Percentage Of Membrane-intact Spermatozoa Research Articles

Application of High Hydrostatic Pressure (HHP) to Improve Cryopreservation of Young Bull Semen

Abstract The objective of the study was to determine the effect of high hydrostatic pressure (HHP) on quality of cryopreserved semen of young bulls. Semen for this study was collected from 8 bulls aged between 13 and 18 months at monthly intervals, from June to September. After collection, semen was diluted in a commercial Bioxcell® extender (one part at 1:1 and a second part to give a sperm concentration of 20 million/0.2 mL), filled into straws and treated with HHP at 30 MPa for 90 min. After HHP treatment, pre-diluted semen (1:1) was diluted to a sperm concentration 20 million/0.2 mL and filled into straws. In addition, part of the semen diluted to a concentration of 20 million/0.2 mL was not treated with HHP (control). All of it was held at +4°C and frozen in a freezer after 2.5-h equilibration. Semen was thawed in a water bath at 38°C and subjected to estimation of the percentage of motile sperm both subjectively and using a computer-assisted semen analyzer and cytometric assessment of sperm cell membrane integrity. Subjective motility and fast progressive motility were significantly higher with pre-diluted (1:1) and HHP treated semen compared to control (P<0.05). No significant differences were observed in percentage of membraneintact spermatozoa between control and experimental groups. Additionally, the influence of HHP on the sperm of individual bulls was assessed. In bull number 2, the HHP treatment after semen pre-dilution significantly improved progressive motility from 54.1 to 63.4 percent (P<0.05). In bull number 4, the HHP treatment after semen pre-dilution significantly improved subjective motility, rapid motility and progressive motility by 12.5, 16.8 and 16.3 percent, respectively (P<0.05). No effect was seen for 6 bulls. It is concluded that for some bulls, the application of HHP before semen freezing may improve the cryopreservation outcome. However, this requires further research in this area, also to determine the fertilizing capacity of bull semen exposed to high hydrostatic pressure.

Measurement of membrane integrity in canine spermatozoa using a fluorescent computer-assisted spermatozoal quantification method after SYBR-14/PI staining compared with manual counting after CFDA/PI staining.

The aim of this study was to compare measurements of spermatozoal membrane status in dogs using computer-assisted spermatozoal quantification (CASQ) after staining with SYBR-14 and propidium iodide (PI) with manual counting after CFDA/PI staining. CASQ was performed on fresh (n=11) and thawed cryopreserved canine semen (n=91) using (1) a red long-pass (LP) filter on an untreated sample (membrane-disrupted spermatozoa, MDS count) and in a sample with all cellular membranes disrupted (total spermatozoal count, TC), (2) green LP filter for a TC and the red filter for an MDS count and (3) a green short-pass filter to obtain a membrane-intact spermatozoa (MIS) count and the red filter to obtain the MDS count, which were added to give a TC (red-green filter CASQ, n=50). Spermatozoa were also stained with CFDA/PI, manually examined and classified as MIS or MDS. All measurements were performed in duplicate. The percentage of membrane-intact spermatozoa (MIS) was calculated. The percentage of progressively motile spermatozoa (PMS) was determined subjectively. The data were analysed to measure the agreement between the CASQ and CFDA/PI methods, repeatability of the methods and correlation between the MIS and PMS percentage. Compared with the CFDA/PI method, the agreement of MIS percentage with red filter CASQ was -12% to 34%, green LP filter CASQ -42% to 47% and red-green filter CASQ -23% to 29%. The repeatability of the CFDA/PI and red-green filter CASQ methods were the highest. The MIS and PMS percentages were always correlated (p<.05). Measurement of MIS percentage using red and red-green filter CASQ appeared to be the most reliable automated methods.

Measurement of membrane integrity in canine spermatozoa using a fluorescent computer‐assisted spermatozoal quantification method and manual counting after eosin‐nigrosin staining compared with manual counting after CFDA/PI staining

The aim of this study was to compare measurement of spermatozoal membrane status using computer-assisted spermatozoal quantification (CASQ) and eosin-nigrosin (EN) staining with manual counting after CFDA/PI staining. Analysis was performed on both fresh and thawed cryopreserved canine semen. Membrane-disrupted spermatozoa (MDS) were counted using CASQ (n=311) in an untreated sample and a completely membrane-disrupted sample, and the percentage of membrane-intact spermatozoa (MIS) calculated: (Total count-Untreated sample count)÷Total count×100. Spermatozoa were stained with a one-step EN stain (n=501), and then, at least 100 spermatozoa were manually examined under ×1,000 magnification and classified as MDS (stained with eosin) or MIS (non-stained). Spermatozoa from the same samples were also stained with CFDA/PI, and then, at least 200 spermatozoa were manually examined under ×1,000 magnification and classified as MIS (completely stained by CFDA) or MDS. The percentage of progressively motile spermatozoa (PMS) was determined by both computer-assisted semen analysis (CASA) and subjective methodologies, and the data were subsequently analysed to measure the agreement between the CASQ and EN methods with the CFDA/PI technique using Bland-Altman methodology. Pearson's correlation was measured between the MIS and PMS percentage samples and correlation coefficients compared. The mean MIS percentage was lower for CASQ and higher for EN than in CFDA/PI for all comparisons. The agreement of MIS percentage between CASQ and CFDA/PI was -20.2% to 32.0%, and between EN and CFDA/PI was -32.9% to 14.9%. In all methods, the MIS and PMS percentages were correlated (p<.001). Measurement of CFDA/PI appeared to be the most reliable and accurate method of determining MIS percentage in dogs. Further investigation is required to determine whether the CASQ technique can be improved. Eosin-nigrosin staining also appeared to be unreliable at MIS <80% and overestimated the MIS percentage.

Effects of a long-day light programme on the motility and membrane integrity of cooled-stored and cyropreserved semen in Shetland pony stallions

Increasing day length in spring stimulates reproductive functions in horses. In this study, we have analysed the effect of artificial long days on the quality of cooled-stored and cryopreserved semen in Shetland stallions. Stallions of the treatment group (AL, n=8) were exposed to 16h light and 8h darkness from 15th December to 20th March while control stallions (CON, n=7) were kept under natural photoperiod. Semen was collected once weekly and processed for cooled-storage and cryopreservation once per month. Total and progressive motility and percentage of membrane intact spermatozoa were analysed at 24, 48 and 72h of cooled-storage and after freezing-thawing, respectively. Total and progressive motility and membrane integrity decreased during cooled-storage for 72h in each month and both groups (p<0.001). All these parameters were lower in CON versus AL stallions (p<0.05) and the decrease was more pronounced in group CON (storage time x group p<0.05). Differences between groups decreased throughout the observation period from January (p<0.05 between groups) to July (e.g. total motility after 72h of cooled-storage in January for group AL 80±3 and group CON 49±12%, respective values in July, 83±2 and 72±6%). Neither total and progressive motility nor percentage of membrane-intact and morphologically defect spermatozoa in frozen–thawed semen differed between groups and months. In conclusion, motility of cooled-stored semen was reduced in January and increased in stallions kept under a long day light programme for at least 30 days.

Cooled Storage of Canine Semen:in vitroEffects of Different Concentrations of an Antibiotic Combination on Growth of Mollicutes

The aim of this study was to examine effects of an antibiotic combination at different concentrations on growth of mycoplasma and ureaplasma during cooled storage of canine semen (n=20). Semen aliquots were diluted with Tris-citric acid-fructose-egg yolk extender containing either 1.0g/l streptomycin and 0.6g/l benzylpenicillin (control) or a combination of gentamycin, tylosin, lincomycin and spectinomycin (GTLS-1: 0.25, 0.05, 0.15 and 0.3; GTLS-2: 0.5, 0.1, 0.3 and 0.6; GTLS-3: 1.0, 0.2, 0.6 and 1.2g/l). Samples were assessed for motility and membrane integrity by computer-assisted sperm analysis immediately after dilution and at 24, 48 and 72h of cooled storage. Morphologically, normal spermatozoa were determined, and bacterial culture was performed at 24 and 72h. Mycoplasma spp. were detected in 14 of 20 ejaculates (70%) with severe growth in 12 samples. A reduction but not total elimination of mycoplasma growth occurred in all GTLS extenders with the most pronounced reduction in group GTLS-3 (control vs GTLS-1 and GTLS-2 p<0.05, control vs GTLS-3 p<0.001). Ureaplasmas were detected in four ejaculates, and growth was reduced to the same extent in GTLS and control extender. Progressive motility in all groups, total motility in groups GTLS 1-3 and percentage of membrane-intact spermatozoa in groups GTLS 2 and 3 decreased slightly (p<0.05) over time. In conclusion, dilution of canine semen with GTLS extender has no major detrimental effects on spermatozoa during cooled storage. It reduced the growth but did not totally eliminate mycoplasmas and ureaplasmas from cooled-stored dog semen.

Dietary Supplementation of Two Varying Sources of n-3 Fatty Acids and Subsequent Effects on Fresh, Cooled, and Frozen Seminal Characteristics of Stallions

The use of cooled and frozen semen offers many advantages to breeders. However, many stallions produce spermatozoa that are unable to endure the stresses associated with these preservation processes. Improving the quality and viability of equine spermatozoa via appropriate dietary alterations may render spermatozoa from these stallions commercially viable for preservation. One way to improve sperm quality may be through increasing dietary intake of PUFA, specifically docosahexaenoic acid (22:3n-3), to enhance the plasma membrane of the sperm cell. To evaluate this possibility, along with analyzing varying supplemental sources of n-3 fatty acids that are high in docosahexaenoic acid, stallions were placed into 1 of 2 treatment groups and fed either an algae- and flaxseed-based or a fish-based dietary supplement. Semen was collected every 2 wk for 112 d, 28 d before and 84 d during PUFA supplementation. Spermatozoa motion characteristics and membrane integrity and morphology of fresh, cooled, and stored (24 and 48 h), and frozen and thawed semen samples were analyzed. When spermatozoa obtained from stallions in each treatment were compared, no overall improvements were found (P > 0.05) in motility, percentage of membrane-intact spermatozoa, and percentage of morphologically normal spermatozoa regardless of supplementation or source of supplementation.

Effects of Equex STM Paste on the quality of frozen-thawed epididymal dog spermatozoa

This study was carried out to investigate the cryoprotective efficacy of Equex STM Paste on the quality of canine post-thaw epididymal spermatozoa. Following castration, spermatozoa were flushed from the cauda epididymides. Epididymal spermatozoa from 13 of 16 dogs with a sperm motility of >70% were frozen in an egg yolk-Tris extender, supplemented with Equex STM Paste (0.5%, v/v); the extender free of Equex STM Paste served as a control cryoprotective diluent. The quality of spermatozoa, judged by its motility, plasma membrane integrity and acrosome integrity, was evaluated on four occasions, immediately after collection, after equilibration and at 0 and 2h post-thaw. Reducing the temperature to 4 degrees C for 2h prior to freezing decreased sperm motility (P=0.001), but had no effects on membrane integrity or acrosome integrity. Immediately after thawing, the percentage of acrosome-intact spermatozoa significantly decreased in samples frozen without Equex STM Paste compared to freshly collected or Equex-treated samples. After incubation at 37 degrees C for 2h post-thaw, a greater percentage of motile spermatozoa (P=0.018) and spermatozoa with intact acrosomes (P=0.001) were observed in Equex-treated samples compared with the control. The percentage of membrane-intact spermatozoa did not differ significantly between Equex-treated and control samples at any time. Supplementation with Equex STM Paste in the semen extender was effective for freezing canine epididymal spermatozoa because it protected acrosome integrity against damage induced by cryopreservation and it prolonged post-thaw sperm motility during in vitro incubation at 37 degrees C.

Influence of bacteria and gentamicin on cooled-stored stallion spermatozoa

This study investigated effects of bacteria from the genital tract of horses and the effect of gentamicin in semen extender on spermatozoal function in cooled-stored stallion semen. Semen was collected from healthy stallions and processed with a milk-based extender with or without gentamicin (1 g/l). Pseudomonas ( Ps.) aeruginosa, Staphylococcus ( St.) aureus, Streptococcus ( Sc.) equi subsp. equi ( Sc. equi), Sc. equi subsp. zooepidemicus ( Sc. zooepidemicus), Sc. dysgalactiae subsp. equisimilis ( Sc. equisimilis) or culture medium alone (control) were added. Immediately after addition of bacteria and after storage at 5 °C for 24, 48 and 72 h, motility, velocity and membrane integrity of diluted semen were determined with a CASA system. After 24 h, semen with Ps. aeruginosa and Sc. equisimilis showed significantly lower motility and velocity compared to all other groups; after 72 h these differences still existed for Ps. aeruginosa ( p < 0.05). The percentage of membrane-intact spermatozoa was significantly lower after 24 h of storage in spermatozoa incubated with Sc. equisimilis and after 72 h with Sc. equisimilis and Ps. aeruginosa. Addition of gentamicin to extender resulted in decreased motility and velocity in semen without addition of bacteria and did not improve motility parameters in semen with bacteria added. In conclusion, certain bacteria may have detrimental effects on semen quality during cooled-storage. These effects are not reduced by addition of gentamicin. Gentamicin can negatively affect spermatozoal function in extended semen during cooled-storage and therefore, optimal concentrations have to be tested for the respective extender medium.

Effects of dead spermatozoa on motion characteristics and membrane integrity of live spermatozoa in fresh and cooled-stored equine semen

The aim of this study was to determine if dead spermatozoa reduced motility or membrane integrity of live spermatozoa in fresh and cooled-stored equine semen. Three ejaculates from each of three stallions were centrifuged and virtually all seminal plasma was removed. Spermatozoa were resuspended to 25×10 6 spermatozoa/ml with EZ-Mixin ® CST extender and 10% autologous seminal plasma, then divided into aliquots to which 0 (control), 10, 25, 50, or 75% (v/v) dead spermatozoa were added. Dead spermatozoa preparations contained 25×10 6 spermatozoa/ml and 10% seminal plasma from pooled ejaculates of the three stallions, in EZ-Mixin ® CST extender. Spermatozoa were killed in the pooled ejaculates by repeated freezing and thawing, then stored at −20 °C until warmed to 37 °C and mixed with aliquots of fresh spermatozoa to be cooled and stored in an Equitainer for 24 h. Motion characteristics (% total motility (MOT), % progressive motility (PMOT), and mean curvilinear velocity (VCL)) for fresh and 24 h cooled samples were determined using a computerized spermatozoal motion analyzer. The presence of up to 75% dead spermatozoa did not adversely affect MOT or PMOT of live spermatozoa in either fresh or cooled-stored semen. However, VCL and the percentage of membrane-intact spermatozoa were reduced compared to control samples when 75% (v/v) dead spermatozoa were added. Membrane integrity, as assessed by staining with carboxyfluoresein diacetate-propidium iodide, was highly correlated ( r>0.8; P<0.001) with MOT and PMOT in both fresh and cooled-stored semen samples. Results of this study have application to the processing of both cooled and frozen equine semen.

Generation of live rat offspring by intrauterine insemination with epididymal spermatozoa cryopreserved at -196 degrees C

This study reports the development of a reliable method for cryopreservation of rat epididymal spermatozoa and the production of live young by artificial insemination using these cryopreserved spermatozoa. The motility and membrane integrity of rat spermatozoa were investigated after spermatozoa had been subjected to physical stress and frozen with various concentrations of glycerol (0, 3 and 6%) either in the presence or absence of Equex Stem as cryoprotective agents. The ability of cryopreserved spermatozoa to generate normal offspring by intrauterine insemination was also evaluated. Rat spermatozoa that had been centrifuged at 700 g for 5 min showed a significant decrease in motility compared with non-centrifuged spermatozoa. In addition, after centrifugation three times the percentage of membrane-intact spermatozoa decreased to approximately 0%. The percentage of membrane-intact spermatozoa was significantly higher (P < 0.01) in semen samples that had been frozen in medium without glycerol than in samples frozen in medium with 3% glycerol. Although the addition of 0.7% Equex Stem to medium without glycerol or with 3% glycerol did not influence rates of sperm motility after freezing and thawing, the percentage of membrane-intact spermatozoa was improved by the presence of 0.7% Equex (P < 0.05). Therefore, rat spermatozoa were handled gently to avoid physical stress and were frozen in medium containing 23% egg yolk, 8% lactose monohydrate and 0.7% Equex Stem, at pH 7.4 adjusted with 10% Tris(hydroxymethyl)aminomethane solution. Thirteen female rats were inseminated into the oviductal end of both uterine horns with frozen-thawed spermatozoa. Forty-one normal live offspring were obtained from nine of the inseminated females. These results indicate that frozen-thawed rat spermatozoa can generate normal offspring. To our knowledge, this procedure is the first successful production of offspring using spermatozoa cryopreserved in liquid nitrogen.

Generation of live rat offspring by intrauterine insemination with epididymal spermatozoa cryopreserved at -196 degrees C.

This study reports the development of a reliable method for cryopreservation of rat epididymal spermatozoa and the production of live young by artificial insemination using these cryopreserved spermatozoa. The motility and membrane integrity of rat spermatozoa were investigated after spermatozoa had been subjected to physical stress and frozen with various concentrations of glycerol (0, 3 and 6%) either in the presence or absence of Equex Stem as cryoprotective agents. The ability of cryopreserved spermatozoa to generate normal offspring by intrauterine insemination was also evaluated. Rat spermatozoa that had been centrifuged at 700 g for 5 min showed a significant decrease in motility compared with non-centrifuged spermatozoa. In addition, after centrifugation three times the percentage of membrane-intact spermatozoa decreased to approximately 0%. The percentage of membrane-intact spermatozoa was significantly higher (P < 0.01) in semen samples that had been frozen in medium without glycerol than in samples frozen in medium with 3% glycerol. Although the addition of 0.7% Equex Stem to medium without glycerol or with 3% glycerol did not influence rates of sperm motility after freezing and thawing, the percentage of membrane-intact spermatozoa was improved by the presence of 0.7% Equex (P < 0.05). Therefore, rat spermatozoa were handled gently to avoid physical stress and were frozen in medium containing 23% egg yolk, 8% lactose monohydrate and 0.7% Equex Stem, at pH 7.4 adjusted with 10% Tris(hydroxymethyl)aminomethane solution. Thirteen female rats were inseminated into the oviductal end of both uterine horns with frozen-thawed spermatozoa. Forty-one normal live offspring were obtained from nine of the inseminated females. These results indicate that frozen-thawed rat spermatozoa can generate normal offspring. To our knowledge, this procedure is the first successful production of offspring using spermatozoa cryopreserved in liquid nitrogen.