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1.
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.  相似文献   

2.
The eastern oyster Crassostrea virginica can change sex which makes self‐fertilization possible if sperm can be cryopreserved. In this study, small (~1 year old) and large (~2–3 years old) oysters were biopsied for sperm collection. Survival of the biopsied oysters after 1 year was 50% for small oysters and 17% for large oysters. Oocytes were collected from sex‐reversed females, and self‐fertilized with cryopreserved sperm. Of the 24 cryopreserved samples, 14 individuals had ≤1% fertility when crossed with oocytes from unrelated females, indicating that the cryopreserved sperm had reduced fertility. The other 10 individuals had a fertility of 39 ± 25% when crossed with oocytes from unrelated females (non‐selfing), but showed a significantly lower success of self‐fertilization (12 ± 16%) (P = 0.008), while aliquots of the same oocytes had a fertilization of 83 ± 11% when crossing with fresh sperm. Larvae were produced at day 3 in the self‐fertilized families (12–94% of the fertilized oocytes), and survived to eyed‐larvae stage at days 11–14. Genotyping with 9 microsatellite markers confirmed that the larvae resulted from self‐fertilization in four families. This study demonstrated the feasibility of creating self‐fertilized inbred lines of oysters by use of non‐lethal sperm collection and cryopreservation.  相似文献   

3.
Three cryoprotectants (dimethyl sulphoxide, propylene glycol and glycerol) and two diluents (sucrose based and saline based) were mixed (9 parts diluent–1 part cryoprotectant) factorially to produce six extenders that were tested to develop an effective sperm cryopreservation protocol for winter flounder Pseudopleuronectes americanus (Walbaum). Sperm were diluted 1:3 with each extender and frozen by flotation on liquid nitrogen before being submerged and stored for 30 days. Sperm left unfrozen in each extender for 20 min showed no toxic effects on motility. Extenders containing propylene glycol (PG) as cryoprotectant yielded higher post‐thaw sperm motilities than those containing dimethyl sulphoxide (DMSO) or glycerol. The sucrose‐based diluent performed better than the saline‐based diluent when DMSO was used as cryoprotectant, but there were no differences in post‐thaw motility between diluents for the other cryoprotectants. Activating sperm with ovarian fluid and sea water instead of sea water alone had no effect on post‐thaw motility. In fertilization trials, no differences were observed between any of the extenders and fresh milt when milt, eggs and sea water were left in contact for 1 h. When sperm were forced to compete for eggs by reducing contact time to 20 s, fertilization results followed those of sperm motility rates. Percentage hatch and morphology of larvae at hatching did not differ for eggs fertilized by cryopreserved and fresh sperm. This study represents the first reported successful attempt at cryopreserving winter flounder sperm and should improve gamete and broodstock management protocols for this species.  相似文献   

4.
Valuable aquaculture and fisheries industries for the North American geoduck, Panopea generosa, have driven interest in developing similar ventures for other geoduck species including P. zelandica from New Zealand. However, little is known about the fertilization kinetics of this species, or the conditions under which the amount and quality of larvae can be maximized. We determined the effects of sperm concentration, gamete age and contact time on the fertilization success of P. zelandica using an extended‐Vogel‐Czihak‐Chang‐Wolf (EVCCW) model. The model provided a good fit to laboratory data when applied to individual contact times. For a contact time of 10 s, optimal fertilization was achieved at concentrations of approximately 104 sperm μL?1, but this decreased to as little as 102 sperm μL?1 for contact times of several minutes. Optimal fertilization was always <100% (max. observed 70%) and the proportion of fertilized eggs decreased rapidly at sperm concentrations above the optimal. According to the model this was due to a slow block to polyspermy. If commercial hatchery facilities ensure that broodstock are in ripe condition, and use sperm <30‐min old, then optimal fertilization can be expected at sperm densities of 102–103 sperm μL?1 at contact times of 5–10 min.  相似文献   

5.
An experiment was conducted to investigate the effects of dietary biotin on zebrafish Danio rerio reproduction. Biotin and avidin (biotin antagonist) were added to two isonitrogenous and isocaloric purified diets to provide molar avidin:biotin ratios of 0:1 (biotin‐sufficient diet) and 120:0 (biotin‐unsufficient diet). Each diet was fed to a group of males mean initial mass 0.262 g) and a group of females (mean initial mass 0.285 g) for 99 days. Following this feeding period, males fed biotin‐sufficient diet exhibited higher gonado‐somatic index, sperm density, sperm motility and sperm viability than those fed biotin‐unsufficient diet (P < 0.05). In the presence of biotin‐sufficient males, biotin‐sufficient females spawned more eggs (222.2 eggs) than biotin‐deficient females (18.8 eggs) (P < 0.05). The same pattern was observed with biotin‐deficient males (7.6 vs. 1.8 eggs) (P < 0.05). Biotin‐sufficient males generated a higher percentage of fertilized eggs (90% vs. 42%), hatching rate (62% vs. 27%), larvae survival (98% vs. 37%) and larvae length at 7 days post fertilization (4.4 mm vs. 4.2 mm) than biotin‐deficient males (P < 0.05) . Biotin status of the male is of high consideration for successful breeding in zebrafish, because it significantly impacts the reproductive performances of the female.  相似文献   

6.
High rates of unexplained mortalities (up to 70%) are anticipated in the cultured juvenile sea bass (Dicentrarchus labrax) in farms in the eastern Black Sea at least for the last 8 years. Diplectanum aequans, often blamed for the observed mortalities, is the only prevalent parasite impacting the health of sea bass in the brackish water of the Black Sea. To investigate the seasonal fluctuation of D. aequans prevalence, mean intensities and the potential effects of D. aequans on the fitness of sea bass, eight cages from one farm (Farm A) and four cages at another distinct site (Farm B) were surveyed monthly from May to October of 2008. Six hundred and sixty‐one juvenile sea bass from Farm A and 236 from Farm B were individually examined for the presence and intensity of the parasite. The prevalence of D. aequans in cultured sea bass (96.3 ± 11.11; Mean ± SD) ranged from 66.7% to 100% in June, the beginning of 6‐month growing season. The mean intensity was 6.08 ± 2.19, significantly higher than that (2.74 ± 0.87; P < 0.05) in December (P < 0.05), the end of the growing season. Fish with low fitness had significantly higher number of parasite than the fish with higher fitness (P < 0.005). Although D. aequans negatively influenced host's condition factor, the impact was not enough to lead mortalities in the cultured sea bass in the Black Sea.  相似文献   

7.
The characterization of sperm motility patterns, particularly post‐activation changes, is the first step in setting up species‐specific protocols involving gamete management and embryo production, for both aquaculture and laboratory research purposes. This study is aimed at the characterization of the sperm motility pattern of the purple sea urchin Paracentrotus lividus. Semen samples were individually diluted in artificial sea water for sperm motility activation. They were then incubated at 18°C for up to 24 hr. Motility was evaluated on dilution, and 1 hr, 3 hr and 24 hr after activation, by computerized analyser. The semen fertilization capacity was also evaluated. Under our experimental conditions (dilution 1:1,000 in artificial sea water plus 0.05% BSA, 18°C, in the dark), P. lividus semen remained viable for up to 24 hr, as the total motile sperm and the fertilization percentages did not change significantly during the incubation time. In contrast, the mean curvilinear velocity and the subpopulation of rapid sperm (those having a curvilinear velocity > 100 µm/s) slightly but significantly decreased after 3 hr, thereafter remaining unchanged for up to 24 hr after activation. In conclusion, our results show that diluted P. lividus semen can be used for a longer period than that of most fish species, with no need for motility inhibition procedures, supporting its wider use in laboratory research. In addition, the development of artificial fertilization protocols for aquaculture production is simplified by long‐lasting sperm motility.  相似文献   

8.
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.  相似文献   

9.
This study developed a technique of sperm cryopreservation using liquid nitrogen (LN) vapour in farmed blacklip abalone Haliotis rubra through evaluating the following five key factors: (1) cryoprotectant agent (CPA) toxicity; (2) cooling temperature; (3) thawing temperature; (4) sperm to egg ratio and (5) sugar addition, using sperm motility or fertilization rate as quality assessment indicators. The results demonstrated that 6% dimethyl sulfoxide (DMSO) was the best single CPA for sperm cryopreservation in this species. The highest post‐thaw sperm motility was achieved when sperm were exposed to LN vapour for 10 min at 5.2 cm above the LN surface and thawed at 60°C and recovered at 16°C in seawater baths. Post‐thaw sperm motility was found to be significantly higher when 6% DMSO was used in combination with 1% or 2% glucose than 6% DMSO alone. Further evaluation of fertilization rate between these CPAs showed that 6% DMSO+2% glucose achieved the highest fertilization rate of 70% at a sperm to egg ratio of 10 000:1.  相似文献   

10.
We developed both a cryopreservation method for Japanese sea cucumber spermatozoa and an artificial fertilization method using post‐thaw spermatozoa. Twenty per cent dimethyl sulfoxide (DMSO), 16% foetal bovine serum, and 64% artificial seawater were suitable cryodiluent, and the diluent was pre‐cooled to 0°C. Semen was diluted with the solution and enclosed in a 250 μl straw, cooled to ?50°C at 10.4 ± 0.4°C/min, and immediately immersed in liquid nitrogen. Although this method showed the highest post‐thaw motility in all the conditions we examined, its post‐thaw motility was still less than approximately 15%. Artificial fertilization was carried out by adding post‐thaw semen with a cryodiluent to the oocytes. The fertilization rate of 200 oocytes/ml seawater increased with the amount of post‐thaw semen from 1 to 5 μl but showed a significant decrease at 25 μl. This decrease was considered to be due to DMSO in the cryodiluent, because the fertilization rate of the fresh semen decreased sharply when the DMSO concentration around the oocytes was 1.0% or more. Further improvement in increasing post‐thaw motility and lowering the cryoprotectant concentration is necessary for commercial‐scale artificial fertilization.  相似文献   

11.
The objective of this study was to assess sperm DNA longevity in blue mussels (Mytilus edulis) using a dynamic assessment of sperm DNA fragmentation (SDF) after sperm activation. Mature blue mussels (n = 57) in Vigo (Galicia, Spain) were obtained, specifically rope farmed blue mussels (n = 38) and wild blue mussels (n = 19). After the sperm collection, a subsample was assessed for SDF (0 h), while the rest of the sample was incubated for 6, 9, 12, 24 and 48 h at 15°C, assessing each time point using the Sperm‐Halomax kit (Halotech DNA, Madrid, Spain). The Kaplan–Meier estimator, log‐rank (Mantel–Cox) test and Mann–Whitney U‐test were used for statistical analyses (spss v. 16.0), α = 0.05. The rate of SDF (r‐SDF) between rope farmed and wild blue mussels over 0–6 h incubation was not significantly different (P = 0.278), but was for 6–24 h (P = 0.004). Differences in r‐SDF were observed when comparing the means between the two groups (P < 0.0001). Individual differences in r‐SDF existed among the rope farmed (P < 0.0001) and wild blue mussels (P < 0.0001). Wild blue mussels presented a higher DNA longevity than the farmed blue mussels. Selection of blue mussel males with a low level of sperm DNA damage and greater sperm DNA longevity may result in better fertilization and seed production.  相似文献   

12.
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.  相似文献   

13.
A 45‐day trial was performed to evaluate the effect of biofloc technology (BFT) with or without fresh food (FF) supplementation during pre‐maturation period on Farfantepenaeus duorarum spawning performance, biochemical composition and fatty acid profile of eggs as compared with conventional clear‐water system (CW+FF). Females raised in biofloc and that received FF supplementation (FLOC+FF) achieved better spawning performance in terms of number of eggs per spawn (49 × 103), number of eggs per spawn per g of spawner's body weight (2.1 × 103) and egg size (~275 μm) as compared with CW+FF (23 × 103, 1.1 × 103 and 263 μm respectively), but both treatments did not vary from FLOC (P > 0.05). High spawning activity was also observed in biofloc system as compared with clear‐water system as shown in number of spawns per ablated female (2.2–3.0 versus 0.6) and percentage of females that spawn at least once (80–82 versus 25%). Biochemical composition of eggs presented no significant differences among treatments. FA profile of eggs indicated that high spawning activity performed by females in FLOC+FF treatment was reflected in lower mean levels of EPA, DHA and sum of polyunsaturated fatty acids (n‐3) and (n‐6). The better reproductive performance demonstrated by females raised in biofloc justified the application of this technology in F. duorarum broodstock.  相似文献   

14.
The oocyte of Sterlet (Acipenser ruthenus) and Siberian sturgeon (Acipenser baeri) was evaluated after incubation with 17α, 20β‐Dihydroxyprogesterone (DHP) in artificial media. Concentration of testosterone (T), progesterone (P4) and 17‐β estradiol (E2) in blood and their relation to fertilization rate were measured during experimental period. The oocytes at the same developmental stages were incubated in SIS (based on sturgeon blood ionic composition), RM2 (Ringer solution modified for sturgeons) and L‐15 (Leibovitz medium) artificial mediums in the presence of 1 μg/ml of 17α, 20β‐Dihydroxyprogesterone for 12, 18 and 24 hr. The result of this study demonstrated that fertilization rate decreased with the increasing duration of incubation in all mediums. In the Siberian and Sterlet sturgeon, the highest fertilization rate was observed in the oocytes incubated in RM2 (0.81 ± 0.4) and SIS (0.44 ± 0.8) mediums for 12 hr respectively. In the Siberian sturgeon, fertilization rate decreased significantly (p < .05) with incubation time in all three mediums. In Starlet, fertilization rate decreased significantly (p < .05) with time and reached a minimum after 24 hr (0.16 ± 0.1) in L‐15 medium. No significant (p > .05) differences were observed between blood plasma hormones and fertilization rate. The results of this study indicated that 17α, 20β‐Dihydroxyprogesterone is essential for induction of in vitro oocyte maturation in these species.  相似文献   

15.
This work was carried out to investigate the effects of injection of Tenacibaculum maritimum formalin‐killed cells (FKC), extracellular products (ECPs) and crude lipopolysaccharide (LPS) as well as 1% feed supplements of oil extracts of Echinacea purpurea and Origanum vulgare on sea bass immunity improvement to the favour of T. maritimum experimental infection control after 4 weeks of the experiment. Tenacibaculum maritimum isolated from naturally infected sea bass showed brown to yellowish‐brown lesions (sores) on gills, skin and/or fins and identified by different biochemical methods and polymerase chain reaction technique. Immune parameters namely, total protein, globulin and lysozyme activity, as well as the relative level of protection were improved by T. maritimum (FKC), (LPS), (ECPs), O. vulgare and E. purpurea, respectively compared with control. Histopathological examination of T. maritimum naturally infected sea bass indicated many pathological changes in gill, skin and musculatures. Present study could be concluded that application of T. maritimum (FKC), (LPS), (ECPs), O. vulgare or E. purpurea improved sea bass immunity to the favour of disease resistance against T. maritimum. Further investigations on the combination between the previous control methods and the vaccine application methods will be needed.  相似文献   

16.
In the present study, methodology of gynogenetic induction in spotted halibut were developed and optimized; the sex ratio of putative meiotic gynogenetic diploids was determined using AFLP-based molecular sexing technique; the homozygosity of gynogenetic population was assessed as opposed to cultivated population. The results showed that high percentage of meiotic gynogenetic diploids were generated when the eggs fertilized with irradiated heterologous sea perch frozen sperm (30–50 mJ cm−2) were cold shocked in sea water of −1°C for 40–75 min at 5 min after fertilization. About 15,200 diploid gynogenetic larvae were achieved and they exhibited normal morphology similar to diploid control. The gynogenetic diploids were 100% female, which first confirmed the female homogamete (XX/XY) sex determination in spotted halibut. The genetic analysis showed that the average H O was, respectively, 0.404 and 0.724 in gynogenetic population and cultivated population, indicating an increase of homozygosity in gynogenetic population.  相似文献   

17.
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.  相似文献   

18.
In this study, a non‐programmable freezing technique has been developed with the strip spawned blue mussel (Mytilus galloprovincialis) sperm. The key parameters optimized including (1) cryoprotectant agents (CPAs); (2) cooling temperature; (3) thawing temperature; (4) sugar and amino acid supplementation and (5) sperm to egg ratio. The fertilization rate and/or integrity of plasma membrane and acrosome were used as sperm quality assessment indicators. The highest post‐thaw sperm fertilization rate of 95% was achieved, when sperm were cryopreservated in 8% dimethyl sulfoxide at 7.8 cm above the liquid nitrogen surface and thawed in a 60°C seawater bath. The addition of glucose, sucrose or trehalose in dimethyl sulfoxide did not, whereas 0.8% glycine did significantly improve the post‐thaw sperm fertilization rates. The fluorescent evaluation has demonstrated that the addition of glycine significantly improved the post‐thaw sperm acrosome integrity, revealing a positive role of glycine in the improvement of post‐thaw sperm quality in blue mussels.  相似文献   

19.
We evaluated the effect of high temperature on Apostichopus japonicus gametes. Gametes were exposed to the optimal temperature (18°C; control) or one of six high temperature treatments (26, 27, 28, 29, 30 and 31°C) and the cleavage rate (CR) of fertilized eggs was measured as an indication of fertilization ability. At control temperatures (18°C), the male and female gametes had high fertilization ability (CR ≥95.0%) in 5 h and the time at which 50% of gametes lost the ability to successfully fertilize (LT50) was 7.03 h. All gametes were non‐viable after 9 h. When male and female gametes were both exposed to high temperature, the LT50 was lower than that when female or male gametes were treated individually at the same high temperature (P < 0.05). The LT50 was lower for male gametes treated alone than for female gametes treated alone. In conclusion, exposure to high temperature results in a decline in fertilization ability of A. japonicus gametes, and this decline was more rapid at higher temperature. The female gametes were more resistant to high temperatures than male gametes.  相似文献   

20.
As adhesion and translocation through fish gut enterocytes of the pathogen Vibrio (Listonella) anguillarum are not well investigated, the effective cause of disease and mortality outbreaks in larval sea bass, Dicentrarchus labrax, suffering from vibriosis is unknown. We detected V. anguillarum within the gut of experimentally infected gnotobiotic sea bass larvae using transmission electron microscopy and immunogold labelling. Intact bacteria were observed in close contact with the apical brush border in the gut lumen. Enterocytes contained lysosomes positive for protein A‐gold particles suggesting intracellular elimination of bacterial fragments. Shed intestinal cells were regularly visualized in the gut lumen in late stages of exposure. Some of the luminal cells showed invagination and putative engulfment of bacterial structures by pseudopod‐like formations. The engulfed structures were positive for protein A‐colloidal gold indicating that these structures were V. anguillarum. Immunogold positive thread‐like structures secreted by V. anguillarum suggested the presence of outer membrane vesicles (MVs) hypothesizing that MVs are potent transporters of active virulence factors to sea bass gut cells suggestive for a substantial role in biofilm formation and pathogenesis. We put forward the hypothesis that MVs are important in the pathogenesis of V. anguillarum in sea bass larvae.  相似文献   

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