Supplementation of the thawing medium with reduced glutathione improves function of frozen-thawed goat spermatozoa

Sperm cryopreservation represents a useful tool in the management of reproduction in goat production. However, freezing and thawing produce physical and chemical stress on the sperm membrane that reduces their viability and fertilizing ability. In this study, firstly we evaluated the effects of reduced glutathione (GSH, 1 and 5 mM) supplementation of the thawing extender on parameters of frozen-thawed goat spermatozoa. We used a set of functional sperm tests that included sperm motility assayed by computer-assisted semen analysis (CASA), membrane lipid packing disorder, spontaneous acrosome reaction, free radical production (ROS generation) and sperm chromatin condensation. The main findings from this study were that addition of GSH to the thawing medium resulted in: (1) a higher motility and progressive motility; (2) a higher number of non-capacitated viable spermatozoa; (3) higher number of viable spermatozoa with intact acrosome; (4) a reduction in ROS generation and (5) lower chromatin condensation. In a second study, the additions of reduced (GSH, 5 mM) or oxidized glutathione (GSSG, 2.5 mM) to the thawing media were evaluated. We confirmed the protective effect of GSH on the sperm functionality. The addition of GSSG to the thawing media was less protective to sperm functions compared to GSH. Addition of GSH to the thawing extender could be of significant benefit in improving the function and fertilizing capacity of frozen goat spermatozoa. The information derived from this study suggests the importance of oxidative stress as responsible for cryo-injury to spermatozoa and opens new windows to explore the practical application of antioxidants to improve the quality of post-thaw goat semen.     

Decrease in glutathione content in boar sperm after cryopreservation. Effect of the addition of reduced glutathione to the freezing and thawing extenders

Although glutathione content in boar spermatozoa has been previously reported, the effect of reduced glutathione (GSH) on semen parameters and the fertilizing ability of boar spermatozoa after cryopreservation has never been evaluated. In this study, GSH content was determined in ejaculated boar spermatozoa before and after cryopreservation. Semen samples were centrifuged and GSH content in the resulting pellet monitored spectrophotometrically. The fertilizing ability of frozen-thawed boar sperm was also tested in vitro by incubating sperm with in vitro matured oocytes obtained from gilts. GSH content in fresh semen was 3.84 +/- 0.21 nM GSH/10(8) sperm. Following semen cryopreservation, there was a 32% decrease in GSH content (P < 0.0001). There were significant differences in sperm GSH content between different boars and after various preservation protocols (P = 0.0102 ). The effect of addition of GSH to the freezing and thawing extenders was also evaluated. Addition of 5 mM GSH to the freezing extender did not have a significant effect on standard semen parameters or sperm fertilizing ability after thawing. In contrast, when GSH was added to the thawing extender, a dose-dependent tendency to increase in sperm fertilizing ability was observed, although no differences were observed in standard semen parameters. In summary, (i) there was a loss in GSH content after cryopreservation of boar semen; (ii) addition of GSH to the freezing extender did not result in any improvement in either standard semen parameters or sperm fertilizing ability; and (iii) addition of GSH to the thawing extender resulted in a significant increase in sperm fertilizing ability. Nevertheless, future studies must conclude if this is the case for all boars. Furthermore, since addition of GSH to the thawing extender did not result in an improvement in standard semen parameters, this suggests that during the thawing process, GSH prevents damage of a sperm property that is critical in the fertilization process but that is not measured in the routine semen analysis.     

Impact of supplementation of semen extender with antioxidants on the quality of chilled or cryopreserved Arabian stallion spermatozoa

The aim of the present study was to evaluate the effects of supplementation of semen extender with various non-enzymatic antioxidants on the quality of cooled or cryopreserved Arabian stallion spermatozoa. Semen collected from four pure Arabian stallions was centrifuged at 600g for 15 min. Spermatozoa were then diluted in INRA-82 extender supplemented with bovine serum albumin (BSA; 0, 10, 15 and 20 mg/mL) or trehalose (0, 75, 100 and 150 mM) or zinc sulphate (0, 100, 150 and 200 μM). The diluted semen was then either cooled at 5 °C or cryopreserved in 0.5–ml plastic straws. After cooling or thawing, sperm motility, viability, sperm abnormalities, viability index, and plasma membrane integrity were evaluated. The results showed that supplementation of semen extender with 150 mM trehalose or with 200 μM zinc sulphate significantly (P < 0.05) improved motility, viability, sperm membrane integrity and acrosome status in Arabian stallion spermatozoa after cooling or after freezing and thawing compared with controls (non-supplemented media) or with those supplemented with other concentrations of trehalose or zinc sulphate. Supplementation of semen extender with BSA did not improve sperm motility or cryosurvival of Arabian stallion spermatozoa after cooling or after freezing and thawing. In conclusion, supplementation of semen extender with non-enzymatic antioxidants (trehalose or zinc sulphate) improved the quality of chilled and frozen/thawed Arabian stallion spermatozoa. The most beneficial effects occur when semen diluent was supplemented with 150 mM trehalose or 200 μM zinc sulphate.     

Quality and fertilizing ability of electroejaculated cat spermatozoa frozen with or without Equex STM Paste

An optimal protocol for cat semen cryopreservation has not yet been defined. Addition of Equex STM Paste has been tested for epididymal cat spermatozoa but not for ejaculated cat spermatozoa. Furthermore, the effect of Equex STM Paste on fertilizing ability of cryopreserved semen has never been evaluated in that species. Therefore, the aims of the current study were to investigate if addition of Equex STM Paste to a freezing extender for electroejaculated cat (Felis catus) semen would improve postthaw sperm quality and if sperm fertilizing ability after cryopreservation with or without Equex STM Paste was preserved. Semen was collected by electroejaculation and frozen in a Tris-glucose-citrate egg yolk extender supplemented with (0.5% vol/vol) or without Equex STM Paste. In Experiment 1, sperm motility, membrane integrity, and acrosomal status were determined immediately after collection and at 0, 3, and 6 h postthaw. In Experiment 2, frozen semen from the two groups was used for in vitro fertilization (IVF) of in vitro-matured cat oocytes. Cleavage rate was recorded 30 h after IVF, and embryo development was evaluated on Days 6 and 7 of culture. In Experiment 1, the rate of motile spermatozoa after freezing-thawing was higher when Equex STM Paste was added to the freezing extender, but progressive motility score was not influenced (P > 0.05). Sperm membrane integrity was positively affected (P < 0.05) by the addition of the detergent. Intact acrosomes after thawing were similar (P > 0.05) between groups. Even if the decreasing rates of motility and membrane integrity were more rapid in presence of Equex than those in controls, total motility and sperm viability were similar at 3 and 6 h after thawing (P > 0.05). In Experiment 2, there was no difference in fertilizing ability and embryo development between the two groups (P > 0.05). The results of this study demonstrate that the addition of Equex STM Paste in the freezing extender avoids the loss of motile spermatozoa and maintains fertilizing ability of frozen-thawed spermatozoa.     

Positive effect of butylated hydroxytoluene (BHT) on the quality of cryopreserved cat spermatozoa

The semen cryopreservation processes are associated with state of oxidative stress induced by high levels of reactive oxygen species (ROS), causing damage to functional spermatozoa. Whereby, antioxidants have been utilized to scavenge or neutralize the elevated levels of ROS. The aim of at the present study was to evaluate the effect of adding BHT to the freezing extenders on post-thaw characteristics of domestic cat spermatozoa. Semen samples were frozen in Tris-fructose-citric acid-based extender, supplemented with different concentrations of BHT (0.5 mM, 1.0 mM and 2.0 mM) and a control sample without antioxidant. After thawing, sperm samples were assessed for motility by computer‐assisted sperm analysis and viability, acrosome integrity, superoxide anion production and membrane lipid peroxidation status by flow cytometry. In the study, the parameters of sperm motility and acrosome integrity were significantly higher in 2.0 mM BHT compared to sperm frozen in the extender with other concentrations and control (P < 0.05), in addition, this concentration reduced significantly the superoxide anion production and lipid peroxidation of the sperm. The results demonstrated that the supplementation of BHT to the freezing extender could protect the function and cellular structure of domestic cat sperm from cryoinjuries.     

Cryopreservation of spermatozoa in cyprinid fishes

The present study investigated semen cryopreservation in cyprinid fish using computer-assisted sperm motility analysis for viability control. Spermatozoa of the bleak, Chalcalbumus chalcoides, were used as a basic model to describe the toxic and cryoprotective effects of internal and external cryoprotectants, their most effective concentrations and combinations, the freezing and thawing conditions, and the effects of equilibration. We also used these data to develop a cryopreservation protocol for Barbus barbus, Chondrostoma nasus, Ctenopharyngodon idella, Cyprinus cario, Hypohtalmichthys molitrix, Leuciscus cephalus, Rutilus meidingerii, and Vimba vimba. For all investigated species the optimal extender composition was a buffered physiological sperm motility-inhibiting saline solution containing 10% DMSO and 0.5% glycin. The optimal sperm equilibration period in the extender was < or = 5 min. Freezing was performed in an insulated box in liquid nitrogen vapor and it was optimal at 4 to 5 cm above the surface of the liquid, depending on the species. Thawing was optimal in a 25 degrees C water bath whereby the thawing time ranged depending on species from 15 to 45 sec. This cryopreservation protocol resulted in frozen-thawed semen with 35 to 65% motile and 5 to 25% locally motile spermatozoa depending on the quality of fresh semen.     

Freeze–thaw induced genotoxicity in buffalo (<Emphasis Type="Italic">Bubalus bubalis</Emphasis>) spermatozoa in relation to total antioxidant status

Use of cryopreserved semen has become an important tool in assisted reproduction but freezing and thawing cause sub-lethal damage to spermatozoa. This is detrimental to sperm because of the membrane damage including permeability and integrity. An excess generation of reactive oxygen species (ROS) creates oxidative stress due to reduced antioxidant status of the cryopreserved spermatozoa. In the present study fresh buffalo semen was collected and divided into two aliquots. One aliquot was used for fresh semen analysis and the other was cryopreserved in Tris-egg yolk-citrate extender. The semen samples were used to study different sperm quality parameters like motility, viability, membrane integrity and total antioxidant status. The DNA integrity in fresh and cryopreserved spermatozoa was also studied using comet assay. The sperm quality parameters like post-thaw sperm motility, viability, membrane integrity and total antioxidant status of cryopreserved spermatozoa were significantly lowered (P < 0.05) compared to fresh spermatozoa. The DNA fragmentation in cryopreserved spermatozoa was significantly higher (P < 0.01) as compared to fresh spermatozoa. The results show that the irreversible DNA damage occurs in spermatozoa during cryopreservation.     

The effect of Equex STM paste and sperm morphology on post-thaw survival of cat epididymal spermatozoa

Cryopreservation of epididymal spermatozoa is a potentially valuable tool for preserving genetic material from individuals of endangered species that die accidentally. Improvement of sperm-freezing protocols would increase the efficacy of gene banking from endangered felids, and the domestic cat can be used as a model for the wild felids. Addition of the detergent Equex STM paste to semen freezing extenders has been found to improve post-thaw survival and longevity of spermatozoa from various species but has never been tested for cat spermatozoa. Spermatozoa from cats with a high percentage of morphologically abnormal spermatozoa are more susceptible for cold injury and osmotic stress than spermatozoa from normozoospermic cats. Therefore, the aims of this study were to investigate: (a) if addition of Equex STM paste to a semen freezing extender would improve post-thaw sperm survival, and (b) if there is a relation between the percentage of morphologically normal spermatozoa and cryopreservation induced damage in cat epididymal spermatozoa. Spermatozoa harvested from epididymides of 10 male cats were frozen in a Tris egg yolk extender with or without the addition of Equex STM paste (0.5%, v/v). Sperm motility, membrane integrity and acrosomal status were evaluated immediately after harvesting, and at 0, 2, 4 and 6 h post-thaw. Sperm membrane integrity and acrosomal status were also evaluated after cooling to 4 degrees C, just before freezing. Cooling did not cause significant damage to the spermatozoa, whereas freezing damaged sperm membranes and acrosomes. Addition of Equex to the freezing extender had a significant positive effect on the percentage of intact acrosomes immediately after thawing (P > 0.05), but had a negative effect on the longevity of the spermatozoa; the percentages of membrane intact and motile spermatozoa being significantly lower in the presence of Equex than in the controls at 6h after thawing. The percentage of morphologically normal spermatozoa was not found to be correlated with either cryopreservation induced acrosome or plasma membrane damage, or with post-thaw motility (P > 0.05). The results clearly show that addition of Equex STM paste in the freezing extender protects the acrosomes of cat epididymal spermatozoa during the freezing--thawing process, but reduces the sperm longevity during in vitro incubation at 38 degrees C. Our results also indicate that the percentage of morphologically normal epididymal spermatozoa is not correlated with cryopreservation induced sperm damage using the described freezing protocol.     

Decrease in glutathione (GSH) content in bovine sperm after cryopreservation: comparison between two extenders

Growing evidence suggests that among the causes which deteriorate qualitative and functional characteristics of semen after freezing and thawing, there are those linked to oxidative stress and decrease detoxification of antioxidants present in the cells and seminal plasma. Overall, cryopreservation reduces the spermatozoal GSH content. In the present study we assessed the motility, capacitation status, ability to undergo acrosome reaction and levels of GSH in bovine spermatozoa after freezing and thawing, and compared the effects of the egg yolk tris-citrate and a commercial extenders (Bioxcell). Six semen samples were collected from each of five bulls and frozen and thawed in the two extenders. The level of total GSH of bovine spermatozoa detected in the raw semen ranged from 246.5 to 776.2 pmoles/mg of protein. The Bioxcell extender was superior in preserving GSH content (647.2+/-300.1 versus 223.6+/-154.0 pmoles/mL; P<0.05), motility levels (44.3+/-4.8 versus 41.8+/-4.0%; P<0.05), non-capacitated state (68.3+/-13.6 versus 61.3+/-17%; P<0.05) and a lower proportion of acrosome reacted spermatozoa post thaw (3.8+/-2.1 versus 6.3+/-3.1%; P<0.001) compared to egg yolk tris-citrate extender. These findings could be partially ascribed to the high GSH level contained in the commercial extender which seem able to alleviate oxidative damages to spermatozoa surviving freezing thawing procedures. Further researches are required in order to validate in vivo these results.     

Improvement of the post-thaw qualities of Okinawan native pig spermatozoa frozen in an extender supplemented with ascorbic acid 2-O-alpha-glucoside

The technical establishment of boar sperm cryopreservation is indispensable for effective breeding of the scarce Okinawan native pig Agu. The objective of the present study was to determine whether ascorbic acid 2-O-α-glucoside (AA-2G), a stable ascorbate derivative, is capable of improving the quality of cryopreserved Agu spermatozoa. Ejaculated Agu sperm frozen in an extender supplemented with 0, 100, 200, 400 or 800 μM AA-2G was thawed, and then evaluated the sperm motility and other qualities. Treatment with 200 μM AA-2G has the most beneficial effect on the sperm motility and the plasmalemma integrity after frozen-thawing among the concentrations tested (P < 0.05). In particular, the incidences of total motile sperm and rapid progressive motility at 1 and 3 h after incubation were markedly increased by treatment with AA-2G at 200 μM. The addition of AA-2G during cooling and freezing efficiently protected spermatozoa against the lipid peroxidation and the DNA damage. Spermatozoa frozen in the presence of AA-2G possessed significantly higher levels (P < 0.05) of ATP even after thawing than those frozen without AA-2G, implying that sperm viability was effectively conserved. Furthermore, higher sperm penetrability to matured oocytes in vitro was maintained in sperm treated with AA-2G during cryopreservation. These effects were observed for all sperm derived from three individuals. These findings demonstrate that the addition of AA-2G to the freezing extender efficiently improves the post-thaw qualities of fragile Agu sperm through the protection of spermatozoa against cell damage caused by oxidative stress during cryopreservation.     

Cryopreservation of Iberian red deer (Cervus elaphus hispanicus) spermatozoa obtained by electroejaculation

We tested extenders and freezing protocols for Iberian red deer semen. Samples were obtained by electroejaculation (10 stags), and analyzed for motility (CASA), viability (propidium ioide), acrosomal (PNA-FITC) and mitochondrial status (JC-1). Samples were diluted 1+1 in extender, cooled and adjusted for glycerol (extender with higher glycerol concentration), brought to 160×10⁶ mL⁻¹ and frozen. Four experiments were carried out, repeating sperm analysis after thawing to compare treatments. In a first experiment, seven samples were frozen using Triladyl^® (20% egg yolk) and UL extender (Tes-Tris-fructose, 15% egg yolk, 4% glycerol). Triladyl^® yielded higher motility after thawing. In a second trial, 17 samples were frozen using Triladyl^®, Andromed^®, Bioxcell^®, and UL with 8% LDL (low-density lipoproteins). Triladyl^® and Andromed^® performed better than Bioxcell^® on motility, and than UL-LDL on viability and acrosomal status. In a third experiment, the performance of freezing the sperm-rich ejaculate fraction versus the whole ejaculate was tested on nine samples. The sperm-rich ejaculate fraction not only rendered more motile and viable spermatozoa but also showed higher freezability (higher motile spermatozoa recovery). In a fourth experiment, we tried three modifications of the freezing protocol, for improving the freezability of low concentration samples: prior removal of seminal plasma; replacing extender (second fraction) for pure glycerol to reduce dilution; and performing only the 1+1 dilution, not the second dilution. No differences were found, although only three samples could be used. Both Triladyl^® and Andromed^® were deemed appropriate for freezing Iberian red deer semen, and the rich fraction should be selected for freezing.     

Improved cryopreservability of stallion sperm using a sorbitol-based freezing extender

Cryopreservation of stallion semen is often associated with poor post-thaw sperm quality. Sugars are among the important components of a freezing extender and act as non-permeating cryoprotectants. This study aimed to compare the quality of stallion sperm frozen with glucose, fructose or sorbitol-containing freezing extenders. Semen was collected from six stallions of proven fertility and cryopreserved using a freezing extender containing different types of monosaccharide sugars (glucose, fructose or sorbitol). After thawing, the semen was examined for sperm motility, viability, acrosome integrity, plasma membrane functionality and sperm longevity. The fertility of semen frozen in the presence of sorbitol was also tested by artificial insemination. Sperm quality was significantly decreased following freezing and thawing (P < 0.05). Fructose was inferior for protecting sperm during cryopreservation when compared to sorbitol and glucose (P < 0.05). Although the viability, motility and acrosome integrity of sperm cryopreserved with a glucose-containing extender did not significantly differ from sperm frozen in the sorbitol-based extender when examined at 2 and 4 h post-thaw, all of these parameters plus plasma membrane functionality were improved for sperm frozen in the sorbitol extender than in the glucose extender when examined 10 min post-thaw. Two of four mares (50%) inseminated with semen frozen with a sorbitol-containing freezing extender became pregnant. It is concluded that different sugars have different abilities to protect against cryoinjury during freezing and thawing of stallion sperm. This study demonstrated that an extender containing sorbitol as primary sugar can be used to successfully cryopreserve equine sperm; moreover, the quality of frozen-thawed sperm appeared to be better than when glucose or fructose was the principle sugar in the freezing extender.     

Effect of seminal plasma on the cryopreservation of equine spermatozoa

Seminal plasma is generally removed from equine spermatozoa prior to cryopreservation. Two experiments were designed to determine if adding seminal plasma back to spermatozoa, prior to cryopreservation, would benefit the spermatozoa. Experiment 1 determined if different concentrations of seminal plasma affected post-thaw sperm motility, viability and acrosomal integrity of frozen/thawed stallion spermatozoa. Semen was washed through 15% Percoll to remove seminal plasma and spermatozoa resuspended to 350 x 10(6)sperm/mL in a clear Hepes buffered diluent containing either 0, 5, 10, 20, 40 or 80% seminal plasma for 15 min, prior to being diluted to a final concentration of 50 x 10(6)sperm/mL in a Lactose-EDTA freezing diluent and cryopreserved. Sperm motility was analyzed at 10 and 90 min after thawing, while sperm viability and acrosomal integrity were analyzed 20 min after thawing. Seminal plasma did not affect sperm motility, viability or acrosomal integrity (P>0.05). Experiment 2 tested the main affects of seminal plasma level (5 or 20%), incubation temperature (5 or 20 degrees C) and incubation time (2, 4 or 6 h) prior to cryopreservation. In this experiment, spermatozoa were incubated with 5 or 20% seminal plasma for up to 6h at either 5 or 20 degrees C prior to cryopreservation in a skim milk, egg yolk freezing extender. Samples cooled immediately to 5 degrees C, prior to freezing had higher percentages of progressively motile spermatozoa than treatments incubated at 20 degrees C (31 versus 25%, respectively; P<0.05), when analyzed 10 min after thawing. At 90 min post-thaw, total motility was higher for samples incubated at 5 degrees C (42%) compared to 20 degrees C (35%; P<0.05). In addition, samples containing 5% seminal plasma had higher percentages of total and progressively motile spermatozoa (45 and 15%) than samples exposed to 20% seminal plasma (33 and 9%; P<0.05). In conclusion, although the short-term exposure of sperm to seminal plasma had no significant effect on the motility of cryopreserved equine spermatozoa, prolonged exposure to seminal plasma, prior to cryopreservation, was deleterious.     

Assessment of the efficacy of Sephadex G-15 filtration of bovine spermatozoa for cryopreservation

Semen from five dairy AI bulls was split-filtered through a Sephadex G-15 filter and frozen in a Tris-citric acid buffer egg yolk-based extender. The effect of filtration was studied morphologically for individual sperm abnormalities. Computer-assisted sperm analysis (CASA) was used for motility and sperm motion assessment. Flow cytometry was used to disclose sperm viability (SYBR-14/PI), mitochondrial membrane potential (Mitotracker Deep Red/SYBR 14), acrosome integrity (SYBR 14/PE-PNA/PI), plasma membrane stability (Merocyanine 540/YO-PRO 1/Hoechst 333342), and chromatin stability (acridine orange staining). Filtration significantly reduced the concentration of recovered spermatozoa (P < 0.01), but improved semen quality, reducing the number of spermatozoa with various forms of morphological defects. Filtration also affected percentages of sperm motility after equilibration and after freezing/thawing. Sperm motion characteristics were, however, not significantly affected by filtration at any stage of the cryopreservation protocol, including post-extension, equilibration, or freezing/thawing. Filtration enhanced sperm viability after thawing (P < 0.05), but had no significant effect (P > 0.05) on recovery of spermatozoa with high mitochondrial potential, intact acrosomes, or preserved sperm chromatin structure. Sperm plasma membrane stability was also not affected by the filtration method used (P > 0.05). It can be concluded that filtration effectively separates weaken or abnormal spermatozoa in pre-freezing semen samples and therefore the procedure could be recommended to improve post-thaw sperm viability of selected, fertile sires.     

Comparison of TNC and standard extender on post-thaw quality and in vivo fertility of Thai native chicken sperm

Semen extender has a vital role in preservation of sperm cells properties in terms of sperm viability, motility, acrosome integrity, and mitochondrial membrane potential. The objective of the present study was to evaluate a new extender, known as Thai native chicken (TNC) extender compared to BHSV-based and modified Sasaki extenders for freezing chicken semen. Semen from Thai native roosters was collected, pooled, and randomly divided into three groups. Semen was frozen with a simple freezing method using nitrogen vapor and dimethylformamide. In the first experiment, post-thaw motion parameters, viability, acrosome integrity, mitochondrial function, and lipid peroxidation levels were analyzed using computer-assisted sperm analysis, propidium iodide, fluorescein isothiocyanate-conjugate peanut agglutinin, JC-1, and the thiobarbituric acid reaction. Results showed that the type of extender had no effect on the percentage of total motile and curvilinear velocity. The percentage of progressive motile, straight-line velocity, and average path velocity of post-thawed semen were significantly lower in TNC compared to the modified Sasaki extender. However, the percentages of post-thawed acrosome integrity and active mitochondria were significantly higher in TNC extender (P < 0.05). For the second experiment, semen was thawed by using each of extenders thereafter, was inseminated to 48-layer breeder hens to determine the fertility rate. Among the three extenders used, the highest fertility rate was found in TNC extender. In conclusion, TNC extender can be recommended as an appropriate and useful cryopreservation media for native chicken semen since it maintains the quality of rooster semen and fertility after freezing and thawing process.     

Addition of superoxide dismutase mimics during cooling process prevents oxidative stress and improves semen quality parameters in frozen/thawed ram spermatozoa

High levels of reactive oxygen species (ROS), which may be related to reduced semen quality, are detected during semen cryopreservation in some species. The objectives of this study were to measure the oxidative stress during ram semen cryopreservation and to evaluate the effect of adding 2 antioxidant mimics of superoxide dismutase (Tempo and Tempol) during the cooling process on sperm motility, viability, acrosomal integrity, capacitation status, ROS levels, and lipid peroxidation in frozen and/or thawed ram spermatozoa. Measuring of ROS levels during the cooling process at 35, 25, 15, and 5 °C and after freezing and/or thawing showed a directly proportional increase (P < 0.05) when temperatures were lowering. Adding antioxidants at 10 °C confered a higher motility and sperm viability after cryopreservation in comparison with adding at 35 °C or at 35 °C/5 °C. After freezing and/or thawing, sperm motility was significantly higher (P < 0.05) in Tempo and Tempol 1 mM than that in control group. Percentage of capacitated spermatozoa was lower (P < 0.05) in Tempo and Tempol 1 mM in comparison with that in control group. In addition, ROS levels and lipid peroxidation in group Tempo 1 mM were lower (P < 0.05) than those in control group. These results demonstrate that ram spermatozoa are exposed to oxidative stress during the cooling process, specifically when maintained at 5 °C and that lipid peroxidation induced by high levels of ROS decreases sperm motility and induces premature sperm capacitation. In contrast, the addition of Tempo or Tempol at 0.5 to 1 mM during the cooling process (10 °C) protects ram spermatozoa from oxidative stress.     

Boar spermatozoa cryopreservation in low glycerol/trehalose enriched freezing media improves cellular integrity

The use of glycerol for boar semen cryopreservation results in low fertility, possibly due to toxicity. This has led to recommend the use of solutions with less than 4% glycerol. Trehalose is a disaccharide known to stabilize proteins and biologic membranes during processes such as cryopreservation. Thus, it was decided to evaluate the cryoprotective effect of glycerol/trehalose mixtures. Effects on motility (M), viability (Vb) and acrosomal integrity (nA) were evaluated. Sperm samples were frozen in three different extenders: G4 contained 4% glycerol; T1 contained 1% glycerol plus 250 mM trehalose and T0.5 was constituted by 0.5% glycerol plus 250 mM trehalose. All extenders yielded similar post-freezing/thawing motility rates. Viability was diminished in T0.5 as compared to the others. In regard to acrosome integrity, it was twice as high (P < 0.05) in the trehalose enriched media as in G4, the glycerol-only extender. Thus, T1 twice as many spermatozoa were alive, motile and intact, than in either T0.5 or G4, i.e. during freeze/thawing the use of T1 resulted in twice as many fertile cells as when using the other extenders. During our study, we noted that there were wide individual variations both in sperm viability and in motility.     

Optimization of the cryopreservation of African clawed frog (Xenopus laevis) sperm

Cryopreservation of testicular sperm in the African clawed frog, Xenopus laevis, was tested using three penetrating cryoprotectants (DMSO, methanol, and glycerol) and three semen diluents (300 mmol/L glucose, 300 mmol/L sucrose, and a motility inhibiting saline [MIS] solution [150 mmol/L NaCl, 3 mmol/L KCL, 1 mmol/L Mg₂SO₄, 1 mmol/L CaCl₂, and 20 mmol/L Tris, pH 8.0]). Three freezing rates and four thawing rates were also tested, and the best freezing/thawing conditions have been determined. The responses of sperm motility, viability, and fertility were assessed. Incubation of the sperm macerates with penetrating cryoprotectants showed that DMSO was the least toxic and methanol the most toxic. Semen in cryodiluents frozen 10 cm above the surface of liquid nitrogen (freezing rate of 20 to 25 °C/min) and thawed at room temperature for 40 sec had significantly higher percentages of motile and viable sperm than that of semen frozen 5 cm or 8 cm above the surface of liquid nitrogen and thawed at 5, 25, or 30 °C for 10, 15, or 60 sec, respectively. Sperm frozen in MIS containing 5% DMSO had a higher hatching rate than that of sperm frozen in sucrose and glucose diluents containing 5% or 10% DMSO and in MIS containing 10% DMSO. Addition of 73 mmol/L sucrose to the sperm extender MIS + 5% DMSO could improve the postthaw sperm motility and fertility. In conclusion, dilution of collected sperm in MIS solution (to have a final concentration of 6.5 × 10⁶ to 8 × 10⁶/mL) containing 5% DMSO and 73 mmol/L sucrose, freezing in a vapor of liquid nitrogen at 10 cm above the surface, and thawing at room temperature for 40 sec was the best cryopreservation protocol. This protocol gave 70% hatching rate, 80% motility rate, and 75% viability rate of fresh hormonally induced sperm.     

Butylated hydroxytoluene inclusion in semen extender improves the post-thawed semen quality of Nili-Ravi buffalo (Bubalus bubalis)

The study was carried out to evaluate the potential impact of butylated hydroxytoluene (BHT) on the frozen-thawed semen quality of Nili-Ravi buffalo bulls. Ejaculated bull semen was extended in a Tris-citrate egg yolk extender containing various concentrations of BHT (0.5, 1.0, 2.0 and 3.0 mM). Semen was frozen at −196 °C using 50 × 10⁶ spermatozoa per 0.5 mL straws. Five straws from each treatment were thawed to assess the semen quality in terms of sperm motility, viability, plasma membrane integrity and acrosomal integrity. Post-thawed sperm motility was determined using a phase-contrast microscope. Viability, plasma membrane integrity and acrosomal integrity were evaluated by the supravital staining, hypo-osmotic swelling test and normal acrosomal reaction, respectively. The highest (P < 0.05) motility, acrosomal integrity and hypo-osmotic swelling response of spermatozoa was achieved by addition of 1.0 and 2.0 mM BHT to semen extender. However, highest (P < 0.05) viability of spermatozoa was achieved by inclusion of 2.0 mM BHT. The higher concentration of BHT (3.0 mM) reduced the motility, acrosomal integrity, viability and hypo-osmotic swelling response of the spermatozoa compared to other concentration used. In conclusion, BHT when added in the semen extender can improve the semen quality of buffalo bulls.     

Cryopreservation of Iberian pig spermatozoa. Comparison of different freezing extenders based on post-thaw sperm quality

The aim of this study was to evaluate the cryoprotective effect of different freezing extenders against cryopreservation injuries on Iberian boar sperm. The sperm-rich fraction was collected and pooled from six sexually mature Iberian boars, and was frozen in different extenders containing glucose, lactose or fructose as sugar source and including Orvus ES Paste only in the freezing extender-2 (Glucose; Lactose and Fructose) or in both freezing extenders (Glucose2; Lactose2 and Fructose2). During the cryopreservation process, the supernatant was removed after the centrifugation step, then was extended with freezing extender-1 for the equilibration period and with freezing extender-2 immediately before freezing. Post-thaw sperm characteristics, such as plasma membrane integrity (SYBR-14/PI), mitochondrial function (Rhodamine 123) and acrosome integrity (NAR), were monitored. Overall sperm motility and the individual kinematic parameters of motile spermatozoa (assessed by the computer-aided sperm analysis system Sperm Class Analyzer [SCA]) were recorded in the different experimental treatments. Measurements were taken at 30 and 150 min post-thaw. The state of the acrosome after thawing did not show significant differences between the freezing extenders studied. Freezing–thawing caused a significant decrease (P < 0.001) in plasma membrane integrity and in mitochondrial activity in the spermatozoa frozen with Orvus ES Paste in both freezing extenders. Furthermore, spermatozoa frozen with Orvus ES Paste in both freezing extenders exhibited lower (P < 0.05) motility and kinematic parameters than those frozen in the absence of Orvus ES Paste in the first freezing extender. The spermatozoa frozen with the Lactose extender and with Orvus ES Paste only in the second freezing extender showed a better evolution of the motility and kinematic characteristics (P < 0.05) over time. The deterioration in post-thaw sperm motility and kinematic parameters were concurrent with reduced sperm characteristics. It can be suggested that in the Iberian pig, the beneficial effects of Orvus ES Paste during the freezing process of spermatozoa is time dependent. The analysis of different sperm characteristics such as motility, plasma membrane integrity and mitochondrial function, determined that the extenders studied in the present experiment affected the quality of frozen-thawed semen in Iberian boar.