Sperm fertility of the subtropical freshwater streaked prochilod Prochilodus lineatus (Characiformes) improved after dilution and cold storage

The aims of this study were to evaluate the efficiency of simple and complex extenders in prolonging the cold storage of sperm (Experiments 1 and 2) and to test the diluted‐cooled sperm in the best extender with regard to sperm quality parameters (Experiment 3) in the streaked prochilod, Prochilodus lineatus. In all the experiments, aliquots of 0.3 mL of sperm were diluted 1:10 in extenders and stored at 4–6 °C. Sperm diluted in simple extenders (NaCl and glucose solutions) yielded 0–26% sperm motility, whereas sperm diluted in complex extenders (BTS^?, M III^? and Androstar^?) yielded 62–81% sperm motility on day 4 after cold storage. When Androstar^? was further investigated, the following was observed on day 4: 53% motility with 94 s of duration; 47% live spermatozoa; 26–61% fertility rate; and 22–60% hatching rate. The use of Androstar^? improves the sperm fertility of the streaked prochilod during a 4‐day storage period and can therefore be used to facilitate artificial reproduction.     

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.     

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.     

Use of ACP‐104 at Different Dilutions for Sperm Cryopreservation of Dourado,Salminus brasiliensis

The dourado, Salminus brasiliensis, is an important fish in South American river basins. The fish‐farming potential and concerns for its conservation in watersheds has stimulated studies with this species. This study compared the effect of different dilutions of two cryoprotectant solutions on the quality of cryopreserved dourado sperm (duration of motility/motility rate) post‐thawing using three different activating solutions. The cryoprotectant solution ACP‐104 was compared with the cryoprotectant solution most commonly used for dourado, which consists of a mixture of dimethyl sulfoxide, glucose, egg yolk, and distilled water. The semen was mixed following dilutions of semen : cryoprotectant solution: 1:3, 1:9, 1:15, 1:21, and 1:27. The cryopreserved samples were activated using distilled water, 0.45% NaCl or 1% NaHCO₃. The results showed differences in thawed semen using the different activating solutions and cryoprotectant solutions. The glucose‐based cryoprotectant not only resulted in sperm motility equal to or better than that produced by ACP‐104, but the quality was not affected by the dilution. However, the sperm quality improved with increasing dilutions for ACP‐104. Under the conditions tested, the standard cryoprotectant solution is recommended for the cryopreservation of dourado semen because it requires a lower solution volume and, therefore, reduced storage space for the same volume of 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.     

A Strategy for Sperm Cryopreservation of Atlantic Salmon,Salmo salar,for Remote Commercial‐scale High‐throughput Processing

Sperm cryopreservation is an essential tool for long‐term storage of genetic resources for aquaculture fishes. The goal of this study was to develop an efficient and streamlined protocol for high‐throughput processing for sperm cryopreservation in Atlantic salmon, Salmo salar. The objectives were to evaluate: (1) osmolality of blood serum for determining extender osmolality, (2) effects of extenders for fresh sperm dilution and refrigerated storage, (3) effects of methanol and dimethyl sulfoxide (DMSO) on fresh sperm motility, and (4) motility and fertility after thawing. In this study, sperm samples were collected at a hatchery site in Canada and shipped to a freezing site located 2200 miles (3550 km) away in the USA. Evaluation of three extenders indicated that Mounib solution was suitable for diluting dry sperm for sample processing. Ten percent of methanol or DMSO was less toxic to sperm cells than was 15% within 30 min. Further testing with methanol at 5, 10, and 15%, and sperm solution : extender dilutions (v:v) of 1:1, 1:3, and 1:19 (at concentrations of 5 × 10⁷, 3 × 10⁸, and 1 × 10⁹ cells/mL) indicated that methanol at 5 and 10% showed less toxicity to fresh sperm within 1 h at sperm:extender dilutions of 1:1 and 1:3. Post‐thaw motility of sperm cryopreserved with 10% methanol was significantly higher than that with 10% DMSO, and fertility reflected those results (0–1% in DMSO vs. 38–55% in methanol). Further evaluation of sperm cryopreservation with 10 and 15% methanol at sperm dilution ratios of 1:1, 1:3, and 1:19 indicated that post‐thaw motility in 10% methanol was significantly higher than that in 15% methanol, and post‐thaw fertility in 10% methanol at 1:1 and 1:3 dilution ratios had fertilization rates similar to that of fresh sperm controls. Sperm samples from 12 males cryopreserved with 10% methanol showed male‐to‐male variation in post‐thaw motility (0–36%). Overall, a simplified standard protocol was established for cryopreservation of shipped sperm of Atlantic salmon using extender without egg yolk and yielded satisfactory post‐thaw motility and fertilization rates. This procedure can be readily adopted by aquaculture facilities to take advantage of high‐throughput cryopreservation capabilities at remote service centers. Most importantly, this approach lays the groundwork for an alternative commercial model for commercial‐scale production, quality control, and development of industrial standards. Control of male variability and sperm quality remain important considerations for future work.     

Characterization of the testicular semen of the African catfish, Clarias gariepinus (Burchell, 1822), and its short-term storage

Semen of the African catfish, Clarias gariepinus (Burchell, 1822), was investigated with respect to its cellular composition, sperm cell density, maturation grade, motility and fertility. Storage conditions were tested, whereby sperm viability was assessed by measurement of the motility after activation and by fertility tests. Testicular semen differed in its composition, i.e. the sperm density and numbers of spermatids, according to the maturity grade of the testis. Two semen types could be distinguished: semen type I was characterized by high sperm densities and low numbers of spermatids and semen type II had lower sperm densities and higher numbers of spermatids. Two semen types did not differ in motility and fertility (when adjusted for differences in sperm density). During storage, the sperm viability was influenced by the sodium concentration of the storage medium, temperature, membrane stabilizers as bovine serum albumen (BSA) or hen egg yolk, antibiotics and oxygen. Semen viability was maintained best when it was diluted at a ratio of 1:5 in storage solution (150 mmol L^?1 NaCl, 2.5 mmol L^?1 KCl, 1 mmol L^?1 CaCl₂, 1 mmol L^?1 MgSO₄, 20 mmol L^?1 Tris (pH 8.5) and 0.5% BSA or 0.5% hen egg yolk) and stored at 4 °C. Oxygen gassing and addition of antibiotics (1 mg mL^?1 gentamycine sulphate) to the storage solution affected the two semen types in different ways. Antibiotics had no effect on type I semen, but had a positive effect on type II semen. Oxygen gassing had a positive effect on type I semen but a negative effect on type II semen.     

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 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.     

Seasonal changes in sperm production and quality in the red porgy Pagrus pagrus (L.)

Cultured red porgy Pagrus pagrus (L.) males (n=6) were sampled every 2 weeks for milt, in order to monitor changes in sperm quality parameters during a whole spawning period. On 11 January 2001, 60% of the fish were spermiating, increasing to 100% in mid‐February and dropping to 30% by mid‐April. Sperm density showed a slight increasing trend, with mean values ranging between 8.6 and 23.7×10⁹ spermatozoa mL^?1. Sperm motility percentage exhibited a significant improvement during the spawning season (analysis of variance (anova ) P=0.0001). The duration of forward motility for the major part of the monitoring period ranged between 2 and 4 min. Red porgy spermatozoa maintained their viability for many days after whole storage of milt at 4°C. During the monitoring period there were significant changes in the mean duration of sperm survival after cold storage, ranging from 5 to 12 days. The total volume of expressible milt was maximal on 28 March, increasing from a mean value of 1.7 mL to 5.3 mL kg^?1. Milt production of captive‐reared red porgy does not appear to be limiting, when compared with the volume of expressible milt produced by other cultured marine fishes.     

Refrigerated Storage of Channel Catfish Sperm

Abstract.— Refrigerated storage of sperm is useful for genetic study and artificial breeding of fishes. Due to the potential loss of donor males, storage is important in species such as channel catfish Icralurus punctatus from which sperm cannot be stripped. This study addresses short-term storage (at 4 C) of channel catfish sperm by evaluation of storage methods employed for other species and for cryopreservation of channel catfish sperm. The objectives were to evaluate: 1) storage of intact testes and storage of sperm suspended in an extender solution; 2) use of various storage containers with and without supplemental oxygen; 3) use of extender solution with and without the addition of an antibiotic/antimycotic cocktail; 4) use of extender solution with and without the addition of methanol; and 5) use of extender solution with and without the addition of glucose and methanol. Sperm suspended in extender solution retained motility significantly longer (9 d) than did sperm in intact testis (2 d). Sperm stored in Zip-loc® plastic bags inflated with pure oxygen retained motility significantly longer (12 d) than did sperm stored in Zip-loc® plastic bags without supplemental oxygen (7 d), or sperm stored in plastic beakers (8 d) or test tubes (8 d) without supplemental oxygen. Sperm stored with the addition of antibiotic/antimycotic cocktail or methanol retained motility significantly longer (10–12 d) than did sperm stored without additives (6–8 d). Sperm stored in extender solution without glucose retained motility significantly longer (19–21 d) than did sperm stored in extender with glucose (13–16 d). Motility was retained for as long as 21 d in sperm stored in extender solution with 5% methanol and without glucose. In each experiment, loss of motility was associated with bacterial growth.     

Effect of cryoprotectants, extenders and freezing rates on the fertilization rate of frozen striped catfish, Pangasius hypophthalmus (Sauvage), sperm

The effects of four cryoprotectants (methanol, MeOH; dimethyl sulphoxide, DMSO; dimethyl acetamide, DMA; and ethylene glycol, EG), three extenders (calcium‐free Hanks' balanced salt solution, C‐F HBSS, Hanks' balanced salt solution, HBSS and sodium chloride, NaCl) and two different freezing procedures (one‐ and two‐step) on the cryopreservation of striped catfish (Pangasius hypophthalmus (Sauvage)) sperm were investigated. Sperm were frozen using a controlled‐rate freezer in 250 μL straws and stored for 2 weeks in a liquid nitrogen (LN₂) container. They were then airthawed at room temperature, and fertilization, motility and viability were assessed. The highest fertilization rate of 41% (81% of control) was achieved with the combination of 12% DMSO and 0.9% NaCl using a one‐step freezing procedure (10°C min^?1). Also, DMA resulted in a higher fertilization rate (30% or 51% of the control) than MeOH (18% or 38% of the control) or EG (8% or 12% of the control). In addition, the three extenders used did not affect fertilization rates after cryopreservation with each cryoprotectant. There were no significant differences among the three cryoprotectant concentrations and between the one‐ and two‐step freezing procedures. However, fertilization rates of cryopreserved sperm were significantly lower than the controls (P<0.05). The results of this study indicate that high fertilization rates of striped catfish eggs can be achieved using cryopreserved sperm when frozen at 10°C min^?1 in DMSO or DMA with either 0.9% NaCl or C‐F HBSS.     

Carbon dioxide reversibly inhibits sperm motility and fertilizing ability in steelhead (Oncorhynchus mykiss)

The effects of different carbon dioxide (CO₂) levels on the short-term storage of semen samples from hatchery-produced steelhead (Oncorhynchus mykiss) were evaluated. Sperm motility and fertilizing ability were significantly reduced following 4 h incubation under a relatively modest (0.9 kPa = 1%) amount of CO₂. The dose-dependent reductions, however, were not the result of cell death as sperm viability was unaltered even at the highest (5.2 kPa = 5.6%) CO₂ exposures. Reductions in sperm motility and fertilizing ability were reversible. Although previous work has indicated a direct relationship between salmonid sperm motility and sperm ATP content, the inhibitory effects of CO₂ on sperm motility were not the result of reduced sperm ATP levels. Decreasing the pH of the seminal fluid (to below 7.5) significantly reduced sperm motility. However, this effect was only observed after prolonged (4 h) exposure; short-term (1 min) exposure to this lowered pH did not alter sperm motility. Moreover, acetazolamide, a carbonic anhydrase inhibitor, attenuated the inhibitory effects of CO₂ on sperm motility. These results suggest that CO₂ inhibits steelhead sperm motility and therefore fertility in a dose-dependent manner, by reversibly lowering intracellular pH.     

Fertilizing capacity and motility of tench Tinca tinca (L., 1758) sperm following cryopreservation

Experiments were carried out to develop an optimal cryopreservation protocol for tench sperm by testing the fertilizing capacity and motility parameters including progressive motility, curvilinear velocity (VCL) and linearity (LIN) of cryopreserved sperm. Three experiments were designed to this aim: first experiment where we tested the effects of two extenders (sugar‐based Grayling and ion‐based Kurokura 180) and two cryoprotectants (DMSO and methanol) on fertilization and hatching success; second where we tested the effect of cryoprotectant type (methanol or DMSO) in different concentrations (5%, 10% and 15%) on fertilization and hatching success; and third where we tested the effect of two cryoprotectants (methanol and DMSO) on sperm motility parameters (progressive motility, VCL and LIN) after 4 h post‐thaw storage (4°C). Sperm prepared with the sugar‐based Grayling extender displayed better fertilization and hatching rates independently of the applied cryoprotectant most likely due to glucose present which acted as an external cryoprotectant. Concerning cryoprotectant concentrations, the use of 10% methanol yielded the highest fertilization (85 ± 15%) and hatching (80 ± 13%) rates, which were significantly higher than in all other groups. During the post‐thaw storage time, 5% methanol, 10% methanol and 5% DMSO groups had significantly higher motility parameters than other groups and we observed no significant decline in any of the parameters during the storage time. Overall, we found that a sugar‐based extender in combination with methanol as cryoprotectant is suitable for the cryopreservation of tench sperm and allows storage of cryopreserved sperm for up to 4 h post thaw.     

Effect of extenders on sperm cryopreservation of African catfish, Clarias gariepinus (Burchell)

The effect of extenders was studied on the cryopreservation of sperm from African catfish, Clarias gariepinus (Burchell). The following six basic extenders were tested: fructose, glucose, sucrose, NaCl, KCl solutions and the artificial seminal plasma of the African catfish. Each of these extenders was tested both with and without buffer systems (i.e. NaHCO₃-CO₂ and Tris-HCl) by using 10% dimethyl-sulphoxide (DMSO) as a cryoprotectant. The two-step freezing was carried out in a programmable freezer by using the following freezing rates: (1) 4 °C min^–1 between 3 and –4 °C; (2) and 11 °C min^–1 between –4 and –80 °C. The best post-thaw motility (25%) was achieved with 333 mmol L^–1 fructose solution and NaHCO₃ buffer. The fertilization experiments were carried out with unbuffered fructose and glucose extenders using various amounts of sperm and two fertilization methods: (1) dry and (2) wet. The best fertilization rates were achieved with 75 μL of sperm and wet fertilization with glucose extender, or 100 μL of sperm and dry fertilization in case of fructose – both methods fertilized 96% of all eggs.     

Antibiotics,Penicillin and Sreptomycin improve semen quality indices of endangered Caspian brown trout,Salmo trutta caspius (Kessler, 1870) during in vitro short‐term storage

In this study, we examined the effects of 500 IU mL^?1 penicillin + 500 μg mL^?1 streptomycin sulphate on semen quality indices of endangered caspian brown during 12 days short‐term storage at 4°C. Twenty‐four millilitre semen samples with good quality were considered for the experiment. The semen samples were then stored in the presence and absence of 500 IU mL^?1 penicillin + 500 μg mL^?1 streptomycin sulphate. The semen quality parameters including percentage and duration of sperm motility were measured 0, 3, 6, 9 and 12 days after storage. In the antibiotic receiving group, the values of percentage and duration of sperm motility reduced 3 and 6 days after storage respectively and reduced to lowest levels at day 12. In the antibiotic‐free group, the duration and percentage of sperm motility decreased significantly after 3 days of storage and reached to lowest values at day 12. Also, percentage and duration of sperm motility in each storage time were significantly higher in the antibiotic receiving group than in the antibiotic‐free group. The overall values of percentage and duration of sperm motility for all storage periods were higher in the antibiotic receiving group than in the antibiotic‐free group. In conclusion, our results demonstrated that 500 IU mL^?1 penicillin + 500 μg mL^?1 streptomycin sulphate improves the viability of caspian brown trout during short‐term storage.     

Production and storage of sperm from the black sea bass Centropristis striata L

Black sea bass Centropristis striata L. are protogynous hermaphrodites that develop and spawn as females before changing sex to male. Since all fish eventually become males, determining the relationship between sperm production, sperm quality and seasonal changes in plasma levels of testosterone (T) and 11‐ketotestosterone (11‐KT) could be useful for identifying appropriate males to maintain as broodstock. Milt and blood samples were collected three times during an 8‐week spawning season. Milt volume (3.5±0.76 mL kg^?1), sperm density (3.2 × 10⁸± 0.31 cells mL^?1), sperm production [11 × 10⁸±3.4 cells kg^?1 body weight (BW)] and sperm motility (80±0.6%) were at their highest during the first sampling interval and coincided with the highest 11‐KT levels (1.0± 0.11 ng mL^?1). All of the sperm indices decreased to their lowest levels during the final 3 weeks of the study. Sperm viability was highly correlated (adjusted R²=0.84) with sperm motility. Sperm cryopreserved in modified Mounib's extender (MME) had the highest post‐thaw motility compared with two other extenders. Post‐thaw motility of sperm cryopreserved in MME was not different from fresh after 90 days of storage. There was no difference in fertilization rates between fresh (69±2.4%) and post‐thaw (67±4.1%) sperm samples taken from the same male or among males. These results demonstrate that the quality of black sea bass spermatozoa is higher earlier in the spawning season and that acceptable post‐thaw fertilization rates can be obtained from cryopreserved sperm.     

The Cryopreservation of Striped Bass Morone saxatilis Semen

Abstract.— Two experiments were designed to improve upon existing methods for cryopreserving striped bass Morone saxatilis , semen. In the first experiment, two extenders, two cryoprotectant concentrations, and two freezing rates were evaluated on the basis of post-thaw semen motility after 1, 7, and 30 d of storage at −196 C. Semen samples cryopreserved at a freezing rate of −40 C/m resulted in a significantly higher percentage of motile sperm ( P < 0.001) and longer duration of spermatozoa motility ( P < 0.001) than samples cryopreserved at a freezing rate of -30 Chin. Also, the cryoprotectant dimethyl-sulfoxide yielded a significantly higher percentage of motile sperm ( P < 0.001) and longer duration of spermatozoa motility ( P < 0.001) when a 5% concentration was used instead of 7.5%. In the second experiment, the two extenders from Experiment I were re-evaluated and a new extender, which was a modified version of Extender 1, was tested. The samples were cryopreserved at -40 C/min with 5% DMSO and thawed in a 25 C water bath. Spermatozoa motility and fertilization ability were evaluated, and semen cryopreserved in Extender 2 yielded the longest duration of spermatozoa motility ( P < 0.001). the highest percentage of motile sperm ( P < 0.001). and the highest percentage of fertilized eggs ( P < 0.002) in comparison to Extenders I and 3.     

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.     

Biochemical Composition of Seminal Plasma and Annual Variations in Semen Characteristics of Jundiá <Emphasis Type="Italic">Rhamdia quelen</Emphasis> (Quoy and Gaimard,Pimelodidae)

This study investigated the composition of milt of the South American silver catfish (Rhamdia quelen) or jundiá. The semen was taken from jundiá in different periods during the four seasons. The biochemical composition of seminal fluid and the characteristics of sperm were analyzed. The semen quantity which can be extracted per fish in one day was 0.95 ± 0.08 ml during spring (maximum) and 0.24 ± 0.03 ml during winter (minimum). Sperm density (spermatocrit) showed higher values in the spring (75.1 ± 1.3%) decreasing slightly afterwards and reaching 63.0 ± 2.4% to 65.0 ± 2.2% in the fall and winter. Immediately after water dilution, 90–100% of the spermatozoa presented vigorous straightforward motility that remained for at least 20 s. The total duration of the motility was 47.9 ± 1.3 s in the spring and 38.6 ± 0.6 s in the other seasons (P < 0.05). This pattern of motility is maintained for more than 2 h after storage of the milt at room temperature. The pH from 5 to 10 of the water dilution does not influence the sperm motility. The mean seminal pH and osmolality values were 8.7 ± 0.07 and 274.8 ± 11.2 (mOsm/kg), respectively. The ion concentration was: Na 153.7 ± 2.4, K 10.7 ± 2.4, Cl 139.4 ± 2.1, Ca 4.2 ± 0.2, Mg 0.9 ± 0.05, P 0.9 ± 0.08 (mEq/l). The total protein was 0.6 ± 0.05 mg/dl and cholesterol concentration was 13.9 ± 0.9 mg/dl.