Catfish Sperm Research Articles

Addition of Cryoprotectant DMSO Reduces Damage to Spermatozoa of Yellow Catfish (Pelteobagrus fulvidraco) during Cryopreservation: Ultrastructural Damage, Oxidative Damage and DNA Damage.

Spermatozoa cryopreservation protocols have been established for yellow catfish (Pelteobagrus fulvidraco), but cryopreservation can still cause cellular damage and affect spermatozoa viability and fertility. Therefore, the aim of this paper was to evaluate the effects of adding or not adding cryoprotectants during low-temperature storage on the ultrastructural damage, oxidative damage, and DNA damage of thawed yellow catfish spermatozoa. The mixed semen of three male yellow catfish was divided into a fresh spermatozoa group, a frozen spermatozoa group (DMSO+) with a cryoprotectant (10% DMSO), and a frozen spermatozoa group without a cryoprotectant (DMSO-). Ultrastructural of the spermatozoa after thawing were observed under an electron microscope and the spermatozoa were assayed for SOD, MDA, and T-AOC enzyme activities, as well as for DNA integrity. In terms of movement parameters, compared with DMSO-, the addition of DMSO has significantly improved sperm motility, curve line velocity (VCL), and straight line velocity (VSL). The ultrastructural results showed that although thawed spermatozoa exhibited increased damage than fresh spermatozoa, 10% DMSO effectively reduced the damage to the plasma membrane, mitochondria, and flagellum of spermatozoa by cryopreservation, and most of the spermatozoa were preserved with intact structure. The results of oxidative damage showed that compared with frozen spermatozoa, 10% DMSO significantly increased the activities of SOD and T-AOC enzymes and clearly reduced the activity of the MDA enzyme. The antioxidant capacity of spermatozoa was improved, lipid peroxidation was reduced, and the oxidative damage caused by cryopreservation was mitigated. The DNA integrity of spermatozoa showed that 10% DMSO clearly reduced the DNA fragmentation rate. In conclusion, 10% DMSO can effectively reduce the ultrastructural damage, oxidative damage, and DNA damage of yellow catfish spermatozoa during cryopreservation; it can also further optimize the cryopreservation protocol for yellow catfish spermatozoa. Meanwhile, it also provides a theoretical basis for the future optimization of the cryopreservation protocols.

Effect of cryoprotectant and concentration on the sperm quality of walking catfish, Clarias batrachus, post-cryopreservation

BACKGROUND: Walking catfish, Clarias batrachus is one of the native and most popular freshwater catfish species in Indonesia. However, cultivation faces challenges, particularly due to the scarcity of larvae resulting from underdeveloped breeding technologies. Cryopreservation is a method of storing sperm to maintain viability for a long period and support the breeding technology of the fish. Cryoprotectant, in this context, plays an important role in determining the success of sperm cryopreservation. OBJECTIVE: To determine the best type and concentration of cryoprotectant for cryopreservation of walking catfish sperm. MATERIALS AND METHODS: A total of five different types of cryoprotectants, namely DMSO, glycerol, ethyl glycol, ethanol, and methanol, were tested at four concentration levels namely 0%, 5%, 10%, 15%, and 20%, each with four replications. RESULTS: The type and concentration of cryoprotectant had a significant effect on sperm motility and viability (P<0.05). The best outcomes were obtained with 5% DMSO and ethyl glycol, 10% glycerol and methanol, as well as 15% ethanol. CONCLUSION: The highest motility and viability values were obtained with 5% DMSO, resulting in its recommendation for cryopreservation of walking catfish sperm.

Molecular biomarkers for sperm quality in an economically important fish: Blue catfish, Ictalurus furcatus

Intraspecies variability in sperm quality is common among aquaculture species, thus necessitating an urgent need to establish reliable molecular biomarkers to screen for males with high-quality sperm. The channel catfish (Ictalurus punctatus) ♀ × blue catfish (I. furcatus) ♂ hybrid accounts for >50% of United States catfish production. Blue catfish sperm cannot be collected using non-lethal methods, and thus, males must be sacrificed to determine reproductive quality. To establish gene expression biomarkers of sperm quality, we characterized important sperm traits, ranked blue catfish males based on the curvilinear velocity of their sperm, and sequenced testicular tissue for mRNAs and sRNAs. Of the 43 males characterized, testicular tissue from 4 individuals with low-velocity (155.70 mm/s ± 1.49 SD) and 4 with high-velocity (205.01 mm/s ± 4.63 SD) were chosen for sequencing. No differentially expressed sRNAs were detected between the two sperm performance groups. However, we identified two differentially expressed mRNA transcripts between the low-velocity and high-velocity groups, aqp3a and ly97.3, both upregulated in the high-velocity group compared to the low-velocity group. These molecular biomarkers of sperm quality in testicular tissue are linked to gamete quality and may be applied for more efficient catfish aquaculture.

Оптимизация состава криопротектора для сохранения репродуктивных клеток африканского сома

The article presents a study on the selection of the optimal composition of the protective medium for cryopres-ervation of the reproductive cells of male African catfish. Glycerol, dimethyl sulfoxide (DMSO) at concentrations of 3, 5 and 10% were studied as the main cryoprotective additives. The influence of these compounds on the quality indicators of catfish sperm (the percentage and time of sperm movement after activation), the level of sperm lipid peroxidation, and the activity of the antioxidant enzyme catalase before and after the cryopreservation process was assessed. A decrease in fish breeding indicators of the quality of African catfish sperm (the proportion of living cells, motility time) was established when DMSO was added to the basic cryomedium. The greatest decrease in the percentage of sperm motility before freezing is observed when DMSO is added at a concentration of 10%. A dose-dependent effect of a decrease in sperm activity after freezing was noted with an increase in the addition of DMSO to the cryogenic medium from 3 to 10%. It has been shown that 3% glycerol in a cryogenic medium does not contribute to the preservation of cells during freezing, cryodamage caused by the formation of ice crystals leads to a significant decrease in the motility of catfish sperm. The best indicators of sperm quality were obtained when using glycerol, the most effective concentration is 5% (40% of mobile reproductive cells within 53 s). The addition of 5% glycerol to the cryomedium contributes to the greatest decrease in the level of peroxidation of sperm lipids and an increase in catalase activity, which is fully consistent with the results on sperm motility. The calculated correlation coefficients confirm the dependence of the reproductive qualities of gametes on the antioxidant status of cells. The results indicate the importance of using antioxidants as cryoprotective additives to improve the quality of African catfish sperm breeding and indicate the need for more research to evaluate the effect of adding new potentially effective and safe antioxidants as cryoprotective additives.

In vitro fertilization with frozen-thawed blue catfish (Ictalurus furcatus) sperm and implications for gene banking

Catfish farming constitutes about 75% of total U.S. finfish aquaculture production, for which the channel catfish female (Ictalurus punctatus) × blue catfish male (I. furcatus) hybrid accounts for nearly 60% of the harvest. Current hatchery technology to produce hybrids is labor intensive and requires the sacrifice of males. This is expensive and time-consuming, as blue catfish males require 4 to 7 years to mature. Sperm, even from mature fish, are often of low quality and quantity and do not necessarily give high fertility and hatch. Therefore, paternal gametes should be used sparingly, using the minimum quantity of sperm for fertilization, while maximizing offspring production. This is particularly important when using cryopreserved cells, as each males' sperm is in limited supply. To optimize the use of available sperm cells, the objectives of this study were to (i) compare sperm swimming kinematics and sperm health metrics before and after cryopreservation; (ii) determine the minimum quantity of frozen-thawed sperm required to maximize hatching success; and (iii) decipher how early offspring development is affected when eggs are sired with fresh and frozen-thawed sperm. Sperm kinematics were evaluated by computer-assisted sperm analysis, while health metrics (i.e., viability, oxidative stress, DNA fragmentation) were assessed using fluorescent imaging and flow cytometry. Over 2 spawning seasons, eggs from 18 females were collected and fertilized using cryopreserved sperm from 36 males at 10 sperm-to-egg ratios (ranging from 1.0 × 104 to 9.0 × 105 sperm per egg). Embryos sired with fresh and frozen-thawed sperm were then incubated under common environmental conditions and growth performance was documented over 40 days post-hatch. Generally, sperm kinematic traits [curvilinear velocity (VCL), motility, progressive VCL, and progressive motility] and health metrics (cell membrane viability, oxidative stress, and DNA fragmentation) declined after cryopreservation. Despite this decrease in sperm quality, hatching success using cryopreserved sperm was high, but dependent on the sperm-to-egg ratio. For instance, at ratios of 1.0 × 104 to 5.0 × 104 sperm per egg, hatch increased from 18.0% ± 8.5 to 43.9% ± 8.2. Adding >5.0 × 104 sperm per egg had no significant effect on hatching success. Furthermore, there were generally no differences in morphology or deformity rates in offspring sired with cryopreserved sperm, compared to a fresh control. These data improve understanding of frozen-thawed sperm quality for blue catfish to improve reproductive sustainability and reduce production costs.

Transcriptome Analysis Reveals Key Gene Expression Changes in Blue Catfish Sperm in Response to Cryopreservation.

The hybrids of female channel catfish (Ictalurus punctatus) and male blue catfish (I. furcatus) account for >50% of US catfish production due to superior growth, feed conversion, and disease resistance compared to both parental species. However, these hybrids can rarely be naturally spawned. Sperm collection is a lethal procedure, and sperm samples are now cryopreserved for fertilization needs. Previous studies showed that variation in sperm quality causes variable embryo hatch rates, which is the limiting factor in hybrid catfish breeding. Biomarkers as indicators for sperm quality and reproductive success are currently lacking. To address this, we investigated expression changes caused by cryopreservation using transcriptome profiles of fresh and cryopreserved sperm. Sperm quality measurements revealed that cryopreservation significantly increased oxidative stress levels and DNA fragmentation, and reduced sperm kinematic parameters. The present RNA-seq study identified 849 upregulated genes after cryopreservation, including members of all five complexes in the mitochondrial electron transport chain, suggesting a boost in oxidative phosphorylation activities, which often lead to excessive production of reactive oxygen species (ROS) associated with cell death. Interestingly, functional enrichment analyses revealed compensatory changes in gene expression after cryopreservation to offset detrimental effects of ultra-cold storage: MnSOD was induced to control ROS production; chaperones and ubiquitin ligases were upregulated to correct misfolded proteins or direct them to degradation; negative regulators of apoptosis, amide biosynthesis, and cilium-related functions were also enriched. Our study provides insight into underlying molecular mechanisms of sperm cryoinjury and lays a foundation to further explore molecular biomarkers on cryo-survival and gamete quality.

In vivo male african catfish sperm obtaining and evaluation for artificial reproduction

African catfish is a valuable unpretentious object of breeding. It is successfully grown in re-circulating aquaculture systems in mild climate areas. However, artificial reproduction of the African catfish has problems with the collecting of high-quality sperm for fertilization of eggs. Abdominal massage does not give results even after appropriate hormonal stimulation: sperm is released in a small volume, often with an admixture of urine and blood. At the same time, most spermatozoa are motionless. This is due to the morphological features of the gonad structure: the seminal tubules are of the percoid type, in which there are seminal vesicles that prevent sperm release. When breeding catfish, the method of slaughtering males is mainly used, followed by the extraction of gonads, their grinding and straining through gauze or a sieve. Therefore, an urgent task is to develop methods for in vivo male African catfish high-quality sperm collecting. This report shows the technique of gonadectomy and surgery techniques for extracting one or both gonads. General anesthesia was performed by immersing the fish in a solution of clove oil. The optimal dose of clove oil for anesthesia was determined as 0.10–0.20 ml/l. The surgical area was treated with an antiseptic solution. The skin was dissected and separated from abdominal muscles. After acutorsion of testicular vessels, the gonad was excised entirely, since with partial resection, the remaining part of the parenchyma oozes, causing inflammation of the abdominal cavity and impaired healing of the surgical wound. Intermittent octal sutures were applied separately to the peritoneum with abdominal muscles and skin to create additional anastomoses and prevent complications.

Simple hygienic procedures during silver catfish (Rhamdia quelen) sperm collection reduce microbial load and provide a new perspective for handling males

Simple hygienic procedures during silver catfish (Rhamdia quelen) sperm collection reduce microbial load and provide a new perspective for handling males

Short Communication: Effect of cryopreservation on ultrastructure and mitochondrial function of albino Pangasius catfish spermatozoa

Abstract. Hasanah U, Binawanto, Alimuddin A, Boediono A, Kursini E. 2020. Short Communication: Effect of cryopreservation on ultrastructure and mitochondrial function of albino Pangasius catfish spermatozoa. Biodiversitas 21: 4524-4528. Cryopreservation techniques have been carried out on many endangered species and animals with unique characteristics. Successful cryopreservation techniques vary between species depending on various factors. The study used cryopreserved spermatozoa of the albino Pangasius catfish as samples. The cryopreserved spermatozoa were analyzed by its ultrastructure, functional mitochondria, and viability. The cryopreservation was performed using a combination of 10% methanol, skim milk, and fish ringer extender. A deep freezer is used for cryopreservation at -80 °C with a storage period of 14 days. Observations were made on fresh spermatozoa, post-equilibration spermatozoa, and frozen-thaw spermatozoa. This study found there were differences in ultrastructure and morphology in the three treatments. Fresh spermatozoa and post-equilibration spermatozoa appeared intact membrane, mitochondrial, and flagellar structures. In contrast, in frozen-thaw spermatozoa, there was damage to the cell membrane. The study showed different percentages of yields on functional mitochondria of fresh spermatozoa (98 ± 2%), spermatozoa post-equilibration (57 ± 7%), frozen-thaw spermatozoa (42 ± 3.21%). Cell viability showed that there were differences in viability of fresh spermatozoa and frozen-thaw (p <0.05), the results of fresh spermatozoa (92 ± 0.57%) spermatozoa post-equilibration (80 ± 3.51%), frozen-thaw spermatozoa (61 ± 2.30%).The study concluded that the spermatozoa cryopreservation affects the ultrastructure, mitochondrial function, and viability in albino Pangasius catfish spermatozoa.

The Effect of Glutathione on The Quality of Aceh Local Catfish (Clarias gariepinus) Spermatozoa After Cryopreservation

The cryopreservation process might reduce the quality of spermatozoa due to an increase in the production of reactive oxygen species (ROS) compounds during cooling, freezing, and thawing. The quality of spermatozoa can be maintained by adding glutathione as an exogenous antioxidant into cryo-diluent. This study aimed to examine the effect of the addition of different doses of glutathione in cryopreservation of Aceh Local catfish (Clarias gariepinus) spermatozoa after freezing. The method used was a completely randomized design (CRD) with four treatments and four replications. Fresh semen was diluted in Ringer, 15% DMSO, and 20% Fetal Bovine Serum (FBS) and then added with glutathione antioxidants of 0.0 mgL-1, 0.5 mgL-1, 1.0 mgL-1, and 2.0 mgL-1. The parameters observed in this study were motility, integrity of the plasma membrane, fertility, and DNA integrity. The results showed that the concentration of glutathione had no effect on motility, integrity ofthe plasma membrane, or fertility, but had an effect on DNA integrity. The highest percentage of motility and plasma membrane integrity respectively was 40.50% (P3) and 70.87% (P2). Furthermore, the assessment of DNA integrity showed that there was no DNA fragmentation both treatments and fresh spermatozoa. This research is the first study regarding glutathione supplementation in cryo-diluent of Aceh Local catfish spermatozoa. Finally, the results obtained can provide information about the exact concentration of glutathione in the extender on the quality of spermatozoa of Aceh Local catfish (C. gariepinus) after the cryopreservation process. These results can also increase the success of fertility be used by the seed hall unit and the aquaculture industry to increase the productivity and supply high quality seeds.

Channel catfish ovarian fluid differentially enhances blue catfish sperm performance

For externally fertilizing fishes, interactions between male and female gametes have been shown to have remarkable impacts on sperm performance. Ovarian fluid (OF) and its ability to alter the swimming behavior of fish sperm makes it a determining factor of fertility. With the expansion of channel catfish (Ictalurus punctatus) ♀ × blue catfish (Ictalurus furcatus) ♂ hybrid aquaculture, it is essential to understand the impacts during fertilization and the magnitude such gametic interactions have on sperm performance and subsequent male fertility potential. This study was conducted to address the following: 1) activate blue catfish sperm with/without channel catfish OF to determine impacts on sperm performance and 2) assess if sperm behave differently when activated in the OF from individual females. Sperm (n = 4 males) were activated without OF (control) and with diluted OF from unique females (n = 6), creating 24 experimental crosses. Sperm motility (%), velocity (VCL), and longevity were analyzed using computer assisted sperm analyses software. With OF incorporated in the activation media, sperm velocity was significantly higher than the control at 10, 20, and 30 s post-activation. OF did not have an impact on motility for any females at 10 s and 20 s post-activation but became significantly higher than the control at 30 s. In all cases, OF treatments greatly increased longevity. Male × female interactions were highly significant, such that motility, velocity, and longevity were dependent on specific male-female pairs. This information shows that OF should be incorporated in aquatic media to simulate natural spawning conditions and accurately assess the fluid mechanics of sperm propulsion for each male. Additionally, there are mechanisms that drive gamete interactions that need to be explored further, which may improve selection of male-female pairs for in-vitro fertilization. On a broad scale, our results also help to shed light on the complexities of fertilization and fish reproduction overall, which may have implications for recruitment variability and recovery strategies of threatened and/or endangered freshwater species.

Optimization of spermatozoa cryopreservation of Albino pangasius catfish: cryoprotectants with various concentrations and different equilibration times

This study was aimed to optimize the spermatozoa cryopreservation protocol in Albino pangasius catfish by assessing the effectiveness of different cryoprotectant concentrations and different time of equilibration. The first experiment was conducted to optimize the combination of 10% methanol with different concentration of skim milk (0%, 5%, 10%, 15%, and 20%). The second experiment was aimed to optimize different equilibration time (10, 20, and 30 minutes). Deep freezer was used for the cryopreservation with a temperature of -80 °C at a storage period of 8 days. The first study found that the viability and motility of sperm after cryopreservation was significantly different (P<0.05) with fresh sperm. The best result was obtained at the 10% skim milk concentration with sperm viability of 78 ± 4.35% and motility of 69.66 ± 1.52%. The second experiment identified a significant different (P<0.05) in sperm viability and motility between fresh sperm and sperm post equilibration. The best result was obtained at 10 minutes equilibration time with sperm viability of 76 ± 1.15% and motility of 66 ± 2.64%. The study concluded that the 10% methanol with 10% skim milk, and 10 minutes equilibration time is the best combination for cryopreservation of Albino pangasius catfish spermatozoa.

Lactate dehydrogenase in fish spermatozoa and its role in sperm cell bioenergetics

Lactate dehydrogenase (LDH) in spermatozoa of three fish species was studied to determine its role in sperm cell bioenergetics in normal and polluted conditions. The adenylate energy charge (AEC) has been used as the measure of energy storage for adenine nucleotide pool of living cells and tributyltin (TBT) as a model toxicant. It was demonstrated that carp Cyprinus carpio Linnaeus, 1758 spermatozoa exhibit higher LDH activity than catfish Clarias gariepinus (Burchell, 1822) and herring Clupea harengus Linnaeus, 1758 spermatozoa. Native electrophoretic pattern of LDH from spermatozoa compared to somatic tissues extract shows nine LDH isoenzymes in carp spermatozoa, two LDH isoenzymes in herring and one LDH isoenzyme in catfish spermatozoa. It was confirmed that lactate plus pyruvate, which are the substrates for LDH seems to maintain AEC value. It indicates ATP synthesis in tricarboxylic acid cycle prevails upon the ATP utilization in catfish spermatozoa. It was noticed that LDH-B4 is more strongly inhibited by TBT in herring than the nine LDH isoenzymes in carp spermatozoa which confirms that herring spermatozoa may be more sensitive for pollution by decreasing their energetic state. LDH role in maintaining physiological bioenergetic state of spermatozoa is also discussed.

Effects of cysteine supplementation on the quality of cryopreserved sperm of South American silver catfish

The cryopreservation promotes cellular damage that could compromise sperm quality in terms of motility and fertility rates, which may be caused by oxidative stress. Thus, the aim of this study was to assess the effects of cysteine addition on post-thaw sperm quality, DNA damage and indices of oxidative stress of the South American silver catfish (Rhamdia quelen) sperm, compared with the cryoprotectant solution without cysteine addition. Sperm collected from five males were cryopreserved in cryoprotectant solution (fructose 50 g/L, powdered milk 50 g/L and methanol 100 ml/L) containing different cysteine concentrations (0, 2.5, 5, 10 and 20 mM). After thawing, the following were measured: sperm motility, morphology, sperm viability, DNA damage, lipid peroxidation, concentration of carbonyl and sulfhydryl groups and the activity of SOD, CAT, GST and GPx enzymes. The lowest sperm motility was determined for semen cryopreserved with addition of 20 mM of cysteine. The control group had the lowest DNA damage and lipid peroxidation. The findings of this study show that cysteine addition had no positive effect on evaluated parameters. Therefore, the concentrations tested are not recommended for the supplementation of cryoprotectant solution for semen of R. quelen.

Comparative transcriptome analyses reveal changes of gene expression in fresh and cryopreserved yellow catfish (Pelteobagrus fulvidraco) sperm and the effects of Cryoprotectant Me2SO

This study, for the first time in fish, compared the transcriptome of fresh and frozen-thawed sperm, and would help to better understand the effect of cryopreservation on fish sperm and then better preserve the aquatic germplasm resources. Here, we employed high–throughput sequencing technology to obtain the transcriptome of yellow catfish from fresh sperm, cryopreserved sperm with and without cryoprotectant. When cryoprotectant (Me2SO) was excluded, down-regulated genes were significantly enriched into calcium ion binding, cytoskeletal protein binding, microfilament motor activity, calmodulin binding and carnitine O-acyltransferase activity, which affected Ca2+ regulation, cellular morphology, motility and metabolism. Moreover, heat shock proteins and genes associated with regulation of cholesterol, HCO3− and protein tyrosine phosphorylation (PTP) were down-regulated, and thus would impair ability against stress, membrane rigidity, pH regulation and signal transduction of cryopreserved sperm. After Me2SO was added, the amounts of DEGs decreased significantly and down-regulation of genes were found mainly in cytoskeleton and heat shock proteins, thereby suggesting that Me2SO effectively reduced the impact caused by low temperature on gene expression. Whether adding Me2SO or not, the up-regulated genes were mainly found in ribosomal proteins genes. However, when Me2SO was added, over-expression of some genes might contribute to maintain normal function of cryopreserved sperm.

Cryopreservation disrupts lipid rafts and heat shock proteins in yellow catfish sperm

Lipid rafts and associated membrane proteins (flotillin, caveolin) play important roles in cell signaling and sperm fertilization while heat shock proteins (Hsp) ensure properly protein folding to fulfill their physiological functions. The markedly reduced fertility in thawed sperm after cryopreservation could result from disrupted membrane lipid rafts and these proteins. To explore the effect of sperm cryopreservation on lipid rafts and heat shock proteins, we compared lipid raft integrity, and the expression levels of lipid raft associated proteins (Flot-1, Flot-2, Cav-1) as well as heat shock proteins (Hsp90, Hsp70) in fresh and thawed sperm cryopreserved under different scenarios in yellow catfish. We found higher lipid raft integrity, higher protein expression levels of Flot-1, Flot-2, Cav-1, Hsp90, and Hsp70 in fresh sperm samples than in thawed sperm samples, in thawed sperm samples cryopreserved with optimal cooling rate than those cryopreserved with sub-optimal cooling rate, and in thawed sperm samples cryopreserved with extenders supplemented with cholesterol than those supplemented with methyl-β-cyclodextrin (for cholesterol removal). Our findings indicate that lipid raft integrity, and expression levels of Flot-1, Flot-2, Cav-1, Hsp90, and Hsp70 are clearly associated with sperm quality, and together they may play a cumulative role in reduced fertility associated with thawed sperm in aquatic species.

Testicular germ line cell identification, isolation, and transplantation in two North American catfish species.

Our aim was to transplant blue catfish germ line stem cells into blastulae of triploid channel catfish embryos to produce interspecific xenogenic catfish. The morphological structure of the gonads of blue catfish (Ictalurus furcatus) in ~ 90- to 100-day-old juveniles, two-year-old juveniles, and mature adults was studied histologically. Both oogonia (12-15μm, diameter with distinct nucleus 7-8μm diameter) and spermatogonia (12-15μm, with distinct nucleus 6-7.5μm diameter) were found in all ages of fish. The percentage of germ line stem cells was higher in younger blue catfish of both sexes. After the testicular tissue was trypsinized, a discontinuous density gradient centrifugation was performed using 70, 45, and 35% Percoll to enrich the percentage of spermatogonial stem cells (SSCs). Four distinct cell bands were generated after the centrifugation. It was estimated that 50% of the total cells in the top band were type A spermatogonia (diameter 12-15μm) and type B spermatogonia (diameter 10-11μm). Germ cells were confirmed with expression of vasa. Blastula-stage embryos of channel catfish (I. punctatus) were injected with freshly dissociated blue catfish testicular germ cells as donor cells for transplantation. Seventeen days after the transplantation, 33.3% of the triploid channel catfish fry were determined to be xenogenic catfish. This transplantation technique was efficient, and these xenogenic channel catfish need to be grown to maturity to verify their reproductive capacity and to verify that for the first time SSCs injected into blastulae were able to migrate to the genital ridge and colonize. These results open the possibility of artificially producing xenogenic channel catfish males that can produce blue catfish sperm and mate with normal channel catfish females naturally. The progeny would be all C × B hybrid catfish, and the efficiency of hybrid catfish production could be improved tremendously in the catfish industry.

Optimization of conditions for the cryopreservation of yellow catfish (Pelteobagrus fulvidraco) sperm

Yellow catfish (Pelteobagrus fulvidraco) is a promising aquaculture species in China with an increasing market demand. To serve the growing demand of male broodstock for artificial fertilization and the preservation of valuable strains for selective breeding, we tried to develop a species-specific cryopreservation protocol for yellow catfish sperm in this study. Important factors such as cryoprotectant, freezing height above the liquid nitrogen (LN) surface, dilution ratio, equilibration time, thawing temperature and cool storage before freezing were standardized. Among the cryoprotectants tested here, 10% Me2SO was the most suitable for sperm cryopreservation. Freezing at 7 cm above the LN surface for 10 min yielded the highest post-thaw motility. Further evaluation showed that dilution ratio of 1:3 and 1:5 produced higher post-thaw motility than semen diluted at 2:1, 1:1, 1:9 or 1:19. Equilibration times from 0 to 30 min did not cause significant differences in both equilibrated and post-thaw motility. Also, cool storage up to 24 h did not affect the suitability of sperm for cryopreservation. After thawing, sperm could be stored at 4 °C for 2 h without a reduction in motility parameters. With the combination of optimized freezing conditions, the fertilization and hatching rate of cryopreserved sperm were 87.1 ± 5.2% and 78.5 ± 7.4%, respectively, which were similar to those of fresh sperm (91.8 ± 3.5% and 83.7 ± 2.5%). In general, the cryopreservation protocol optimized here would facilitate breeding practice and hatchery operation in this economically important fish.

Effects of Cryoprotectants and Diluents on the Cryopreservation of Spermatozoa from Far Eastern Catfish, Silurus asotus.

ABSTRACTThe aim of this study was to compare the efficacy of cryopreservation methods for ex situ conservation of spermatozoa from far eastern catfish, Silurus asotus. The spermatozoa activity index (SAI) and hatching rates were higher in spermatozoa stored in Alserver’s solution than those of spermatozoa stored in glucose solution. The SAI and hatching rates in all experimental groups gradually decreased with increasing duration of storage. Additionally, the SAI and hatching rates gradually decreased with increasing thawing temperatures at all storage durations (P<0.05). Based on the SAI and hatching rates, our results suggest that the optimal cryopreservation conditions of catfish spermatozoa involve storage in Alserver’s solution with 15% ethylene glycol, and thawing at 25℃. Cryopreservation of spermatozoa is a useful and reliable technique for conserving gene resources and for artificial propagation of far eastern catfish.

Effect of Extender Compositions, Glycerol Levels, and Thawing Rates on Motility and Fertility of Cryopreserved Wild African Catfish (Clarias gariepinus) Sperm

The aim of this study was to determine the effect of different extenders, glycerol levels, and thawing rates on post-thaw sperm motility and fertilization ability of cryopreserved African catfish (Clarias gariepinus) sperm. Having determined the main spermatological properties (volume, motility, motility duration, spermatozoa concentration and pH), the pooled ejaculates were diluted with 3 different extenders containing different glycerol levels (5, 10 and 15%) respectively. Dilution ratio was 1:10 and the diluted sperm was packaged in 0.25 ml straws and left for 10 min equilibration at 4ºC. Following equilibration, the straws were exposed to liquid nitrogen vapor for 10 min and plunged into liquid nitrogen (-196ºC), and then exposed to different thawing rates (30ºC/20s and 40ºC/20s) to determine sperm motility and post-thaw motility duration. The highest post-thaw sperm motility, motility duration, and fertilization rate was 85%, 81s, and 95% respectively when sperm was frozen with the extender (ACSE 3) containing 15% glycerol (p&lt;0.05). The protocol reported in this study can be successfully used for cryopreservation of African catfish sperm.