as a way to overcome the lack of mating of the closed thelycum Farfantepenaeus paulensis under large scale conditions is reported. The performance of artificially inseminated females (49.4±11.6 g) maintained in conventional versus unisex maturation systems was compared. Groups of unilaterally eyestalk-ablated females were artificially inseminated with one compound spermatophore soon after molting. The percentage of fertilized spawns increased from 26% before the use of artificial insemination to 57% afterwards. The reproductive performance of inseminated females held in conventional or unisex maturation systems showed no significant differences. Artificial insemination was considered a practical approach to overcome the lack of mating of F. paulensis under large-scale conditions with acceptable reproductive performance. The results also indicate the possibility of holding females separately from males, which could mean significant improvements in maturation systems through the increase in the stocking density of spawning females. [Copyright &y& Elsevier]

Eggs of the Japanese eel, Anguilla japonica, were obtained by hormonally induced maturation and ovulation, and then categorized as buoyant and non-buoyant types in seawater after insemination. The water content of the buoyant eggs, 93.1%, was significantly higher than that of the non-buoyant eggs, 87.5%. Furthermore, the buoyant eggs had 1.5 times more free amino acids (FAA), 573.5 μmol/g dry weight (DW), than the non-buoyant eggs, 385.8 μmol/g DW, while no significant difference in egg lipid content or protein content was found. Compared to other pelagic eggs reported in the literature, the FAA content of the buoyant eggs was relatively low, although the water content was similar. These results suggest that there is a close relation between the quality of Japanese eel eggs obtained by typical hormonal inducement and the FAA content of the eggs, but the roles of FAA in the acquisition of egg buoyancy and fulfilling the early nutritional requirements are comparatively minor. [Copyright &y& Elsevier]

Abstract: Artificial inseminations using sperm masses were applied to intra-specific crosses of Penaeus (Litopenaeus) occidentalis, as validation of the technique. Reciprocal, inter-specific crosses of P. occidentalis and P. (Litopenaeus) stylirostris were performed; complementary, inseminations of both species were also accomplished with sperm masses of P. (Litopenaeus) vannamei. Intra-specific inseminations generated viable spawns with 54.2% of normal embryos; on the contrary, inter-specific spawns were all unfertile, with a high rate of eggs showing abnormal intracellular corpuscles. Based on recent discoveries about sperm maturation/capacitation, a new approach to study inter-specific sperm–egg compatibility in the sub-genus Litopenaeus is discussed. [ABSTRACT FROM AUTHOR]