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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]

The present study was carried out to investigate the embryonic and larval development of freshwater catfish Rita rita. The mature eggs and sperms were collected by using artificial insemination technique and fertilized eggs were incubated in mini circular hatchery with provision of continuous water supply. The fertilized eggs were transparent, demarsal, spherical, non-adhesive and brownish in colour with a diameter ranging between 1.3 to 1.6 mm. First cleavage occurred within 25-30 min post-fertilization at temperature of 28±1°C. Hatching started 22 h post-fertilization and completed within 24 h at the same temperature range. Newly hatched larvae were 2.0 mm in length devoid of mouth and pigmentation and started feeding within 48-60 h post-hatching. To date, this is the first time the early embryonic and larval development of freshwater catfish R. rita is described. Thus the findings of the present study provide valuable information that may help establishing the large scale seed production technique of Rita. [ABSTRACT FROM AUTHOR]

Biodiversity is the basic indicator, which expresses the durability and stability zone in direct relationship with the environment and life. Biodiversity monitoring both quantitatively and qualitatively puts us in contact with reality environment and its stability, because biodiversity is constantly changing. Only by measuring the biodiversity we can perceive the sudden chances, which influence directly our life in order to interfere with redesigning the media by ecological reconstruction. The application of biotechnologies to buffalo breeding is paramount to remedy the existing situation. The most numerous Romanian buffalos are the Carpathian type, with valuable gene resource, well adapted to the cold climate for buffalo. Results of artificial insemination in this species are influenced by the quality of semen used. We know that there are periods in which sperm are immobile, so it is important to test the ability of sperm fertilized. This test can be performed in vitro using direct or indirect methods. Utilization of Polamski scale principle remains present, but can be improve and combine with Milovanov resistance and "in vitro" capacitating methods. For determination of fertilizing power we use "in vitro" capacitation methods: direct method, indirect method and "swim-up" technique who are obligatory in E.U. We use this tests because Polamski scale can't be applied, knowing that an immobility of buffalo semen in some conditions is a characteristic of this semen, semen that can be recovery on the normal parameters. Milovanov resistance test have restricted efficiency because NaCl solution have a negative effect over the sperms. Semen charges testing with these new methods they present a good fertilizing power. This test is easier to realize and takes of decision is faster. [ABSTRACT FROM AUTHOR]

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]

The role of monovalent (Na+, K+) and divalent (Ca2+, Mg2+) cations in Bufo arenarum fertilization was analysed. Our results showed that the highest fertilization percentages were obtained when strings of uterine oocytes (UO) were inseminated. Under these conditions, full jelly (FJ), which represents the jelly coats surrounding the oocytes at the time of deposition, contained 68.5 ± 7.0 mM Na+, 27.4 ± 2.4 mM K+, 6.3 ± 0.9 mM Ca2+ and 6.9 ± 0.9 mM Mg2+. When the strings of oocytes were washed in deionized water, these cations diffused into the liquid medium surrounding them. There was a marked similarity between the loss of Ca2+ in the jelly and the decrease in the fertilizability of the UO. Furthermore, the use of chelating agents of divalent cations showed the importance of the Ca2+ contained in the jelly. When Ca2+ was sequestered from the jelly coats by the addition of the chelating agents to the insemination medium as well as by pretreatment of the UO strings, a decrease in fertilization percentages occurred, this effect being dose dependent and more markedwith EGTA. These results demonstrate that the Ca2+ in the jelly plays a role in fertilization. Nevertheless, taking into account that during the washing of the jelly other jelly coat components were diffused and considering that the addition of Ca2+ to the insemination medium reverted significantly, but only partially, the loss of fertilizability of jellied UO (washed), the participation of other components in the fertilization mechanism is suggested. [ABSTRACT FROM AUTHOR]

The present study investigated the embryonic and larval development of Indian catfish, Heteropneustes fossilis from fertilization until metamorphosis. The fully matured eggs and sperm were obtained by artificial insemination. The fertilized eggs were demersal, nonadhesive, spherical, and brownish green in color. The average diameter of the fertilized eggs ranged from 1.30 to 1.50 mm. The incubation period was from 23-24 h at an average temperature of 29±1°C. The newly hatched larvae were 2.5±0.2 mm in length. The yolk absorption was completed within three d after hatching. The aerial breathing behaviour of the larvae was observed 10 d after hatching. The larva resembled the adult in its external features and was metamorphosed to young juveniles within 20 d post-hatching. [ABSTRACT FROM AUTHOR]

Experimental insemination was performed using artificially produced low-motility sperm. A mathematical model was applied to the results of the insemination in order to clarify the relationship between sperm motility, the density of sperm and the fertilization rate of eggs. In the model, the probability of fertilization by individual spermatozoa was a function of sperm density in the insemination solution. The results showed that the probability of fertilization clearly decreased with increased sperm density, and the maximum possible fertilizing rate by increasing the sperm density was constrained by the proportion of motile sperm (% motility). The model was also applied to the results of insemination tests of cryopreserved sperm in order to evaluate the fertilizing capacity of cryopreserved sperm. It was proven that cryopreserved sperm needed a higher density to obtain the maximum fertilization rate compared with fresh sperm, and it was anticipated that the ratio of the motile inseminated cryopreserved sperm should be more than 5.0% to achieve an egg fertilization rate greater than 90%. [ABSTRACT FROM AUTHOR]

The authors detail an experiment establishing the impact of insemination times on the fertilization rate and hatch rate of eggs of obscure puffer fish, Takifugu obscurus. Fertilization rates in highly matured ova are low. The authors conclude that insemination must be done within 4 hours of ovulation for better results.

To develop an appropriate cryopreservation protocol for spermatophores of black tiger shrimp, Penaeus monodon, three cryoprotectants (dimethyl sulphoxide (DMSO), methanol (MeOH) and ethylene glycol (EG)) at two concentrations (5% and 10%) were examined. Artificial implantation of spermatophores was also carried out to assess the fertilizing ability of fresh and post-thaw spermatophores. Spermatophores were collected during consecutive regenerations (15-day intervals) and assessed for qualitative and quantitative changes and also for fertilizing ability by implantation. The mean fertilization rate for artificial insemination using post-thaw spermatophore was 79.9±3.7%, lower than the fertilization rates observed for artificial implantation using fresh spermatophore and natural mating. Mean hatch rates for fresh spermatophore, frozen-thawed spermatophore and natural mating were 88.8±0.6%, 87.8±0.4% and 88.3±0.5%, respectively; and there was no difference among the three groups. The mean fertilization rate of spermatophores collected during the first stripping was higher (90.6±0.6) than during the second stripping (85.7±2.6), but the mean hatch rate was not different between the two strippings. The highest mean sperm viability (79.7±0.4%) was obtained from DMSO (5%), with no survival observed in the 10% MeOH treatment. Spermatophore weight, total sperm count and percentage of abnormal sperm were not different between spermatophores collected at the first and second stripping. This is the first study to report high fertilization and hatch rates from cryopreserved spermatophore using artificial implantation of spermatophore before spawning. [ABSTRACT FROM AUTHOR]

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]

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The silkworm, Bombyx mori, has a dimorphic sperm system. The eupyrene sperm is the sperm to fertilize eggs and the apyrene sperm plays a crucial role for assisting fertilization. Heat-treated (33°C for 96h) Daizo (DH) males, one of the strains in the silkworm, produce only eupyrene sperm, while in triploid males only apyrene sperm are functional. Though both types of males are found to be sterile, double copulation of the two males with a single female greatly increases fertility. Here we examined the fertilizing ability of eupyrene and apyrene sperm by means of an artificial insemination technique previously established in B. mori. Neither the eupyrene sperm collected from DH males, nor the apyrene sperm from triploid males have the ability to fertilize eggs. Artificial insemination with the mixture of eupyrene and apyrene sperm leveled up the frequency of fertilized eggs to more than 80%. When cryopreserved DH sperm (eupyrene sperm) were subjected to the same experiment, more than 95% fertilized eggs were obtained. These results confirmed that apyrene sperm play an important and indispensable role in fertilization in B. mori. Separate collection of functional eupyrene sperm and functional apyrene sperm and success of fertilization by means of the artificial insemination technique are applicable for further studies to elucidate the function of apyrene sperm. J. Exp. Zool. 297A:196–200, 2003. © 2003 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Triploidy was induced in the fighting fish, Betta splendens Regan, by varying all possible combinations of temperature (37–41°C), time after insemination (2–3 min) and shock duration (2–4 min). Heat shock at 39°C for 3 min duration initiated 2.5 min after insemination gave high frequencies of triploids (86%) as assessed from chromosome number and red blood cell nuclear volume. There was no significant difference in the growth rate of triploid and diploid fish. Gonadal development in both sexes was retarded in triploids at 5 months of age. Eggs fertilized with milt from triploids developed to gastrulation. Beyond gastrulation there was increasing mortality associated with abnormalities and none of them hatched. The display frequencies of air gulping, erection of operculum and fins, striking and biting, and undulating movements were fewer in triploids compared to diploids. It appears that triploids are less aggressive than diploids. The aggressive behaviour of fighting fish may be related Io their reproductive activity. [ABSTRACT FROM AUTHOR]

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]

In addition to gametes, mammalian internal fertilisation has required the evolution of assorted anatomical, physiological and biochemical devices to deal with intra- and inter-sexual conflict such as sperm competition and female cryptic choice respectively. The seminal coagulum of primates and other mammals is viewed as one of such devices. Among primates, the seminal coagulum characteristically occurs in multi-male and multi-female species, leading us to suppose that it intervenes in sperm competition. However, it can also provide cues to the female reproductive tract about male desired or undesired traits, and therefore deter or favour sperm survival and migration. The present work investigates whether the seminal coagulum of the black-handed spider monkey enhances sperm fertilisation chances by improving the female reproductive tract conditions, and if the female reproductive tract is 'blind' to semen or behaves selectively towards ejaculates of different males. A series of artificial inseminations were done in five females, using the ejaculates of three different males, one at a time, and measuring the presence of distinct types of sperm inside the uteri at 10, 30 and 60 min following the insemination. The presence of coagulum, menstrual phase, and male and female identity only affected fast, straight-moving sperm, with larger amounts of fast sperm appearing inside the uteri when ejaculates had seminal coagulum, as well as when in the periovulatory phase. There was great intra-uterine fast-sperm variation regarding which male's semen inseminated which female. The results provide evidence to account for sexual conflict in the spider monkey as well as a methodological approach to this kind of study. [ABSTRACT FROM AUTHOR]