Idebenone improves quality of ram sperm by mitigating oxidative stress during cryopreservation

The present study was designed to test the effect of different levels of idebenone, a potent antioxidant on the quality of ram semen at post thaw. Eighteen (18) ejaculates were collected and extended with tris extender supplemented with no antioxidant (CON), with 2 μM idebenone (Id2), 5 μM idebenone (Id5), 7.5 μM idebenone (Id7.5) and 10 μM idebenone (Id10). The sperm quality was determined in terms of percent sperm motility, live sperm percentage, percent hypoosmotic swelling test (HOST) positive spermatozoa and percent intact acrosome (PIA). Moreover, malondialdehyde (MDA) level, an end product of lipid peroxidation (LPO) was also measured at post thaw both in seminal plasma and sperm cell. At post thaw, the percent sperm motility was significantly higher (p < 0.05) for Id10 as compared to Id2, Id5, Id7.5 and control. The live sperm percentage was non-significantly (p > 0.05) higher for Id10 as compared to control, Id5 and Id7.5 but significantly higher than Id2. The percent HOST positive spermatozoa was significantly higher (p < 0.05) for Id10 than control, Id2 and Id5. The MDA level in seminal plasma was significantly lower (p < 0.05) for Id10 than control and Id2. The MDA level in spermatozoa did show similar trend as in seminal plasma. Further, all the sperm parameters at all idebenone levels declined significantly from pre freeze to post thaw. In conclusion, idebenone at 10 μM level improved post thaw sperm quality by mitigating peroxidative stress, hence could be considered as a promising antioxidant additive for cryopreservation of ram semen.     

The role of osmotic resistance on equine spermatozoal function

Cryopreservation requires exposure of sperm to extreme variations in temperature and osmolality. The goal of this experiment was to determine the osmotic tolerance levels of equine sperm by analyzing motility, viability, mitochondrial membrane potential (MMP), and mean cell volume (MCV). Spermatozoa were incubated at 22 degrees C for 10 min in isosmolal TALP (300 mOsm/kg), or a range of anisosmolal TALP solutions (75-900 mOsm/kg), for initial analysis, and then returned to isosmolal conditions for 10 min for further analysis. Total sperm motility was lower (P < 0.05) in anisosmolal conditions compared to sperm motility in control medium. When cells were returned to isosmolal conditions, only sperm previously incubated in 450 mOsm/kg TALP were able to recover to control levels of motility. Sperm viability and MMP were lower (P < 0.05) when exposed to hypotonic solutions in comparison to control solutions. Sperm suspensions that were returned to isosmolal conditions from 75, 150, and 900 mOsm/kg had lower (P < 0.05) percentages of viable sperm than control suspensions (300 mOsm/kg). MMP was lower (P < 0.05) in cells previously incubated in 75 and 900 mOsm/kg when returned to isosmolal, as compared to control cells. MCV differed (P < 0.05) from control cell volume in all anisosmolal solutions. Cells in all treatments were able to recover initial volume when returned to isosmolal medium. Although most spermatozoa are able to recover initial volume after osmotic stress, irreversible damage to cell membranes may render some sperm incapable of fertilizing an oocyte following cryopreservation.     

Pre-conditioning with Xanthine oxidase to improve post thawed quality of bull sperm

The purpose of this study was to examine the effects of sub-lethal concentration of Xanthine oxidase (XO) on the post-thawed bull sperm quality. Semen samples were collected from four Holstein bulls, twice a week and during three consecutive weeks (n = 24 total ejaculates). After collection in each replicate, semen samples were pooled and then frozen by semen extender containing different concentrations [0 (XO-0), 0.05 (XO-0.05), 0.5 (XO-0.5), 5 (XO-5), 50 (XO-50) and 500 (XO-500) μM] of XO. After thawing, motion parameters (SCA), plasma membrane functionality (HOST), apoptosis status (Phosphatidylserine translocation assay), mitochondrial activity (Rhodamine 123), and acrosome integrity (PSA), were evaluated. The results showed that total motility, VAP, VSL, VCL, STR, and LIN were lower in XO-50 and XO-500 compared to other groups (P < 0.05). Progressive motility were higher in XO-0.05 and XO-0.5 compared to XO-0, XO-50, and XO-500 (P < 0.05). Mitochondrial activity was highest in XO-0.05 and XO-0.5 groups. Sperm plasma membrane functionality was significantly greater in XO-0, XO-0.05, XO-0.5, and XO-5 than that of XO-50 and XO-500. Xanthine oxidase had not significant effects on acrosome integrity and dead spermatozoa. Higher percentage of live spermatozoa was recorded for XO-0, XO-0.05, XO-0.5, and XO-5; however, the lower amount of apoptotic spermatozoa was detected in the aforementioned groups (P < 0.05). In conclusion, it seems that XO at lower doses may have beneficial effects on post-thawed bull sperm quality.     

Effects of radiographic contrast media on domestic cat epididymidal sperm

Laparoscopic artificial insemination has an important role in felid conservation but it is costly and includes surgical risk. Therefore, radiographic contrast medium combined with non-surgical transcervical AI to verify intrauterine gamete placement could be a viable alternative. Gamete-rescued fresh and frozen-thawed sperm were extended with one of two commercial contrast media (nonionic and ionic), with osmolarity adjusted to 320-330 mOsm, or feline optimized culture medium (control). Percent motility, forward progression status, and acrosomal integrity were recorded every 30 min for 4 h. Sperm penetration abilities were assessed by coincubating treated sperm with conspecific in vitro matured oocytes for 18 to 20 h, and presumptive zygotes and embryos were fixed and stained to determine sperm penetration and fertilization rate. There was reduced motility and acrosomal integrity in frozen-thawed versus fresh sperm (P < 0.05). Neither radiographic contrast medium induced adverse effects on fresh sperm motility relative to control medium (P > 0.05), but motility of frozen-thawed sperm decreased when treated with nonionic radiographic contrast medium compared to control medium (P < 0.05). There were no differences in acrosomal integrity between radiographic contrast and control media in fresh (P > 0.05) or frozen sperm (P > 0.05). Neither radiographic contrast media decreased the numbers of morphologically normal sperm (P > 0.05) or reduced the ability of domestic cat sperm to penetrate (P > 0.05) or fertilize (P > 0.05) conspecific oocytes. Ionic radiographic contrast medium can be added to fresh or frozen-thawed domestic cat sperm with no adverse effect on motility, morphology, acrosomal integrity or oocyte penetration rates, and thus may be used to facilitate further development of transcervical AI procedures.     

Hypoosmotic swelling of boar spermatozoa compared to other methods for analysing the sperm membrane

The aims of this work were to adapt the hypoosmotic swelling test (HOST) to boar spermatozoa and to compare this method with other tests which evaluate the integrity of the sperm membrane. The spermatozoa were incubated in 50, 100, 150 or 200 mOsm/L solutions for 5, 30, 60 or 120 min. An easily identifiable swelling and coiling of the tails occurred when the boar spermatozoa were incubated at 37 degrees C for 30 to 120 min in a mixture of fructose and Na-citrate (100-150 mOsm/L). Transmission electron microscopy showed that the hypoosmotic swelling reaction of the spermatozoa was caused by coiling of the flagellum inside the plasma membrane. When used as described, HOST was found to be highly reliable when known populations of live spermatozoa were tested. We also compared the results obtained with HOST with those obtained using eosin Y and carboxyfluorescein diacetate. The percentage of spermatozoa unstained with eosin Y and the percentage of spermatozoa which fluoresced with carboxyfluorescein diacetate were similar. However, the hypoosmotic swelling values were significantly below those of the other tests. This may be because either HOST evaluates different aspects of sperm membrane than other sperm membrane tests or the membranes of some spermatozoa are inactivated by contact with the hypoosmotic solution. In short, our findings suggest that HOST is a sensitive and reproducible test to assess the functional integrity of boar sperm membranes after incubation under hypoosmotic stress conditions and may be a useful tool for detecting subpopulations of subviable spermatozoa when used in conjunction with another type of membrane integrity test.     

Effect of feeding a docosahexaenoic acid-enriched nutriceutical on the quality of fresh and frozen-thawed semen in Holstein bulls

The aim of the current study was to investigate the effect of feeding a DHA-enriched nutriceutical on the in vitro quality and sperm motility parameters of fresh and frozen-thawed bull semen assessed by CASA. Samples were obtained from nineteen Holstein bulls used for semen collection at Semen Production Center, Karaj, Iran. Control group (n = 10) were fed a standard concentrate feed while treatment group bulls (n = 9) had this standard feed top dressed with 100 g of a commercially available DHA-enriched nutriceutical. Semen quality was assessed on ejaculates collected at the baseline and after 5, 9, and 12 weeks of supplementation. Classical semen evaluation, assessment of sperm motility (subjective and computer-assisted), viability (eosin-nigrosin), and hypo-osmotic swelling test (HOST) were conducted. Semen volume, sperm concentration, and consequently total sperm output were not affected by dietary treatment (P > 0.05). Feeding the nutriceutical was indeed found to affect sperm motility parameters assessed by CASA after 9 weeks of trial. The treatment has improved total motility (P < 0.01), progressive motility (P < 0.05), average path velocity (P < 0.05), HOST-positive (P < 0.01), and proportion of rapid spermatozoa (P < 0.01) in the fresh semen of bulls. Moreover, the proportion of viable spermatozoa increased (P < 0.05) in the ejaculates collected from nutriceutical-fed bulls compared to the control after 12 weeks of feeding trial. The post-thawed HOST and sperm motility data obtained by CASA did not differ between two groups (P > 0.05). On the other hand, dietary supplementation did not affect body weight, BCS and scrotal circumference. Consequently, it can be concluded that dietary DHA supplementation or its precursors, improve in vitro quality and motility parameters of fresh semen assessed by CASA in Holstein bulls. However, this effect was not pronounced in frozen-thawed semen.     

Effect of feeding a docosahexaenoic acid-enriched nutriceutical on the quality of fresh and frozen-thawed semen in Holstein bulls

The aim of the current study was to investigate the effect of feeding a DHA-enriched nutriceutical on the in vitro quality and sperm motility parameters of fresh and frozen-thawed bull semen assessed by CASA. Samples were obtained from nineteen Holstein bulls used for semen collection at Semen Production Center, Karaj, Iran. Control group (n = 10) were fed a standard concentrate feed while treatment group bulls (n = 9) had this standard feed top dressed with 100 g of a commercially available DHA-enriched nutriceutical. Semen quality was assessed on ejaculates collected at the baseline and after 5, 9, and 12 weeks of supplementation. Classical semen evaluation, assessment of sperm motility (subjective and computer-assisted), viability (eosin-nigrosin), and hypo-osmotic swelling test (HOST) were conducted. Semen volume, sperm concentration, and consequently total sperm output were not affected by dietary treatment (P > 0.05). Feeding the nutriceutical was indeed found to affect sperm motility parameters assessed by CASA after 9 weeks of trial. The treatment has improved total motility (P < 0.01), progressive motility (P < 0.05), average path velocity (P < 0.05), HOST-positive (P < 0.01), and proportion of rapid spermatozoa (P < 0.01) in the fresh semen of bulls. Moreover, the proportion of viable spermatozoa increased (P < 0.05) in the ejaculates collected from nutriceutical-fed bulls compared to the control after 12 weeks of feeding trial. The post-thawed HOST and sperm motility data obtained by CASA did not differ between two groups (P > 0.05). On the other hand, dietary supplementation did not affect body weight, BCS and scrotal circumference. Consequently, it can be concluded that dietary DHA supplementation or its precursors, improve in vitro quality and motility parameters of fresh semen assessed by CASA in Holstein bulls. However, this effect was not pronounced in frozen-thawed semen.     

The hypoosmotic swelling test in fresh rabbit spermatozoa

The hypoosmotic swelling test (HOST) has proved to be a good tool for evaluating the membrane integrity of spermatozoa of various domestic animals including cattle, horses, and swine. However, the best approach for using this technique in rabbit semen has not been tested. The present study aimed to establish the best hypoosmotic solution (HS) for testing membrane integrity in fresh rabbit semen. Sucrose solutions with the following osmolarities were used: 50, 60, 75, 100, 125 and 150mOsm/L. Semen samples (n=30) were collected from five mature White New Zealand rabbits (six collections per rabbit) at 72h intervals. After macroscopic evaluation, 10microL of semen was immediately added to 2mL of each solution and incubated for 1h at 37 degrees C. Sequentially, 20microL of semen diluted in HS were evaluated with oil immersion using a phase-contrast microscope. A total of 200 spermatozoa were counted in at least five different fields, and sperm tails were classified as non-coiled, coiled, and strongly coiled. The respective percentages of spermatozoa with coiled tails (coiled plus strongly coiled) in the six solutions listed above were 54.8, 65.2, 54.3, 53.9, 38.9 and 29.4%. Percentage of strongly coiled spermatozoa was: 40.2, 51.0, 43.2, 41.5, 32.7 and 26.9 for the six solutions, respectively. According to total and strong coiling 60mOsm/L was superior to others treatments (P<0.05). Results suggest that the 60mOsm/L solution would be most desirable for use in HOST in fresh rabbit spermatozoa.     

Use of antioxidants reduce lipid peroxidation and improve quality of crossbred ram sperm during its cryopreservation

Ram sperm are subjected to extreme oxidative stress during their preservation at −196 °C resulting in reduced quality at post thaw. Therefore, the main objective of this study was to evaluate the effect of antioxidants taurine, quercetin and reduced glutathione on the post thaw quality of crossbred ram sperm. A total of twenty four ejaculates from six crossbred rams were collected and extended with tris-based extender with no antioxidant (Control), with taurine (40 mM), quercetin (5 μg/ml) and reduced glutathione (5 mM). The post thaw sperm quality was determined by percent sperm motility, live sperm count, intact acrosome and hypo-osmotic swelling test (HOST) reacted spermatozoa and lipid peroxidation was measured in terms of malondialdehyde (MDA) level both in seminal plasma and sperm cell. At post thaw, percent sperm motility and live sperm count were significantly (p < 0.05) higher for taurine than control and reduced glutathione but did not differ significantly (p > 0.05) from quercetin. The percent HOST reacted spermatozoa were significantly higher for taurine than control, quercetin and reduced glutathione. Seminal plasma MDA level was significantly (p < 0.05) lower for taurine than control and non-significantly lower than quercetin and reduced glutathione. However, spermatic MDA level did not differ significantly (p > 0.05) among the control and antioxidants. In conclusion, taurine at 40 mM reduced lipid peroxidation and improved post thaw sperm quality of cryopreserved crossbred ram semen. Further, transportation time of semen samples in an ice chest at 4–5 °C may be included as a part of equilibration period, when collection shed and frozen semen unit are located at a distance.     

Head morphometric changes in cryopreserved ram spermatozoa are related to sexual maturity

The importance of understanding the sperm changes after the cryopreservation process has been emphasized in human and veterinary andrology. In previous studies, we have shown that the morphometric characteristics assessed by computer-assisted analysis following the freeze-thawing process revealed differences in terms of dimension and shape between individuals that may be related to bio-physiologic factors such as sexual maturity. The purpose of this study was to determine if there are differences associated with cryoresistence and sperm head morphometric dimensions in individuals with different sexual maturity ratings (SMRs; 12, 30 and 96 months of age). Ejaculates from nine normospermic fertile rams with different SMRs were analyzed in an attempt to quantify the morphometric dimensions and the shape of sperm heads from each group after the cryopreservation process. The mean values of sperm concentration among individuals with different SMRs were significantly different (P < 0.01). Cryopreservation substantially reduced sperm motility and plasma membrane integrity irrespective of SMR assessed, with young animals being the most affected (P < 0.01). Sperm quality at thawing for all sperm parameters evaluated was significantly higher for old individuals than for middle-aged or young individuals (P < 0.01). There were no significant differences in the sperm head dimension or shape among middle-aged and old individuals (P > 0.05). However, significant differences were detected in area, perimeter and width (lower values) and length, ellipticity and elongation (higher values) in old or middle-aged individuals compared with young individuals (P < 0.01). In conclusion, this study confirms that ram age is related to sperm morphometric dimensions, and sperm size and shape may affect spermatozoa survival, being good indicators of freezability. Therefore, the present study provides information on the morphometric maturation of ram sperm and supports the idea that the dimensions of spermatozoa may be taken as an approximate indication of its relative maturity.     

Arbutin's suppression of cryodamage in goat sperm and its mechanism of cryoprotection

Arbutin (4-hydroxyphenyl-glucopyranoside) is a glycosylated hydroquinone present in high concentrations in the leaves of several plants capable of surviving prolonged, extreme dehydration. Two experiments were conducted to determine the effects of arbutin on cryopreservation of goat sperm. In Experiment 1, goat sperm were frozen in extenders with various ratios of Tris-citric acid-glucose (TCG) and arbutin; concentrations of the latter were 0.0 (only TCG), 0.1, 0.2, 0.3, and 0.4 M (only arbutin)]. All extenders had 20% (v/v) egg yolk (EY) and 4% (v/v) glycerol (osmolality = 370 mOsm, pH = 7.0). Sperm motility and acrosome integrity were assessed using CASA, and fluorescein isothiocyanate-labeled peanut agglutinin (FITC-PNA), respectively. Percentages of motile and progressively motile sperm improved with the addition of arbutin; results were optimal (89.0 and 70.0%, respectively; P < 0.05), with 0.4 M arbutin. Furthermore, arbutin improved (P < 0.05) post-thaw recovery rates for both motility and progressive motility. After incubation for 3 h, motility of frozen-thawed washed sperm improved (70%, P < 0.05) with arbutin in the extender. The percentage of sperm with an intact acrosome peaked (77.2%, P < 0.05) with 0.4 M arbutin in the extender. In Experiment 2, the percentage of cells with merocyanine 540/Yo-Pro staining was higher in sperm treated with arbutin than with TCG (P < 0.05), with the best result (58.0%) with 0.4 M arbutin; therefore, arbutin increased membrane fluidity. In conclusion, substitution of a TCG-EY diluent composition with arbutin improved freezability of goat sperm (apparently due to increased membrane fluidity). Furthermore removal of arbutin by centrifugation after freezing and thawing increased sperm longevity.     

Relationship between sperm motility, morphology and the fertility of stallions

Sperm quality has an important role in determining fertility. Although there have been numerous studies to document the relationship between sperm quality and fertility, the methods of determining this association and conclusions vary. In the present study, computer-assisted sperm analysis (CASA) was used for evaluation of sperm motility, and differential interference contrast (DIC) microscopy was used for evaluating sperm morphologic features of breeding stallions. Fertility was measured using three endpoints: seasonal pregnancy rate (PR), percent pregnant/cycle (PC), and percent pregnant/first cycle (FCP). Increased total sperm motility (P = 0.08) and progressive path velocity (P = 0.06) tended to be associated with higher PR, whereas percent coiled tails (P = 0.02) was associated with a lower PR. Sperm motility variables associated with an increase in PC and FCP included total, progressive, and rapid sperm motility, and increased path and progressive velocity. Percent pregnant/first cycle was the only fertility measure able to discriminate among high, average, and low fertility groups, based on total and progressive sperm motility. Percent normal sperm was the only morphology variable associated with an increased PC and FCP, whereas increased levels of most sperm morphologic abnormalities (including abnormal and detached heads, proximal and distal droplets, general midpiece abnormality, and coiled tails) were associated with a decline in PC and FCP. Sperm quality variables most highly correlated with fertility included percent total sperm motility (PR, r = 0.37, P < 0.05; PC, r = 0.59, P < 0.05; and FCP, r = 0.64, P < 0.05), and percent morphologically normal sperm (PC, r = 0.42, P < 0.05; and FCP, r = 0.39, P < 0.05).     

Quality of canine semen submitted to single or fractionated glycerol addition during the freezing process

Glycerol is the cryoprotector most frequently used to freeze semen from different species. The objective of the present study was to compare the effect of single and fractionated glycerol addition on canine semen quality after thawing. Sperm fractions from 12 stud dogs were collected, evaluated, extended in Tris plus egg-yolk and separated into two aliquots to which glycerol was added in one step (single) or in three steps at 5-min intervals (fractionated). Semen was frozen and stored in liquid nitrogen and thawed after 1 week. A thermoresistance test was performed over a period of 120 min at 37-39 degrees C to evaluate the percentage of mobile spermatozoa (motility) and the status of motility (vigor) after thawing. There were no significant differences between the two methods of glycerol addition-immediately after thawing, and during the thermoresistance test-in sperm motility, vigor or morphology. A significant reduction in motility and vigor was found at 15 min after thawing and these parameters continued to decline until 120 min. In conclusion, glycerol can be added to canine semen in single or fractionated manner, but the single addition method is the easiest and the most practical to use.     

Fertility of frozen-thawed stallion semen cannot be predicted by the currently used laboratory methods

The aim of the project was to use current simple and practical laboratory tests and compare results with the foaling rates of mares inseminated with commercially produced frozen semen. In Exp. 1, semen was tested from 27 and in Exp. 2 from 23 stallions; 19 stallions participated in both experiments. The mean number of mares per stallion in both experiments was 37 (min. 7, max. 121). Sperm morphology was assessed and bacterial culture performed once per stallion. In Exp. 1, progressive motility after 0, 1, 2, 3, and 4 h of incubation using light microscopy, motility characteristics measured with an automatic sperm analyzer, plasma membrane integrity using carboxyfluorescein diacetate/propidium iodide (CFDA/PI) staining and light microscopy, plasma membrane integrity using PI staining and a fluorometer, plasma membrane integrity using a resazurin reduction test, and sperm concentration were evaluated. In Exp. 2, the same tests as in Exp. 1 and a hypo-osmotic swelling test (HOST) using both light microscopy and a fluorometer were performed immediately after thawing and after a 3-h incubation. Statistical analysis was done separately to all stallions and to those having ≥ 20 mares; in addition, stallions with foaling rates < 60 or ≥ 60% were compared. In Exp. 1, progressive motility for all stallions after a 2 – 4-h incubation correlated with the foaling rate (correlation coefficients 0.39 – 0.51), (p < 0.05). In stallions with > 20 mares, the artificial insemination dose showed a correlation coefficient of -0.58 (p < 0.05). In Exp. 2, the HOST immediately after thawing showed a negative correlation with foaling rate (p < 0.05). No single test was consistently reliable for predicting the fertilizing capacity of semen, since the 2 experiments yielded conflicting results, although the same stallions sometimes participated in both. This shows the difficulty of frozen semen quality control in commercially produced stallion semen, and on the other hand, the difficulty of conducting fertility trials in horses.     

Motile sperm subpopulations in frozen-thawed dog semen: Changes after incubation in capacitating conditions and relationship with sperm survival after osmotic stress

In this study we investigated the changes that in vitro incubation under capacitating conditions could induce on the motile sperm subpopulations present in frozen-thawed dog semen samples. In addition, cryopreserved dog spermatozoa were exposed to CCM (canine capacitating medium) solutions of 300, 150, 100 and 75mOsm and the proportions of live spermatozoa with swollen tails were recorded (HOST+). Finally, frozen-thawed dog semen samples were submitted to a second cycle of freezing and thawing and the overall sperm motility, as well as the motile sperm subpopulations structure, was determined. Cryopreserved dog semen samples were structured in four sperm subpopulations with different motility characteristics: Subpopulation (Sp) 1 contained moderately rapid and progressive spermatozoa (25.2±8.5%), Sp 2 included poorly motile and non progressive sperm (15.3±8.1%), Sp 3 was represented by moderately slow non progressive sperm (14.9±5.9%), and Sp 4 contained the most rapid and progressive sperm (20.8±14.7%). After 3h of incubation under capacitating conditions, percentages of spermatozoa assigned to Sp 2 (6.1±3.4%) and 3 (4.9±2.8%) significantly decreased, whereas those assigned to Sp 1 (17.0±11.2%) and 4 (16.2±12.8%) did not significantly change. Significant correlations were found between percentages of HOST+, for the 3 osmolarities tested, and percentages of spermatozoa included in Sp 1 and 4 after 3h of incubation in capacitating conditions or in Sp 4 after double freezing and thawing. These results indicated that subpopulations with the most rapid and progressive sperm seemed to be highly resistant to in vitro incubation in capacitating conditions and to osmotic stress, suggesting they are likely to be the source of the fertilizing population.     

Osmotic tolerance of avian spermatozoa: influence of time, temperature, cryoprotectant and membrane ion pump function on sperm viability

Potential factors influencing sperm survival under hypertonic conditions were evaluated in the Sandhill crane (Grus canadensis) and turkey (Meleagridis gallopavo). Sperm osmotolerance (300-3000 mOsm/kg) was evaluated after: (1) equilibration times of 2, 10, 45 and 60 min at 4 °C versus 21 °C; (2) pre-equilibrating with dimethylacetamide (DMA) or dimethylsulfoxide (Me₂SO) at either 4 °C or 21 °C; and (3) inhibition of the Na⁺/K⁺ and the Na⁺/H⁺ antiporter membrane ionic pumps. Sperm viability was assessed using the eosin-nigrosin live/dead stain. Species-specific differences occurred in response to hypertonic conditions with crane sperm remaining viable under extreme hypertonicity (3000 mOsm/kg), whereas turkey sperm viability was compromised with only slightly hypertonic (500 mOsm/kg) conditions. The timing of spermolysis under hypertonic conditions was also species-specific, with a shorter interval for turkey (2 min) than crane (10 min) sperm. Turkey sperm osmotolerance was slightly improved by lowering the incubation temperature from 21 to 4 °C. Pre-equilibrating sperm with DMA reduced the incidence of hypertonic spermolysis only in the crane, at both room and refrigeration temperature. Inhibiting the Na⁺/K⁺ and the Na⁺/H⁺ antiporter membrane ion pumps did not impair resistance of crane and turkey spermatozoa to hypertonic stress; pump inhibition actually increased turkey sperm survival compared to control sperm. Results demonstrate marked species specificity in osmotolerance between crane and turkey sperm, as well as in the way temperature and time of exposure affect sperm survival under hypertonic conditions. Differences are independent of the role of osmotic pumps in these species.     

Effects of reduced glutathione and catalase on the kinematics and membrane functionality of sperm during liquid storage of ram semen

The objective of this study was to evaluate the effects of reduced glutathione (GSH) and catalase (CAT) supplementation on the kinematics and membrane functionality of sperm during the liquid storage of ram semen, cooled at 5 °C, for up to 24 h. Semen samples from four rams were pooled, diluted with Tris-egg yolk extender without antioxidants (control) or supplemented with either CAT (100, 200, and 400 U/mL) or GSH (100, 200, and 400 mM) at a final concentration of 50 × 10⁶ sperm/mL. Sperm kinematics, which was analyzed by computer-assisted sperm analysis (CASA), and membrane functionality, which was analyzed using the hypo-osmotic swelling test (HOST), were determined after the addition of the semen samples at different processing times (fresh/diluted, 1.5, 6, 12, and 24 h, at 5 °C). No significant differences were recorded in the kinematics or membrane functionality between treatments at different times. The supplementation of diluents with 100 and 200 U/mL of CAT prevented the harmful effects of cooling on total sperm motility. No significant differences were observed in progressive sperm motility throughout processing, regardless of the treatment and time of evaluation. Supplementation with 400 mM GSH resulted in an earlier reduction (P < 0.05) of total sperm motility, a decrease in rapid sperm rate and a reduction in curvilinear velocity during incubation, at 5 °C. The cooling induced a reduction (P < 0.05) in the percentage of sperm with a functional plasma membrane (HOST), especially after 1.5 h of incubation. Based on the results of the present study, the addition of CAT (100 and 200 U/mL) reduced the deleterious effects of cooling on total motility in ram sperm maintained at 5 °C for 24 h, although it did not affect the functionality of the sperm membranes. However, the addition of 400 mM GSH caused negative effects on the velocity parameters of the sperm.     

Can amides be alternative cryoprotectors for the preservation of feline semen?

Sperm cryopreservation is a tool for the conservation of the genetic material of animals of genetic importance or for species preservation. In the case of domestic cats, this can be used to generate information about seminal harvest, evaluation and preservation, which is especially important due to its applicability to wild felids. This study evaluated seminal samples harvested by urethral catheterisation from 13 adult domestic cats. Samples were cryopreserved with experimental groups of extenders were defined by the penetrating cryoprotectant: 6% glycerol (GLY6%), 3% dimethylacetamide (DMA3%) and 3% dimethylformamide (DMF3%). The samples were thawed and evaluated by conventional microscopy and by computer-assisted sperm analysis (CASA). The structural and functional membrane integrity was assessed by supravital tests (EOS), hypoosmotic swelling tests (HOST) and flow cytometry (FC). There was a correlation (P < 0.05) between total motility and EOS (r = 0.54), HOST and FC (r = −0.62) and total motility and flow cytometry (r = 0.63), indicating that these are complementary parameters that increase the accuracy of the feline sperm quality evaluation post-thaw. The results regarding the structural and functional integrity of the sperm plasma membrane did not differ (P > 0.05) among groups. However, the DMA3% group had a lower (P < 0.05) percentage of morphological changes in the sperm tail compared to samples cryopreserved with GLY6% and DMF3%. Additionally, DMA3% provided lower values of immobile sperm post-thaw when compared to DMF3%. DMA is an interesting alternative to GLY and superior to DMF for the cryopreservation of feline semen at the studied concentrations.     

Intra- and interspecies comparison of sperm migration through polyacrylamide gel as an index of spermatozoal viability

Sperm migration distance through a polyacrylamide gel mimicking cervical mucus was compared among seven species: dog, cat, goat, ram, clouded leopard, dorcas gazelle, and Eld's deer. Freshly collected spermatozoa were evaluated microscopically and then assayed for migration distance in gel-filled capillary tubes in raw or diluted concentrations or after longevity declines in sperm motility and progressive status. Sperm count per milliliter of ejaculate and sperm percent motility ratings varied (P < 0.05) among selected species. Undiluted ejaculated spermatozoa from all species, except the clouded leopard, penetrated the polyacrylamide gel. Migration distance (mm) was different (P < 0.05) among species and not clearly correlated to concentration or motility factors. Although sperm concentrations in the dog and Eld's deer were similar, average migration distance of deer sperm was more than fourfold greater (P < 0.01) than that of dog sperm. The sperm motility rating for the gazelle was greater (P < 0.05) than that for the cat; however, penetration distance in the cat was more than twice as great (P < 0.05) as that in the gazelle. Species also varied in migration response after altering sperm concentration: Halving the sperm count of the dog, gazelle, and Eld's deer had no effect, but the same procedure decreased (P < 0.05) penetration distance in the goat and ram. No migration was observed in any species at a sperm concentration of 25 × 10⁶ cells or less. Within species a decline in sperm percent motility-progressive status ratings was correlated (P < 0.05) to a subsequent decrease in penetration distance. These results provide a comparative assessment of sperm cell migration through a synthetic cervical mucus and suggest that this test may be a useful adjunct in evaluating reproductive potential. However, the assay is specific in interpretive merit, as sperm penetration distance within a single batch of gel is markedly variant among species and in some species dependent on sperm concentration. This interspecies specificity in sperm migration through a homogeneous gel suggests intrinsic species variance in sperm cell-cervical mucus interaction, suggesting that this assay could be valuable in future studies of the mechanism of sperm transport.     

Effect of insulin-like growth factor-I on some quality traits and fertility of cryopreserved ovine semen

The objective was to evaluate the effects of insulin-like growth factor-I (IGF-I) on the quality and fertility of frozen/thawed ovine semen. Five rams (five ejaculates/ram) were used for evaluation of semen parameters. Before cryopreservation, ejaculates were divided into four aliquots and extended with Tris alone or supplemented with human IGF-I (50, 100, or 250 ng/mL). Semen was evaluated immediately after thawing (T0), after 1 h (T1) and 2 h (T2) post-incubation at 37 °C. The percentage of live cells (fluorescence analysis-calcein and ethidium), acrosome integrity (NAR) and motility were analyzed, and hypo-osmotic swelling tests (HOST) were used to evaluate membrane resistance. In addition, AI was performed using 121 ewes to compare the optimal concentration of IGF-I vs. Tris alone on pregnancy rates after laparoscopic insemination. Pregnancy diagnosis was performed by transrectal ultrasonography. After 1 and 2 h post-incubation, in every group, percentage motile sperm, NAR and HOST decreased compared to semen at T0. Motility was higher (P < 0.05) in the IGF-I 100 and IGF-I 250 groups when compared to the IGF-I 50 and Tris groups (76.2 and 74.4% vs. 66.2 and 64.4 percent, respectively) at T0, after 1 h (67 and 63.6% vs. 56.2 and 54.7%) and 2 h post-incubation (58.2 and 55.8% vs. 48 and 47.2%). Furthermore, viability was higher (P < 0.05) in the insulin-like growth factor-I (IGF-I) 100 and IGF-I 250 groups than in the IGF-I 50 and Tris groups (88.7 and 88.3% vs. 76.6 and 77.6%, respectively) at T0. There was no difference (P > 0.05) in NAR or hypo-osmotic swelling tests (HOST) among groups. There were no differences (P > 0.05) in fertility between the IGF-I 100 and Tris groups. In conclusion, IGF-I improved subjective sperm motility and structural integrity of the plasma membrane without a significant effect on 45-day pregnancy rates after laparoscopic insemination of ewes with frozen-thawed semen.