Post-thaw Motility Research Articles

Development of a sperm cryopreservation protocol of Gangetic Mystus (Mystus cavasius)

Cryopreservation is considered as one of the most useful techniques for long-term preservation of genetic material specially sperm of fish. This study focused on the development of a sperm cryopreservation protocol for indigenous near threatened gulsha (Mystus cavasius) and a number of experiments were conducted for the purpose. To collect milt, male gulsha were sacrificed and milt was suspended in extenders. Different concentrations of NaCl were used to evaluate the activation of sperm motility and it decreased as the concentration of the extending media increased, therefore, motility was completely inhibited at 0.8% and 1.2% NaCl solution when sperm suspended in Kurokura-2 and Alsever’s solution, respectively. The toxicity of cryoprotectants to sperm were evaluated using two cryoprotectants, dimethyl sulfoxide (DMSO) and methanol along with the extenders, Alsever’s solution and Kurokura-2 solution. DMSO and methanol with 5% and 10% concentrations produced significantly higher motility during 5 and 10 min incubation and their 15% concentration found toxic to sperm. Alsever’s solution with 10% DMSO produced best equilibration (83.75±2.39%) as well as post-thaw motility (67.5±3.23%) while Kurokura-2 solution with DMSO produced similar equilibration motility (81.25±2.39%) but the post-thaw motility (50.0±6.12%) was significantly much lower than that of Alsever’s solution. Sperm preserved with Alsever’s solution plus DMSO produced highest fertilization, 72.5±7.5% and hatching, 56.8±5.6% while fresh sperm yielded 85.0±5.0% and 74.8±3.6% fertilization and hatching, respectively. The protocols that have been developed can be used for conservation of genetic materials of M. cavasius and other endangered fish species and new generations of them can be propagated using the cryopreserved sperm.Progressive Agriculture 27 (4): 517-529, 2016

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

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

THE QUALITY OF BOAR FROZEN SEMEN DILUTED IN BTS® AND MII® WITH DIFFERENT CRYOPROTECTANT SUPPLEMENTED WITH SODIUM DODECYL SULPHATE

This research was aimed to study the effect of administration of glycerol and dimetilacetamida (DMA) in BTS® and MIII®extender supplemented with sodium dodecyl sulphate (SDS) on boar frozen semen. A number of four boars were used in this study for semen collection (n=20). The collected semen was evaluated both macroscopically and microscopically. In this study, only the semen that demonstrated>70% sperm motility, >200.106/mL sperm concentration, and<20% sperm abnormalities were used and divided into eight tubes. A number of 4 tubes were diluted with 5 mL of BTS, and the rest with 5 mL MIII. The sampel was stored at 20-22° C for 2 hours, followed by centrifugation for 15 minutes (at 2000 rpm), and taken of pellet with 1 ml supernatant. The pellet that was resulted from centrifugation using BTS, then re-diluted with BTS-glycerol 5% (BTSG), BTS DMA 5% (BTSD), BTS-glycerol 5% and SDS (BTSG-S), BTS-DMA 5% and SDS (BTSD-S). Four other pellets that were centrifuged with MIII also re-diluted with MIII-glycerol 5% (MIIIG), MIII-DMA 5% (MIIID), MIII-glycerol 5% and SDS (MIIIG-S), MIII-DMA 5% and SDS (MIIID-S). Next, all of diluted semen were inserted into 0.5 mL straw and equilibrated for 2 hours (4° C), then frozen and stored in liquid nitrogen. The evaluation of frozen semen quality was conducted at 24 hours after frozen. The result of this study showed that post-thawing motility of spermatozoa in BTSD-S (40.17±0.2%) was found higher (P<0.05) compared to seven other dilution processes. Therefore, it is concluded that the concentration of 5% DMA that supplemented with SDS in BTS dilution much better for maintaining boars frozen semen quality.

Metabolic changes in droplet vitrified semen of wild endangered Persian sturgeon Acipenser persicus (Borodin, 1997)

Comparative quantitative metabolite profiling can be used for better understanding of cell functions and dysfunctions in particular circumstances such as sperm banking which is an important approach for cryopreservation of endangered species. Cryopreservation techniques have some deleterious effects on spermatozoa which put the obtained results in controversy. Therefore, in the present study, quantitative 1H NMR (Nuclear Magnetic Resonance) based metabolite profiling was conducted to evaluate metabolite changes related to energetics and some other detected metabolites in vitrified semen of critically endangered wild Acipenser persicus. The semen was diluted with extenders containing 0, 5, 10, and 15 μM of fish antifreeze protein (AFP) type III as a cryoprotectant. Semen-extenders were vitrified and stored for two days. Based on post-thaw motility duration and motility percentage assessments, two treatments with 10 μM and 0 μM of AFP had the highest and the lowest motility percentages respectively and they were objected to 1H NMR spectroscopy investigations in order to reveal the extremes of the metabolites dynamic range. Univariate (ANOVA) and multivariate (PCA) analysis of the resulting metabolic profiles indicated significant changes (P > 0.05) in metabolites. The level of some metabolites including acetate, adenine, creatine, creatine phosphate, lactate, betaine, sarcosine, β-alanine and trimethylamine N-oxide significantly decreased in vitrified semen while some others such as creatinine, guanidinoacetate, N, N-dimethylglycine, and glycine significantly increased. There were also significant differences between vitrified treatments in levels of creatine, creatine phosphate, creatinine, glucose, guanidinoacetate, lactate, N, N-dimethylglycine, and glycine, suggesting how fish AFP type III can be effective as a cryoprotectant.

Sperm cryopreservation in the spermcasting Australian flat oyster Ostrea angasi by a programmable freezing method

Cryopreservation offers long-term storage of gametes without constraint from seasonal gamete maturation, provides opportunities to improve the efficiency of breeding and genetic programs, and protects endangered species from extinction due to epidemic diseases and natural disasters. In this study, a protocol for cryopreserving sperm of the spermcasting Australian flat oyster Ostrea angasi was developed by optimizing key factors influencing the quality of cryopreserved sperm. Dimethyl sulfoxide (DMSO) was non-toxic to sperm within the concentration and duration assessed in the toxicity experiment whereas 10% methanol or a higher concentration was toxic to sperm from the exposure duration of 30 min onwards. DMSO produced higher post-thaw sperm motility among the treatments with a single cryoprotectant. The inclusion of trehalose or glucose with DMSO further increased the post-thaw sperm motility (%) and plasma membrane integrity (PMI). Sperm equilibrated for 30 min showed higher post-thaw motility and PMI than those for 10 or 50 min. Higher post-thaw sperm motility and PMI were achieved at the freezing rate of −3 °C/min than at −7 °C/min. Sperm packaged in 0.5 ml straws had a higher post-thaw motility and PMI than those packaged in 0.25 ml straws. In this study, 44.4% post-thaw sperm motility and 49.2% PMI were achieved when sperm were equilibrated in 10% DMSO +0.45 M trehalose for 30 min, packaged in 0.5 ml straws, frozen at −3 °C/min from 4 °C to −80 °C, and thawed at 40 °C for 8 s. The availability of viable cryopreserved sperm would open an option for future breeding and genetic improvement programs for the spermcasting Australian flat oyster.

Consequences of uncontrolled cooling during sterlet (Acipenser ruthenus) sperm cryopreservation on post-thaw motility and fertilizing ability

The significant influence of the number and position of fish sperm sample straws in uncontrolled cooling devices on post-thaw spermatozoa parameters, such as motility and fertilizing ability, is presented in this study. The two most popular uncontrolled cooling devices were used in this study: a Styrofoam box setup with a polystyrene floating raft on liquid nitrogen and the dry shipper setup with a straw holder. We tested the effect of different quantities of straws (6 or 60) placed on the polystyrene floating raft and the position of the straws in the holder (on the periphery or in the centre). Using these cooling methods, sperm of 10 male sterlets diluted with methanol containing cryoprotective medium was frozen. All temperature changes were recorded by a thermocouple inside the straw, and the thermogram intervals were analysed. Spermatozoa motility was evaluated by video microscopy with integrated computer-assisted sperm analysis software. Fertilization trials were conducted at a 105 spermatozoa/egg ratio. Post-thaw spermatozoa parameters, including the percent of motile spermatozoa, curvilinear velocity, velocity according to the smoothed path, linearity of track, beat-cross frequency and fertilization rate, were significantly decreased in the 60-straw floating raft setup in comparison to all of the other cooling methods. The freezing rate between −10 °C and −30 °C was significantly decreased by up to 18.6 ± 0.61 °C/min for the 60-straw floating raft setup in comparison to the other freezing conditions. Considering the above results, efforts to standardize cryopreservation protocols using uncontrolled cooling devices should take into account the amount of straws subjected to freezing.

Vitrification as an Alternative Approach for Sperm Cryopreservation in Marine Fishes.

The Southern Flounder Paralichthys lethostigma is a high-value species and a promising aquaculture candidate. Because sperm volume can be limited in this species (<500 μL), new sperm cryopreservation methods need to be evaluated. Vitrification is an alternative to conventional slow-rate freezing, whereby small volumes are cryopreserved at high cooling rates (>1,000°C/min). The goal of this work was to develop a standardized approach for vitrification of Southern Flounder sperm. The specific objectives were to (1) evaluate thawing methods and vitrification solutions, (2) evaluate the postthaw membrane integrity of sperm vitrified in different cryoprotectant solutions, (3) examine the relationship between membrane integrity and motility, and (4) evaluate the ability of vitrified sperm to fertilize eggs. From the vitrification solutions tested, the highest postthaw motility (28 ± 9% [mean ± SD]) and membrane integrity (11 ± 4%) was observed for 20% ethylene glycol plus 20% glycerol. There was no significant difference in postthaw motility of sperm thawed at 21°C or at 37°C. Fertilization from vitrified sperm in one trial yielded the same fertilization rate (50 ± 20%) as the fresh sperm control, while the sperm from the other two males yielded 3%. This is the first report of fertilization by vitrified sperm in a marine fish. Vitrification can be simple, fast, inexpensive, performed in the field, and, at least for small fishes, offers an alternative to conventional cryopreservation. Because of the minute volumes needed for ultrarapid cooling, vitrification is not presently suited as a production method for large fishes. Vitrification can be used to reconstitute lines from valuable culture species and biomedical models, conserve mutants for development of novel lines for ornamental aquaculture, and transport frozen sperm from the field to the repository to expand genetic resources.

Study on Keeping Quality and Cryopreservability of Buffalo Semen in Egg Yolk and Soybean Based Extenders

The study was aimed to compare efficacy of egg yolk based diluent (TFYG) with soybean based commercial diluents (Bioxcell® and Optixcell®, IMV, France) for refrigeration and cryopreservation (-196°C) of six Surti buffalo semen. Each qualified ejaculate (n=8/bull, &gt;70% initial motility) was splitdiluted @ 100 ×106 sperm ml-1 at 34°C with 3 diluents. Part of each aliquot was filled in French mini straws and rest transferred to refrigerator for gradual cooling to 4-5°C. After 4 h of equilibration at 4-5°C in cold handling cabinet, the straws were frozen using programmable bio-freezer. The initial mean motility, acrosome integrity and hypo-osmotic reactive sperm per cent of fresh semen were 78.54±0.30, 94.40±0.20 and 79.35±0.42%, respectively. Sperm progressive motility at 24 hrs of refrigeration storage was 68.33±0.45, 66.25±0.46 and 70.31±0.46 %, and at 72 hrs of refrigeration storage 52.50±0.68, 48.54±0.84 and 53.85±0.75 % in TFYG, Bioxcell and Optixcell diluents, respectively. Acrosomal integrity and hypo-osmotic reactivity at 24 hrs of refrigeration storage were 88.15±0.18, 87.25±0.21, 89.27±0.20% and 66.04±0.50, 63.81±0.45, 68.10±0.46%, whereas at 72 hrs of refrigeration, the values were 81.67±0.23, 80.60±0.30, 82.83±0.27% and 51.35±0.60, 47.35±0.68, 53.40±0.68%, respectively, in above 3 diluents. The pre-freeze sperm progressive motility recorded in TFYG, Bioxcell and Optixcell diluents was 69.48±0.37, 68.02±0.49 and 70.94±0.38%, and post-thaw motility was 47.71±0.79, 44.38±0.85 and 49.90±0.90%, respectively. Per cent sperm acrosomal integrity and hypo-osmotic reactivity in TFYG, Bioxcell and Optixcell diluents were 89.54±0.18, 88.58±0.22, 90.52±0.21% and 67.96±0.32, 65.65±0.42, 70.23±0.37% at pre-freeze stage, whereas 76.83±0.23, 75.90 ±0.27, 78.50±0.25% and 45.02±0.84, 42.31±0.82, 47.81±0.90% at post-thaw stage, respectively. The post-thaw longevity after 60 minutes of incubation (37°C) was 35.52±0.79, 31.15±0.85, 37.19±0.81%, respectively, in above 3 diluents. Among the three semen diluent percent sperm progressive motility, acrosomal integrity and hypo-osmotic reactivity at particular stage were higher in Optixcell showing at par results in TFYG, whereas Bioxcell showed significantly (P less than 0.05) lower findings than other two diluents. Optixcell is a commercial product much costlier than TFYG, hence there is a need to develop local soybean based semen extender which can give comparable results with TFYG.

EFFECTS OF L-CARNITINE ON MORPHOLOGY AND ANTIOXIDANT ENZYMES AND DNA INTEGRITY OF CRYOPRESERVED BUFFALO SPERMATOZOA

Cryopreservation induces sub lethal damage to the spermatozoa, thereby reduce their fertile life. There are some biochemical additives that may enhance buffalo semen freezability; L-carnitine is one of these biochemical semen additives. Till now, the exact effects of L-carnitine on buffalo semen processing outcomes haven’t been discovered. The current study aimed to clarify L-carnitine roles during buffalo semen cryopreservation. Semen was cryopreserved in tris-based extender supplemented with different concentrations of L- carnitine (0.01, 0.05 and 0.1mg/ml)Vs.Tris-based extender only (control). Then they were processed to cryopreservation and thawing to assess different semen characteristics. Cryopreserved semen was assessed for percentage post- thawing motility, acrosomal and plasma membrane integrity, viability, DNA damage, antioxidant enzymes concentration, lipid peroxidation, in vitro fertilizing potentials and conception rate. Current results indicated that addition of 0.05 mg/ml L- carnitine to semen extender significantly (P<0.05) improved post-thawing motility, viability and acrosomal integrity (63.33±9.28 %, 133.33±9.40 and 12.33± 2.02 %, respectively) compared with control (43.33±6.01 %, 74.16± 10.93 and 24.67±2.03 %, respectively). Moreover, 0.05mg/ml L- carnitine significantly increased (P<0.05) total antioxidant capacity (TAC), superoxide dismutase (SOD) and glutathione peroxidase (GPx) concentrations (0.50±0.07 mµ/ml, 73.67±5.37 U/ml and 106.66±12.03 U/L, respectively) with respect to the control (0.19±0.01 mµ/ml, 27.33±3.49 U/ml and52.33±4.09 U/L, respectively). Furthermore, 0.05mg/ml L- carnitine significantly decreased (P<0.05) lipid peroxidation of the cryopreserved spermatozoa compared with the control semen (11.00±1.73 vs 24.82±4.90nmol/ml). Likewise, at this concentration sperm DNA damage, tail length and tail moment of the cryopreserved semen significantly (P<0.05) reduced (1.87±0.36 %, 1.83±0.33 µm and 3.72±1.44, respectively) compared with control (3.47±0.13 %, 3.48±0.17 µm and 11.91±0.87, respectively). Additionally, 0.05 mg/ml L- carnitine significantly (P<0.05) improved in vitro fertilization rate (57.45%) and conception rate (61.76%) compared with the control (33.33 and 37.93%, respectively). In conclusion the use of 0.05 mg/ml L-carnitine in the freezing extender improves DNA integrity through enhancing the antioxidant defense of buffalo sperm cells and decreasing the rate of lipid peroxidation. Therefore, L-carnitine may improve sperm cryopreservation quality, reduce cryodamage and improve sperm fertilizing potential

Effect of season on semen quality parameters in Murrah buffalo

Seasonal influence on frozen semen quality in Murrah buffalo breeding bulls was determined. Frozen semen samples of 6 Murrah buffalo bulls were collected and semen frozen in 4 different seasons, viz. winter (Dec-Feb), spring (mid Feb-Apr), summer (May-Jun) and rainy (Jul-Aug) were assessed. Samples (12) of each bull, in a season, were evaluated for sperm motility, viability and acrosome integrity. Motility and other kinematics of spermatozoa during incubation (37°C) at 0, 30, 60, 90 and 120 min of thawing were assessed with computer assisted semen analyzer. Post-thaw sperm total motility and viability differed significantly among the seasons, the highest was in winter. Sperm plasma membrane integrity, acrosome integrity, progressive motility, rapid motility and other CASA evaluated parameters did not differ significantly among the seasons. Higher values of plasma membrane integrity (PMI), progressive motility, rapid motility, average path velocity (VAP), straight line velocity (VSL), curvilinear velocity (VCL), beat cross frequency (BCF), linearity (LIN) and straightness (STR) were obtained in winter season as compared to other seasons. Post-thaw motility at 0 min and 60 min of post-thaw incubation varied significantly between seasons and higher sperm motility was sustained for a longer period in semen cryopreserved in winter followed by rainy season, summer and spring. It can be concluded from this study that buffalo bull semen produced and frozen during winter season resulted in higher sperm motility, viability and postthaw longer survivability in comparison to other seasons.

Clinical freeze preservation of whole human testicular tissue: the practicality of pre-freeze in vitro culture (IVC) to optimize post-thaw sperm motility

Clinical freeze preservation of whole human testicular tissue: the practicality of pre-freeze in vitro culture (IVC) to optimize post-thaw sperm motility

Effect of Supplementation of Butylated Hydroxytoluene on Post-Thaw Viability, Motility and Membrane Integrity of Crossbred Bovine Spermatozoa

Effect of Supplementation of Butylated Hydroxytoluene on Post-Thaw Viability, Motility and Membrane Integrity of Crossbred Bovine Spermatozoa

Effect of seminal plasma from high- and low-fertility bulls on cauda epididymal sperm function.

The aim of this study was to characterise the effect of seminal plasma (SP) from bulls of high or low fertility on sperm function. First, the effect of SP on the motility of fresh cauda epididymal spermatozoa (CES) and frozen-thawed ejaculated spermatozoa was assessed (Experiment 1a). Seminal plasma was then collected from bulls of known high and low fertility. Pooled CES were incubated in the SP from each bull, diluted and assessed for motility and viability on Days 1, 2, 3 and 5 after packaging as fresh semen (Experiment 1b). Also assessed were motility, kinematics, viability and mitochondrial membrane potential after thawing (Experiment 1c) as well as hypotonic resistance (Experiment 2) and fertilisation potential using in vitro fertilisation (Experiment 3). Seminal plasma increased the motility of CES (P<0.05); however, there was no effect of SP on the motility and viability of fresh CES or on CES post-thaw motility, viability and mitochondrial membrane potential (P>0.05). The hypotonic resistance of CES was reduced by SP (P<0.05), irrespective of whether the SP was from high- or low-fertility bulls. Seminal plasma from high- or low-fertility bulls had no effect on cleavage or blastocyst rates (P>0.05). In conclusion, SP affects the physiological function of CES but there is no difference between SP from high- or low-fertility bulls.

Cryopreservation of saltwater crocodile (Crocodylus porosus) spermatozoa.

The aim of the present study was to develop a protocol for the successful cryopreservation of Saltwater crocodile spermatozoa. Sperm cells were frozen above liquid nitrogen vapour in phosphate-buffered saline (PBS) containing either 0.3M trehalose, 0.3M raffinose or 0.3M sucrose and compared with glycerol (0.3-2.7M). Although the highest levels of mean post-thaw motility were observed following cryopreservation in 0.3M trehalose (7.6%) and 0.3M sucrose (7.3%), plasma membrane integrity (PI) was best following cryopreservation in 2.7M glycerol (52.5%). A pilot study then assessed the cytotoxicity of glycerol and sucrose prior to cryopreservation and revealed no loss of survival when spermatozoa were diluted in 0.68M glycerol or 0.2-0.3M sucrose once cryoprotectants were washed out with PBS or Biggers, Whitten and Whittingham medium containing sperm capacitation agents (BWWCAP). A final study refined the combined use of permeating (0.68 or 1.35M glycerol) and non-permeating (0.2 or 0.3M sucrose) cryoprotectants. Spermatozoa were cryopreserved in liquid nitrogen vapour at rates of approximately -21°Cmin-1 (fast freeze) or -6.0°Cmin-1 (slow freeze). Post-thaw survival was highest with a combination of 0.2M sucrose and 0.68M glycerol and when these cryoprotectants were washed out with BWWCAP, regardless of whether spermatozoa were frozen using a fast (motility 14.2±4.7%; PI 20.7±2.0%) or slow (motility 12.0±2.7%; PI 22±4%) cryopreservation rate.

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.

Factors affecting in-vivo fertility of crossbred Egyptian - Italian buffalo semen

Objective: To assess the effect of region, season and year of insemination on in-vivo fertility of Italian-Egyptian crossbred buffalo semen. Methods: A total number of 4 799 female buffaloes were inseminated by frozen semen with at least 50% post-thaw motility of Egyptian-Italian crossbred bulls in three localities in Delta, lower Egypt (El-Behira, El-Sharkia and Damietta) during the period of 2013, 2014 and 2015. The pregnancy rate after two months was evaluated during the four seasons. Results: The rate of pregnancy was significantly (P<0.000 1) differ among the three localities. The effect of year of insemination on pregnancy rate was significantly higher during 2014 and 2015 than 2013 in El-Sharkia and El-Behira. But in Damietta, the rate of pregnancy was significantly higher in 2014 than 2013 and 2015. There were no significant differences among seasons in El-Behira and Damietta governorates but there was significant (P<0.05) differences in pregnancy rate in El-Sharkia. It was higher in summer, spring and autumn than in winter. Conclusions: Localities, year of insemination and season of the year have effects on fertility of crossbred Egyptian-Italian buffalo semen.

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.

Supplementing rooster sperm with Cholesterol-Loaded-Cyclodextrin improves fertility after cryopreservation

Little is known about the effects of Cholesterol-Loaded Cyclodextrin (CLC) on post-thaw semen quality in chicken. The aim of the present study is to investigate the efficacy of CLC levels (0, 1, 2 and 3 mg/mL Schramm diluent) on post-thawed semen quality and fertility in two breeds of chicken Pradu Hang Dum (native chicken) and Rhode Island Red. Semen samples of each breed were pooled, divided into 4 aliquots and diluted with Schramm diluents, cooled to 5 °C when DMF was added (6% of final volume). Semen straws were subjected to cryopreservation using the liquid nitrogen vapor method. Post-thawed sperm motility, viability, acrosome integrity, mitochondrial function, and the Malondialdehyde (MDA) level were determined. The fertility of frozen semen was tested by inseminating laying hens. Post-thaw motility between Pradu Hang Dum and Rhode Island Red was no different; but Rhode Island Red had a higher semen viability and live cell intact acrosomes than Pradu Hang Dum (P < 0.05). The percentage of high functioning mitochondria in the Pradu Hang Dum was higher than the Rhode Island Red. CLC at 2 and 3 mg/mL supplementation was associated with improved viability of frozen semen; that is, acrosome integrity and mitochondrial function (P < 0.01), albeit having no effect on MDA levels. The sperm with 1 mg/mL CLC yielded a significantly better fertility (P < 0.01). CLC (1 mg/mL) improved the quality of frozen rooster semen. There was no interaction among breeds and CLC on post-thaw semen quality and fertility.

Cryosurvival of Marwari stallion sperm in different extenders

Extenders (3) were compared for frozen-thawed semen quality of Marwari horses in the present study. Ejaculates (36) including 6 ejaculates from each of 6 stallions were collected using artificial vagina (AV) method. The ejaculates were cryopreserved either with glucose-EDTA-lactose, HF-20 or egg yolk-skim milk extender using custom freezing technique. Sperm characteristics, viz. motility, viability, acrosome intactness and plasma membrane integrity were studied in frozen-thawed semen besides record of fresh semen quality as a prerequisite. Present study indicated a non-significant difference in frozen-thawed semen parameters with glucose-EDTA-lactose and HF-20 extenders, however, both the extenders resulted in a higher percentage of post-thaw motility (PTM), viability, intact acrosome and plasma membrane integrity of sperm than the egg yolk-skim milk extender. It was concluded that glucose-EDTA-lactose and HF-20 are better semen extenders as compared to egg yolk-skim milk and they can be used effectively for semen cryopreservation in horses.

164 CARNITINE IMPROVES POST-THAWING SPERM MOTILITY BY INCREASING ADENOSINE TRIPHOSPHATE CONTENT IN BUFFALO (Bubalus bubalis)

Semen cryopreservation plays a critical role for a wide application of both AI and in vitro embryo production in buffalo. In this species, spermatozoa are more susceptible to hazards during freezing and thawing than cattle spermatozoa, thus resulting in lower fertilizing potential (Andrabi et al. 2008 Anim. Reprod. Sci. 104, 427–433). Carnitine is a quaternary ammonium compound with antioxidant capacities, able to reduce the availability of lipids for peroxidation by transporting fatty acids into the mitochondria for β-oxidation to generate adenosine triphosphate (ATP) energy (Tanphaichitr and Leelahagul 1993 Nutrition 9, 246–54). It is known that cryopreservation processes decreases the intracellular concentration of carnitine in spermatozoa (Reyes-Moreno et al. 2000 J. Androl. 21, 876–86). In cattle, supplementation of semen extender with carnitine improves sperm motility and DNA integrity (Bucak et al. 2010 Cryobiology 61, 248–53). The aim of this study was to evaluate whether supplementation of semen extender with carnitine would increase ATP content in buffalo sperm and affect post-thawing motility. Eight ejaculates from 4 bulls were used for the trial. Each ejaculate was split into 3 equal aliquots and diluted at 37°C with BioXcell extender containing 0 (control), 2.5, and 7.5 mM carnitine to a final concentration of 30 × 106 spermatozoa/mL. After 4 h at 4°C, the straws were frozen in an automated system. At thawing, sperm motility was evaluated by phase contrast microscopy at 40× magnification (Gillan et al. 2008 Anim. Reprod. Sci. 103, 201–204). Adenosine triphosphate content was measured using a Colourimetric ATP Assay Kit (Biovision, Milpitas, CA, USA). Briefly, Percoll-separated spermatozoa were homogenised and then deproteinized using 10-kDa spin columns. Samples were incubated at RT for 30 min and absorbance was measured at 570 nM in a microplate reader. Differences in sperm motility and ATP content among groups were analysed by ANOVA. Both concentrations of carnitine increased post-thawing sperm motility compared with the control (44.4 ± 3.5, 53.1 ± 3.9, and 52.5 ± 3.6, respectively, with 0, 2.5, and 7.5 mM carnitine; P &lt; 0.05). Interestingly, carnitine increased ATP content of buffalo frozen–thawed sperm in a dose-dependent manner (4.1 ± 0.1, 5.3 ± 0.1, and 8.2 ± 0.4 nM × 108 sperm, respectively, with 0, 2.5, and 7.5 mM carnitine; P &lt; 0.01). In conclusion, the enrichment of semen extender with carnitine improved post-thawing motility of buffalo sperm by boosting mitochondrial ATP production, hence providing energy for use by spermatozoa.