The effects of antioxidants on semen traits and in vitro fertilizing ability of sperm from the flat-headed cat (Prionailurus planiceps)

Since antioxidants can overcome the negative effects of reactive oxygen species (ROS) during sperm cryopreservation, post-thaw sperm quality in flat-headed cats (Prionailurus planiceps), an endangered species, might benefit from the addition of antioxidants to semen extender. The objectives of this study were to: 1) investigate semen traits; and 2) evaluate effects of the vitamin E analogue Trolox (vitamin E) and glutathione peroxidase (GPx) on the quality of frozen sperm from captive flat-headed cats in Thailand. Eight ejaculates were collected by electroejaculation from four flat-headed cats. Each semen sample was divided into three aliquots and re-suspended in a semen extender as follows: 1) without antioxidant supplementation (control); 2) supplemented with 5 mM vitamin E; or 3) supplemented with 10 U/mL GPx. All samples were cryopreserved and thawed. Subjective sperm motility, progressive motility, and the integrity of the sperm membrane, acrosome and DNA were evaluated at semen collection, after 1 h cold storage, and at 0 and 6 h after thawing. Mitochondrial membrane potential, early apoptotic cells, and embryo development by heterologous in vitro fertilization were evaluated after thawing. Captive flat-headed cats were affected by teratozoospermia. After 1 h cold storage, sperm membrane integrity in samples supplemented with GPx was higher than the control group (54.5 ± 13.7 vs 51.3 ± 13.9; P < 0.05; mean ± SD). Sperm frozen in extender with GPx had higher motility at 6 h and greater mitochondrial membrane potential at 0 and 6 h post-thaw incubation than the other groups (P < 0.05). In conclusion, GPx improved the quality of frozen-thawed sperm in flat-headed cats.     

Effect of cryopreservation on sperm parameters, lipid peroxidation and antioxidant enzymes activity in fowl semen

The aim of the present study was to determine the influence of chicken semen cryopreservation on sperm parameters, lipid peroxidation and antioxidant enzymes activities. Pooled semen from 10 Black Minorca roosters was used in the study. Semen samples were subjected to cryopreservation using the “pellet” method and dimethylacetamide (DMA) as a cryoprotectant. In the fresh and the frozen-thawed semen sperm membrane integrity (SYBR-14/propidium iodide (PI)), acrosomal damage (PNA-Alexa Fluor^®488) and mitochondrial activity (Rhodamine 123) were assessed using flow cytometry. Malondialdehyde (MDA) concentration, catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities were determined in sperm cells and seminal plasma by spectrophotometry. All sperm characteristics evaluated using flow cytometry were affected by cryopreservation. After freezing-thawing, there was significant (P < 0.01) reduction in sperm membrane integrity, sperm acrosome integrity and mitochondrial activity. Following cryopreservation, MDA concentration significantly increased in chicken seminal plasma and spermatozoa (P < 0.01, P < 0.05). The CAT activity in seminal plasma significantly decreased (P < 0.05), while intracellular activity of this enzyme did not significantly change in frozen-thawed semen. In seminal plasma of frozen-thawed semen the significant increase (P < 0.01) in GPx activity was detected. Whereas GPx activity in spermatozoa remained statistically unchanged after thawing. The SOD activity significantly increased (P < 0.01) in cryopreserved seminal plasma with simultaneous decrease (P < 0.01) of its activity in cells. In conclusion, this is probably the first report describing the level of antioxidant enzymes in frozen-thawed avian semen. The present study showed that the activity of CAT, GPx and SOD in chicken semen was affected by cryopreservation, what increased the intensity of lipid peroxidation (LPO). Catalase appeared to play an important role in the sperm antioxidant defense strategy at cryopreservation since, opposite to SOD and GPx, its content was clearly reduced by the cryopreservation process. Change in the antioxidant defense status of the chicken spermatozoa and surrounding seminal plasma might affect the semen quality and sperm fertilizing ability.     

Pre-freezing equilibration for 22 h improves post-thaw sperm functions in cryopreserved ram semen by reducing cholesterol efflux

Failure of cervical insemination with cryopreserved semen is hindering implementation of AI in sheep in field condition. Here the effect of equilibration time and catalase on post-thaw qualities of ram semen was investigated. Pooled semen was diluted (800 × 10⁶ sperm mL⁻¹) with a TES-Tris-fructose extender with 6% glycerol, 15% egg yolk and supplemented with 0, 50, 100 and 200 U mL⁻¹ catalase and packaged into 0.25 mL straws. In experiment 1, straws were equilibrated at 5 °C either for 3 h in a cold cabinet (E3) or for 10 (E10) and 22 h (E22) inside a refrigerator. In experiment 2, all straws were equilibrated for 22 h inside refrigerator. Straws were frozen at −25 °C min⁻¹ up to −125 °C using a cell freezer and finally plunged into liquid nitrogen. The post-thaw total and rapid motility were higher (P < 0.05) in E22 compared to E3 and E10. Sperm kinetics was comparable between E3 and E22, but lower in E10. Similarly, acrosome integrity, functional membrane integrity, percent high cholesterol (mCHO) and live-high mitochondrial membrane potential (MMP) were higher (P < 0.05) while live-high intracellular calcium and acrosome-reacted sperm were lower in E22 compared to E3 and E10. The percent rapid motile, high mCHO and live-high MMP were significantly (P < 0.05) lower in catalase-treated samples compared to the control, while the membrane integrity was comparable within the groups. In conclusion, pre-freezing equilibration for 22 h compared to 3 or 10 h resulted in higher post-thaw sperm functions while catalase had negative impact on cryopreservation of ram semen.     

Trehalose improves rabbit sperm quality during cryopreservation

High levels of reactive oxygen species are associated with spermatozoa cryopreservation, which bring damage to functional spermatozoa. The aim of the present study was to investigate whether and how the freezing extenders supplemented with trehalose was beneficial for the survival of rabbit spermatozoa. semen was diluted with Tris-citrate-glucose extender addition of different concentrations of trehalose. Addition of 100 mM trehaose significantly improved post-thaw rabbit sperm parameters, such as motility, acrosome integriy, membrane integrity and mitochondrial membrane potential. Moreover, when freezing extenders supplemented with trehalose, activities of catalase (CAT), superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) of post-thaw spermatozoa were enhanced, meanwhile, reactive oxygen species (ROS) level and Malondialdehyde (MDA) content were decreased. The results suggest that freezing extenders supplemented with 100 mM trehalose resulted in less ROS level and MDA content, higher motility and mitochondrial membrane potential as well as the integrity of acrosome and plasma membrane. Supplementation of trehalose with freezing extenders is beneficial to the rabbit breeding industry.     

Natural, but not lyophilized, low density lypoproteins were an acceptable alternative to egg yolk for cryopreservation of ram semen

The objective was to evaluate the suitability of using natural or lyophilized low density lipoproteins (LDL), in lieu of whole egg yolk, in extenders for cryopreserving ram semen. Once extragonadal sperm reserves were depleted in 10 fertile Santa Inês cross rams, two ejaculates per ram were collected for cryopreservation. Nine extenders were used: Tris-16% egg yolk extender with 5% glycerol as a control (T1), and substitution of whole egg yolk with 8, 12, 16 or 20% natural LDL (T2-T5, respectively), or with 8, 12, 16, or 20% lyophilized LDL (T6-T9). Semen was diluted to 100 × 10⁶ sperm/mL, packaged into 0.25 mL straws, cooled, held at 5 °C for 3 h, and then frozen in liquid nitrogen vapor. Immediately after thawing (37 °C for 30 s), sperm total and progressive motility, and kinetic parameters were analyzed with computer assisted semen analysis (CASA). Percentage of sperm with plasma membrane functional integrity was assessed by the hypoosmotic swelling test (HOST), sperm membrane physical integrity with propidium iodide (PI), and acrosome integrity with FITC-PSA using an epifluorescent microscope. For all sperm end points, there was no difference between the control and natural LDL treatments (P > 0.05): total motility (T1: 20.9 ± 11.9 and average of T2-T5: 25.9 ± 13.6%; mean ± SD), progressive motility (T1: 6.6 ± 4.2 and average of T2-T5: 11.7 ± 7.5%), HOST⁺ (T1: 23.7 ± 6.9 and average of T2-T5: 23.2 ± 8.7 %) and PI⁻/PSA⁻ (T1: 13.8 ± 7.8 and average of T2-T5: 18.1 ± 7.8%). However, lyophilization was apparently unable to preserve the protective function of LDL; every sperm end point was significantly worse than in the control and natural LDL groups. We concluded that natural LDL was appropriate for cryopreserving ram semen, as it yielded results similar to those obtained with whole egg yolk.     

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

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

Effects of different cryoprotective agents on ram sperm morphology and DNAintegrity

This study investigates the effects of glycerol, 1,2 propanediol, sucrose, and trehalose on post-thaw motility, morphology, and genome integrity of Awassi ram semen. Ejaculates of thick consistency with rapid wave motion (>+++) and >70% initial motility were pooled. Sperm were diluted to a final concentration of 1/5 (semen/extender) in 0% cryoprotectant, 6% glycerol, 6% 1,2 propanediol, 62.5 mM sucrose or 62.5 mM trehalose using a two-step dilution method. The equilibrated semen was frozen in 0.25-ml straws. Semen samples were examined for sperm motility, defective acrosomes (FITC-Pisum sativum agglutinin (FITC PSA)), DNA integrity (acridine orange staining (AO)) and apoptotic activity (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and Caspase-3 activity) at four time points: after dilution with extender A, after cooling to 5 °C, after equilibration and post-thaw. Freezing and thawing procedures (cooling at 5 °C, dilution, equilibration, and thawing) had negative effects on motility (P < 0.001), acrosome integrity (P < 0.001), and DNA integrity as determined by AO (P < 0.001) and TUNEL (P < 0.001) assays. There were positive correlations between sperm with defective acrosomes and apoptotic (AO- and TUNEL-positive) spermatozoa. In contrast, a significant negative correlation was found between sperm motility and defective acrosomes and AO- and TUNEL positivity (P < 0.01). The cryopreservation process acts as an apoptotic inducer in ram semen; all cryoprotectants used in the present study allowed apoptosis to some extent, with negative effects on sperm morphology and DNA integrity. The glycerol group performed better than the propanediol, sucrose, trehalose, and control groups in terms of post-thaw sperm motility but not DNA integrity.     

Effect of bioactive peptide on ram semen cryopreservation

This present study investigated the effect of bioactive peptide (BAPT) (BAPT) on the quality of ram semen during cryopreservation. Ram ejaculates were extended with Tris buffer supplemented with no antioxidants (as control group), 20 μg/mL BAPT (as BAPT20 group), 40 μg/mL BAPT (as BAPT40 group) and 60 μg/mL BAPT (as BAPT60 group). After cryopreservation, sperm quality including motility, vitality, the percentage of hypoosmotic swelling test (HOST)-positive spermatozoa and the percentage of intact acrosomes was assessed. Furthermore, the malondialdehyde (MDA) in seminal plasma and spermatozoa were analyzed, followed by the measurement of superoxide dismutase (SOD), catalase (CAT) and glutathione-peroxidase (GSH-Px) levels in seminal plasma. After in vitro fertilization, the embryonic cleavage rates and development rates of different groups were analyzed to compare the developmental abilities of spermatozoa. The results showed that the post-thaw sperm motility was significantly higher in the BAPT60 group compared to those in the BAPT20, BAPT40 and control groups (P < 0.05). The percentage of live sperms significantly increased from 48.12 ± 2.35% for the BAPT20 group, 55.43 ± 2.16% for the BAPT40 group to 57.53 ± 3.15% for the BAPT60 group. The percentage of HOST-positive spermatozoa was significantly higher in the BAPT60 group than those in BAPT20, BAPT40 and control groups (P < 0.05). The MDA levels in seminal plasma and spermatozoa were significantly reduced with BAPT supplement (P < 0.05). Additionally, the SOD, CAT and GSH-Px levels in the BAPT experimental groups were significantly higher than those of the control group, which further indicated that BAPT significantly inhibit the reactive oxygen species (ROS) production during the cryopreservation of ram semen. Furthermore, the embryonic cleavage rates and development rates of the BAPT40 and BAPT60 groups were significantly increased in comparison with the BAPT20 and control groups (P < 0.05).In conclusion, BAPT improved the ram sperm quality via inhibiting the ROS production during cryopreservation, and could be applied as a promising supplement for ram semen cryopreservation.     

Quercetin supplemented casein-based extender improves the post-thaw quality of rooster semen

The advantageous influence of quercetin (Q) supplementation in an extender has not yet been evaluated for rooster semen cryopreservation. This research was purposely conducted in order to assess the effect of different quercetin concentrations added into an extender on the sperm quality of the rooster subsequent to a freezing-thawing process. After the freezing-thawing process, spermatozoa quality parameters (membrane functionality, acrosome integrity, motility, viability, and abnormal morphology), endogenous enzymes (SOD, CAT, and GPx), mitochondrial activity, DNA fragmentation index, lipid peroxidation (MDA), and ROS were all evaluated. A total of 75 neat pooled ejaculates (3 ejaculates/rooster) were collected from 25 arbor acres roosters (24 wks) twice a week using abdominal massage technique, then divided into five equal aliquots and diluted with an extender containing different doses of Q (CS-Q) as follows: casein extender without Q (control only), casein extender containing 0.040 mg/mL quercetin (CS-Q 0.040), 0.020 mg/mL quercetin (CS-Q 0.020), 0.010 mg/mL quercetin (CS-Q 0.010), and 0.005 mg/mL quercetin (CS-Q 0.005). Our results depicted that adding to the extender with a 0.010 mg/mL Q enhanced (P < 0.01) sperm motility, membrane function, viability, mitochondrial activity, intact acrosome (P < 0.05), SOD (P < 0.001), CAT, and GPx (P < 0.01) compared to the control group at post-thaw. Compared to the control group and other treatment groups after the freeze-thawing process, the addition of 0.005 mg/mL Q into the extender also showed higher (P < 0.05) improvement in the quality of sperm parameters and a higher (P < 0.01) SOD and CAT but did not improve mitochondrial activity and sperm viability. In addition, there was a lower degree of DNA fragmentation index, lower (P < 0.05) lipid peroxidation and ROS in frozen-thawed sperm treated with 0.010 mg/mL and 0.005 mg/mL Q than in control and the other treatment groups. In addition, 0.020 mg/mL Q supplementation into the extender also reduced DNA fragmentation and improved GPx activity compared to the control group at post-thaw. Different concentrations of Q 0.010 and 0.005 mg/mL added to the extender reduced the percentage of abnormal spermatozoa compared to the other groups. The results of this study showed for the first time that the inclusion of an extender with a suitable quercetin concentration of 0.010 mg/mL improved the post-thawed quality of rooster semen.     

Antioxidative effect of melatonin on cryopreserved chicken semen

This is a unique study because is the first time we are adding melatonin into an extender in order to determine its influence on cryopreserved chicken semen. The primary focus of our present study was to evaluate the influence of different concentrations of Melatonin on cryopreserved chicken semen. Semen samples were allocated into four treatments, being one control and three different combinations of antioxidants and after the freeze-thaw operation, the sperm motility, plasma membrane integrity, acrosome integrity, endogenous enzymes (GSH-Px, CAT, SOD), MDA and ROS of chicken spermatozoa were all evaluated. The collection of the semen samples was from 40 Arbor Acre roosters and this procedure was repeated twice a week and then mixed in an extender that contained different MEL treatments as follows: a diluent without MEL (control, M 0), a diluent comprising 0.125 mg/mL (M 0.125) 0.25 mg/mL, (M 0.25) and 0.5 mg/mL (M 0.5). It was revealed that the supplementation of the base extender with an optimal 0.25 mg/mL MEL led to a higher significant difference in the motility of chicken sperm (P < 0.01), higher acrosome integrity (P < 0.05) and a higher plasma membrane integrity (P < 0.01) when compared to the control group at post-thaw. Furthermore, when compared to the control group, 0.25 mg/mL MEL addition into the extender significantly enhanced the activity of endogenous enzymes (GSH-Px, CAT, and SOD) in the chicken spermatozoa at post-thaw (P < 0.05). Moreover, 0.5 mg/mL MEL supplementation into the extender enhanced the GSH-Px activity in the chicken spermatozoa when compared with the control group (P < 0.05) at post-thaw. In contrast, the addition of 0.25 mg/mL MEL into the extender resulted in a significantly lower MDA in comparison to the 0.125 mg/mL, 0.5 mg/mL MEL treatment group and the control group (P < 0.05). Also, compared to the control group, MEL concentration of 0.125 mg/mL and 0.5 mg/mL MEL into the extender resulted in a significantly low ROS concentration (P < 0.05) but the addition of 0.25 mg/mL MEL concentration resulted in a significantly lower ROS level when compared to the control group (P < 0.01). In summary, MEL improved the quality of cryopreserved chicken sperm quality by decreasing oxidative stress level and the most optimal concentration was 0.25 mg/mL.     

Effect of relaxin on cryopreserved beef bull semen characteristics

This study aimed to improve the quality of cryopreserved beef bull (Piedmontese) semen by incorporation of relaxin in diluted semen before cryopreservation procedures. Semen samples were collected from 4 proven fertile bulls, using artificial vagina, once per week for 8 consecutive weeks and pooled together then diluted with Bullxcell® extender, and supplemented with different concentrations of relaxin (0 (control), 25, 50 and 100 ng/ml) before cooling, equilibration and freezing procedures. Frozen semen was thawed and assessed for motility by Computer-Assisted Sperm Analysis and vitality parameters such as acrosome, plasma membrane and DNA integrities, apoptosis, mitochondrial membrane potential, mucus penetration and SOD activity. The developmental potential of bovine embryos produced in vitro by using relaxin-treated was also investigated. In the present study, 50 and 100 ng/ml relaxin incorporation in extended bull semen before cryopreservation induced a reduction of sperm motility immediately after thawing (0h), whereas, during long incubation periods (1–2 h), relaxin showed a significant positive effect on sperm quality by improving the sperm motility and velocity parameters. Interestingly, sperm vitality was improved by 25 and 100 ng/ml relaxin and the blastocyst developmental rate was significantly increased in the 25 ng/ml relaxin group compared with controls (52/118, 44.0% vs. 32/116, 27.6%, respectively). These findings suggest a potential use of relaxin at the doses tested in the present study as an additive in the cryopreservation media of bull semen to improve sperm quality.     

Total and specific activities of superoxide dismutase (SOD) in seminal plasma are related with the cryotolerance of jackass spermatozoa

This study investigated whether the activities of four antioxidant enzymes present in jackass seminal plasma (SP), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) and glutathione reductase (GSR), are related to the sperm ability to withstand cryopreservation. Eighteen ejaculates from 16 healthy jackasses were collected and split into two aliquots. The first one was centrifuged (3,000×g, 4 °C for 10 min) and used to determine the activities of these four enzymes in SP, whereas the other was diluted in a skim-milk extender and then cryopreserved. Assessment of sperm motility and membrane integrity was performed before and after cryopreservation. Based on the percentages of total motile and viable spermatozoa at post-thaw, samples were classified as good (GFE) or poor (PFE) freezability ejaculates through cluster analyses. Total and specific activities of SOD in seminal plasma were higher (P < 0.05) in GFE than in PFE, whereas no significant differences between GFE and PFE were observed regarding total and specific activities of CAT, GPX and GSR. However, post-thaw sperm parameters were positively correlated with total and specific activities of CAT and negatively correlated with those of GSR. In conclusion, determination of total and specific activities of SOD in the seminal plasma of a given jackass ejaculate may predict the sperm ability to withstand cryopreservation. In addition, our results warrant further research on addressing whether SOD activity in seminal plasma does not only allow predicting the sperm cryotolerance of a given ejaculate but also that of all ejaculates from a given jackass.     

Effects of mint,thyme, and curcumin extract nanoformulations on the sperm quality,apoptosis, chromatin decondensation,enzyme activity,and oxidative status of cryopreserved goat semen

Goat semen cryopreservation is a challenging process as it results in reduced motility, vitality, and fertility of spermatozoa after freezing. In this study, we evaluated the effects of different herbal extract nanoformulations (NFs) [mint (MENFs), thyme (TENFs), and curcumin (CENFs)], supplemented at either 50 or 100 μg into Tris-extender on the cryopreserved goat semen quality. The hydrothermal squeezing method was used for the preparation of the NFs extracts. The morphological evaluation of the NFs extracts was conducted by transmission electron microscopy. All NFs supplements improved (p < 0.05) the progressive motility, vitality, and plasma membrane integrity of sperm compared with the control extender after equilibration (5 °C for 2 h) and thawing (37 °C for 30 s), but had no effect on sperm abnormality and acrosome integrity. All NFs supplements decreased (p < 0.05) the apoptosis, malondialdehyde level, and chromatin decondensation of sperm cells, while increased (p < 0.05) the total antioxidant capacity and catalase activity in the frozen/thawed extender. Particularly, CENFs at a level of 100 μg showed improvement of sperm parameters and antioxidant status during cryopreservation of goat semen more than TENFs and MENFs. The CENFs improved the quality of goat spermatozoa in post-thawed semen in terms of preventing cryodamage and promoting the cryotolerance of spermatozoa when compared with TENFs and MENFs. Therefore, supplementation of Tris-extender with CENFs could enhance goat semen processing during cryopreservation.     

The advantages of low-density lipoproteins in the cryopreservation of bull semen

Egg low-density lipoprotein (LDL) was added at concentrations (w/v) of 7%, 8% or 9% to the extenders used to freeze bull semen and its effects on seminal parameters and anti-oxidant activities of frozen–thawed sperm were assessed. Analysis of data showed that sperm exposed to 8% LDL exhibited the greatest percentages of sperm motility, acrosome integrity and membrane integrity, compared to the control which differed from the treatment groups by replacing LDL with 20% egg yolk (P < 0.05). No difference was observed for membrane integrity between 8% and 9% LDL groups (P > 0.05). The extender supplemented with LDL did not exhibit improvement in SOD levels. However, 8% LDL group favored the highest anti-oxidant activities of CAT, GSH-Px and GSH in comparison to other groups (7%, 9% LDL and the control) (P < 0.05). No difference was observed for CAT activity between 9% LDL and the control group. In conclusion, sperm cryopreserved in the extender containing 8% LDL in place of egg yolk exhibited the greatest percentages of post-thaw sperm motility, acrosome integrity and membrane integrity, in comparison with the control, and favored the highest anti-oxidant activities of CAT, GSH-Px and GSH in comparison with other groups. The replacement of egg yolk by LDL in the composition of extenders was beneficial for bull sperm cryopreservation.     

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.     

Effects of platelet-activating factor and platelet-activating factor: acetylhydrolase on in vitro post-thaw boar sperm parameters

Cryopreservation of boar sperm compromises fertility after thawing by reducing sperm longevity and inducing acrosome reaction-like changes. In an attempt to improve the post-thaw motility and acrosome integrity of boar sperm, semen was frozen using a modified Westendorf method in which the medium was supplemented with either platelet-activating factor (PAF) or a recombinant platelet-activating factor:acetylhydrolase (PAF:AH; Pafase) before or after freezing. Platelet-activating factor is a phospholipid that is present in boar semen and PAF:AH is the naturally occurring enzyme that converts PAF to biologically inactive Lyso-PAF. Addition of PAF to the cryopreservation medium improved post-thaw motility immediately after thawing and after 3h incubation at 37 degrees C (60.0+/-0.0% and 25.0+/-2.9%; mean+/-S.E.M.) compared to the control sperm (41.7+/-1.7% and 10.0+/-2.9%; P<0.05). Acrosome integrity was higher immediately after thawing and after 3 and 6h incubation at 37 degrees C when sperm were frozen in the presence of Pafase (55.7+/-3.2%, 45.7+/-3.7% and 23.0+/-3.1%), compared to the control sperm (42.7+/-1.5%, 25.7+/-5.7% and 12.3+/-2.7%) and sperm frozen in the presence of PAF (33.0+/-3.7%, 26.3+/-2.2% and 11.7+/-0.3%; P<0.05). Addition of PAF to sperm after thawing improved motility immediately post-thaw (41.6+/-2.6%), compared with addition of Pafase (23.3+/-2.2%) or the control sperm with no supplementation of the medium (26.7+/-2.2%; P<0.05). However, this beneficial effect was lost by 3h post-thaw. Supplementation of boar semen cryopreservation medium with PAF and Pafase appeared to have beneficial effects on the in vitro quality of the sperm post-thaw.     

The influence of trehalose, taurine, cysteamine and hyaluronan on ram semen Microscopic and oxidative stress parameters after freeze-thawing process

There is a lack of information regarding lipid peroxidation and antioxidant capacity in cryopreserved ram semen, and cryopreservation is associated with the production of reactive oxygen species (ROS) which lead to lipid peroxidation (LPO) of sperm membranes, resulting in a loss of motility, viability and fertility of sperm. The aim of this study was to determine the influence of certain additives and their different doses on standard semen parameters, lipid peroxidation and antioxidant activities after the cryopreservation/thawing of ram semen. Ejaculates collected from four Akkaraman rams, a native breed of sheep, were evaluated and pooled at 33 degrees C. Semen samples which were diluted with a Tris-based extender containing additives including trehalose (50, 100mM), taurine (25, 50mM), cysteamine (5, 10mM), and hyaluronan (0.5, 1mg/ml), and an extender containing no additives (control) were cooled to 5 degrees C and frozen in 0.25ml French straws, being stored in liquid nitrogen. Frozen straws were thawed individually at 37 degrees C for 20s in a water bath for evaluation. The use of a Tris-based extender supplemented with 50mM trehalose, 25mM taurine, and 5 and 10mM cysteamine led to higher percentages of post-thaw motility, in comparison to the control group (P<0.01). No significant differences were observed in the percentages of acrosome and total abnormalities, and the hypoosmotic swelling test upon the supplementation of the freezing extender with antioxidants after the thawing of semen. In biochemical assays, the addition of antioxidants did not cause significant differences in levels of malondialdehyde (MDA), glutathione (GSH), and glutathione peroxidase (GSH-Px), after thawing, when compared to groups with no additives. In this study, catalase (CAT) activities were higher in the group that was applied 25mM taurine as an antioxidant, than in all of the other groups (P<0.001). Compared to the controls, antioxidant treatment with 100mM trehalose, 50mM taurine, 5mM cysteamine and 0.5mg/ml hyaluronan, significantly elevated vitamin E (vit E) levels in samples (P<0.001).     

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.     

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

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

The effect of sperm and cryoprotectant concentration on the freezing success of sex sorted ram sperm for in vitro fertilization

The current method used to sex-sort ram sperm resulted in a dilute end product. The obligatory removal of cryopreservation medium during preparation of sperm IVF further reduced sperm number. This study aimed to increase the number of viable, sex-sorted sperm available for IVF by increasing their pre-freeze concentration and assessing the cryodiluent concentration used to accommodate this change. In Experiment 1, semen was collected from Merino rams (n = 3), sex-sorted, and then frozen at concentrations of 20, 40, or 80 × 10⁶ sperm/mL in three forms of tris-citrate-glucose cryodiluent containing 5% (L-Cryo), 6% (M-Cryo), and 8% (H-Cryo) (v/v) glycerol. Motility, plasma membrane and acrosome integrity, and mitochondrial activity were assessed at 0, 2, 4, and 6 h post thaw. In Experiment 2, cleavage and blastocyst development rates were compared between non-sorted and sex-sorted sperm frozen at the aforementioned concentrations (in the cryodiluent most effective in Experiment 1). In Experiment 1, total motility between 0 and 6 h was similar for all sperm concentrations when frozen using L-Cryo. Mitochondrial activity was elevated in samples frozen in L-Cryo and M-Cryo at 0 h compared to those preserved in H-Cryo for all concentrations (P < 0.05). In Experiment 2, sex-sorted sperm with a higher pre-freeze concentration yielded a higher sperm concentration after preparation for IVF (8.57 ± 1.22 sperm/mL), compared to the lowest group (2.96 ± 0.18 sperm/mL; P < 0.05). There were no significant differences between non-sorted and sex-sorted sperm for rates of embryo cleavage or development. Therefore, sex-sorted sperm was effectively cryopreserved at a higher concentration than conventionally practiced. Although this yielded a higher sperm concentration for IVF, reduced insemination volume, and increased the number of potentially fertile gametes from which to select, fertilisation rate was not significantly improved.