Highest Post-thaw Sperm Motility Research Articles

Cryopreservation of sperm from the gudgeon, Microphysogobio rapidus (Cyprinidae): Effects of cryoprotectant, diluents, and dilution ratio

We investigated methods for cryopreserving sperm from the endangered gudgeon, Microphysogobio rapidus, by examining the effects of cryoprotective agent (CPA) concentration, diluent, and dilution ratio on post-thaw sperm quality. The quality of frozen sperm was evaluated in terms of motility and kinematic parameters, viability, DNA damage, and fertilization rate. We evaluated methanol, glycerol, dimethyl sulfoxide (DMSO), and ethylene glycol as CPAs. Sperm motility, velocity, and viability were significantly higher when methanol was used as the CPA (p < 0.05). The diluents tested were Ringer's solution, Kurokura's Extender, Common Carp Sperm Extender (CCSE), and buffered sperm motility-inhibiting saline solution (BSMIS); post-thaw motility was highest when Ringer's solution was used as the diluent. Next, various quantities of methanol were combined with Ringer's solution to identify the optimal dose of methanol. The dilution ratios tested ranged from 1:1 to 1:7. Cryopreserved sperm was thawed at 20 °C for 15 s. The use of 10% methanol with Ringer's solution at a dilution ratio of 1:5 resulted in the highest post-thaw sperm motility, viability, and velocity including VAP, VCL, and VSL. Post-thaw sperm showed significantly greater DNA damage than the control (fresh sperm) (p < 0.05). The fertilization rate was highest with fresh sperm (p < 0.05), followed by sperm frozen with 10% methanol + Ringer's solution. We recommend that the best way to preserve sperm in the studied species is to use a combination of Ringer's solution and 10% methanol at a 1:5 dilution ratio. Our findings will facilitate the artificial fertilization of M. rapidus.

Evaluation of the effects of glucose concentration in extender and sperm concentration in straw for cryopreservation of Atlantic sturgeon (Acipenser oxyrinchus) semen

The Atlantic sturgeon (Acipenser oxyrinchus) faces unique conservation challenges due to its extended maturation period and irregular spawning patterns, which restrict its reproductive capacity. Therefore, in order to overcome these difficulties we have made a first attempt to develop effective cryopreservation procedure for Atlantic sturgeon through evaluation of the effects of the glucose concentration in extender and final sperm concentration in straw on sperm motility parameters. A remarkably high (81 ± 8%) postthaw sperm motility was obtained. The other sperm velocity and trajectory parameters were differentially affected both by concentrations of glucose or sperm and cryopreservation process. Our results undoubtedly show that the post-thaw sperm motility of Atlantic sturgeon is highly influenced by the final glucose and sperm concentration. The recommended cryopreservation procedure includes 0.05 M glucose and 7.5% methanol at 1.0×109 spermatozoa ml−1. We have confirmed that our elaborated protocol can be used for cryopreservation of Atlantic sturgeon semen which can be subsequently used for long-term conservation of this endangered species, offering a valuable tool in efforts to safeguard its genetic diversity and contribute to its survival.

The optimum concentration of soya-lecithin for replacing egg yolk in INRA-82-modified semen extenders improved the cryopreserved semen quality and DNA integrity of Arabian stallions

The cryopreservation of stallion spermatozoa varies greatly between animals. Different equine semen extender supplements were invented to maximize the post-thaw semen quality. Soy lecithin is one of those additives that contain plant origin phospholipids used to replace egg yolk to minimize disease transmission. This study aimed to determine which concentrations of soy lecithin can replace egg-yolk in modified INRA-82 semen extenders for the cryopreservation of horse semen. Semen was collected from six Arabian stallions and extended with modified INRA-82 semen extender supplemented with 0.0, 1.25, 2.50, and 5.0% soya lecithin. Frozen-thawed sperm motility was measured at 0, 1, 2, and 3 h. Viability index, sperm plasma membrane integrity (SPMI), and acrosome integrity in addition to the comet assay non-fragmented DNA, head DNA, tail DNA, tail length, and tail moment were statistically analyzed. The addition of 2.5% soya lecithin had the highest post-thaw sperm motility at 0, 1, 2 h (P &lt; 0.001) and 3 h (P &lt; 0.0001), the optimum viability index (P &lt; 0.0001), SPMI (P &lt; 0.05), acrosome integrity (P &lt; 0.0001), non-fragmented DNA (P &lt; 0.01), DNA in the comet head (P &lt; 0.05), minimum DNA in the comet tail (P &lt; 0.05), shortest comet tail length (P &lt; 0.0001), and lowest comet tail moment (P &lt; 0.01). Soy lecithin at concentrations other than 2.5% deteriorated all post-thaw semen parameters. Soy lecithin (2.5%) can replace egg yolk in the modified INRA-82 semen extender for the cryopreservation of good-quality stallion semen.

Proteomic analysis reveals the potential positive effects of Mito-TEMPO on ram sperm motility and fertility during cryopreservation

The aim of this study was to investigate the effects of mitochondria-targeted antioxidant Mito-TEMPO on the protein profile of ram sperm during cryopreservation and evaluate the cryoprotective roles of Mito-TEMPO on ram sperm quality and fertilization capacity. Semen collected from 8 Dorper rams was cryopreserved in TCG-egg yolk extender supplemented with various concentrations of Mito-TEMPO (0, 20, 40 and 60 μM). After thawing, sperm characteristics, antioxidant status and the abundance of hexose transporters (GLUT 3 and 8) were analyzed. The cervical artificial insemination (AI) was performed to evaluate the fertilization ability of cryopreserved ram sperm. The alterations of sperm proteomic profile between the control and MT40 groups were determined using iTRAQ-coupled LC-MS. Supplementation with 40 μM of Mito-TEMPO resulted in the highest post-thaw sperm motility and kinematics. Sperm quality, antioxidant capacity and glucose transporter abundance of frozen-thawed ram sperm were elevated in the MT40 group. The inclusion of 40 μM Mito-TEMPO in freezing extender also resulted in the higher pregnancy rate of ewes. A total of 457 proteins including 179 upregulated proteins and 278 downregulated proteins were defied as differentially expressed proteins (DEPs) using fold change (FC) > 1.2 with P < 0.05. Sixty-one DEPs with (FC > 1.5) were dramatically regulated by Mito-TEMPO. These DEPs are mainly involved in sperm motility, energy metabolism and capacitation. Our data suggest that the beneficial effects of Mito-TEMPO on sperm motility and fertility potential of cryopreserved ram semen are achieved by regulating sperm antioxidant capacity and sperm proteins related to energy metabolism and fertility.

EPICATECHIN IMPROVES FROZEN SPERM VITALITY BY ITS ANTIOXIDANT AND CRYOPROTECTIVE ACTIONS

This study served to elucidate the effects of three selected doses of epicatechin (EPI; 25, 50 or 100 μmol/L) on the motion and oxidative profile of bovine spermatozoa subjected to the process of cryopreservation. Furthermore, we focused on the impact of EPI on the expression patterns of heat shock proteins 90 and 70 as well as the BAX and Bcl-2 proteins involved in the process of apoptosis. The experimental samples were compared with a native control as well as specimens cryopreserved in the absence of any supplement. Our dada indicates that particularly 50 and 100 μmol/L EPI assured a higher preservation of the sperm motility (p&lt;0.001) in comparison with the cryopreserved control. The best protection of the sperm DNA (p&lt;0.05), lipids (p&lt;0.05) and proteins (p&lt;0.01) were observed in the samples exposed to 100 μmol/L EPI in comparison to untreated frozen samples. The presence of higher EPI concentrations in the cryopreservation medium led to significant stabilization of the heat shock protein 90 (p&lt;0.01 in case of 50 μmol/L EPI; p&lt;0.001 with respect to 100 μmol/L EPI) as well as a higher maintenance of the BAX:Bcl-2 ratio (p&lt;0.001) when compared to the untreated frozen control. In summary, we may assume that EPI exhibits antioxidant properties which enable the molecule to prevent excessive damage to biomolecules essential for the cell survival and offer an additional layer of protection to spermatozoa exposed to low temperatures, all of which may be translated into a higher post-thaw sperm motility and activity.

Effects of mitoquinone (MitoQ) supplementation during boar semen cryopreservation on sperm quality, antioxidant status and mitochondrial proteomics

Mitochondria are the most important organelles and the main reactive oxygen species producers in spermatozoa. The objective of this study was to investigate the effects of mitochondria-targeted antioxidant mitoquinone (MitoQ) supplementation in boar semen extender during cryopreservation on sperm quality, antioxidant status and the changes of sperm mitochondrial proteomic profile. Semen collected from 10 Large White boars was cryopreserved in lactose-egg yolk extender supplemented with various concentrations of MitoQ (0, 5, 10, 20 and 40 μM). After thawing, sperm characteristics, antioxidant status and the abundance of hexose transporters (GLUT 3 and 8) were analyzed. The comprehensive mitochondrial proteomic profiling was performed on spermatozoa in the control and MitoQ10 groups. Supplementation with 10 μM of MitoQ resulted in the highest post-thaw sperm motility and kinematics. Sperm quality, antioxidant capacity and glucose transporter abundance of frozen-thawed boar sperm were also elevated in the MitoQ10 group. Excessive MitoQ (40 μM) supplementation induced a reduction of sperm motility parameters, sperm quality and antioxidant status and the abundance of GLUT3 and 8 proteins. A total of 189 proteins were defied as differentially expressed proteins (DEPs) using fold change (FC) > 1.2 with P < 0.05, and 33 of them were dramatically (FC > 1.5) regulated by MitoQ. These DEPs are mainly involved in sperm motility, energy metabolism and oxidative phosphorylation. Our data suggest that the beneficial effect of MitoQ on cryopreserved boar semen is achieved by regulating sperm antioxidant capacity and the mitochondrial proteins related to motility and metabolism.

Optimization of sterlet sperm concentration for cryopreservation

One of the critical points in low temperature storage is the expense of storage capacities. Moreover, sperm concentration in a sample is not only a parameter for effective use of a cryobanks but also a parameter affecting cryopreservation outcomes. Sturgeon sperm in comparison to other species is significantly less concentrated due to the specificity of sperm maturation process, during which sperm is mixed with urine. In this study, we evaluated whether the artificial modification of the sterlet spermatozoa concentration before freezing can be useful during the routine application of sturgeon sperm cryopreservation.The sperm was first concentrated by centrifugation of native sperm samples. Then, the excess of seminal fluid was collected. The concentrated sperm samples were further diluted with the same seminal fluid to obtain different sperm concentrations in suspension, and they were subjected to freeze-thawing. The percentage of post-thaw sperm motility depended on the sperm concentration in the samples. The highest post-thaw sperm motility was found in sperm samples with concentration of 0.2 and 1 × 109 spz mL−1. The sperm concentration of 3 × 109 spz mL−1, which is higher than the native sperm concentration in sterlet, was found to be appropriate for use in cryopreservation procedures since the sperm fertilizing ability remained at a high level even though a significant decline in percentage of sperm motility was observed. These findings supported the conclusion that the procedure of artificially increasing the sperm concentration before freezing could be useful for the reduction of the volume kept in cryostorage and to decrease the sample volume required for artificial fertilization.

Cryopreservation of sperm from farmed Pacific abalone, Haliotis discus hannai

This study aimed to improve a sperm cryopreservation protocol for farmed Pacific abalone, Haliotis discus hannai. Dimethyl sulfoxide (Me2SO), glycerol, ethylene glycol (EG), propylene glycol (PG), and methanol were chosen as cryoprotectants (CPAs). Four different equilibration time (5, 10, 30, and 60 min), and two types of equilibration temperature (4 °C and 20 °C) were selected at the present experiment. Most equilibration temperatures with each CPA showed significant differences among different equilibration time. Post-thaw sperm motility of five CPAs showed no significant difference at two equilibration temperature. Based on these results, 8% Me2SO, 8% EG, 6% PG, 2% glycerol, and 2% methanol were chosen to determine optimal conditions for sperm cryopreservation of H. discus hannai. The highest post-thaw sperm motility (8% Me2SO: 50.6%, 8% EG: 45.6%, 2% glycerol: 44.5%, 6% PG: 28.7%, 2% methanol: 25.4%) was achieved after exposing sperm to liquid nitrogen (LN2) vapor for 10 min at 5 cm above the LN2 surface and then submerging them in LN2 for at least 2 h followed by thawing at 60 °C with seawater and recovering them at 20 °C with seawater. In this study, 8% Me2SO and 2% glycerol were chosen to check post-thaw sperm quality to estimate percentages of plasma membrane integrity (PMI), mitochondrial potential analysis (MP), and acrosome integrity (AI) using fluorescent techniques. No significant difference in PMI, MP, and AI was found between sperm cryopreserved with 8% Me2SO and those cryopreserved with 2% glycerol. The current study has demonstrated that 8% Me2SO was optimal for sperm cryopreservation for H. discus hannai with 5 min of equilibration time, 5 cm of rack height and 60 °C of thawing temperature. The present research provides more effective cryopreservation methods for H. discus hannai sperm than previous studies.

Development of an efficient and standardized method for the cryopreservation of Arctic charr milt and its use in the fertilization of brook trout eggs to produce ‘sparctic’ hybrids

The general objective of this study was to establish a standardized procedure for the cryopreservation of Arctic charr milt and its use in the production of ‘sparctic’ hybrids. The specific objectives of this study were to determine: (i) the optimal final sperm concentration as well as (ii) the final glucose concentration, (iii) the effect of post-thaw storage time on sperm motility characteristics, and (iv) the fertilization ability of cryopreserved Arctic charr milt at 0 and 60 min after thawing at sperm:egg ratios of 500,000:1 and 1,000,000:1. We observed high post-thaw sperm motility (49–57%) at final sperm concentrations ranging from 0.5 to 2.0 × 109 spermatozoa ml−1 in straw. We determined that a final glucose concentration of 0.15–0.19 M in 7.5% methanol was optimal (post-thaw sperm motility 48–60%). Milt cryopreserved at optimal conditions (final concentrations: 0.17 M of glucose, 7.5% methanol and 1.5 × 109 spermatozoa ml−1) could be stored for up to 15 min after thawing without the loss of sperm motility. The ability of cryopreserved milt to fertilize brook trout eggs was high (68–72%) and did not differ between the tested sperm:egg ratios (500,000:1 and 1,000,000:1) when milt was used immediately after thawing or after 60 min of post-thaw storage. The milt used in this study was obtained from two hatcheries and was characterized by significantly different values of sperm concentrations. In conclusion, a standardized and efficient cryopreservation method for Arctic charr milt has been developed and used for the production of ‘sparctic’ hybrids. Species-specific requirements for final sperm and glucose concentrations and the post-thaw storage time were determined. The hatchery of origin of Arctic charr should be taken into consideration, as high variability in sperm characteristics may be expected. The development of this method and the positive results of the fertilization trials indicate promising potential for the implementation of cryopreserved milt in hatchery practice.

Comparative analysis of sperm freezability of sex-reversed female brook trout and sex-reversed female rainbow trout semen

The aim of our study was to compare quality of fresh and cryopreserved semen of sex-reversed female rainbow trout and sex-reversed female brook trout. The effect of different final sperm concentrations in straws with a constant extender concentration and final glucose concentration on the sperm motility parameters of cryopreserved semen was tested. Furthermore, we examined the effect of post-thaw storage time on sperm motility parameters at optimal sperm and glucose concentrations. The effects of semen cryopreservation from sex-reversed female rainbow trout and sex-reversed female brook trout on sperm fertilizing ability at the sperm-to-egg ratios 500,000:1 and 1,000,000:1 after 0 and 60 min of post-thaw storage of semen were investigated. We have demonstrated that final sperm concentrations in straws as well as final glucose concentrations in extended semen influenced post-thaw sperm motility parameters in both species. The high post-thaw sperm motility was recorded for sperm concentrations within the range of 1.0–4.0 × 109 spermatozoa ml−1 for both species. Furthermore, the glucose concentration appeared to be very important for cryopreservation success and its effect was species-specific. The final glucose concentrations of 0.15 M and 0.19 M, for sex-reversed female rainbow trout and sex-reversed female brook trout, respectively, produced the highest results for sperm motility after cryopreservation. The post-thaw sperm motility was unaffected by 60 and 360 min after thawing for sex-reversed female rainbow trout and sex-reversed female brook trout, respectively. The fertilization rates of cryopreserved semen of sex-reversed female rainbow trout were high and did not differ between the investigated sperm:egg ratios after 0 and 60 min of post-thaw storage. However, fertilization rates of cryopreserved semen of sex-reversed females brook trout were lower and decreased after 60 min of post-thaw storage of semen at sperm: egg ratio 1,000,000:1. In conclusion, our results demonstrated differences in freezability between the semen of sex-reversed female rainbow trout and sex-reversed female brook trout. The semen of both species can be cryopreserved at the final sperm concentration of 3.0 × 109 spermatozoa ml−1. However, the influence of final glucose concentration on cryopreservation success, as well as the duration of post-thaw storage time and fertilization rates, are species-specific. In our opinion, standardizing the procedure of semen cryopreservation presented in this study is a prerequisite for the development of repeatable procedures and the future implementation of cryopreserved semen from sex-reversed females into hatchery practice.

Standardized cryopreservation protocol of European perch (Perca fluviatilis) semen allows to obtain high fertilization rates with the use of frozen/thawed semen

The general aim of this study was to develop an efficient and standardized cryopreservation procedure for European perch semen. The specific aims of this study were: to test the effects of (i) final glucose concentration, (ii) final sperm concentration in the extended semen and (iii) the storage time of post-thaw semen on sperm motility parameters. Moreover, the effects of cryopreservation of semen on fertilization rates were tested at sperm:egg ratios ranging between 50,000:1 and 500,000:1 (iv), as well as the fertilization of 25 g portions of eggs (v). The range of optimal sperm concentrations in straws with high post-thaw sperm motility (66–73%) was quite wide, from 1.0 to 6.0 × 109 spermatozoa ml−1. The final glucose concentration of 0.30–0.34 M in 7.5% methanol produced the highest results of sperm motility (76 ± 5%) after cryopreservation. Storage time of post-thaw semen negatively influenced sperm motility parameters. The fertilization rates while using cryopreserved semen were high (79%) and did not differ from fresh semen (85%). Similar results (73–77%) were obtained between sperm:egg ratio ranged from 50,000:1 to 500,000:1. Moreover, cryopreserved semen was successfully used for fertilization as much as 12,500 eggs (25 g). However, semen has to be used for fertilization within 2 min after thawing. In our opinion, standardizing the procedure of semen cryopreservation presented in this study is a prerequisite for the development of repeatable procedures and the future implementation of cryopreserved semen in hatchery practice. Further studies should focus on extension of time of post-thaw sperm motility, determination of optimal sperm:egg ratio at the conditions of large-scale fertilization and scaling up procedure to fertilize 50–150 g of eggs (one ribbon) at single application.

Cryopreserved rainbow trout semen can be used for the fertilization of up to 8000 eggs in a single application

Here we aimed to test the fertilizing ability of cryopreserved semen of rainbow trout (Oncorhynchus mykiss) during the fertilization of between 500 and 8000 eggs, thus establishing the optimal number of eggs to use in a single application of cryopreserved semen. The cryopreservation procedure resulted in a high post-thaw sperm motility (~70%). The fertilization rates ranged from 29 to 92% and did not differ depending on the number of eggs used. However, we did detect high and significant variability in fertilization rate among the pools of cryopreserved semen. Our results show that the cryopreserved semen of rainbow trout can be used to fertilize up to 8000 eggs in a single application. Cryopreserved sperm can be potentially implemented into breeding programs based on the crossing of selected males with individual females after confirmation of the rearing performance of larvae. Further studies should now focus on the cause of the between male variability in fertilizing ability observed in this study.

Optimal sperm concentration in straws and final glucose concentration in extender are crucial for improving the cryopreservation protocol of salmonid spermatozoa

Ensuring the precise conditions during cryopreservation relies on constant sperm concentration in straws with constant cryoprotectant concentrations, which are essential for standardizing cryopreservation protocols. The aim of our study was to test the effect of different sperm concentrations in straws with constant extender concentration on sperm motility parameters of cryopreserved semen of brown trout (Salmo trutta m. fario), sea trout (Salmo trutta m. trutta), brook trout (Salvelinus fontinalis) and Atlantic salmon (Salmo salar). At the same time, the usefulness of different straw sizes (0.25 and 0.5ml) for cryopreservation of the semen of these species was examined. Additionally, the effect of the final glucose concentration at a constant sperm concentration on post-thaw sperm motility was evaluated. The final sperm concentration in straws at a constant cryoprotectant concentration influenced the post-thaw sperm motility parameters of salmonids. The optimal sperm concentration with high post-thaw sperm motility was 2.0×109 spermatozoa ml−1 for brook trout, 3.0×109 spermatozoa ml−1 for brown trout and sea trout and 4.0×109 spermatozoa ml−1 for Atlantic salmon. The use of 0.5ml straws led to higher or similar values of post-thaw sperm motility than the 0.25ml straws. The final glucose concentration of 0.15M produced the highest results of sperm motility after cryopreservation for all tested species. Our results demonstrated that the influence of sperm concentration in straws on cryopreservation success is species-specific within salmonids. On the other hand, the effect of glucose concentration did not appear to be species-specific. In our opinion, standardization of the semen cryopreservation protocol presented in this study is a prerequisite for the development of a repeatable procedure and hence the future implementation of cryopreserved semen in hatchery practice.

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.

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.

The effect of supplementation of a trehalose-based extender with KCl on rainbow trout (Oncorhynchus mykiss) sperm freezability and post-thaw motility

The aim of this study was to test if a cryopreservation procedure using methanol as an internal cryoprotectant and trehalose as an external cryoprotectant could be applied to rainbow trout sperm (Oncorhynchus mykiss). Additionally, the effects of potassium ions and Tris-HCl on post-thaw sperm motility, viability, and fertilizing capacity were analyzed. In this study, we demonstrate for the first time that rainbow trout sperm can be cryopreserved using a trehalose and methanol based extender. Trehalose was found to be effective in rainbow trout sperm cryopreservation at concentrations ranging between 175 and 200mM when supplemented with 10% methanol (v/v). The most effective dilution ratio was 1:11.5, which resulted in a final sperm concentration ranging from 0.8 to 1.6×109 spermatozoa mL−1. A 15-min equilibration period of sperm in the extenders did not influence sperm motility parameters. The highest post-thaw sperm motility was found using extenders containing 175mM trehalose (77.4±10.8) and 175mM trehalose with 40mM KCl (79.3±9.2). The supplementation of the extender with Tris-HCl and the adjustment of the pH to 7.5 and 8.5 resulted in a decrease in post-thaw motility. The post-thaw sperm motility in samples cryopreserved in an extender containing 40mM of KCl did not decrease 60min after thawing. The fertilizing capacity of sperm cryopreserved in 175mM trehalose and 40mM KCl was the highest and did not differ from fresh sperm at a sperm-to-egg ratio of 200,000:1. In conclusion, our results indicate that rainbow trout sperm can be successfully cryopreserved with the application of a trehalose based extender. The supplementation of the cryopreservation extender with 40mM KCl resulted in prolonged post-thaw storage of the sperm, allowing prolonged use of the sperm over time for common fishery practices. Statement of relevanceThis work is related to fish production and conservation.

Effect of dilution in sperm maturation media and time of storage on sperm motility and fertilizing capacity of cryopreserved semen of sex-reversed female rainbow trout

Masculinized females, also called neomales or sex-reversed females have a male phenotype but retain the female genotype (XX). Therefore, all spermatozoa produced in their functional testes carry an X chromosome, which is desired for the production of all-female rainbow trout populations. Semen of sex-reversed female rainbow trout is of low quality and in vitro maturation is required, which includes dilution of sperm suspensions with specially formulated maturation solutions. The aim of this study was to determine the effect of dilution in different maturation media on sperm quality (sperm motility characteristics and fertilizing capacity) of frozen/thawed sperm of sex-reversed female rainbow trout. The effect of time of post-thaw storage (0, 15, 60 and 120min) on semen quality was also tested. Sperm motility parameters and fertilization rate at the eyed and hatching stages were assessed for post-thaw semen diluted in different media. The cryopreservation procedure resulted in high post-thaw sperm motility of about 57% and did not differ from fresh semen. Unexpectedly, maturation media decreased sperm activation capacity immediately after dilution; however, sperm motility increased over time. Fertilization rates of frozen/thawed semen were high (71–87%) and did not differ significantly between experimental variants at any of tested periods of storage. Our results demonstrated that the effect of the maturation media on frozen/thawed sperm is different from that of fresh sperm. The progressive increase in post-thaw sperm motility in maturation media can potentially be applied to routine hatchery practice.

Greenlip abalone (Haliotis laevigata Donovan, 1808) sperm cryopreservation using a programmable freezing technique and testing the addition of amino acid and vitamin

This study investigated factors key to the development of sperm cryopreservation in the greenlip abalone Haliotis laevigata using a programmable freezing technique, including (1) permeable cryoprotectant agent (CPA) selection; (2) cooling rate; (3) endpoint temperature; (4) thawing temperature; (5) sperm to egg ratio and (6) sugar, vitamin and amino acid supplementation, using sperm motility, fertilization rate, plasma membrane integrity, mitochondrial membrane potential or acrosome integrity as quality assessment indicators. Results showed that among the permeable CPAs evaluated, 10% dimethyl sulfoxide was the most suitable for greenlip abalone sperm cryopreservation. The highest post-thaw sperm motility was achieved with the sperm being frozen at a cooling rate of 5°C min 1 to 30°C from 0°C and thawed and recovered in 40°C and 18°C seawater baths respectively. The addition of sugars in 10% dimethyl sulfoxide did not significantly improve the post-thaw sperm motility and fertilization rate. The addition of 0.6% glycine, 0.2% taurine or 0.02% L-ascorbic acid, on the other hand, significantly improved the postthaw sperm motility. However, only the addition of 0.6% glycine improved the post-thaw sperm fertilization rate, which was further confirmed by the improvement of the post-thaw sperm mitochondrial membrane potential and acrosome integrity through flow cytometry analysis.

Effect of postthaw storage time and sperm-to-egg ratio on fertility of cryopreserved brook trout sperm

The aim of this study was to test the influence of postthaw storage time on sperm motility parameters of brook trout (n = 9). Furthermore, we examined the effect of sperm-to-egg ratios of 300,000:1 and 600,000:1 on fertility of postthaw, cryopreserved, brook trout sperm. The application of a cryopreservation procedure produced very high postthaw sperm motility (56.8 ± 4.0%). The cryopreserved sperm of brook trout could be stored up to 60 minutes without loss of the percentage of sperm motility (52.0 ± 9.0%). The fertilization capacity of brook trout postthaw sperm was comparable with the fertilization rate of fresh semen at a sperm-to-egg ratio as low as 300,000:1 (42.4 ± 14.0% and 36.5 ± 11.0% for eyed and hatched stages, respectively). The possibility of postthaw semen storage for the prolonged time and the obtainment of high fertilization rate at low sperm-to-egg ratio can lead to the significant improvement of brook trout semen cryopreservation procedure.

Application of glucose–methanol extender to cryopreservation of semen of sex-reversed females rainbow trout results in high post-thaw sperm motility and fertilizing ability

Abstract We tested the usefulness of 0.18 M glucose in 9% methanol as an extender for the cryopreservation of semen of sex-reversed rainbow trout females (n = 9). The semen-to-extender ratio of 1:9 and 15 min equilibration were used. Motility and viability parameters as well as the fertilization rate at the eyed stage and hatching were assessed for post-thaw sperm. The fertilization success of cryopreserved semen was about 80% for both 500,000 and 1,000,000:1 sperm-to-egg ratios. The application of the cryopreservation procedure resulted in remarkably high post-thaw sperm motility and viability (55 and 56%, respectively). Our results for the first time show that the post-thaw fertilization ability of rainbow trout sex-reversed female semen can be similar to that of fresh semen at a sperm-to-egg ratio as low as 500,000:1. The sperm motility pattern characterized by low linearity, high amplitude of lateral head displacement and curvilinear velocity seems to be beneficial for the fertilizing ability of thawed sperm. Sperm viability appears to be a reliable indicator of the quality of thawed semen of rainbow trout sex-reversed females. The cryopreservation procedure using glucose–methanol extender after scaling up can be recommended for routine hatchery practice.