Effect of Different Permeable Cryoprotectants on the Quality of Cat Epididymal Spermatozoa.

BACKGROUND: Permeable cryoprotectants (CPAs) are required for successful sperm cryopreservation. OBJECTIVE: To investigate the efficacy of adding different CPAs to a freezing extender on cat epididymal sperm quality. METHODS: Epididymal spermatozoa were suspended in Tris-glucose-citrate-egg yolk extender supplemented either with glycerol, methanol, formamide, ethylene glycol (EG), propylene glycol (PG) and dimethylsulfoxide (DMSO), all at 5% (v/v), and then cryopreserved. Sperm motility, viability, functional membrane integrity, morphology and acrosome integrity were examined at post-thaw. RESULTS: Glycerol, formamide, EG and DMSO exhibited good, comparable cryoprotective effects, whereas PG showed moderate cryoprotection. Sperm viability in PG was lower than that in glycerol and EG, but was not different in formamide and DMSO. Contrarily, the least efficacy was observed in methanol. Interestingly, the CPA type has no effect on functional membrane integrity and morphology. CONCLUSION: Using Tris-glucose-citrate-egg yolk extender, formamide, EG and DMSO could substitute glycerol as permeable CPAs for cat epididymal sperm cryopreservation.

Cryopreservation and storage of cat epididymal sperm using ‒75 °C freezer vs liquid nitrogen.

The quality of cat epididymal sperm cryopreserved and stored by four methods was assessed. Epididymal sperm were suspended in Tris-glucose-citrate egg yolk extender, loaded in 0.25 mL straws and then cryopreserved. The samples in a standard protocol (LN) were cryopreserved and stored in liquid nitrogen (LN2). The sperm straws in the LN-Fr-LN group were cryopreserved in LN2 and stored in a -75 °C freezer; the straws were returned to LN2 prior to thawing. The loaded straws in the Fr group were transferred directly from 4 °C to the freezer and maintained in the freezer until thawing. The Fr-LN samples were cryopreserved and stored in the freezer and were introduced into LN2 before thawing. The sperm thawing was conducted on days 30, 60, 90 and 120 of cryopreservation. The sperm motility, viability, membrane integrity and acrosome integrity were evaluated at 15 and 180 min after thawing. The quality of post-thaw sperm in all three modified protocols was comparable (P > 0.05) and did not differ from that in the standard protocol except the membrane integrity of the 60 days stored samples evaluated at 15 min after thawing, which was significantly higher for the LN-Fr-LN than the Fr-LN groups (P = 0.04). The length of cryopreservation time had no effect (P > 0.05) on the sperm parameters assessed at 15 min after thawing. The sperm motility was significantly greater (P = 0.01 to P = 0.02) for the 15 min than the 180 min incubation. In conclusion, cat epididymal sperm could alternatively be cryopreserved and/or stored by using the -75 °C freezer for 120 days. To use, the cryopreserved sperm in the freezer could be thawed immediately or after being transferred to LN2. This was useful for the application of the -75 °C cryopreserved sperm in remote areas.

Protocols for sperm cryopreservation in the domestic cat: A review.

The main objectives of sperm cryopreservation in domestic cats are to preserve these gametes for future use, especially in valuable domestic cat breeds and to use knowledge-gained for developing sperm preservation techniques in wild felids that are threatened with extinction. To achieve acceptable quality of post-thaw sperm and results after insemination, sperm samples must be properly handled, cryopreserved and thawed by using appropriate protocols. In this paper, cryopreservation protocols of domestic cat sperm that have been reported previously are described. The subtopics include sources of sperm, freezing extenders, methods of sperm dilution, freezing storage vessels, methods of sperm cryopreservation, thawing temperature, and thawing extenders. In addition, comparisons of sperm quality results for different treatments within the same studies and between different studies are also presented.

Lipid and protein oxidation levels in spermatozoa and seminal plasma of Asian Elephants (Elephas maximus) and their relationship with semen parameters.

Peroxidation damage to spermatozoa and seminal plasma has an important role in sperm quality. Thus, the objective of this study was to determine the levels of lipid and protein oxidation in spermatozoa and seminal plasma of Asian elephants (Elephas maximus) with varying percentage of progressive motility. Lipid and protein oxidation was measured by the thiobarbituric acid-reactive species (TBARS) assay and the 2, 4-dinitrophenylhydrazine (DNPH) carbonyl groups assay, respectively. Fresh semen samples were collected from Asian elephants and classified according to the percentage of motile spermatozoa into good (>60%) and poor (≤20%) motility. Results revealed that seminal plasma malondialdehyde (MDA) and seminal plasma protein carbonyls (PCs) were significantly higher in poor motility than in good motility (p < .05). The MDA and PC levels in seminal plasma were negatively correlated with the percentages of progressive motility (p < .05). In addition, the negative correlation between sperm concentration and seminal plasma MDA level was investigated (p < .05). The sperm viability was also negatively correlated with sperm PC level (p < .05). This study indicated that lipid and protein oxidation has deleterious effect on semen quality of Asian elephants.

Preliminary study on effects of bovine frozen semen storage using a liquid nitrogen-independent method on the quality of post-thaw spermatozoa.

Frozen semen of eight bulls was used to assess effects of storage temperature and length of storage time on frozen-thawed bovine sperm quality. In experiment 1, 25 straws of frozen semen of each bull were allocated to 3 groups. The control was still maintained in liquid nitrogen (LN2). The rest were abruptly moved from LN2 to -80°C and -30°C mechanical freezers, respectively. After thawing, it was found that the sperm motility, vitality and membrane integrity were comparable (P>0.05) between the control and the -80°C samples and were significantly inferior (P<0.001) in the -30°C samples, irrespective of storage time (1-day, 1-week and 1-month). In experiment 2, two out of the three parts (16-18 straws) of frozen semen of each bull were rapidly removed from LN2 and further kept in the freezer (-80°C). One day before being thawed, half of the samples in the freezer were promptly put back to LN2. The results showed that the frozen-thawed sperm quality was not significantly affected (P>0.05) both by storage temperature (-196°C, -80°C and -80 & -196°C) and storage time [day-2, day-8 (1-week) and day-31 (1-month)]. At the same storage times, the quality measures at different temperatures were not significantly different from one another (P>0.05). In conclusion, a -80°C mechanical freezer was as effective as LN2 in preserving in vitro quality of frozen-thawed bovine spermatozoa throughout 1-month of storage. When required for use, frozen semen stored in the freezer could be thawed immediately or transferred to the LN2 tank for thawing elsewhere.

Determination of appropriate cryopreservation protocols for epididymal cat spermatozoa.

Effects of Equex and glycerol additions and sample dilution step on frozen-thawed epididymal cat spermatozoa were investigated. The epididymal sperm pellets were resuspended in extenders using one- (groups III and IV) or two- (groups I, II, V and VI) step dilution. For one-step dilution, the pellets were resuspended in plain egg yolk-Tris medium (EYT) + 5% glycerol with (IV)/without (III) 0.5% Equex and cooled (4(°) C, 1 h). For two-step dilution, the pellets were resuspended in EYT (I and V) and in EYT + 3% glycerol (II and VI), cooled and further diluted with EYT + 10% glycerol with (I)/without (V) 1% Equex and with EYT + 7% glycerol with (II)/without (VI) 1% Equex. Immediately after freeze-thawing, no differences (p > 0.05) were found in the motility, viability and membrane integrity (HOST) among the groups except the lowest HOST in IV (p = 0.005 to p = 0.04). The acrosome integrity (FITC) in group I was comparable to that in group II (p > 0.05) and was higher than the rest (p < 0.001 to p = 0.02). At 2 h after thawing, the motility, viability and HOST were comparable among the groups (p > 0.05) except the lower percentages of viability in III (p = 0.008 to p = 0.3) and of HOST in IV (p = 0.005 to p = 0.2). Two-step dilutions with Equex (I, II) were more beneficial for the FITC at 2 h than without Equex (V) (p = 0.005 and p = 0.02) and than one-step dilutions (III, IV) (p < 0.001 to p = 0.02). In conclusion, epididymal cat sperm quality after freeze-thawing could be improved when Equex was added and two-step dilution was performed during freezing. The extenders prepared for the first step of dilution could be with (3%) or without (0%) glycerol.

Sperm-TALP: an alternative extender for retrieving and diluting epididymal sperm in the domestic cat.

Effects of sperm-TALP (TALP) on the quality of fresh-extended and frozen-thawed epididymal cat sperm were evaluated. The epididymides suspended in Tris-glucose-citrate solution (Tris), a conventional medium, and TALP were cut into small pieces to recover epididymal sperm. In experiment 1, the sperm pellets remained after centrifugation were re-suspended (1 : 2, v/v) in Tris and TALP. The sperm quality in all four groups, that is, sperm retrieved with Tris (I and II) or TALP (III and IV) and diluted with Tris (I and III) or TALP (II and IV) was assessed. The sperm motility at the 0-h incubation in TALP-TALP was superior to that of the rest (p < 0.001 to p = 0.04). At the 2-h incubation, the motility in Tris/TALP-TALP was greater than that in Tris/TALP-Tris (p ≤ 0.001). In experiment 2, after centrifugation, the sperm pellets were added with freezing extenders and frozen. The thawed sperm previously retrieved from the epididymides with Tris and TALP were allotted so as not to further diluted (Tris/TALP-O) and to further diluted (1 : 1, v/v) with Tris (Tris/TALP-Tris) and TALP (Tris/TALP-TALP) and were evaluated the quality. At both incubation times, the motility of frozen-thawed sperm recovered with TALP (TALP-O/Tris/TALP) was comparable with or significantly higher than that in the Tris groups (Tris-O/Tris/TALP; p = 0.003 to p > 0.05). The motility and viability of thawed sperm in Tris-Tris were significantly decreased during the 2-h incubation (p = 0.007 for the motility and p = 0.01 for the viability). In both experiments, neither type of diluent (Tris vs TALP) nor incubation time (0 vs 2 h) significantly affected the sperm membrane integrity under hypo-osmotic condition (p > 0.05). According to beneficial effects on the quality of fresh-extended and frozen-thawed sperm demonstrated, sperm-TALP could be used as an alternative medium for recovering sperm from the epididymides and for diluting epididymal sperm in the domestic cat.

Influence of cryoprotectants glycerol and amides, combined with antioxidants on quality of frozen-thawed boar sperm.

The present study was undertaken to examine whether the cooling and freezing extenders containing a mixture of antioxidants (AOs) catalase, Na-pyruvate and mercaptoethanol and one of three types of cryoprotectants (CPs) would be able to improve the quality of frozen-thawed boar sperm. The collected semen, only the sperm-rich fraction, was diluted 1:1 with Androhep plus™ extender, stored at 15°C for 2 h and centrifuged. The centrifuged sperm pellet was re-suspended in lactose-egg yolk extender and divided into four groups for mixing with freezing extenders containing different kinds of CPs at 5°C: (I) glycerol (GLY) as control; (II) GLY with AOs; (III) dimethyl formamide (DMF) with AOs and (IV) dimethyl acetamide (DMA) with AOs. Processed sperm were packaged in 0.25-mL straws and frozen using a controlled rate freezer. After thawing, the diluted thawed sperm were incubated at 38°C for 10 min and was assessed for motility by CASA, membrane/acrosome integrity by FITC-PNA/PI and DNA integrity (DFI) by SCSA. All sperm parameters evaluated, except DFI, were negatively affected (P<0.001) when using DMF (III) or DMA (IV) as CPs instead of GLY (I and II). Total sperm motility was lower (P<0.001) in the samples supplemented with AOs (32.4 ± 1.2, 23.9 ± 1.5, 6.9 ± 0.7, and 10.3 ± 0.9%, for treatments I, II, III and IV, respectively). The quality of sperm frozen in DMF was not different from DMA (P>0.05). There was no difference in DFI among the studied groups (P>0.05). In conclusion, based on the present results, addition of AOs to cooling and freezing extenders and/or replacement of GLY with DMF or DMA could not improve quality of frozen-thawed boar sperm.

Effects of straw volume and Equex-STM on boar sperm quality after cryopreservation.

The present experiments were designed to study the effect of adding the detergent Equex-STM to freezing extender, and of straw volume (0.25 ml vs 0.5 ml), on boar sperm quality after cryopreservation. Three ejaculates from each of four purebred boars (three Landrace and one Yorkshire) were collected and frozen with a lactose-egg yolk extender containing glycerol with or without 1.5% Equex-STM. The extended semen was loaded into either 0.25- or 0.5-ml straws. The straws were placed in liquid nitrogen (LN(2)) vapour approximately 3 cm above the level of LN(2) for 20 min and then were plunged into LN(2). Thawing was achieved in warm water at 50 degrees C for 12 s and then was incubated in a 38 degrees C water-bath for 30 min before evaluating sperm quality. Results showed that the individual motility, viability and acrosomal normal apical ridge (NAR) were improved (p < 0.001) when Equex-STM was added to the freezing extender. There was no difference (p = 0.48) in sperm motility between 0.25- and 0.5-ml straws when Equex-STM was added. The percentages of viable and of NAR sperm in 0.5-ml straws were higher than those in 0.25-ml straws (p = 0.02, p = 0.0003 respectively). The percentages of membrane intact sperm evaluated using the short hypo-osmotic swelling test were not affected by straw volume or the adding of Equex-STM (p > 0.05). The results of these investigations suggested that Equex-STM exerts a beneficial effect on the quality of cryopreserved boar semen and this cryopreservation protocol was favourable for a 0.5-ml straw.

Assessment of motility of ejaculated, liquid-stored boar spermatozoa using computerized instruments.

Visual-motility assessment is a tool used to determine the quality of boar ejaculates. This method is subjective by nature, and consequently, computer-assisted sperm analysis (CASA), with different software designs, has been developed for more objective assessment using conventional image analysis or particle counting. In the present study, we compared the results of sperm analysis using a conventional CASA system (Cell Motion Analyzer, SM-CMA), with those using a novel software (QualiSperm) and those of visual assessment performed by two operators. Ejaculates were collected weekly from four Swedish Landrace boars for 4 weeks. Each ejaculate was divided into three aliquots of different sperm concentration (300, 125, and 40 million spermatozoa/mL) and stored at approximately 17 degrees C for 96h. Only samples at 40 million spermatozoa/mL concentration were analyzed using both automated systems; for the remaining concentrations, the SM-CMA was not used due to its inability to examine higher sperm concentrations. The number of spermatozoa analyzed was highest for the QualiSperm ( approximately 300-5000 spermatozoa), followed by the SM-CMA ( approximately 200 spermatozoa), and lastly, by subjective motility evaluation ( approximately 100 spermatozoa). There was a time-course decrease in motility of the liquid-stored semen, detectable by either computerized method. Although the percentage of motile spermatozoa measured by the two automated systems correlated well (r > or = 0.75), there was disagreement between operators. In conclusion, because of the lower degree of variation, the numbers of spermatozoa analyzed, and the speed of analysis ( approximately 1 min per sample), QualiSperm appears to be a suitable instrument for routine work, provided it maintains stability and is available at an affordable price.