Cryopreservation of summer flounder,Paralichthys dentatus L., sperm

The summer flounder, Paralichthys dentatus L., is a high‐value species and considerable research has been conducted to determine practices conducive for its culture. As milt can be limited in this species, experiments were conducted to develop a practical sperm cryopreservation protocol for hatchery use. Two dilution ratios (1:2 and 1:4; sperm:extender), 2 diluents (saline and sucrose‐based), 2 cryoprotectants (10% DMSO and 12% glycerol) and 3 freezing rates (?5, ?10 and ?15°C min^?1) were evaluated using differential staining to assess post‐thaw sperm survival. Seven combinations of the factors examined reduced post‐thaw viability by less than 30%. The average viability of sperm from fresh, pooled flounder milt (67.2 ± 2.9%) was not different from that of thawed milt diluted 1:4 with sucrose diluent (10% DMSO) frozen at ?5°C min^?1 (38.4 ± 7.7%) and fertilization and hatch success were not different in trials using fresh or thawed, cryopreserved sperm. From these experiments a practical sperm cryopreservation method was developed, but further refinement of the freezing protocol is necessary to optimize results.     

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.     

Cryopreservation of Mekong catfish,Pangasius bocourti Sauvage, 1880 spermatozoa

The effects of three extenders (Ginzburg fish ringer, Calcium‐free Hank's balanced salt solution, C‐F HBSS and sodium chloride, 0.9% NaCl) and four cryoprotectants (dimethyl sulphoxide, DMSO; dimethyl acetamide, DMA; methanol, MeOH and glycerol) in different concentrations (5%, 10% and 15%) on the motility, viability and fertilization rates of Mekong catfish (Pagasius bocourti) sperm were investigated. Sperm samples were transferred into 250‐μL French straws and sealed with a heated haemostat. The straws were then placed in a cryochamber. A computer‐controlled rate freezer (CL 3300) and programmable Cryogenesis, version 4 were used to regulate the freezing rate. The sperm samples were frozen at a rate of 10°C min^?1 from 4 to ?80°C and then evaluated after 72 h. Of the three extenders used with each cryoprotectant, C‐F HBSS had the highest fertilization rate of 75% (93% of control). This was not significantly different from the control treatment (fresh sperm) when tested with DMSO as the cryoprotectant. The lowest fertilization rate of 27% (38% of control) was resulting from the combination of 15% glycerol and C‐F HBSS. This study found that fertilization, motility and viability rates in all of the experiments had a positive significant correlation (P < 0.001).     

Effect of vitamin E on spermatophore regeneration and quality of pond‐reared,black tiger shrimp (Penaeus monodon)

The objective of the present work was to evaluate the effects of different levels of vitamin E in feed on the spermatophore regeneration and quality of male Penaeus monodon. The experiment was carried out with the four following treatments: the basal diet no added vitamin E, the diet added 200, 600 and 1,000 mg/kg respectively. Spermatophore regeneration and quality were evaluated by spermatophore weight, sperm count and spermatophore absence rates, which male P. monodon were extruded spermatophore for feeding 20 and 40 days. In the experiment, the weight of the twice regenerated spermatophore of the males added to the vitamin E group was higher than that of the untreated control group. The weight of the first regenerated spermatophore with the addition of 1,000 mg/kg group was the highest and significantly higher than the control group (p < .05), but there was no significant difference among the three groups with different levels of vitamin E. The weight of the second regenerated spermatophore with the addition of 600 mg/kg group was the highest, followed by 1,000 mg/kg group, both of which were higher than the control group and the addition of 200 mg/kg group. Within the same group, the regeneration spermatophore weight showed overall upward trend as the feeding time, twice regenerate experiment spermatophore weight with added to the vitamin E groups were significantly higher than the initial value (p < .05), but three spermatophore weight of male shrimp at the control group had no significant difference. The sperm quantity and the percentage of normal sperm of the twice regenerated spermatophore of the males with added to the vitamin E group was higher than that of the untreated control group, and those of the addition of 200 mg/kg group was significantly higher than that of control group (p < .05). The total number of sperm and the percentage of living sperm of male shrimp in the experimental group decreased with the increase of vitamin E in the feed. Within the same group, the total number of sperm and the percentage of living sperm of male shrimp with added to the vitamin E groups showed overall upward trend as the feeding time and were significantly higher than the initial value (p < .05), but the control group was slightly down and had no significant difference. Comprehensive sperm weight, sperm quantity and living sperm percentage of three indicators, that adding 200 mg/kg of vitamin E in feed could effectively promote the spermatophore regeneration in the male P. monodon and improve the sperm quantity. The experimental results provide a scientific basis for the breeding of P. monodon.     

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.     

Cryopreservation of sperm in farmed blacklip abalone (Haliotis rubra Leach, 1814)

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.     

The urogenital papillae as an indicator of sexual maturity in male African catfish,Clarias gariepinus, (Burchell, 1822)

The present study investigated a non‐invasive method based on macroscopic morphological features of male Clarias gariepinus for the assessment of sexual maturity stage. As African catfish cannot be stripped for semen as in most other fish species; they have to be killed to obtain semen from the testes. This method could be used to ensure that males used to obtain sperm are sexually mature to increase the concentration and quality of mature sperm for egg fertilization in the production of C. gariepinus. Morphological features including the length and width of the urogenital papillae (UGP), an external secondary sexual characteristic of C. gariepinus (n = 89) were measured and calculated as a percentage of total body length. The urogenital papillae length (L_UGP) and width (W_UGP) index of each fish were used and compared with the macroscopic and microscopic maturity stage of the testes. The results revealed that although positive, a significant correlation does not exist between the L_UGP or W_UGP and maturity stage of the testes. Therefore, although this procedure would be valuable as an initial non‐invasive assessment of maturity, it does not provide an accurate or reliable indication of the maturity stage of the fish.     

Sperm cryopreservation of silver barb (Barbodes gonionotus): cryoprotectants,cooling rate and storage time on sperm quality

Effectiveness and efficiency of frozen sperm on fertilization and hatching success of eggs from silver barb was examined in relation to cryoprotectants, freezing rate and storage period. Sperm was diluted in calcium‐free Hank's balanced salt solution, equilibrated with dimethylsulphoxide (DMSO), propylene glycol, sucrose or methanol at 5%, 10%, 15% or 20% final concentrations, and frozen in 250‐μL straws using a one‐step freezing procedure (1, 5 and 8°C min^?1 from 25 to ?40°C). Highest post‐thaw sperm motility was found from a treatment using 10% DMSO and 5°C min^?1 (82.2 ± 2.1%), similar to that of 10% DMSO and 8°C min^?1 (87.8 ± 3.2%). Post‐thaw motility of sperm frozen at 5 or 8°C min^?1 was significantly higher than 1°C min^?1. Relative sperm motility declined significantly after 10 months of cryostorage while viability did not change during a 12‐month cryostorage. Average fertilization rates of sperm after 1 and 4 months of storage were 64.5 ± 4.6% and 61.3 ± 3.4%, respectively, similar to those of fresh sperm (69.6–72.3%). Hatching rates of cryopreserved sperm (45.4–51.2%) were similar to those of fresh sperm (51.8–57.8%). This study developed suitable methods for cryopreservation of silver barb sperm that can be used to facilitate hatchery operation.     

Development of cryopreservation methods for silver barb (Barbodes gonionotus,Bleeker) spermatozoa by manipulation of cooling rates in dry shipper

The aim of this study was to develop a simple cryopreservation protocol for silver barb, Barbodes gonionotus, semen using a dry shipper. Freezing rates within the upper and lower chambers of dry shipper were recorded for 14 days post liquid nitrogen loading (dpl). To regulate freezing rates, straws (250 and 500 µl) wrapped with various insulators (polystyrene foam box, oxygen tube, silicone tube and electric wire) were frozen within the upper chamber. Straws containing semen diluted with Calcium‐free Hank's Balanced Salt Solution (Ca‐F HBSS) and 10% dimethyl sulphoxide were cryopreserved with or without insulators. Appropriate protocols were selected based on sperm quality during a 45‐day cryostorage. The upper chamber had potential as a freezing chamber within 9 dpl due to no significant (p > 0.05) change in freezing rates. High percentages of sperm motility and viability (p < 0.05) were observed when 250 µl straws with silicone tube (T4) frozen for 5 min, non‐insulated 500 µl straws (T9) and 500 µl straws with polystyrene foam box (T12) frozen for 1–5 min, having freezing rates of 43.1 ± 1.3, 71.3 ± 1.4 and 14.7 ± 0.4°C/min respectively. Dry shipper can be used as a freezing tool to cryopreserve silver barb semen.     

Spermatological research of experimentally farmed Patagonian blenny (Eleginops maclovinus) (Perciformes: Eleginopsidae) in Chile

Spermatological research of the Patagonian blennie was carried, specifically biometric parameters, sperm density, sperm count and motility in different activation mediums (815, 716, 590 and 0 mOSm kg^?1), at different temperatures (5, 10 and 15°C) and pH levels (5, 7 and 9). The results indicate that Patagonian blennie spermatozoa have a primitive form, with a total length of 44.09 ± 3.36 μm, with a head length of 2.15 ± 0.28 μm and head width of 2.5 ± 0.31 μm. The mid‐piece had a length of 0.72 ± 0.12 μm, and its tail measures 41.21 ± 3.21 μm long. The motility pattern indicates that the spermatozoa are found immobile in the seminal plasma and only initiates its movement in a hypertonic medium from 590 to 815 mOsm kg^?1. The longest motility time that was registered at 10°C in 716 mOSm kg^?1 was of 245 ± 39 s and an optimum pH of 7 was observed.     

An extender solution for the short‐term storage of Litopenaeus vannamei sperm to be used in artificial insemination

Some shrimp hatcheries use artificial insemination (AI) to improve the male to female ratio in their breeding populations. We describe a sperm extender solution, which allows the short‐term storage of diluted sperm in Litopenaeus vannamei, and its use in an artificial insemination process. We also evaluate its fertilization capacity. An AI experiment was designed using two, one, or half spermatophore segments. We tested four treatments involving three different male:female ratios: Natural mating (1:1), Regular and Regular diluted (1:2) and Half diluted (1:4). Data analysis revealed that the number of nauplii produced per mating was affected by treatment, with Regular (158 420) performing better than Half diluted (112 864) (P < 0.05), but with no differences between the latter and Regular diluted (130 340) (P > 0.05). A binomial variable named female success (FS) was defined as successful when the number of nauplii obtained per mate was ≥25 000. Analysis showed differences for FS across treatments (P < 0.001), but not between Regular (79.2%), the hatchery conventional AI technique and Half diluted (60.4%), maybe due to sample size. Since the number of nauplii per mate is crucial to consider AI successful, it is necessary to improve this AI technique before it can be used in the shrimp industry.     

Spermatophore production and sperm quality of the river prawn Macrobrachium americanum Spence Bate, 1868 fed with different diets

The effects of different diets on spermatophore production and sperm quality were investigated in the river prawn Macrobrachium americanum. River prawns were cultured and fed with three diets for 244 days: fresh food (50% squid meat, Dosidicus gigas and 50% sardine muscle, Sardinops sagax); commercial pellets (35 Purina®); and a 50:50 mixture of both diets. Spermatophore production was recorded every 24 days on average as the percentage of spermatophores produced per extraction per diet, weight and biochemical composition. Sperm quality was measured as the total number of sperm, the proportion of live/dead sperm and normal/abnormal sperm morphology. There were no significant differences in the mean biochemical composition of M. americanum spermatophores for any of the diets. Biochemical composition was 36.3% protein, 25.8% carbohydrate and 4.6% lipids for all data pooled. The weight of spermatophores and sperm counts was not significantly different among diets, nor were there any differences as a function of the male initial total length (p > .05). Male river prawn reproductive exhaustion was observed as a decline in spermatophore production, weight of the spermatophores and the number of sperm cells per spermatophore, with an increasing proportion of dead and abnormal sperm seen throughout the experiment. The recommended period of maintenance in captivity for male broodstock is less than 115 days. It is recommended to feed broodstock males of M. americanum with commercial pellets because no significant differences were detected with the diets tested; pellets are easier to use, ensuring the same spermatophore production and sperm quality that was obtained with fresh food.     

Properties and activities of blood‐ or seawater‐contaminated wild‐caught Striped Jewfish (Stereolepis doederleini) sperm

Understanding the effects of environmental factors in sperm qualities will be helpful in the development of optimal artificial reproduction methods and contributes towards the knowledge base of better short‐ and long‐term fish semen preservation conditions The objectives of this study were to determine properties and activities of wild‐caught striped jewfish Stereolepis doederleini sperm contaminated with blood or seawater and compare them with data reported in the literature on other freshwater and marine fish species, for effective short‐ and long‐term storage of fish semen. Overall, we observed that the sodium, chloride, glucose, total protein concentrations of normal sperm were not significantly different from blood‐ or seawater‐contaminated sperm. The salinity and osmolality concentration of sperm contaminated with blood were lower than sperm contaminated with seawater and were not significantly different from normal sperm. In addition, the spermatozoa motility (SM) and duration of spermatozoa motility (DSM) in blood‐contaminated sperm were higher than seawater‐contaminated sperm and also not significantly different from normal sperm. The best condition for SM and DSM in normal sperm was dilution rate of 1:50. Sperm was immotile in distilled water, and cationic factors were shown to stimulate the initiation of spermatozoa activation. The maximum SM and DSM were observed in solution containing 0.4 M NaCl, 0.6 M KCl, 0.6 M CaCl₂ and 0.4 M MgCl₂. This study provides some basic and important knowledge about striped jewfish sperm sensitivity to a cationic condition. In this regard, Na⁺ is the major inhibitory factor of spermatozoa motility in this fish species.     

Spermiation time affect the milt quality indices of the Russian sturgeon,Acipenser gueldenstaedtii,Brandt & Ratzeburg, 1833

In this study, the effects of spermiation time are investigated on milt quality of Russian sturgeon over the course of the spawning season. The milt samples were collected from three broodstock batches at three time points including: the beginning, middle and at the end of the spawning season. According to the results, the milt quality parameters including pH, sperm density, spermatocrit, duration of sperm motility and percentage of sperm motility were significantly low in the beginning and end of season than middle of season. The values of milt quality parameters in the middle of season were as follows: (motility percentage: 69.6 ± 3.5, motility duration: 460.3 ± 37.2 s, sperm density: 8.7 ± 0.4 × 10⁹, milt volume: 86.3 ± 8.1 and milt pH: 8.3 ± 0.15). Significant positive correlations were also found between milt pH and sperm motility as well as between sperm density and spermatocrit. In conclusion, our study showed that the middle of season is the best time for collection of milt with appropriate quality in Russian sturgeon. Selection of milt with good quality is necessary aim to cryopreservation of spermatozoa in endangered fish species including Russian sturgeon.     

DNA fragmentation in blue mussel (Mytilus edulis) sperm: aquaculture and fisheries implications

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.     

Sperm characteristics of chub Leuciscus cephalus (L.) collected in artificial condition after Ovopel and Ovaprim treatment

The effect of two commercial preparations containing different GnRH analogues with dopamine antagonists on quantitative and qualitative parameters of semen from chub Leuciscus cephalus L. collected in artificial conditions were examined. Semen was collected after the application of [(D‐Ala⁶, Pro⁹ NEt)‐mGnRH + metoclopramide] (Ovopel, n = 9), [(D‐Arg⁶, Pro⁹ Net)‐sGnR + domperidone] (Ovaprim, n = 9) and from the control group (0.9% NaCl, n = 9). Afterwards, semen volume, sperm concentration, total sperm production and semen pH were determined. Osmolality and pH of seminal plasma were also determined. Using the Computer‐assisted sperm analysis system (CASA), selected sperm parameters such as sperm motility (MOT %), progressively motile sperm (PRG, %), curvilinear velocity (VCL, μm s^?1), straight‐line velocity (VSL, μm s^?1), movement linearity (LIN, %), wobbling index (WOB, %), amplitude of lateral head displacement (ALH, μm) and beat cross frequency (BCF, Hz) were analysed. While Ovopel can also be used to stimulate chub spermation, the application of Ovaprim was much more effective for obtaining higher amounts of semen.     

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.     

Embryonic and larval development of a hybrid between kelp grouper Epinephelus moara ♀ × giant grouper E. lanceolatus ♂ using cryopreserved sperm

Hybridization was used to take advantage of desirable traits in offspring. In the present study, we applied the cryopreserved Epinephelus lanceolatus sperm into interspecific hybridization with E. moara. Successful hybridization between these two species was achieved and cultured in 23–24°C seawater (34‰). There was no difference in survival rate between hybrid (E. moara ♀ × E. lanceolatus ♂) and non‐hybrid (E. moara ♀ × E. moara ♂) at 24 hrs post hatch (HPH), but less hybrid (14.35 ± 8.02%) hatched than non‐hybrid (93.60 ± 1.65%), which might be due to irregularity cell cleavage and skeletal deformities from the formation of embryonic body to the later embryonic development. Similar phenomenon was found in hybrid embryos from fertilization with fresh sperm, indicating that species variations between parents, rather than cryopreserved sperm, resulted in deformities in embryos. Mean hatch time of the hybrid was 1 hr faster than that of E. moara. The hybrid was 1.95 ± 0.06 mm in total length when newly hatched and reached 38.00 mm at 58 days post hatch (DPH), which showed faster growth than E. moara (recorded in the previous study). Considering its faster growth, E. moara × E. lanceolatus hybrid was a potential breeding production in aquaculture. Over 212,000,000 larvae have been produced and launched in the market since 2015. The results of this study also shed some lights on further comparative studies in grouper hybrid performance.