Effects of anti-oxidant additives on microscopic and oxidative parameters of Angora goat semen following the freeze–thawing process

The anti-oxidant system of reduced glutathione (GSH), glutathione peroxidase (GSH-PX), catalase (CAT), and superoxide dismutase (SOD) has been described as a defense functioning mechanism against lipid peroxidation (LPO) in semen, and is important in maintaining sperm motility and viability. This anti-oxidant capacity of sperm cells may be insufficient in preventing LPO during the freeze–thawing process. The aim of this study was thus to determine the influence of varying doses of anti-oxidant additives on standard semen parameters, lipid peroxidation and anti-oxidant activities after the freeze–thawing of goat semen. Ejaculate samples (artificial vagina) obtained from 4 mature Angora goats were evaluated and pooled at 37 °C. The semen samples diluted with a Tris-based extender, containing taurine (25, 50, 75 mM), trehalose (25, 50, 75 mM), and cysteine (5, 10, 15 mM), and an extender containing no anti-oxidant additives (control) were again evaluated. Diluted semen was cooled down to 5 °C and frozen in 0.25 ml French straws, prior to being stored in liquid nitrogen. Frozen straws were thawed in a water bath (37 °C) for 30 s for microscopic sperm evaluation. Upon evaluation of parameters for semen quality, the use of a Tris-based extender supplemented with anti-oxidant additives was found to cause no significant improvement in sperm mortality, when compared to the controls. Increasing doses of taurine and trehalose decreased (P < 0.05) the sperm motility following the freeze–thawing of the goat semen. In biochemical assays, the application of taurine (75 mM) produced the lowest level of malondialdehyde (MDA) (4.46 ± 0.31 nmol/ml), compared to the controls (P < 0.001). Lower GSH levels were higher in the groups in which cysteine was included at 10 and 15 mM (3.27 ± 0.11 and 3.45 ± 0.28 nmol/ml) – compared to the group which received 5 mM cysteine, as well as the controls (2.27 ± 0.08 and 2.50 ± 0.08 nmol/ml respectively, P < 0.001). Compared to the controls, taurine at a concentration of 25 and 75 mM, and increasing doses (50 and 75 mM) of trehalose, significantly increased the GSH-PX activity (P < 0.01). The maintenance of CAT activity was demonstrated to be higher with the addition of 10 and 15 mM cysteine, compared to the other groups (P < 0.001). Vitamin A (VitA) levels were significantly higher, compared to the controls (267.34 ± 9.68 mg/dl and 267.34 ± 9.68 mg/dl, respectively), when 25 mM taurine (329.61 ± 6.35 mg/dl) and 10 mM (318.64 ± 6.34 mg/dl) cysteine was added to the extender (P < 0.001). The results of this study provide a new approach to the cryopreservation of Angora goat semen and could contribute to the improvement of this technology in the goat industry.     

Ultramicroscopic and biochemical changes in ram spermatozoa cryopreserved with trehalose-based hypertonic extenders

The ability of a range of extenders to cryopreserve ram spermatozoa was tested. The extenders were modified by the inclusion of citrate, Tris buffer, trehalose, and EDTA. Ejaculates from three Pampinta rams were evaluated and pooled at 30 degrees C. The semen was diluted to contain 1 x 10(9) cells/mL, cooled to 5 degrees C, loaded into 0.25-mL straws, frozen and stored in liquid nitrogen. Evaluation was based on the hypoosmotic swelling test (HOS test), electron microscopy, and biochemical parameters such as lipid peroxidation and reduced and total glutathione levels, all measured after thawing. The HOS test indicated that the percentage of intact plasma membranes after freezing and thawing was significantly higher for the hypertonic extender containing trehalose (T), compared with an extender containing trehalose+EDTA (TE) or an isotonic Tris extender (B) (p < 0.05). Membrane evaluation by ultramicroscopy also indicated better sperm cryopreservation in extender T compared with the others, and there was a significant reduction in the number of damaged membranes (27%, p < 0.0002). The level of reduced glutathione was significantly higher after sperm cryopreservation in either hypertonic diluent (T and TE) with respect to the isotonic extender B, immediately after thawing (12%) and after a 3-h post-thawing thermotolerance test at 37 degrees C (17%, p = 0.007). Total glutathione levels did not show statistical differences among the extenders. After 3h post-thawing incubation at 37 degrees C, lipid peroxide levels in spermatozoa were statistically lower for T than TE (35%) or isotonic extender B (44%) (p = 0.002). Taken together these results indicate a reduction in the oxidative stress provoked by freezing and thawing when semen is cryopreserved in extender T. The antioxidant properties of extender T may be related to its effectiveness in membrane cryopreservation.     

Different concentrations of cysteamine and ergothioneine improve microscopic and oxidative parameters in ram semen frozen with a soybean lecithin extender

The aim of this study was to evaluate the effects of ergothioneine and cysteamine as antioxidant supplements in a soybean lecithin extender for freezing ram semen. Twenty-four ejaculates were collected from four rams and diluted with extenders (1.5% soybean lecithin, 7% glycerol) containing no supplements (control) and cysteamine or ergothioneine (2, 4, 6 or 8 mM). Motility by CASA, viability, plasma membrane functionality (HOS test), total abnormality, lipid peroxidation, glutathione peroxidase (GPx) activity and capacitation status (CTC staining) were assessed after thawing. Using 6 mM of either antioxidant improved total motility. Cysteamine at 6 mM and ergothioneine at 4 and 6 mM improved viability and reduced lipid peroxidation (malondialdehyde concentration). Both antioxidants improved membrane functionality significantly, except at 8 mM. Progressive motility, kinematic parameters, GPx activity, capacitation status and sperm abnormalities were not influenced by the antioxidant supplements. In conclusion, cysteamine at 6 mM and ergothioneine at 4 or 6 mM seem to improve the post-thawing quality of ram semen cryopreserved in a soybean lecithin extender.     

Effect of lecithin nanoliposome or soybean lecithin supplemented by pomegranate extract on post-thaw flow cytometric,microscopic and oxidative parameters in ram semen

This investigation was carried out to study the effect of soybean lecithin 1.5% (wt/vol) (0, 2.5, 5 and 7.5 mg l⁻¹ pomegranate extract (PE)) or PE-loaded lecithin nanoliposome (0, 2.5, 5 and 7.5 mg l⁻¹) to Tris-based extender. Sperm motility (CASA), viability, membrane integrity (HOS test), abnormalities, mitochondrial activity, apoptosis status, lipid peroxidation, total antioxidant capacity (TAC)) and antioxidant activities (GPX, SOD) were investigated following freeze-thawing. No significant differences were detected in motility parameters, viability, membrane integrity, and mitochondria activity after thawing sperm between soybean lecithin and lecithin nanoliposomes. It was shown that PE5 significantly improved sperm total and progressive motility, membrane integrity, viability, mitochondria activity, TAC and reduced lipid peroxidation (malondialdehyde concentration). Moreover, the percentage of apoptotic sperm in PE5 extenders was significantly the lowest among other treatments. Sperm abnormalities, SOD and GPX were not affected by the antioxidant supplements. For apoptotic status, no differences were observed between soybean lecithin and lecithin nanoliposome. We showed that lecithin nanoliposome extender can be a beneficial alternative extender to protect ram sperm during cryopreservation without any adverse effects. It was also observed that regarding pomegranate concentration, PE5 can improve the quality of ram semen after thawing.     

Effects of dithioerythritol on ram semen after the freeze-thawing process

The aim of this study was to evaluate the effects of dithioerythritol added to cryopreservation extender on the post-thawed sperm parameters, lipid peroxidation and antioxidant activities of Merino ram sperm. Semen samples from 5 mature Merino rams (1 and 2 years of age) were used in the study. Semen samples, which were diluted with a Tris-based extender containing 0.5, 1, and 2 mM dithiothreitol and no antioxidant (control), were cooled to 5 °C and frozen in 0.25 ml French straws. Frozen straws were then thawed individually at 37 °C for 20 s in a water bath for evaluation.The addition of dithioerythritol at 0.5 and 2 mM doses led to higher percentages of subjective motility (62.9 ± 4.2% and 63.6 ± 1.8%) compared to control (52.0 ± 4.9%, P < 0.05). As regards CASA motility, dithioerythritol 0.25 and 2 mM (60.2 ± 4.5% and 59.6 ± 1.2%) groups were higher from that of control (44.2 ± 8.7%, P < 0.05). For the CASA progressive motility, 0.25, 0.5 and 2 mM doses of dithioerythritol (22.0 ± 2.1%, 21.7 ± 2.5% and 24.0 ± 1.2%) had increasing effect in comparison to control (15.0 ± 2.5%). Dithioerythritol at 1 and 2 mM doses for ALH provided higher values compared to the control (P < 0.001) following the freeze–thawing process. Supplementation with dithiothreitol did not significantly affect the integrities of sperm membrane and acrosome, and mitochondrial activities. No significant differences were observed in biochemical parameters among the groups (P > 0.05). Findings of this study showed that dithioerythritol supplementation in semen extenders, was of greater benefit to sperm motility of frozen–thawed ram sperm.     

Effect of antioxidants on microscopic semen parameters,lipid peroxidation and antioxidant activities in Angora goat semen following cryopreservation

The aim of this study was to determine the effects of the antioxidants glutamine and hyaluronan and the inclusion of different levels on microscopic semen parameters, lipid peroxidation and the antioxidant activities following the freeze–thawing of Angora goat semen. Ejaculates collected from three Angora goat bucks, were evaluated and pooled at 37 °C. The semen samples which were diluted with a Tris-based extender containing additives including glutamine (2.5; 5 mM) and hyaluronan (500; 1000 μl/ml), and an extender containing no antioxidants (control) were cooled to 5 °C and frozen in 0.25 ml French straws and stored in liquid nitrogen. Frozen straws were thawed individually (37 °C) for 20 s in a water bath for microscopic evaluation. Freezing extenders supplemented with 2.5 and 5 mM glutamine led to higher sperm motility and hypo-osmotic swelling test (HOST) values, compared to the control (P < 0.05) following the freeze–thawing process. The addition of 500 μl/ml hyaluronan resulted in a higher HOST percentage, compared to the addition of 1000 μl/ml hyaluronan and the control (P < 0.001). No significant difference was recorded in the percentage acrosome and total sperm abnormalities, following supplementation with antioxidants. The addition of antioxidants did not prevent malondialdehyde (MDA) formation, compared to the controls. Antioxidant treatment however decreased (P < 0.01) the superoxide dismutase (SOD) activity. The maintenance of catalase (CAT) activity was demonstrated to be insignificant following addition of antioxidants. Further studies are required to obtain more repeatable results regarding the characterization of the enzymatic and non-enzymatic antioxidant systems in cryopreserved goat sperm.     

Raffinose and hypotaurine improve the post-thawed Merino ram sperm parameters

The aim of this study was to determine the effects of raffinose and hypotaurine on sperm parameters after the freeze–thawing of Merino ram sperm. Totally 40 ejaculates of five Merino ram were used in the study. Semen samples, which were diluted with a Tris-based extender containing 10 mM raffinose, 5 mM hypotaurine, 5 mM raffinose +2.5 mM hypotaurine (H + R) and no antioxidant (control), were cooled to 5 °C and frozen in 0.25 ml French straws and stored in liquid nitrogen. Frozen straws were then thawed individually at 37 °C for 25 s in a water bath for evaluation.     

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.     

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 anti-oxidants and oxidative stress parameters on ram semen after the freeze–thawing process

Oxidative damage to sperm resulting from reactive oxygen species generated by the cellular components of semen is one of the main causes for the decline in motility and fertility of sperm during the freeze–thawing process. The aim of this study was thus to determine the effects of anti-oxidants on standard semen parameters, lipid peroxidation (LPO) and anti-oxidant activities after the freeze–thawing of ram semen. Ejaculates collected from four Akkaraman rams, were pooled and evaluated at 33 °C. Semen samples were diluted in a Tris-based extender containing the anti-oxidants glutathione (GSH) (5 mM), oxidized glutathione (GSSG) (5 mM) or cysteine (5 mM) and an extender containing no anti-oxidants (control), cooled to 5 °C and frozen in 0.25 ml French straws. Frozen straws were thawed individually for 20 s in a water bath (37 °C) for microscopic evaluation. The use of an extender supplemented with cysteine led to the highest (P < 0.01) post-thaw motility (61.0 ± 1.9%), compared to the other treatment groups. No significant differences were observed in viability, acrosome damage and total abnormalities, and following the hypo-osmotic swelling test (HOST), following supplementation with anti-oxidants after the thawing of the semen. Following the thawing process, the levels of malondialdehyde (MDA) did not change with the addition of anti-oxidants, compared to the control. The GSH level and glutathione peroxidase (GSH-PX) activity remained significantly higher upon the addition of GSH (3.33 ± 0.14 nmol/ml and 22.02 ± 1.27 IU/g protein) and GSSG (3.24 ± 0.08 nmol/ml and 20.17 ± 3.38 IU/g protein) compared to the other treatment (P < 0.001) groups. Only cysteine significantly elevated the activity of catalase (CAT, 842.40 ± 90.42 kU/l) following the freeze–thawing process. The Vitamin E (VitE) level was significantly higher, when compared to GSSG, cysteine and the control, when GSH (4.21 ± 0.20 mg/dl) was added to the freezing extender (P < 0.001). It could be concluded that future efforts aimed on improving the efficiency of cryopreservation of ram sperm should concentrate on the use of anti-oxidant additives. The results obtained provide a new approach to the cryopreservation of ram semen, and could positively contribute to intensive sheep production.     

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.     

Comparison of the effects of glutamine and an amino acid solution on post-thawed ram sperm parameters,lipid peroxidation and anti-oxidant activities

Ram semen contains sufficient quantities of superoxide dismutase (SOD) and much lower concentrations of glutathione peroxidase (GSH-PX) and catalase (CAT) to prevent oxidative damage. The anti-oxidant capacity of the sperm cell is limited, due to a small cytoplasmic component, which contains these anti-oxidants to scavenge the oxidants. However, the concentration of these anti-oxidants may decrease considerably by the dilution of the semen. The aim of the present work was to study the effect of two anti-oxidants, namely, glutamine and an amino acid solution (BME) in a Tris-based extender on ram sperm parameters, lipid peroxidation and anti-oxidant capacity after the cryopreservation/thawing process. Ejaculates collected from 4 Akkaraman rams were evaluated and pooled at 37 °C. Semen samples which were diluted with the tris-based extender containing glutamine (2.5 or 5 mM), BME (13 or 26%), and no anti-oxidants (control) were cooled to 5 °C and frozen in 0.25-ml French straws and stored in liquid nitrogen. Frozen straws were thawed individually at 37 °C for 20 s in a water bath for evaluation. The freezing extender supplemented with 5 mM glutamine led to higher motility rate (68.0 ± 4.4%) and hypo-osmotic swelling test (HOST) (64.1 ± 5.5%), when compared to glutamine (2.5 mM) and BME (13 and 26%) (P < 0.05). No significant differences were observed regarding sperm motility and HOST, following the supplementation of the freezing extender with glutamine 2.5 mM and BME (13 and 26%) after thawing. CAT activity remained significantly higher following the addition of glutamine 5 mM (6.4 ± 0.9 kU/g protein), compared to the other treatments (P < 0.01). The anti-oxidants at different levels were not effective in the elimination of malondialdehyde (MDA) formation and maintenance of SOD activities, when compared to the control (P < 0.05). Findings showed that glutamine (5 mM) supplementation in semen extenders, was of greater benefit to frozen–thawed ram sperm. Future efforts are needed to find the appropriate anti-oxidants and their effective concentrations to improve post-thaw sperm parameters (e.g. motility, membrane integrity, fertility) and anti-oxidant activities when frozen–thawed ram sperm is used.     

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.     

The effect of raffinose and methionine on frozen/thawed Angora buck (Capra hircus ancryrensis) semen quality, lipid peroxidation and antioxidant enzyme activities

The aim of the present study was to determine the effects of different doses of raffinose and methionine on post-thawed semen quality, lipid peroxidation and antioxidant enzyme activities of Angora buck (Capra hircus ancryrensis) sperm following cryopreservation.Ejaculates collected from three Angora bucks were evaluated and pooled at 37 °C. Semen samples, which were diluted with a Tris-based extender containing the additives raffinose (2.5, 5, 10 mM) and methionine (2.5, 5, 10 mM) and an extender containing no antioxidants (control), were cooled to 5 °C and frozen in 0.25 ml French straws. Frozen straws were thawed individually at 37 °C for 20 s in a water bath for evaluation. The freezing extender supplemented with 2.5 and 5 mM methionine led to higher percentages of CASA motility (63.6 ± 7.0; 63.4 ± 3.1%, respectively), in comparison to the controls (P < 0.01) following the freeze-thawing process. The addition of antioxidants did not provide any significant effect on the percentages of post-thaw subjective and CASA progressive motilities as well as sperm motion characteristics (VSL and VCL), compared to the control groups (P > 0.05). The freezing extender with raffinose (5 and 10 mM) and methionine at three different doses (2.5, 5 and 10 mM) led to lower percentages of acrosome abnormalities, in comparison to the controls (P < 0.001). In the comet test, raffinose (5 and 10 mM) and methionine (10 mM) gave scores lower than those of the controls, and thereby reduced DNA damage (P < 0.05). Malondialdehyde formation was found to be lower (1.8 ± 0.1 nmol/L) in the group of 5 mM raffinose, compared to the controls following the freeze-thawing process (P < 0.01). The additives did not show any effectiveness on the maintenance of SOD, GSH-PX and GSH activities, when compared to the controls (P > 0.05). In conclusion, methionine and raffinose play a cryoprotective role against sperm CASA motility, acrosome abnormality and DNA damage. Raffinose 5 mM exhibited antioxidative properties, decreasing MDA levels. Further studies are required to obtain more concrete results on the characterization of microscopic parameters and antioxidant activities in cryopreserved goat sperm with different additives.     

The effect of supplementation of cryopreservation diluents with sugars on the post-thawing fertility of ram semen

This study was conducted to elucidate the effect of increasing the osmolality of a basic Tris, extender supplemented with sucrose, trehalose or raffinose on post-thawing ram semen quality (sperm motility, viability, acrosome integrity, total sperm abnormalities and membrane integrity). After primary evaluation of the collected ejaculates, only semen samples with more than 70% motile sperm, and a sperm concentration of higher than 3 × 10⁹ sperm/ml were used for cryopreservation. The semen samples were pooled and diluted (1:4) with a Tris-citric acid-fructose-yolk extender, supplemented with different concentrations (50, 70 or 100 mM) of sucrose, trehalose or raffinose. As control, semen was diluted and frozen in the base diluent, without additional sugars. Pooled semen samples were aspirated into 0.25 ml straws, cooled to 5 °C within 90 min and frozen by exposure to liquid nitrogen vapor (4-5 cm above the liquid nitrogen surface) for 10 min - before plunging into liquid nitrogen, for storage. After 24 h, straws were thawed in a water bath (37 °C) for 30 s. The frozen-thawed sperm characteristics were improved significantly (P < 0.05) by increasing the level of the sugars. Optimal results being obtained with 70 and 100 mM trehalose or raffinose. All extenders containing supplemental sugars were superior in terms of sperm quality to the control (P < 0.01) group. The highest sperm motility (60.6 ± 1.9%), viability (60.6 ± 2.5%) and membrane integrity (58.2 ± 2.1%) were recorded using 100 mM trehalose and the lowest with 50 mM sucrose (48.6 ± 1.9%, 51.4 ± 2.5% and 47.9 ± 2.1%, respectively). All sugar concentrations decreased the percentage of acrosomal and total sperm abnormalities (P < 0.05). The extenders containing 100 mM trehalose or raffinose significantly (P < 0.05) decreased the occurrence of sperm abnormalities, compared to the other treatments. The fertility rates obtained after cervical insemination of the frozen-thawed sperm were 46.8%, 44.1% and 16.7% for 100 mM trehalose, 100 mM raffinose and the control with supplementation of the diluents, respectively. The study showed that ram sperm can tolerate hyperosmotic diluents, and that a range of sugar concentrations (50-100 mM) may successfully be incorporated in the ram semen cryopreservation diluents, although further research is warranted.     

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

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

Freeze-dried egg yolk based extenders containing various antioxidants improve post-thawing quality and incubation resilience of goat spermatozoa

The aim of this study was to evaluate different antioxidants-supplemented freeze-dried egg yolk based extenders for the post-thawing quality and incubation resilience of goat spermatozoa. Pooled semen were diluted in a two-step dilution method to a final concentration of 1/5 (semen/extender) in control and antoxidant supplemented freeze-dried extenders (methionine, cysteamine and butylated hydroxytoluene). Semen samples were assessed for sperm motility, plasma membrane functional integrity using hypoosmotic swelling test (HOST), damaged acrosome using FITC-Pisum sativum agglutinin (PSA-FITC) and DNA integrity using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). Membrane lipid peroxidation status was also analyzed using the malondialdehyde (MDA) concentration. In the study, antioxidant supplemented freeze-dried egg yolk based extenders have beneficial effect on goat sperm parameters. In addition, we achieved a higher quality in post thawed goat semen even after 6 h incubation when the extender was supplemented by 5 mM BHT or cysteamine.     

Effects of supplementation of tris-egg yolk extender with different sugars and antioxidants on freezability of ram semen

This study was designed to determine the effects of the combined addition of different levels of certain sugars (trehalose, sucrose and raffinose) and antioxidants (vitamin E, C and taurine), in Tris-egg yolk extender on frozen-thawed ram semen parameters. Semen samples were collected from five healthy, mature and fertile Iranian Afshari rams, twice a week for 8 weeks. Selected samples were pooled and diluted with a Tris-egg yolk extender containing different levels of sugars and antioxidants. In Experiment 1, different levels of trehalose (0, 50 and 100 mM) were tested with different levels of taurine (0, 25 and 50 mM), vitamin E and C (0, 1 and 2 mM). In Experiment 2, different levels of sucrose (0, 60 and 80 mM) were tested with different levels of taurine (0, 25 and 50 mM), vitamin E and C (0, 1 and 2 mM). In Experiment 3, different levels of raffinose (0, 5, 10 mM) were tested with different levels of taurine (0, 25 and 50 mM), and vitamin E and C (0, 1 and 2 mM). In Experiment 4, the selected extenders of experiments 1, 2 and 3 were compared statistically with control (no selected sugar and antioxidant) extender. The results of experiments 1, 2 and 3 revealed that the highest frozen–thawed sperm parameters were recorded for the selected extenders containing 100 mM trehalose +2 mM vitamin E (T100E2), 60 mM sucrose + 2 mM vitamin E (S60E2) and 10 mM raffinose + 2 mM vitamin E (R10E2), respectively. The results of experiment 4 revealed that the post-thaw sperm total motility in T100E2 (62.41 ± 2.41%), S60E2 (59.52 ± 1.91%) and R10E2 (58.33 ± 2.00%) was higher than that of the control extender (46.00 ± 1.79%; P ≤ 0.05). Similarly, the progressive sperm motility in T100E2 (57.18 ± 1.96%), S60E2 (57.49 ± 1.94%) and R10E2 (55.03 ± 2.99%) was also higher than that of the control extender (41.20 ± 1.70%; P ≤ 0.05). Post-thaw sperm viability in selected extenders of T100E2 (65.20 ± 2.67%), S60E2 (62.00 ± 2.07%) and R10E2 (61.80 ± 2.46%) was higher than that of control extender (51.00 ± 1.88%; P ≤ 0.05). In conclusion, the addition of 100 mM trehalose, 60 mM sucrose and 10 mM raffinose combined with 2 mM vitamin E in Tris-egg yolk extender significantly improved frozen-thawed ram semen parameters.     

Effect of extender supplementation with various antimicrobial agents on viability of Brucella ovis and Actinobacillus seminis in cryopreserved ovine semen

The objective was to determine the effectiveness of various antimicrobial agents added to semen extender for inactivation of B. ovis or A. seminis in ovine semen after cryopreservation. In Experiment 1, 20 ejaculates from a crossbred ram infected with B. ovis were cryopreserved in Tris-based extenders with various antimicrobial agents: (I) control without antibiotics, (II) with penicillin and streptomycin (1000 IU/mL and 1 mg/mL, respectively), (III) lincomycin (0.15 mg/mL), (IV) sulphadiazine (0.60 mg/mL), and (V) gentamicin sulphate (0.25 mg/mL). Semen was stored in 0.25 mL straws at a final concentration of 150 × 10⁶ spermatozoa/mL. After thawing (37 °C for 30 s), sperm total motility (TM), sperm morphology, integrity of sperm membranes, and bacterial growth were assessed. In Experiment 2, six B. ovis isolates were separately inoculated into aliquots of a fresh ejaculate from a B. ovis-free ram. Mock inoculated semen was processed for cryopreservation using the five extenders described above, and bacteriologically evaluated after thawing. In Experiment 3, sensitivity of A. seminis to the same antimicrobial agents was evaluated by inoculating an ejaculate from an A. seminis and B. ovis-free ram. There were no significant differences among treatments in post-thawing sperm parameters. B. ovis was isolated from 100% (20/20), 0% (0/20), 95% (19/20), 100% (20/20), and 5% (1/20) of semen samples diluted in tris-based extender of untreated (I) and treated semen samples with antimicrobial agents II, III, IV, and V, respectively. Frequencies of isolation from samples treated with antimicrobial agent II and V were significantly lower than untreated ones (P < 0.05). There were no significant differences in the profile of antimicrobial resistance of different B. ovis isolates. A. seminis had a similar sensitivity to the antimicrobial agents. We concluded that addition of a combination of penicillin and streptomycin or gentamicin alone to ram semen cryo-extenders inactivated B. ovis and A. seminis.     

Assessment of ram sperm membrane integrity following different thawing procedures

Semen from five 2.5-yr-old rams selected for use in an AI program was collected over 3 consecutive days using an artificial vagina. The semen was diluted with a skim milk extender containing 7% glycerol (v/v), packed in French mini-straws (approx. 100 mill/straw), and frozen in a programmable freezer. Three freezing operations were carried out per ram. Three straws per freezing operation were subjected to the following thawing procedures: 1) 70 degrees C, 5 sec; 2) 50 degrees C, 9 sec and 3) 35 degrees C, 12 sec. Post-thaw sperm motility was subjectively assessed using a phase contrast microscope; while the combined fluorochromes carboxyfluorescein diacetate and propidium iodide (CFDA/PI), the hypo-osmotic swelling test (HOS) and the presence of normal apical ridges (NAR's) were used to determine the degree of sperm membrane integrity. Significant differences between thawing treatments were found for post-thaw motility (P < .05) and membrane integrity (P < 0.01), and variation among rams was statistically significant. Post-thaw sperm motility as well as the percentage of spermatozoa showing intact membranes were significantly higher (P < 0.01) for straws thawed at 70 degrees C than for those thawed at 35 degrees C (67.0 +/- 1.1 and 63.0 +/- 1.1%, and 50.5 +/- 1.5 and 41.7 +/- 1.5%, respectively). However, no corresponding statistically significant difference could be found for these parameters when 70 degrees C and 50 degrees C thawing were compared. It was concluded that sperm can be thawed at 50 degrees C for 9 sec instead of 70 degrees C for 5 sec without further reducing sperm motility or membrane integrity. This lower thawing temperature would facilitate the widespread use of frozen/thawed ram semen under farm conditions in Sweden.