Chimpanzee (Pan troglodytes) spermatozoa osmotic tolerance and cryoprotectant permeability characteristics

Osmotic properties of chimpanzee spermatozoa were studied at 22 degrees C. An electronic particle counter was used to determine the isosmotic cell volume, and the volume response after exposure to four commonly used cryoprotectants: dimethyl sulfoxide, glycerol, propylene glycol, and ethylene glycol. The data were analyzed to determine the hydraulic conductivity and the permeability coefficients for the four cryoprotectants. The osmotically inactive volume fraction was determined using a Boyle van't Hoff plot of cells exposed to sodium chloride solutions. A computer-assisted semen analysis system was used to determine the osmotic tolerance of chimp spermatozoa, as well as the effects of a one-step addition and removal of 1 M permeating cryoprotectant on sperm motility. The isosmotic volume of chimpanzee sperm is 27.7 microm3. The osmotically inactive cell fraction is 69%. Hydraulic conductivity was higher in the presence of ethylene glycol: 4.09 +/- 0.76 (mean +/- SEM) and propylene glycol: 3.91 +/- 0.71 as compared to dimethyl sulfoxide: 3.49 +/- 0.79 and glycerol: 2.83 +/- 0.40 microm/min per atmosphere. The permeability of chimpanzee sperm in ethylene glycol (2.18 +/- 0.40 x 10(-3) cm/min) and propylene glycol (1.75 +/- 0.17 x 10(-3) cm/min) was higher than in glycerol (1.42 +/- 0.12 x 10(-3) cm/min) and dimethyl sulfoxide (0.82 +/- 0.015 x 10(-3) cm/min). Although chimpanzee sperm tolerated osmotic stress in the range of 169-400 mOsm very well, loss of motility was observed as the solution concentrations diverged from isosmotic condition. Exposure to the four cryoprotectants at 1 M did not cause a significant reduction in sperm motility. This information on membrane permeability characteristics and cryoprotectant tolerance will aid in designing more reliable cryopreservation protocols for chimpanzee sperm.     

Effects of various extenders and permeating cryoprotectants on cryopreservation of cynomolgus monkey (Macaca fascicularis) spermatozoa

The cryoprotective effects of 11 different extenders, TTE, DM, mDM, LG-DM, G-DM, TCG, TEST, TSM, Test-M, Test-H, and LM, on sperm cryopreservation of cynomolgus monkey (Macaca fascicularis) have been compared with glycerol as cryoprotectant. Sperm motility, plasma membrane, and acrosomal integrity were examined to evaluate frozen-thawed sperm function. The results showed that TTE, DM, mDM, LG-DM, G-DM, and TCG exhibited the best and similar protective efficiencies for cynomolgus monkey sperm cryopreservation in terms of sperm motility and plasma membrane integrity (P > .05). The acrosomal integrity for spermatozoa cryopreserved in TCG was statistically lower than that of TTE, DM, mDM, LG-DM, and G-DM (P < .05) but was significantly higher than that of TEST, TSM, Test-M, Test-H, and LM (P < .05). The postthaw sperm motility for 5 other extenders (TEST, TSM, Test-M, Test-H, and LM) did not exceed 30%, and the 3 sperm parameters evaluated for them were significantly lower than that of TTE, DM, mDM, LG-DM, G-DM, and TCG (P < .05). On the basis of these findings, 5 commonly used permeating cryoprotectants, glycerol, ethylene glycol, dimethyl sulfoxide, acetamide and propylene glycol have further been tested for their effectiveness on sperm cryopreservation in extenders of TTE, DM, mDM, LG-DM, G-DM, and TCG. The results showed that the sperm cryoprotective efficiencies of glycerol and ethylene glycol were similar and best among 5 permeating cryoprotectant treatments (P > .05). Dimethyl sulfoxide or acetamide resulted in average cryoprotection for cynomolgus monkey spermatozoa: poorer than glycerol or ethylene glycol but better than that of propylene glycol (P < .05). In addition, the action of permeating cryoprotectant appeared to be independent of extenders. The results in the present study demonstrate that 1) TTE, DM, mDM, LG-DM, G-DM, and TCG are excellent extenders and suitable for cynomolgus monkey sperm cryopreservation; 2) the mechanism of action of permeating cryoprotectants are not affected by extender composition; 3) ethylene glycol has a similar cryoprotective efficacy to glycerol that makes it a successful cryoprotectant for sperm cryopreservation in cynomolgus monkeys.     

Effects of bovine sperm cryopreservation using different freezing techniques and cryoprotective agents on plasma, acrosomal and mitochondrial membranes

The success of semen cryopreservation is influenced by several factors, such as freezing curves and cryoprotectants. These two factors are of special interest once they may lead to many important physical-chemical changes resulting in different degrees of damage in spermatozoa structure. This experiment was designed to compare the effect of bull semen cryopreservation using two freezing techniques: conventional (CT--cooling rate of -0.55 °C min(-1) and freezing rate of -19.1 °C min(-1) and automated (AT--cooling rate of -0.23 °C min(-1) and freezing rate of -15 °C min(-1)), performed with different curves, and with three cryoprotectants (glycerol, ethylene glycol and dimethyl formamide) on bovine sperm motility and integrity of plasma, acrosomal and mitochondrial membranes. These variables were simultaneously evaluated using the fluorescence probes propidium iodide, fluorescein-conjugated Pisum sativum agglutinin and MitoTracker Green FM. The effects of freezing techniques, as well as of different cryoprotectants were analysed by the analysis of variance. The means were compared by Fisher's test. There were no significant differences between freezing techniques (P > 0.05). Glycerol showed higher percentages of motility, vigour and integrity of plasma, acrosomal and mitochondrial membranes than other two cryoprotectants (P < 0.05). Ethylene glycol preserved higher motility and integrity of plasma and mitochondrial membranes than dimethyl formamide (P < 0.05). Sperm motility with glycerol was 30.67 ± 1.41% and 30.50 ± 1.06%, with ethylene glycol was 21.17 ± 1.66% and 21.67 ± 1.13% and with dimethyl formamide was 8.33 ± 0.65% and 9.17 ± 0.72% to CT and AT curves, respectively. The percentage of spermatozoa with simultaneously intact plasma membrane, intact acrosome and mitochondrial function (IPIAH) was 14.82 ± 1.49% (CT) and 15.83 ± 1.26% (AT) to glycerol, 9.20 ± 1.31% (CT) and 9.92 ± 1.29% (AT) to ethylene glycol 4.65 ± 0.93% (CT) and 5.17 ± 0.87% (AT) to dimethyl formamide. Glycerol provided the best results, although nearly 85% of spermatozoa showed some degree of injury in their membranes, suggesting that further studies are required to improve the results of cryopreservation of bovine semen.     

The effect of reactive oxygen species on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential, and membrane lipid peroxidation

The objective of this study was to examine the influence of reactive oxygen species (ROS), generated through the use of the xanthine (X)-xanthine oxidase (XO) system, on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential, and membrane lipid peroxidation. Equine spermatozoa were separated from seminal plasma on a discontinuous Percoll gradient, and spermatozoa were incubated with 0.6 mM X and 0.05 U/mL XO for 30 minutes. Catalase (150 U/mL), superoxide dismutase (SOD, 150 U/mL), or glutathione (GSH, 1.5 mM) were evaluated for their ability to preserve sperm function in the presence of the induced oxidative stress. At the end of the 30-minute incubation, sperm motility was determined by computer-assisted semen analysis. Viability and acrosomal integrity were determined by Hoechst-Pisum sativum staining, and mitochondrial membrane potential was determined by staining with JC-1. Incubation with the X-XO system led to a significant (P < .01) increase in hydrogen peroxide production and an associated decrease (P < .01) in motility parameters. Total motility was significantly (P < .01) lower in the presence of X-XO compared with the case of the control (29%+/-9% vs 73%+/-1%, respectively). Catalase, but not SOD, prevented a decline in motility secondary to oxidative stress (71%+/-4% vs 30%+/-3%, respectively). The addition of glutathione had an intermediate effect in preserving sperm motility at the end of the 30-minute incubation (53%+/-3%). No influence of X-XO could be determined on viability, acrosomal integrity, or mitochondrial membrane potential. In order to promote lipid peroxidation, samples were incubated with ferrous sulfate (0.64 mM) and sodium ascorbate (20 mM) for 2 hours after the X-XO incubation. No increase in membrane lipid peroxidation was detected. This study indicates that hydrogen peroxide is the major ROS responsible for damage to equine spermatozoa. The decrease in sperm motility associated with ROS occurs in the absence of any detectable decrease in viability, acrosomal integrity, or mitochondrial membrane potential or of any detectable increase in lipid peroxidation.     

Conventional slow freezing cryopreserves mouflon spermatozoa better than vitrification

This work examines the effectiveness of a TCG (Tris, citric acid, glucose, 6% egg yolk and 5% glycerol) and a TEST (TES, Tris, glucose, 6% egg yolk and 5% glycerol) sperm extender in the freezing of mouflon spermatozoa at slow cooling rates, using different pre‐freezing equilibration times (2–3 hr). It also examines the tolerance of mouflon spermatozoa to different concentrations of cryoprotectants (5, 10, 20% glycerol; 5%, 10%, 20% dimethyl sulfoxide; 6% polyvinylpyrrolidone) and/or sucrose (100, 300, 500 mm ). The highest quality (p < .01) thawed spermatozoa were obtained when using the TEST extender and an equilibration time of 3 hr. Sperm motility and membrane integrity were strongly reduced when using rapid freezing rates (60–85°C min^?1), independent of the concentration of cryoprotectants. The lowest sucrose concentration (100 mm ) provided the highest (p < .05) percentage of motile spermatozoa and live spermatozoa with an intact acrosome. Vitrified–warmed sperm variables were at their best when the spermatozoa was diluted in TCG–6% egg yolk + 100 mm sucrose and warmed at 60°C. Slow warming at 37°C strongly reduced (p < .05) sperm motility and viability. However, sperm vitrification returned lower fertility, sperm motility and sperm viability values than conventional sperm freezing.     

Osmotic tolerance limits and properties of rhesus monkey (Macaca mulatta) spermatozoa

Fundamental cryobiological characteristics of rhesus spermatozoa must be determined for successful cryopreservation techniques to be established. The main objectives of the present study were to determine the osmotic behavior and osmotic tolerance limits of rhesus macaque spermatozoa. Cell volume changes over anisotonic conditions were assessed using an electronic particle counter and sperm motility was evaluated with a computer-assisted sperm analysis system. Analysis of membrane integrity and mitochondrial membrane potential was performed using flow cytometry. Rhesus monkey spermatozoa behave as linear osmometers in the osmotic range tested (75-900 mOsmol kg(-1)), as shown by the Boyle van't Hoff plot (r(2) =.99). Rhesus spermatozoa have a mean cell volume of 36.8 +/- 0.5 micro m(3) at 22 degrees C, with 77.2% of the intracellular volume being osmotically inactive. Results regarding sperm tolerance to osmotic stress showed that sperm motility was more sensitive than membrane integrity to deviations from isotonicity and, in addition, that rhesus sperm motility and membrane integrity were more sensitive to hypertonic than hypotonic conditions. Mitochondrial membrane potential did not explain the lack of sperm motility observed under anisosmolal conditions in our study. Although most spermatozoa were able to recover initial volume after osmotic stress, they were not able to recover initial motility.     

Effects of accessory sex gland fluid on viability, capacitation, and the acrosome reaction of cauda epididymal bull spermatozoa

The effect of accessory sex gland fluid (AGF) on viability and acrosomal integrity of spermatozoa was examined with cauda epididymal spermatozoa and AGF from the same Holstein bull (n = 6). Surgical cannulation of the vasa deferentia enabled the separate collection of cauda epididymal effluent and AGF from each bull. Cauda epididymal effluent was incubated with either AGF collected from the same bull or medium alone. Following coincubation, spermatozoa (5 x 10(7) sperm/mL) were incubated in medium alone or under capacitating conditions (10 microg/mL heparin) for 16 hours. Every 2 hours, an aliquot of spermatozoa was exposed to lysophosphatidylcholine (100 microg/mL) to induce the acrosome reaction in capacitated spermatozoa. Sperm motility decreased over time regardless of treatment. Overall, spermatozoa incubated in AGF had fewer acrosome-intact live spermatozoa than did those not incubated in AGF. Viability was significantly (P < .05) compromised over time when spermatozoa were exposed to AGF, compared with those not preincubated in AGF. Significantly more (P < .05) acrosome-reacted live spermatozoa were seen following exposure to heparin and lysophosphatidylcholine when spermatozoa were not preincubated in AGF. We conclude that exposure of spermatozoa to AGF accelerates cell death and that rapid removal of spermatozoa from seminal plasma is critical for maximal viability.     

冷冻保护剂对牛精子功能和渗透压的影响

本文验证了一个假设：在精子中添加和去除冷冻保护剂可能导致精子的调节容量减少,渗透物质损失,精子功能降低。不进行冷冻和解冻的情况下,常用的冷冻保护剂可以降低培养基中牛精子的总活力和前进运动力,降低精子通过宫颈粘液的能力,破坏精子头膜,并导致精子尾巴卷曲。如果在很多小的步骤中而非仅在一个步骤中添加和去除冷冻保护剂,可适当保护精子的功能。特定的冷冻保护剂能降低细胞内的左旋肉碱和谷氨酸的浓度,而不是肌醇和山梨醇的浓度。在多步中添加或者去除冷冻保护剂比在一步中进行好,因为粘液的渗透依赖于精子中原始细胞内左旋肉碱和谷氨酸的总量。总之,在多步中添加或者去除丙三醇和丙二醇可以最好地保持精子的质量。因为丙三醇和丙二醇与降低的细胞内谷氨酸浓度有关。在牛精子中,谷氨酸看来可以调控冷冻保护剂导致的细胞膨胀。     

Porcine sperm vitrification I: cryoloops method

The aims of this study were to evaluate porcine sperm vitrification in cryoloops, with and without two different cryoprotectants and assess two warming procedures. Extended (n = 3; r = 4) and raw (n = 5; r = 2) semen was diluted in media without and with cryoprotectants (4% dimethylformamide and 4% glycerol) to a final concentration of 20 × 10⁶ spermatozoa ml^?1 and vitrified using the cryoloops method. Two warming procedures were evaluated: rapid method (30 s at 37°C) and an ultra‐rapid method (7 s at 75°C, followed by 30 s at 37°C). Total motility (phase contrast), sperm viability (6‐carboxifluorescein diacetate and propidium iodide stain), membrane function (hypo‐osmotic swelling test), acrosome integrity (phase contrast), chromatin condensation (toluidine blue stain) and chromatin susceptibility to acid denaturation (acridine orange stain) were evaluated before and after vitrification and analysed using Friedman's test. In all media, the only seminal parameters that were maintained after vitrification were chromatin condensation and integrity. Vitrification of porcine spermatozoon using cryoloops, both in the presence or absence of cryoprotectants and independent of the warming procedure used, permits conservation of sperm chromatin condensation and integrity. It would be interesting to further verify this by producing porcine embryos using vitrified spermatozoon with intracytoplasmic sperm injection.     

Porcine sperm vitrification II: Spheres method

Owing to current problems in boar sperm cryopreservation, this study proposes to evaluate vitrification in spheres as an alternative cryopreservation procedure, comparing the use or not of permeable cryoprotectants and two warming methods. Extended (n = 3; r = 4) and raw (n = 5; r = 2) porcine spermatozoa were diluted in media, in the absence or presence of either 4% dimethylformamide or 4% glycerol, to a final concentration of 5 × 10⁶ spermatozoa/ml and vitrified using the spheres method. Two warming procedures were evaluated: a rapid method (30 s at 37°C) and an ultrarapid method (7 s at 75°C, followed by 30 s at 37°C). Percentages of total motility (phase contrast), membrane function (hypo‐osmotic swelling test), acrosome integrity (phase contrast), sperm viability (6‐carboxyfluorescein diacetate and propidium iodide stain), chromatin condensation (toluidine blue stain) and chromatin susceptibility to acid denaturation (acridine orange stain) were evaluated in the samples before and after vitrification. Results, analysed using Friedman's test, suggest that rapid warming of raw porcine spermatozoa vitrified without permeable cryoprotectants may preserve DNA condensation and integrity better than the other processing methods studied in this work. Hence, porcine sperm vitrification using spheres could be used to produce embryos with ICSI to further validate this method.     

Effect of Cryoprotective Media and Dilution Methods on the Preservation of Human Spermatozoa

Human Sperm Preservation Medium (HSPM) containing 15% glycerol, 0.05 M sucrose and 1% glycine had an equal or better capacity to preserve sperm motility and vitality than egg yolk-citrate medium (ECM) containing 15% glycerol. Insemination of patients with semen frozen in HSPM resulted in a higher pregnancy rate than semen frozen in ECM. Glycerol was superior to dimethyl sulphoxide (DMSO) or ethylene glycol (EG) as a cryoprotectant, although the cryoprotective ability of 7.5% glycerol and 7.5% EG did not differ significantly. Neither the addition temperature nor the method of addition of the media containing glycerol, DMSO or EG had any influence on the sperm cryosurvival. Raffinose (0.025 to 0.05 M) or sucrose (0.05 to 0.1 M) improved cryosurvival of spermatozoa. The pH of HSPM had no significant influence on the cryosurvival of spermatozoa but the optimal pH was 6.5 to 7. Ratios of 1:1, 1:2, 1:3, and 1:4 of diluent to semen did not affect sperm cryosurvival.     

Interaction among extenders,cryoprotectants and Orvus Es Paste supplementation and freezing rate for sperm cryopreservation in the dromedary camel

This study evaluated the effects of freezing extenders, cryoprotectants and their concentrations, presence of Orvus Es Paste and freezing rates for cryopreserving dromedary camel sperm. Semen (five males; 2 ejaculates/male) was frozen in one of the following extenders (Green Buffer^® or INRA96^®), cryoprotectants (3 and 6% glycerol or ethylene glycol), with or without Orvus Es Paste and freezing at two different heights (1 and 4 cm) above liquid nitrogen. Sperm motility recovery parameters were evaluated post-thaw (0 and 1 hr), vitality and acrosome integrity (0 hr). Green Buffer showed higher total motility recoveries (p < .001). Higher percentage of cryoprotectant improved both total and progressive motility at 0 hr (p < .001; p = .003) and 1 hr (p < .001; p = .005). Acrosome integrity at thawing increased in the presence of ethylene glycol (p < .001) and Orvus Es Paste (p = .001). Kinematics were affected by extender, cryoprotectant concentration and Orvus Es Paste at 0 and 1 hr, and type of cryoprotectant only influenced them at 0h. Our findings showed strong interactions among type of cryoprotectant and concentration, and extender and Orvus Es Paste. Generally, combining Green Buffer, 3%–6% ethylene glycol or 6% glycerol without Orvus Es Paste, regardless of the freezing rates, yielded the highest post-thaw parameters for camel sperm.     

Vitrification of human ICSI/IVF spermatozoa without cryoprotectants: new capillary technology

The aim of this study was to develop and to test the standardized aseptic technology of permeable cryoprotectant-free vitrification of human spermatozoa in capillaries (for intracytoplasmic sperm injection [ICSI] or in vitro fertilization [IVF]). To test the effect of vitrification on basic sperm parameters, each of 68 swim-up-prepared ejaculates from oligo-astheno-terato-zoospermic patients were aliquoted and distributed into 3 groups: 1) nontreated control, 2) 10 μL of spermatozoa cryopreserved by slow conventional freezing with glycerol-contented medium, and 3) 10 μL of spermatozoa vitrified in 50-μL plastic capillaries in culture medium with 0.25 M sucrose. Spermatozoa motility (1, 24, and 48 hours after warming), plasma membrane integrity, acrosomal integrity, and spontaneous capacitation-like changes were determined after warming. Aseptic cryoprotectant-free vitrification showed a significantly stronger cryoprotective effect compared with conventional freezing. One hour after warming, motility, plasma membrane integrity, and acrosomal integrity were significantly higher than is observed for conventionally frozen spermatozoa (28% vs 18%, 56% vs 22%, and 55% vs 21%, respectively; P < .05), although lower than in fresh spermatozoa (35%, 96%, and 84%, respectively; P < .05). Capacitation-like changes did not differ significantly between vitrified and conventionally frozen samples (8% vs 9%, respectively; P > .1) (2% in fresh spermatozoa). The newly developed technology of aseptic vitrification of human spermatozoa in capillaries can effectively preserve these cells from cryo-injures. Spermatozoa, vitrified by this technology, are free from seminal plasma owing to swim-up preceding vitrification and are free from permeable cryoprotectants. They are ready for further use immediately after warming without any additional treatment. Therefore, the reported technology has a great potential for use in ICSI/IVF.     

Comparison and evaluation of capacitation and acrosomal reaction in freeze‐thawed human ejaculated spermatozoa treated with L‐carnitine and pentoxifylline

Cryopreservation is used to preserve the spermatozoa; however, it leads to a reduction in sperm quality. L‐carnitine (LC) influences sperm motility and preserves the sperm membrane and DNA integrity. The objectives of this study were to evaluate the protective effects of LC on the membrane integrity of normal human spermatozoa and compare it with pentoxifylline (PT) during cryopreservation. Thirty normal semen samples, prepared by swim‐up procedure, were divided into three aliquots: a control without any treatment and two experimental aliquots that were incubated in PT or LC for 30 min. All aliquots were cryopreserved and thawed after 48 hr. To evaluate the percentages of intact, acrosomal‐reacted and capacitated spermatozoa, lectin histochemistry and flow cytometry were performed by wheat germ agglutinin, peanut agglutinin and Con A. Statistical analyses were performed using ANOVA. LC supplementation elevated the percentage of noncapacitated spermatozoa compared with control and PT‐treated samples and the percentages of acrosomal intact spermatozoa compared with PT‐treated samples. PT pre‐treatment improved the motility but not membrane integrity. LC supplementation reduced the percentages of acrosomal‐reacted spermatozoa compared with the control and PT‐treated samples. Although LC did not improve motility, it protected the plasma membrane and acrosomal integrity. Therefore, LC may be the superior choice compared to PT for maintaining the sperm integrity.     

冻存后大鼠精子活力变化与其结构的关系

目的　观察冷冻对大鼠精子活动能力的影响 ,并探讨其活动能力的变化与细胞膜完整性、DNA结构、线粒体鞘和顶体的关系。　方法　使用计算机辅助精子活动分析仪 (CASA)检测冻存前后大鼠精子活动力的变化 ;荧光素乙酰乙酸盐 (FDA)染色法检测大鼠精子细胞膜的完整性 ;双氢鲁丹明荧光反应观察精子线粒体鞘的变化 ;金霉素 (CTC)荧光检测法观察冻存对大鼠精子顶体的影响。　结果　复苏后精子的活动力下降 ,为冻存前的 39.7% ;FDA检测显示冻存后的大鼠精子细胞膜完整 ,未受到破坏 ;冻存后的大鼠精子线粒体鞘荧光强度下降 ,且荧光不连续、甚至消失 ;CTC荧光反应显示冻存后精子顶体反应 (AR)的类型发生改变 ,AR型精子比例明显下降 ,由冻存前的 6 8.6 %降至冻存后的 13.4 %。　结论　冻存后大鼠精子细胞膜的完整性未受到破坏 ;精子的线粒体鞘和顶体受到较明显的破坏。大鼠精子冻存复苏后活力下降与线粒体鞘和AR存在相关性。     

In vitro effects of nonylphenol on motility,mitochondrial, acrosomal and chromatin integrity of ram and boar spermatozoa

The objective of this study was to determine the effects of nonylphenol (NP) on viability of ram and boar sperm in vitro. Ram or boar spermatozoa were exposed to 1, 10, 100, 250 and 500 μg NP ml^?1 for 1, 2, 3 or 4 h. Computer‐assisted sperm motility analysis (CASA) system was used to evaluate sperm motility characteristics. Flow cytometry was used to determine mitochondrial membrane potential (MMP) and chromatin integrity, while epifluorescent microscopy was used to determine sperm acrosomal status. Exposure of both species spermatozoa to 250 and 500 μg NP ml^?1 was detrimental to progressive motility (P < 0.05), and its adverse effect was significant at lower (100 μg NP ml^?1) concentration (P < 0.05). The percentages of ram and boar spermatozoa with high MMP declined drastically after exposures to ≥250 μg ml^?1 NP (P < 0.05). Unlike chromatin integrity, which did not appear to be altered by NP exposure, there were dose‐dependent NP effects (P < 0.05) on acrosomal integrity of both species at as low as 1 μg ml^?1 NP for boar spermatozoa and 10 μg ml^?1 NP for ram spermatozoa. These data show adverse effects of NP on ram and boar spermatozoa and thus its potential harmful effects on male reproduction as NP is found in fruits, vegetables, human milk, fish and livestock products.     

Metabolic evaluation of cooled equine spermatozoa

Microscopy has been used in the routine evaluation of sperm metabolism. Nevertheless, it has limited capacity to preview male fertility. As calorimetry may be used to evaluate directly the metabolic activity of a biological system, the aim of this study was to use microcalorimetry as an additive method for sperm metabolism evaluation of cooled equine semen. Two ejaculates of four stallions were collected and motility, viability (eosin 3%) and membrane functional integrity (hyposmotic swelling test) of spermatozoa were evaluated. Sperm samples were processed following different protocols and the metabolism of these samples was accessed by calorimetry. Centrifugation is part of some of these processing protocols and although this procedure has been deleterious for sperm viability and plasma membrane integrity, no decrease in sperm motility was observed. Microcalorimetry was capable of detecting the positive effect of re‐suspending the sperm pellet with Kenney extender. Thus, the use of microcalorimetry offered additional information for equine sperm metabolism evaluation and was efficient in detecting important information from sperm cell metabolism.     

Catalase supplementation on thawed bull spermatozoa abolishes the detrimental effect of oxidative stress on motility and DNA integrity

The potential protective effect of catalase supplementation during in vitro culture of frozen/thawed bull spermatozoa was investigated. Frozen/thawed semen collected from three fighting bulls was diluted in phosphate buffered saline (PBS) and incubated at 37 °C under different experimental conditions: Control, Catalase (CAT) (200 U/mL), Oxidant (OXI) (100 μ m Fe²⁺/1 m m ascorbate), and Catalase + Oxidant (CAT/OXI). We assessed sperm motility, acrosomal integrity, viability and chromatin status (SCSA®) at 0, 2 and 6 h of incubation. Our results showed that catalase abolished the effect of the oxidant, protecting spermatozoa against reactive oxygen species, and improving both sperm motility and chromatin status during incubation. The OXI treatment significantly reduced the percentage of motile sperm after 6 h of incubation. The statistical model also showed that there were differences in sperm motility between CAT/OXI (20.8 ± 2.9%) and OXI (11.6 ± 7.6%) ( p  < 0.001). There were no significant effects of OXI on sperm viability, acrosomal status or proportion of abnormal tails. %DFI (spermatozoa with moderate or high DNA Fragmentation Index) was significantly higher on OXI ( p  < 0.001). Catalase prevented DNA fragmentation even in the presence of the oxidant (%DFI: 30.3 ± 0.8% OXI vs. 17.4 ± 0.7% CAT/OXI). We conclude that catalase supplementation after thawing could protect bull spermatozoa against oxidative stress, and it could improve media used for processing thawed spermatozoa.     

Ejaculate traits and sperm cryopreservation in the endangered Baird's tapir (Tapirus bairdii)

There is little information on the reproductive biology of the male Baird's tapir (Tapirus bairdii). In this study, we characterized the ejaculate traits and evaluated the efficacy of 2 cryodiluents on sperm cryosurvival. Ejaculates were assessed for volume, pH, sperm motility, forward progression, osmolality, sperm concentration, sperm morphology, and acrosomal integrity. For cryopreservation, ejaculates with >50% total sperm motility were washed, and sperm pellets were resuspended in either Botu-Crio (CryoVital, Grandau, Germany) or INRA 96 containing 2% egg yolk and 2.5% each of methyl- and dimethylformamide (INRA 96), and they were cryopreserved over liquid nitrogen vapor. Thawed samples were incubated in vitro (25 °C) and evaluated for percent total sperm motility, forward progression, and acrosomal integrity at hourly intervals for 4 hours. Spermic ejaculates were obtained from all males, and the mean seminal volume, sperm concentration per milliliter, percent sperm motility, progressive status, and percent morphologically normal cells were 20.4 ± 4.3 mL, 101.2 ± 24.0 × 10(6)/mL, 46.1% ± 5.0%, 2.9 ± 0.1, and 6.9% ± 1.4%, respectively. There was a positive significant correlation between percent normal sperm and animal age (r = 0.66; P < .004). Cryopreservation in either Botu-Crio or INRA 96 resulted in a decline (P < .05) in percent sperm motility and acrosomal integrity. Sperm forward progression remained unaffected immediately after thawing in INRA 96 but continued to decline over time. These results characterize, for the first time, the ejaculate traits of the tapir; demonstrate that tapir spermatozoa can be cryopreserved in diluents containing amides alone or in combination with glycerol; and provide fundamental information critical for development of assisted reproductive technologies for the Baird's tapir.     

The fix vital stain method. Simultaneous determination of viability and acrosomal status of bovine spermatozoa.

A rapid, accurate and precise method for simultaneous determination of acrosomal status and viability of bull spermatozoa is introduced and evaluated. The method involves fixation of semen with glutaraldehyde and subsequent addition of the fluorescent dye Hoechst bisbenzimide 33258 (H33258). Wet mounts were examined using a combination of phase-contrast and fluorescence microscopy (x500) for simultaneous visualization of the acrosomal apical ridge, which is indicative of the presence of an intact acrosome, and H33258-labeled nuclei, which is indicative of membrane-damaged cells. This fix-vital stain method allows differentiation between true acrosome reactions and degenerative postmortem loss of acrosomal membranes. Incubation of frozen-thawed spermatozoa for 60 minutes at 37 degrees C in the presence of calcium ionophore A23187 resulted in an increase in the percentage of true acrosome-reacted spermatozoa. The fix-vital stain method does not contain any processing steps that result in loss, selection, or damage of spermatozoa and therefore allows evaluation of representative semen samples.