Novel droplet vitrification combined with fish antifreeze protein type III enhances cryoprotection of semen in wild endangered Persian sturgeon Acipenser persicus (Borodin, 1897)

In this study, the efficiency of a novel droplet vitrification technique along with different doses of fish antifreeze protein (AFP) type III on Persian sturgeon thawed spermatozoa quality (motility duration and motility percentage) was investigated. Semen of seven male individuals was pooled in equal volumes and diluted with 4°C Tris‐Hcl (100 mM), pH = 8 extenders containing 0, 5, 10, 15 μM of AFP type III in a ratio of 1:1 (semen/extenders). Treated semen was dropped into liquid nitrogen. Solidified droplets were stored for 2, 60 and 120 days and thawed by plunging them into a tube containing 5 mL Tris‐Hcl (100 mM), pH=8 with 1% BSA at 37°C. Motility duration in all treatments had no significant difference comparing to fresh sperm (P > 0.05), but their motility percentage was significantly lower. Treatment with 10 μM of AFP had significantly higher motility percentage (16.11 ± 0.5%) comparing to other treatments (P < 0.05). There was no significant difference between 0, 5, 15 μM of antifreeze protein treatments (P > 0.05), suggesting that antifreeze protein effectiveness are highly dose dependent, and dose of 10 μM is appropriate in Persian sturgeon spermatozoa droplet vitrification. Besides, the present technique obtained higher quality of spermatozoa comparing to its analogue techniques.     

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.     

Preservation of black sharkminnow,Labeo chrysophekadion (Bleeker, 1849) spermatozoa

The objective of this study was to investigate the effects of extenders and storage time on motility, viability and fertilization of preserved black sharkminnow, Labeo chrysophekadion spermatozoa. Sperm were diluted 1:3 in one of five extenders: modified Cortland solution (MC); Hanks' balanced salt solution (HBSS); 0.9% sodium chloride (NaCl); Kurokura solution (KU); and modified extender, and undiluted sperm samples were used as control and stored at 4°C for 5 days. Motility, viability and fertilization rates were evaluated every day. After a storage time of three days, the highest motility, viability and fertilization rates (61.27 ± 2.26%, 58.60 ± 2.29% and 40.58 ± 0.57, respectively) were achieved with sperm diluted with modified extender. Motility, viability and fertilization rates decreased significantly (P < 0.05) with increasing storage time in all treatments. In addition, this study found that motility, viability and fertilization had a positive significant correlation (P < 0.01). The results indicate that isotonic extender is suitable for the short‐term preservation of black sharkminnow spermatozoa.     

Cryopreservation of Arctic charr, Salvelinus alpinus (L.), semen in various extenders and in three sizes of straw

Cryopreservation of Arctic charr Salvelinus alpinus (L.) semen was investigated using three diluents, three cryoprotectants [10% dimethyl sulphoxide (DMSO), 10% dimethylacetamide (DMA) or 20% glycerol] and three sizes of straw. The three diluents and three cryoprotectants were combined, resulting in nine extenders. One part semen was added to three parts extender, and motility was evaluated to assess the toxicity of six of the extenders. Semen in nine extenders was frozen in 0.5‐mL straws using liquid nitrogen vapour. Semen extended in 0.3 m glucose and each of the cryoprotectants was also frozen in 0.5‐mL, 1.7‐mL (flat) or 2.5‐mL straws. The freezing rate in each size of straw was measured. Fertility trials were conducted to determine the post‐thaw viability of the frozen semen. The motility of activated spermatozoa was higher in the DMA and DMSO extenders than in the glycerol extender. For the trial using 0.5‐mL straws, post‐thaw fertility results were higher for all extenders containing DMSO, or 0.3 m glucose and DMA, than for all other combinations of diluent and cryoprotectant. For the straw size comparison, the highest fertility was obtained for the 1.7‐mL straw using either DMSO or DMA and for the 2.5‐mL straw using DMSO. For all cryopreservation trials, fertility was low for extenders containing glycerol.     

Cryopreservation of silver barb Puntius gonionotus (Bleeker) spermatozoa: effect of extender composition,cryoprotective agents and freezing rate on their postthawing fertilization ability

The effects of extender composition, cryoprotectant concentration and freezing and thawing on the fertilization efficiency of cryopreserved spermatozoa of Puntius gonionotus were evaluated. Computer‐aided motility analysis of semen was conducted to check the suitability of spermatozoa for cryopreservation after mixing with different extenders and cryoprotective agents (CPAs). Extender‐4 with an osmolality 260 mOsmol kg⁻¹and pH 7.6 was used for the cryopreservation study. Among the CPAs, dimethyl sulphoxide (DMSO) was least toxic and more than 60% fertilization was achieved when used at 1.4 M at 0 °C for 10 and 30 min, whereas the toxicity of all CPAs to spermatozoa was evident when tested at 30 °C. Semen frozen at −16 °C min⁻¹ with 1.4 M DMSO showed 70% fertilization, which was significantly higher (P<0.05) than other freezing rates. Samples thawed at 35 °C water showed a fertilization rate comparable with that of fresh semen. Computer‐assisted semen analysis of fresh and frozen semen after thawing showed variations in different types of motility in spermatozoa and in their class. There was no significant difference in motility before or after cryopreservation; however, significant differences could be observed in the average path velocity (VAP), straight line velocity (VSL) and curve linear velocity (VCL). Semen of silver barb could be cryopreserved with extender‐4 by addition of 1.4 M DMSO to a final cryopreservation medium (MED 2) cooled at a rate of −16 °C min⁻¹, stored in liquid nitrogen (−196 °C) and utilized after thawing at 35±2 °C.     

Effect of extender composition and freezing rate on post-thaw motility and fertility of Arctic char, Salvelinus alpinus (L.), spermatozoa

The effects of extender composition and freezing rate on motility and fertility of frozen‐thawed Arctic char, Salvelinus alpinus, spermatozoa were investigated. Three freezing rates, two semen diluents and three cryoprotectants were tested. Semen frozen in 0.3 mol L^?1 glucose diluent with 10% methanol as a cryoprotectant or in a diluent described by Lahnsteiner with 10%N,N‐dimethylacetamide (DMA) resulted in the highest sperm motility. Fertility was the highest for semen frozen in a glucose–methanol extender but was not significantly different than that for semen frozen in Lahnsteiner's diluent with 10% DMA. Dimethyl sulphoxide (DMSO) at 10% was a relatively ineffective cryoprotectant with either semen diluent. Semen frozen at 6 cm above the surface of liquid nitrogen resulted in a higher post‐thaw sperm motility and fertility than semen frozen at 5 cm. The addition of 7% fresh egg yolk to glucose diluent containing methanol or DMSO did not improve the fertility of frozen‐thawed spermatozoa. However, the addition of 7% fresh egg yolk to glucose–DMA extender significantly improved the fertilization percentages of frozen‐thawed spermatozoa. In conclusion, dilution of semen 1:3 in 0.3 mol L^?1 glucose with 10% methanol and freezing 6 cm above the surface of liquid nitrogen (freezing rate of 40±8°C min^?1, mean±SD from ?5 to ?55°C) is a promising protocol for cryopreservation of Arctic char semen.     

Sperm cryopreservation of tiete tetra Brycon insignis (Characiformes): effects of cryoprotectants,extenders, thawing temperatures and activating agents on motility features

The aim of this study was to test the effects of cryoprotectants [dimethyl sulphoxide (DMSO) and methylglycol], extenders (0.9% NaCl, 5% glucose, Beltsville Thawing Solution^? and Merck III^?), thawing temperatures (30 and 60 °C) and activating agents (0.29% NaCl and 1% NaHCO₃) on the cryopreservation process of tiete tetra Brycon insignis sperm. Sperm was loaded in 0.5 mL straws, frozen in nitrogen vapour at ?170 °C and stored in liquid nitrogen. Post‐thaw sperm quality was evaluated in terms of subjective motility rate, quality motility score (0=no movement; 5=rapidly swimming spermatozoa), duration of motility and vitality (eosin–nigrosin staining). Post‐thaw sperm motility rate was greater in methylglycol (76–88%), compared with DMSO (23–59%). In general, the highest quality motility scores were observed when sperm was thawed at 30 °C and triggered in 1% NaHCO₃ (3.5–4.3). Duration of motility was longer when triggered in 1% NaHCO₃ (95–120 s) compared with 0.29% NaCl (69–107 s). Sperm vitality was not affected by any of the parameters tested and varied from 51% to 69% intact sperm. Brycon insignis sperm frozen in methylglycol combined with any of the extenders tested and using the methods described above yields motility above 57% and that should last long enough to fertilize oocytes.     

Effect of methanol concentration and thaw rate on the viability and fertility of cryopreserved Arctic char,Salvelinus alpinus (L.), spermatozoa

In two trials, Arctic char (Salvelinus alpinus) semen was frozen in 0.5 mL straws using extenders consisting of 0.3 M glucose and 10%, 12.5% or 15% methanol. Cryopreserved semen was thawed by immersing straws in 25 °C water for 17 s (11.6 °C s^?1) or in 5 °C water for 60 s (3.3 °C s^?1). The viability of the frozen–thawed semen was measured by determining post‐thaw motility and sperm membrane integrity. Two fertility trials were also conducted. There was no effect of trial or thaw rate on post‐thaw sperm viability or fertility. Use of 15% methanol in the extender resulted in the highest overall percentage of sperm motility and fertility. Use of 12.5% methanol as a cryoprotectant resulted in a higher per cent post‐thaw motility and a lower percentage of dead cells than did 10% methanol. Thus, levels of methanol higher than the commonly used 10% are beneficial for cryopreserving Arctic char sperm.     

Motility and fertility of cryopreserved spermatozoa of the Japanese sea cucumber Apostichopus japonicus

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.     

Effects of cryopreservation on sperm structure in Japanese pearl osyter <Emphasis Type="Italic">Pinctada fucata martensii</Emphasis>

To clarify factors reducing the motility and fertility of cryopreserved spermatozoa of the Japanese pearl oyster Pinctada fucata martensii, the structure of spermatozoa before and after cryopreservation was observed by scanning electron microscopy. Testicular spermatozoa were diluted with cryopreservation diluent (10% methanol+18% fetal bovine serum+72% sea water), and dispensed into 0.25-mL straws. The straws were cooled at a rate of approximately −20 °C/min to −50°C, and subsequently immersed in liquid nitrogen. Percentage motility of spermatozoa before cryopreservation was 69.9±4.2%, and that of cryopreserved spermatozoa was 24.0±1.8%, respectively. In cryopreserved spermatozoa, the percentage that lacked or had a deformed flagellum was 56.6±3.9%, while in fresh spermatozoa this was 8.7±2.0%. In cryopreserved spermatozoa, the percentage of deformed acrosomes was 76.6±5.2%, while in fresh spermatozoa this was only 0.9±0.3%. Cryopreserved spermatozoa with a normal acrosome and flagellum were only 15.4±3.5% of those in fresh spermatozoa. These results indicate that lesion of the flagellum and deformation of the acrosome occurred through the cryopreservation procedure, and both types of damage lead to loss of the motility and fertility in thawed spermatozoa.     

Comparative gonad histology and semen quality of normal (XY) and neo‐males (XX) of Atlantic salmon (Salmo salar)

With an overarching objective of improving the hatchery production of Atlantic salmon (Salmo salar) all‐female progeny, this study comparatively evaluated the reproductive parameters between normal (genotype XY) and neo‐males (genotype XX). Four normal (XY) and seven neo‐ (XX) males, from the same brood stock, distinguished by their ability to or lack of expressing semen, respectively, were comparatively evaluated. The left testicular lobe was used for histomorphometric analyses, while the right for semen collection and sperm quality analyses. Histomorphometric observations revealed that neo‐male testes are irregularly shaped, and have poorly formed seminiferous ducts, higher proportions of interstitial tissue and lower gonadosomatic index (p < .05). In addition, hypertrophied and cyst forming Sertoli cells were found in these individuals which collectively appear to form a physical barrier, precluding the semen collection by standard stripping techniques and reducing sperm quality. Particularly, semen motility (80.69 ± 2.4% and 57.2 ± 36.5% for XY and XX respectively) and duration of motility (99.31 ± 28.03 s and 66.84 ± 23.83 s for XY and XX respectively) of neo‐males were most compromised (p < .05). Interestingly, the TUNEL assay indicated no signs of apoptotic tissue suggesting that the histological differences may relate to delayed physiological/sexual maturity of neo‐males.     

Effects of cooling rate on post-thaw motility and fertility of Japanese pearl oyster <Emphasis Type="Italic">Pinctada fucata martensii</Emphasis> spermatozoa

The post-thaw motility and fertility of Japanese pearl oyster sperm show large variances, even among sperm samples obtained from the same individuals. This study aimed to clarify the factors that cause such differences. Spermatozoa were diluted 50 times with diluent comprising 10 % methanol, 18 % fetal bovine serum, and 72 % seawater, and dispensed into 0.25 ml straws. A total of 59 straws were cooled, one by one, at 11 different heights from the surface of liquid nitrogen (LN) to −50 °C, and then immediately immersed in LN. After thawing the straws, the relationships between the cooling rate and the post-thaw motility and post-thaw fertility of the spermatozoa were examined. Both the post-thaw motility and the post-thaw fertility showed a sharp peak when the straws were cooled at around −20 °C/min. There was a strong correlation between post-thaw motility and fertility (P < 0.001). There was a large difference in the cooling rates and the post-thaw motilities and fertilities of the spermatozoa, even between straws cooled at the same height. These results indicate that the optimum range for the cooling rate of oyster spermatozoa is quite narrow, and the method of cooling straws at a fixed distance from the LN surface is unsuitable for the cryopreservation of Japanese pearl oyster spermatozoa.     

Sperm motility and seminal plasma characteristics in Barbus sharpeyi (Günther, 1874)

Spermatozoa concentration, ionic composition, osmolality, glucose and total protein contents of seminal plasma and sperm motility were determined in Barbus sharpeyi (Cyprinidae, Teleosotei). Spermatozoa concentration ranged from 9.77 to 20.20 × 10⁹ spermatozoa mL^?1. Osmolality (mOsmol kg^?1) and ionic contents (mM L^?1) of the seminal plasma were 274.5±9.0, 70.0±3.4 Na⁺, 28.8±0.9 K⁺, 101.7±3.1 Cl^?, 0.9±0.1 Mg²⁺ and 2.1±0.1 Ca²⁺ respectively. Total protein and glucose were 5.3±0.2 g L^?1 and 76.7±4.3 mM L^?1 respectively. Sperm motility was initiated in a hypo‐osmotic condition, composed of either an ionic (KCl or NaCl) or a non‐ionic (sucrose) activation medium. Duration of sperm motility was very short: <2 min after activation in distilled water. Percentage of motile spermatozoa was significantly higher in an activation medium containing NaCl compared with that of distilled water. An activating medium containing NaCl or KCl higher than 150 mM or sucrose higher than 275 mM totally inhibited the activation of sperm motility. Immediately after sperm activation, wave(s) propagated along the flagellum, but waves were restricted to the proximal part of the flagellum (close to the head) at 1 min post activation. Studied characteristics in the present study were compared with those of other cyprinids for understanding inter‐species differences.     

Extenders and cryoprotectants for cooling and freezing of piracanjuba (Brycon orbignyanus) semen, an endangered Brazilian teleost fish

The aim of this study was to develop a protocol for semen storage of piracanjuba (Brycon orbignyanus) by both cool storage at 4 °C and cryopreservation at − 196 °C. Semen was diluted in some fish semen extenders (Exp. 1) or in extenders combined with the antibiotic gentamycin sulfate (Exp. 2) and stored at 4 °C. Sperm motility was estimated every 24 h. Then, the effects of egg yolk (0 and 5%), cryoprotectants (dimethyl sulphoxide — DMSO, methanol, and methylglycol) and extenders (NaCl 154 mM, BTS™ Minitub and M III™ Minitub) on semen cryopreservation were evaluated (Exp. 3). Semen was added to each of eighteen cryosolutions (2 yolk concentrations × 3 cryoprotectants × 3 extenders), aspirated into 0.5-mL straws, frozen in nitrogen vapor (Taylor-Wharton, CP 300, “dry shipper”) and stored at − 196 °C. Sperm motility was evaluated after thawing at 60 °C-water bath for 8 s. The three cryosolutions that produced the highest post-thaw sperm motility were used again to freeze semen. Post-thaw semen quality was then evaluated under three tests: sperm motility, the percentage of live spermatozoa and hatching rate (Exp. 4). Piracanjuba semen diluted (1:10 total volume) in NaCl 200 mM or in Saad solution (NaCl 200 mM, Tris 30 mM) maintained motility above 35% for as long as 7 days, at 4 °C. Motility of only 7% was observed on undiluted semen after 3 days at 4 °C. There was neither beneficial nor detrimental effect of gentamycin on sperm motility at 250 μg/mL. Egg yolk addition to the cryosolution was beneficial in samples cryopreserved in NaCl 154 mM and in M III™, but detrimental for samples cryopreserved in BTS™. Methylglycol was the most effective cryoprotector compared to DMSO and methanol. Motility and percentage of live spermatozoa were similar among semen cryopreserved in NaCl–yolk, M III™–yolk and BTS™, all containing 10% methylglycol, but lower than fresh control. Hatching rates of eggs fertilized with sperm cryopreserved in NaCl–yolk or BTS™ were higher than for eggs fertilized with sperm cryopreserved in M III™–yolk, but lower than control fertilizations. The semen cryopreservation protocols developed here will be used to set up a gene bank for endangered piracanjuba populations.     

A fructose‐based extender protects Colossoma macropomum spermatozoa against chilling injuries

Our study assessed the efficiency of a formulated new extender in maintaining viability and morphological integrity of Colossoma macropomum spermatozoa under chilling storage. Semen was diluted in the test extender and BTS? (Beltsville Thawing Solution) and exposed to a short‐term storage at 4.6 ± 0.6°C for 96 hr. Both extenders were able to maintain 17% ± 8% motile spermatozoa by the end of experiment. Sperm dilution in test extender did not affect the morphologically normal cells (61% ± 6%) up to 48 hr of chilling, being higher than in BTS? (50% ± 6%) (p < 0.05). After 96 hr, samples kept in the test extender had 50% of normal spermatozoa, whereas those kept in BTS? presented only 38% of normal cells. Chilling storage increased the incidence of cells with strongly coiled flagella in BTS^?. Our study is the first to evaluate in detail the spermatozoa morphology as indicative of C. macropomum semen viability. The new extender was able to protect the spermatozoa against increase in coiled flagellum injuries.     

Studies on the semen biology and sperm cryopreservation in the sterlet, Acipenser ruthenus L.

The present study investigated motility, acrosome reaction, fertility and cryobiological parameters of the semen of the sterlet, Acipenser ruthenus L. Sperm motility persisted for about 4 min in water, and the main swimming type was the linear motion. Motility was prolonged at osmolalities of 12.5 mosmol kg^?1 and in the presence of magnesium ions, while calcium had no effect. Also a pH in the range of 7.0–9.0 had no effect on ` motility. At osmolalities of 25–50 mosmol kg^?1 the sperm motility was partly inhibited, at osmolalities of 100 mosmol kg^?1, completely and irreversibly. In 50 mosmol kg^?1 solutions with 2.5–5 mM L^?1 KCl the motility inhibition was total, but reversible. The acrosome reaction was not induced by one of the described solutions, but the percentage of spermatozoa with reacted acrosomes was low (<20%) and highly variable in all experiments. The optimal extender base for cryopreservation was a solution consisting of 50 mM L^?1 NaCl, 5 mM L^?1 KCl, 10 mM L^?1 Tris (pH 8.5). From the tested cryoprotectants only dimethylsulphoxide (DMSO) and methanol provided sufficient cryoprotection. After freezing and thawing, the motility rates and swimming velocities were higher with DMSO than with methanol. However, the fertility was very significantly reduced with DMSO (10.3±0.5%) while with methanol fertilization rates in a similar range (32.7±4.4%) as with fresh semen (33.90±0.8%) could be obtained. Optimal freezing conditions for sterlet semen were in the vapour of liquid nitrogen 3–5 cm (?95°C to ?85°C) above its surface, the optimal thawing conditions at 25°C for 30 s. The acrosome reaction was not induced by these cryopreservation protocols.     

Motility of sea urchin Paracentrotus lividus spermatozoa in the post‐activation phase

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.     

Dry shipper cryopreservation of seven‐band grouper (Epinephelus septemfasciatus Thunberg) spermatozoa

This study examined the usage of a dry shipper for cryopreservation of Epinephelus septemfasciatus (Thunberg) spermatozoa. Milt was diluted 1:49 with 5% dimethyl sulfoxide plus 95% foetal bovine serum for cryopreservation. Computer‐assisted sperm analysis was used to analyse sperm motility, while fertilization and hatching trials were conducted to gauge the applicability of the cryopreservation method for aquaculture. We showed that cooling rates of the dry shipper were stable for 14 days and could be manipulated by the use of different sized freezing straws and use of a simple polystyrene foam container (5 × 5 × 12 cm and 1 cm thickness on all sides with the upper layer exposed). Dry shipper cryopreserved spermatozoa had significantly lower post‐thaw per cent motility and velocity than fresh sperm, but linearity of movement was unchanged. Fertilization and hatching rates were not significantly different at all tested sperm to egg ratios (3000:1–243000:1). The results indicated that 0.33 mL of milt when cryopreserved was sufficient to fertilize up to 450 g of oocytes. Application of this technology will help improve seed production in aquaculture and further develop breeding and genetics studies.     

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.     

The Use of Dairy Protocols for Sperm Cryopreservation of Blue Catfish Ictalurus furcatus

The commercial‐scale production of fish by use of artificial (induced) spawning would require reliable, large‐volume sources of sperm. Cryopreservation can be used to preserve and store sperm within commercial and research germplasm repositories, but is limited in its application to aquaculture. Straw volume and cooling chamber size restrict the quantity of sperm that can be frozen, and straws must be filled by hand. In contrast, the dairy industry has refined methods for freezing of bull sperm, including automation of straw filling and the use of large cooling chambers. These methods could be used for commercial‐scale cryopreservation of fish sperm, although application would require testing. To supply sperm in large volumes, bags originally developed for swine semen could be cooled using dairy protocols and used as a container for fish sperm. The current study documented the use of commercial‐scale dairy cryopreservation techniques for the production of hybrids of channel catfish Ictalurus punctatus (female) by blue catfish Ictalurus furcarus. Four cryoprotectants (methanol, dimethyl sulfoxide, dimethyl acetamide, and glycerol) were initially evaluated for use with blue catfish sperm. During May 2000 and March to April 2001, suspensions of blue catfish sperm were cryopreserved with 10% methanol in 0.5‐mL French straws and in commercial swine semen bags (Cochette^* bags, IMV International. Minneapolis, Minnesota, USA). Cryopreservation took place at a dairy breeding cooperative, using technology employed for bull semen. Sperm motility before freezing was 26 ± 18% during Year 1 (2000) and 62 ± 30% during 2001. Sperm were thawed at 40 C and used to fertilize the eggs of channel catfish (yielding hybrids). Motility after thawing for sperm frozen in 0.5‐mL straws was 11 ± 10% during 2000 and 50 ± 24% during 2001. Motility after thawing was 41 ± 17% for sperm frozen in swine semen bags in 5‐mL aliquots and 43 ± 10% for sperm frozen in 10‐mL aliquots. Neurulation of eggs fertilized with thawed sperm from straws was 83 ± 13% during 2000 and 54 ± 27% during 2001. Neurulation was 57 ± 24% using sperm frozen in swine semen bags in 5‐mL aliquots and 55 ± 10% using sperm frozen in 10‐mL aliquots. There was no correlation between sperm motility before freezing (in 0.5‐mL straws) and after thawing during 2000 (r= 0.52) or during 2001 (r= 0.49). In addition, there was no correlation between initial motility and neurulation of channel catfish eggs fertilized using thawed sperm during 2000 (r= 0.14) or during 2001 (r= 0.29). Sperm of blue catfish can thus be cryopreserved at a commercial scale using dairy protocols and can be made available for the production of hybrid catfish when viable eggs are available.