A comparison between freezing methods for the cryopreservation of stallion spermatozoa

The effects of sperm freezing concentration (40 × 10⁶ mL⁻¹ vs. 400 × 10⁶ mL⁻¹), straw size (0.25 mL vs. 0.5 mL) and freezing method (liquid nitrogen vapour in a Styrofoam^® box vs. programmable freezing machine) were evaluated in a 2 × 2 × 2 factorial experimental design using 3 split ejaculates from each of 4 stallions. Immediately after thawing, the total motility and forward progressive motility of spermatozoa frozen at a concentration of 40 × 10⁶ mL⁻¹ was higher than for spermatozoa frozen at 400 × 10⁶ mL⁻¹. No significant differences were observed in the semen parameters assessed after cryopreservation in either 0.25 or 0.5 mL straws. However, the programmable freezer provided a more consistent and reliable freezing rate than liquid nitrogen vapour. We conclude that an effective protocol for the cryopreservation of stallion spermatozoa at low concentrations would include concentrations of 40 × 10⁶ mL⁻¹ in 0.25 mL straws using a programmable freezer. This freezing protocol would be suitable for emerging sperm technologies such as sex-preselection of stallion spermatozoa as the sorting process yields only low numbers of spermatozoa in a small volume available for either immediate insemination or cryopreservation.     

Effects of post-thaw incubation on motility,acrosomal integrity and in vitro fertilizing capacity of boar spermatozoa cryopreserved in 0.5 and 5 ml straws

The effect of post-thaw incubation (0 vs. 5 h at 15 °C) and straw size (5 vs. 0.5 ml) on motility, acrosomal integrity and in vitro fertilizing (IVF) capacity of cryopreserved boar spermatozoa was studied. In samples assessed immediately after thawing, no differences were found between the two straw sizes. After 5 h post-thaw incubation, all parameters, except polyspermy, decreased and, spermatozoa packaged in 5 ml straws showed better functional and IVF parameters than these in 0.5 ml straws.     

Effects of taurine and hypotaurine supplementation and ionophore concentrations on post-thaw acrosome reaction of dog spermatozoa

The aim of this work was to study of the effect of the amino acids (AA) taurine (T) and hypotaurine (H) and of different calcium ionophore concentrations on the ability of capacitated frozen-thawed dog sperm to undergo the acrosome reaction (AR). Fifteen ejaculates grouped into five pools were used. Sperm was frozen at a concentration of 80 × 10⁶ sperm cells/mL in the Uppsala Equex extender (UE) supplemented with 25, 50 and 75 mM of either AA. The UE extender without T or H was used as control. After thawing, sperm was capacitated with Canine Capacitation Medium for 20 min. Sperm was then challenged with calcium ionophore A23178 at 0, 2.5 and 10 μM concentration and evaluated for integrity of plasma and acrosome membranes after 5, 15 and 30 min of incubation, utilizing PI/Fitc-PNA fluorescent staining and flow cytometry.Sperm cryopreserved in UE supplemented with 50 mM T (UE 50T) had higher AR rates than sperm cryopreserved with UE 75T, UE 25H and UE 50H, but AR rates were similar to semen frozen with the control extender. Challenges with 2.5 and 10 μM/L of calcium ionophore increased AR in frozen-thawed sperm incubated for 5, 15 and 30 min. The combination of calcium ionophore concentration and incubation time resulting in the highest AR rate was 10 μM and 15 min.     

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.     

Detection of Y-bearing spermatozoa by DNA-DNA in situ hybridisation

In situ hybridisation of a Y chromosome-specific DNA probe to preparations of decondensed spermatozoa revealed approximately 46.7% labelled spermatozoa among 3,900 scored. This is not significantly different from the 50% expected if only the Y chromosome-bearing spermatozoa are hybridised. Control hybridizations of Escherichia coli DNA and salmon testis DNA to decondensed sperm produced no significant labelling, whereas more than 99% of the spermatozoa were heavily labelled after hybridisation to total human DNA. These controls indicate that the methodology described in this paper renders the chromatin accessible for hybridisation and that the 50% hybridisation observed with the Y chromosome DNA probe was specific. In situ hybridisation with the Y probe therefore identifies the Y-bearing spermatozoa, and the protocol described should prove useful in evaluating methods of separating Y-bearing and X-bearing spermatozoa.     

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.     

Addition of butylated hydroxytoluene (BHT) in tris-based extender improves post-thaw quality and motion dynamics of dog spermatozoa

The aim of this study was to evaluate the interaction of different concentrations of butylated hydroxytoluene (BHT) in a tris-based extender on semen quality parameters in post-thawed dog semen. Twenty-four ejaculates were collected from eight male Beagle dogs using an artificial vagina. Pooled semen was diluted with a tris-based extender supplemented with 0 (control), 0.5, 1.0, 1.5, and 2.0 mM BHT, at a final concentration of 200 × 10⁶ spermatozoa/mL. After thawing, sperm samples were assessed for motility parameters (CASA), membrane integrity (SYBR-14/PI), acrosome integrity (FITC-PNA), mitochondrial activity (JC-1/PI), malondialdehyde (MDA) concentration, and glutathione peroxidase (GPx) activity. The total motility, progressive motility, and average path velocity of the frozen-thawed sperm were significantly higher in the BHT1.5 group than in the control and the other sample groups (P < 0.05). Higher values of straight-line velocity, curvilinear velocity, amplitude of the lateral head displacement, and linearity were observed in the BHT1.0, BHT1.5, and BHT2.0 groups than in the control (P < 0.05). The BHT1.0 and BHT1.5 groups had higher percentages of straightness and acrosome integrity than the other groups (P < 0.05). Beat cross frequency, plasma membrane integrity, and GPx activity of the BHT1.5 and BHT2.0 groups were higher than those of the control (P < 0.05). A lower concentration of MDA was observed in the BHT1.0, BHT1.5, and BHT2.0 groups than in the control (BHT0) (P < 0.05).Our results indicate that 1.5 mM BHT is the optimal concentration for improving the post-thaw quality of canine spermatozoa.     

Mitochondria-targeted antioxidant MitoTEMPO improves the post-thaw sperm quality

Human spermatozoa cryopreservation is an important means of assisted reproductive technology and male fertility preservation. Although this technique is particularly useful, sperm cryopreservation significantly reduces the quality of spermatozoa after freezing and thawing. The objective of the study is to examine the efficacy of mitochondria-targeted antioxidant MitoTEMPO in improving sperm quality during semen cryopreservation processes. Semen samples were collected and cryopreserved in extenders containing different concentrations (0.0, 0.5, 5, 50, and 500 μM) of MitoTEMPO. Sperm motility, viability, membrane integrity, mitochondrial membrane potential and antioxidant activities were measured and analyzed. The results showed that the addition of MitoTEMPO (5–50 μM) significantly improved post-thaw sperm motility, viability, membrane integrity and mitochondrial membrane potential (P < .05). Meanwhile, antioxidant enzymes activities were enhanced and MDA content were decreased in the group supplemented with MitoTEMPO. In conclusion, mitochondria-targeted antioxidant MitoTEMPO improves the post-thaw sperm quality and antioxidant enzymes profile.     

Elamipretide as a potential candidate for relieving cryodamage to human spermatozoa during cryopreservation

As a technique widely used in assisted reproduction, human spermatozoa cryopreservation makes it possible to conserve functional sperm for a long time, but the impact of cryodamage on sperm during the process could not be ignored. The objective of the present study was to investigate the efficacy of Elamipretide, a novel small mitochondrial targeting short cytoprotective peptide, in attenuating cryodamage during spermatozoa cryopreservation. Semen samples were collected and cryopreserved in freeze solution containing different concentrations (0.0, 0.1, 1, and 10 μM) of Elamipretide. Sperm motility, viability, membrane integrity, mitochondrial membrane potential, DNA fragmentation, antioxidant profiles, and acrosome reaction were measured and analyzed. The results showed that supplementation of the freeze media with Elamipretide (1 and 10 μM) significantly improved post-thaw sperm parameters including motility and viability, stability of the plasma membrane, and mitochondria and chromosomes. In addition, by adding Elamipretide, excessive oxidation and acrosome dysfunction in sperm cells undergoing freeze-thaw were also significantly attenuated. Therefore, Elamipretide may be a potential candidate for relieving cryodamage to human spermatozoa during cryopreservation.     

Fatty acids and plasmalogens of the phospholipids of the sperm membranes and their relation with the post-thaw quality of stallion spermatozoa

Fatty acids and plasmalogens were extracted from the phospholipids of the plasma membrane of stallion spermatozoa, to determine their relation with sperm quality after freezing and thawing. Sperm quality was rated using a quality index that combined the results of the analysis of sperm motility and velocity (CASA analysis), membrane status and mitochondrial membrane potential (flow cytometry) post thaw. Receiving operating system (ROC) curves were used to evaluate the value of specific lipid components of the sperm membrane herein studied as forecast of potential freezeability. From all parameters studied the ratio of percentage of C16 plasmalogens related to total phospholipids was the one with the better diagnostic value. For potentially bad freezers, the significant area under the ROC-curve was 0.74, with 75% sensitivity and 79.9% specificity for a cut off value of 26.9. Also the percentage of plasmalogens respect to total phospholipids gave good diagnostic value for bad freezers. On the other hand, the percentage of C18 fatty aldehydes related to total phospholipids of the sperm membrane properly forecasted freezeability with an area under the ROC curve of 0.70 with 70% sensitivity and 62.5% specificity for a cut off value of 0.32.     

Assessment of two thawing processes of cryopreserved human sperm in pellets

In this study, we evaluated the effects of the thawing methodology on sperm function after cryopreservation in pellets. We compared the use of two thawing procedures: method (1) maintaining pellet for 10 min in air at room temperature, then another 10-min period in air at 37 °C followed by dilution in a thawing medium; and method (2) immersing the pellets directly in thawing medium at 37 °C for 20 min. This procedure leads to a higher rate of temperature increase and a dilution of the glycerol present in the freezing medium. We analyzed the effect of the thawing procedure on sperm motility, viability, membrane lipid packing disorder, acrosome status, reactive oxygen species (ROS) level and sperm chromatin condensation. This study revealed a positive effect of the M2 thawing methodology on sperm parameters. The percentage of spermatozoa with fast-linear movement is increased (M1: 17.26% vs. M2: 28.05%, p < 0.01), with higher viability (M1: 37.81% vs. M2: 40.15%, p < 0.01) and less acrosome damage (M1: 40.44% vs. M2: 35.45%, p = 0.02). We also detected an increase in the percentage of viable spermatozoa with low membrane lipid disorder (M1: 31.36% vs. M2: 33.17%, p = 0.03) and a reduction in chromatin condensation (44.62 vs. 46.62 arbitrary units, p = 0.02). Further studies will be necessary to evaluate the possible clinical applications.     

Vitrification induces critical subcellular damages in ram spermatozoa

The aim of the present study was to analyse morphological variations in ovine spermatozoa subjected to different cryopreservation protocols using high resolution imaging techniques. Ejaculates were pooled and diluted in Tris-based extender. Aliquots containing 300 × 10⁶ spz/ml were prepared and evaluated a) after the semen collection and pooling, b) after conventional freezing, c) after vitrification of samples maintained at room temperature (22 °C) prior to vitrification, and d) after vitrification of samples maintained at 5 °C prior to vitrification. Sperm motility, acrosome integrity, DNA fragmentation and morphology were assessed. Subcellular sperm changes were assessed and described by light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The maintenance of spermatozoa at 5 °C prior to vitrification and the use of 0.4 M sucrose pointed out lower dimensions of area, length and width than fresh, frozen and sperm maintained at 22 °C prior to vitrification. It was observed that the head width and length are significantly higher (P < 0.0001) in fresh spermatozoa than in the vitrified sperm samples. It could be hypothesized that greater intracellular fluid loss during vitrification could prevent damages in the spermatozoon throughout the reduced ice crystals formation, but mainly by the reduction of extracellular ice crystals due to the physical properties modification obtained when high concentrations of sugars are added. This is the first ultramicroscopic study carried out in ovine vitrified spermatozoa, which confirms the functional sperm alterations previously detected.     

Improvement of post-thaw sperm survivals using liquid nitrogen vapor in a spermcasting oyster Ostrea angasi

Low survival of cryopreserved sperm impedes the application of cryopreservation technique in spermcasting oyster species. This study developed a simple method of liquid nitrogen vapor freezing to improve post-thaw sperm survival in the spermcasting oyster Ostrea angasi. The results indicate that the permeable cryoprotectants, dimethyl sulfoxide (DMSO), ethylene glycol (EG) and propylene glycol (PG) were non-toxic to sperm up to 20% concentration and 90 min exposure whereas methanol at 10% or higher was toxic to sperm for any exposure over 30 min. Among the treatments with permeable cryoprotectants, 15% EG produced the highest post-thaw sperm motility. Sperm motility was further improved by the addition of non-permeable cryoprotectants (trehalose and glucose), with 15% EG + 0.2 M trehalose resulting in the highest post-thaw sperm motility among all the combinations evaluated. The durations of 20, 30 and 60 min equilibrations produced a higher post-thaw sperm motility and plasma membrane integrity (PMI) than 10 min. Higher post-thaw motility and PMI were achieved by freezing sperm at the 8 cm height from the liquid nitrogen surface than at the 2, 4, 6, 10 or 12 cm height. Holding sperm for 10 min in liquid nitrogen vapor produced higher post-thaw motility and PMI than for 2, 5 or 20 min. The cryopreservation protocol developed in this study improved both post-thaw motility and PMI of O. angasi sperm at least 15% higher than those cryopreserved using programmable freezing method. Liquid nitrogen vapor freezing might have greater applicability in improving post-thaw sperm quality of spermcasting oyster species.     

The cryobiology of spermatozoa

The impact of successful cryopreservation of spermatozoa can be found in many fields, including agriculture, laboratory animal medicine, and human assisted reproduction, providing a cost-effective and efficient method to preserve genetic material for decades. The success of any cryobiologic protocol depends critically on understanding the fundamentals that underlie the process. In this review, we summarize the biophysical fundamentals critical to much of the research in sperm cryobiology, provide a synopsis of the development of sperm cryobiology as a discipline, and present the current state and directions for future research in sperm cryobiology in the three major areas outlined above—agriculture, laboratory animal medicine, and human clinical assisted reproduction. There is much room for new research, both empiric and fundamental, in all areas, including refinement of mathematical models, optimization of cryoprotective agent addition and removal procedures for spermatozoa from many species, development of effective, efficient, and facile cryopreservation protocols and freezing containers for agricultural sperm cryopreservation, and tailoring cryopreservation protocols for individual human samples.     

Effect of quercetin on the motility of cryopreserved canine spermatozoa

The addition of an antioxidant to cryopreservation solutions for preventing oxidative stress to sperm from several species, including that from humans, has been studied previously. Quercetin is a flavonoid contained in subarctic trees with freeze resistance and is known to be a strong antioxidant. Therefore, the effect of quercetin on the cryopreservation of dog spermatozoa was examined in this study. The proportions of total motile spermatozoa were significantly higher at 30, 60, 90, 120, and 150 min and at 60, 120, and 150 min after thawing in groups treated with 5 μg/ml and 10 μg/ml of quercetin dissolved in 0.1% DMSO added to the second extender based on skim milk compared to that in the control group, respectively. There were no differences between the experimental groups in the proportion of total motile spermatozoa during the observation periods. The proportion of total motile spermatozoa among those treated with 5 μg/ml of quercetin in 0.1% DMSO was improved by approximately 10–20% at 30–180 min after thawing compared to that in the control group. To evaluate the fertility of cryopreserved spermatozoa treated with quercetin, 2 × 10⁸ spermatozoa were transcervically inseminated into bitches, and a total of 18 puppies were delivered in three bitches. These results indicated that supplementation of quercetin as a cryoprotectant to the skim milk-based extender improved the motility of cryopreserved spermatozoa from dogs compared to those of the control group. And fertility of cryopreserved spermatozoa with quercetin supplementation was proven with higher efficiency.     

Effects of cryopreservation methods on post-thaw motility of spermatozoa from the Japanese pearl oyster, Pinctada fucata martensii

In order to develop cryopreservation techniques for Japanese pearl oyster spermatozoa, the effects of various cryopreservation conditions on post-thaw motility were examined. Spermatozoa cryopreserved with 10% methanol (MET), dimethylformamide or dimethylacetamide plus 90% diluent comprising 80% seawater and 20% fetal bovine serum (FBS) showed higher percentages of post-thaw motility than those cryopreserved with 10% dimethylsulfoxide or glycerol. When spermatozoa were cryopreserved with various concentrations (0-20%) of MET and 100-80% diluent, 10% MET showed the highest percentages of post-thaw motility. When spermatozoa were cryopreserved with 10% MET and 90% diluent comprising various concentrations (0-100%) of FBS or Ringer solution mixed with seawater, the percentages of post-thaw motility peaked at 20% FBS or Ringer solution, and were significantly higher for 20% FBS than for 20% Ringer solution. The percentages of post-thaw motility increased with increasing dilution ratios from 2.5- to 50-fold. Spermatozoa cooled to -50 degrees C and then immersed in liquid nitrogen (LN) showed higher post-thaw motility than those cooled to -30 degrees C or -40 degrees C. When spermatozoa were cryopreserved to -50 degrees C at various cooling rates by changing the sample height above the LN surface, the post-thaw motilities of spermatozoa cooled at 10 cm (cooling rate: -21.3 degrees C/min) and 12.5 cm (-15.6 degrees C/min) from the LN surface were higher than those at 5, 7.5 or 15 cm. These results indicate that 10% MET plus 90% diluent comprising 80% seawater and 20% FBS is a suitable extender for cryopreservation of Japanese pearl oyster spermatozoa and that samples should be cooled to -50 degrees C at a cooling rate between -15 and -20 degrees C/min for efficient storage.     

Alpha-mannosidase activity in stallion epididymal fluid and spermatozoa

The expression of α-D-mannosidase activity was fluorometrically and electrophoretically assessed in spermatozoa, epididymal fluid and homogenates of stallion epididymal tissue. Enzyme activity had regional differences; it was higher (P < 0.05) in samples from the cauda epididymal region than in samples from the proximal caput region (largely composed of efferent ducts). Based on enzyme activity, as a function of pH of the assay substrate, electrophoretic analysis in native and native/SDS-PAGE conditions, and the effect of inhibitors or activators, we inferred the presence of at least two catalytically active forms of α-D-mannosidase. The neutral form of the enzyme (α-mannosidase II) was activated by Co²⁺, whereas the acid form (optimum pH 3.5 to 4.0) was sensitive to swainsonine (an inhibitor of α-mannosidase I), stabilized or stimulated by Zn²⁺, and not activated by Co²⁺ (activator of the neutral form). The activity of the acid form of the enzyme was highest in the epididymal fluid, where it seemed to be mainly in a secretory form. This form of the enzyme may have a role in plasma membrane remodeling associated with sperm maturation. In contrast, the activity of α-mannosidase II was higher in mature spermatozoa. It has been postulated that α-mannosidase II may act as a receptor in the recognition and binding of the complementary carbohydrate moieties present on the zona pellucida. With non-denaturing electrophoresis, α-D-mannosidase had an electrophoretic mobility of 0.35 and 0.24. When resolved by 1D and 2D SDS-PAGE (under denaturing conditions) the enzyme had a major protein band of molecular weight 154 kDa in spermatozoa and epididymal samples. Based on its properties under native conditions, we inferred that this enzyme might interact with other proteins and form transitory aggregates.     

Maintenance of fertility in cryopreserved Indian gerbil (Tatera indica) spermatozoa

This study is the first attempt at sperm cryopreservation, as well as a further examination of frozen sperm fertility by the hamster test, applied to the maintenance of an Indian gerbil (Tatera indica) colony, which is a newly developing experimental animal.The osmotic tolerance of the spermatozoa was initially investigated by subjection to hypertonicity, up to 620 mOsm/kg, for 5 min at room temperature prior to freezing. Although the percentage of total motile sperm was not affected, that of progressive motile spermatozoa began to drop at 400 mOsm/kg, and a significant decrease was observed at 620 mOsm/kg (p < 0.01). According to these results, the osmolality of the solutions for the freezing experiment, in which 6–22% raffinose was present, was fixed at approximately 400 mOsm/kg. Sperm, suspended in a plastic straw, were frozen in liquid nitrogen vapor for 5 min, followed by immersion in liquid nitrogen. Motile sperm were recovered from all freezing conditions, and high survival was obtained when sperm were frozen in the presence of 14% and 18% raffinose, with a normalized motility higher than 40%. Fertility of cryopreserved Indian gerbil sperm was examined by the zona-free hamster test. Thawed sperm adhered to 88% of the zona-free hamster oocyte surface, and some oocytes were penetrated and exhibited swollen sperm heads or male pronuclei, which we used to define fertilization. Although the fertilization rate of cryopreserved sperm to zona-free hamster eggs was significantly lower than that of fresh sperm (6% vs. 30%, p < 0.01), we demonstrated that thawed Indian gerbil spermatozoa have the ability to maintain their fertility.     

Season of ejaculate collection influences the freezability of boar spermatozoa

The aim of this retrospective study was to evaluate whether the season of ejaculate collection influences the freezability of porcine sperm. A total of 434 ejaculates were collected from boars of six different breeds over three years (2008–2011) and throughout the four seasons of the year identified in the northern hemisphere (winter, spring, summer and autumn). The ejaculates were cryopreserved using a standard 0.5 mL straw freezing protocol. Sperm quality was assessed before (fresh semen samples kept 24 h at 17 °C) and after freezing and thawing (at 30 and 150 min post-thawing in semen samples kept in a water bath at 37 °C), according to the percentages of total motility, as assessed by the CASA system, and viability, as assessed by flow cytometry after staining with SYBR-14, PI and PE-PNA. The data, in percentages, on sperm motility and viability after freezing and thawing were obtained at each evaluation time (recovered) and were normalized to the values before freezing (normalized). The season of ejaculate collection influenced (P < 0.01) sperm quality before freezing and after thawing (recovered and normalized), irrespective of the breed of boar. Sperm quality was lower in summer, both in terms of motility and viability, and in autumn, in terms of motility, than in winter and spring. Seasonality in the normalized data indicates that the season of ejaculate collection influences sperm freezability, regardless of the season’s influence on sperm quality before freezing. Consequently, the spermatozoa from ejaculates collected during summer and, to a lesser extent, also in autumn, are more sensitive to cryopreservation than those from ejaculates collected during winter and spring.     

Mito-Tempo alleviates cryodamage by regulating intracellular oxidative metabolism in spermatozoa from asthenozoospermic patients

As the largest proportion of male infertility population, asthenozoospermia patients often resort to sperm cryopreservation to preserve fertility as well as to enrich motile sperm for assisted reproductive techniques (ART), although it may cause some cryodamage during the freezing–thawing process. The objective of this study was to investigate whether mitochondrial antioxidant Mito-Tempo was effective in preventing cryodamage of asthenozoospermic spermatozoa. Asthenozoospermic semen samples were collected and cryopreserved in media supplemented with different concentrations (0.0, 1.0, 10 and 100 μM) of Mito-Tempo. We measured sperm motility, viability, membrane integrity, DNA fragmentation, mitochondrial membrane potential, oxidation product, and antioxidant enzymes activities. Supplementation of the cryopreservation media with Mito-Tempo (10 and 100 μM) induced a significant improvement in sperm viability, motility, membrane integrity, mitochondrial membrane potential and chromatin integrity (P < 0.05). Significant enhancement of antioxidant enzymes activities accompanied by the decreased formation of oxidation products (ROS and MDA) was also observed in groups supplemented with Mito-Tempo (10 and 100 μM). It is concluded that mitochondria targeted antioxidant Mito-Tempo alleviates cryodamage by regulating intracellular oxidative metabolism in spermatozoa from asthenozoospermic patients after cryopreservation.