Influence of glucose and fructose in the extender during long-term storage of chilled canine semen

The use of chilled, extended semen in dog breeding is becoming increasingly popular as preparation and transportation is less expensive and regulations are often less complicated than for frozen semen. Sugar is one of the main constituents in semen extenders, and glucose and fructose are metabolized in separate pathways by freshly ejaculated dog sperm. In this study, glucose, fructose or an equal mixture of both were used in an egg-yolk-tris (EYT) extender at two different concentrations (10 and 70 mM). EYT extender without sugar supplementation, providing only the glucose (3-4 mM) originating from the egg-yolk, served as a control. The longevity of the chilled semen at 5 degrees C was 23 days: the quality of physical and functional characteristics decreasing with time. Glucose and fructose had a strong influence on motility and movement patterns of chilled canine semen. The beneficial effect of 70 mM sugar concentrations compared to 10 mM and the control was pronounced, and maintained sperm motility > or = 70% for 8 days of storage, compared to for 4 days in the control extender. Fructose maintained higher sperm motility than did glucose and the mixture. VAP values were higher in sugar-supplemented extenders (P < 0.05). Neither type nor concentration of the two sugars influenced sperm plasma membrane, acrosome integrity or the acrosome reaction following ionophore challenge (ARIC). Sugar consumption by dog sperm varied between the different periods of storage and with sugar concentrations provided in the extenders. Glucose consumption by dog sperm was greater than fructose consumption when both sugars were present in equal amounts, indicating that dog sperm used glucose in preference to fructose. In conclusion, the major influence of the two sugars on chilled semen was to support motility. EYT extender supplemented with fructose at a concentration of 70 mM was found to be the best of the tested extenders for long-term preservation of chilled canine semen.     

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.     

Short-term storage sperm of coho salmon (Oncorhynchus kisutch) at 4 °C: Effect of sperm: Extender dilution ratios and antioxidant butyl-hydroxytoluene (BHT) on sperm function

Short-term storage of semen is a necessary key procedure in fish; it allows maximizing the use of gametes. Nevertheless, sperm quality decreases during storage has been associated with oxidative stress damage due to an increase in reactive oxygen species (ROS) during storage. This study was designed to optimize a short-term storage protocol for Coho salmon (Oncorhynchus kisutch) spermatozoa, evaluating the effect of extender dilution and the addition of butyl-hydroxytoluene (BHT) antioxidant on sperm function parameters. In the first experiment, fresh semen was diluted in Storfish®: extender dilution (1:2 and 1:3) and a control sample undiluted and stored at 4 °C for 7-days. In both experiments motility (MO), viability and integrity of plasma membrane, mitochondrial membrane potential (MMP) and superoxide anion level (O₂⁻) were evaluated at 0, 3 and 7 days. Result shows that, 1:3 dilution maintained a higher sperm function for a longer period time. In the second experiment, spermatozoa were suspended in Storfish® (1:3) supplemented with two different concentrations of BHT (1.0 mM and 2.0 mM) and a control sample without antioxidant and stored at 4 °C for 7 days. The results demonstrated that, antioxidant-supplemented samples greater MO than control samples (P < 0.05). The viability remained >75% during storage in all groups. MMP was higher in 2.0 mM BHT compared to 1.0 mM and control (P < 0.05), in addition, this concentration reduced O₂⁻ level (P < 0.05). In conclusion, sperm: extender dilution 1:3 and adding of 2.0 mM BHT in sperm storage extender may enhance protection sperm function in Oncorhynchus kisutch against effects harmful of the oxidative stress during the in vitro storage.     

Cryopreservation of ram semen with antioxidant supplemented soybean lecithin-based extenders and impacts on incubation resilience

The scope of this study was investigation the affects of various antioxidants on 1% soybean lecithin-based semen extenders for ram semen cryopreservation. Ejaculates, collected via electrically stimulated ejaculation, that have a thick consistency, rapid wave motion (3–5 on a 0–5 scale) and >75% initial motility were pooled. The pooled samples were split into four equal aliquots as 5 mM Methionine, 5 mM Cysteamine, 1 mM Cysteine and a sample of antioxidant-free control group. Each sample group was diluted to a ratio of 1/5 (semen/extender, v/v) as final concentration and two step dilution method was used for cryopreservation. Extender groups 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). Semen samples also incubated for 6 h in humidified air with 5% CO₂ at 39 °C to evaluate post-thaw incubation resilience of semen characteristics. The results showed that freezing and thawing procedures had negative effects on motility (P < 0.05), plasma membrane integrity (P < 0.05) and acrosomal integrity (P < 0.05). After 6 h of incubation time, the Cysteine supplemented extender group yielded significantly higher results than other extender groups in terms of spermatological parameters. Furthermore MDA levels in the antioxidant groups were lower than control group (P < 0.05). Nevertheless, there were no significant differences among antioxidant groups.     

Quality of chilled and cold-stored (5 °C) canine spermatozoa submitted to different rapid cooling rates

The aim of this study was to evaluate the sperm quality in chilled canine semen using di?erent cooling rates from room temperature (23 °C) to 5 °C and subsequently cold-stored at 5 °C for up to 96 hours. In experiment 1, semen samples from five dogs were pooled, diluted in Tris-fructose-citrate extender with 20% egg yolk and split into four aliquots that were chilled to 5 °C using di?erent cooling rates of 2.25, 0.9, 0.45, and 0.2 (control) °C/min. In experiment 2, semen from five dogs was processed individually as described above and split into two aliquots that were chilled to 5 °C using rates of either 2.25 °C/min or 0.2 °C/min. In both experiments, the sperm quality (i.e., sperm motility and viability) was evaluated before cooling and after 0, 24, 48, 72, and 96 hours of storage at 5 °C. The total motility, progressive motility, and quality of movement parameters were assessed using computer-assisted analysis system, and the percentage of viable spermatozoa was determined using ?ow cytometry (H-42/PI//FITC-PNA). The cooling rate did not in?uence the sperm quality parameters at any of the evaluation times. All evaluated males showed the same response to chilling semen at a rapid cooling rate. Storage time negatively in?uenced (P < 0.05) sperm motility, regardless of the cooling rate used. In conclusion, canine sperm could be chilled and stored for 96 hours at 5 °C in a Tris-fructose extender with 20% egg yolk using rapid cooling rates, with values for sperm quality similar to those from a conventional protocol.     

Optimization of short-term storage of pikeperch semen: an applicable approach

Contamination of semen with urine and asynchronous maturation of males and females are main obstacles in artificial reproduction of pikeperch Sander lucioperca. The objective of this study was to overcome these obstacles using optimization of a procedure for short-term storage of pikeperch semen at 4 °C using two immobilizing media (IM): (a) IM1, 180 mM NaCl, 2.68 mM KCl, 1.36 mM CaCl₂ ⋅ 2H₂O and 2.38 mM NaHCO₃, 343 mOsm/kg; and (b) IM2, 200 mM NaCl, 2.68 mM KCl, 1.36 mM CaCl₂ ⋅ 2H₂O and 2.38 mM NaHCO₃, 381 mOsm/kg. Undiluted sperm was used as the control. At 6 h poststorage, there were no substantial changes in spermatozoa motility and velocity at 30 s postactivation in all groups. Over 48 h of storage, the highest spermatozoa motility and velocity were obtained in sperm diluted in IM2 compared to the other groups. IM2 could maintain a significantly higher ATP content of diluted sperm than IM1 and undiluted treatment for 2 days. Similarly, the highest values of eyeing and hatching rates were observed in sperm diluted in IM2 compared to sperm in the other studied groups. It can be concluded that the obtained result is a novel and applicable approach to maintain semen quality of pikeperch during short-term storage, suggesting IM2 as a promising medium for short-term storage. The present study also opens possibilities for ensuring a reliable source of semen as a convenient approach for increasing genetic diversity in hatcheries.     

Effect of dilution rate on feline urethral sperm motility,viability, and DNA integrity

This study was designed to investigate if the characteristics of feline urethral sperm can be affected by high dilution in an artificial medium. The semen collected by urethral catheterization from eight male cats was evaluated for sperm concentration and motility and subsequently diluted with a TRIS-based extender to the concentration of spermatozoa 10 × 10⁶/mL, 5 × 10⁶/mL, and 1 × 10⁶/mL. Immediately after the extension samples were assessed for motility, cell viability using SYBR-14 and propidium iodide, acrosome integrity using lectin from Arachis hypogaea Alexa Fluor 488 Conjugate, and propidium iodide and chromatin status by acridine orange. Compared with 10 × 10⁶/mL dilution rate, spermatozoa diluted to 1 × 10⁶ sperm/mL had a significantly lower proportion of motile (31.1% ± 19.8 and 0.7% ± 1.6, respectively, P < 0.05) and viable spermatozoa (88.3% ± 3.1 and 69.1% ± 12.8, respectively, P < 0.01). There was no dilution-related difference in the acrosome integrity (76.7% ± 11.9 vs. 75.9% ± 10.6) and chromatin status (defragmentation index, 3.3% ± 0.97 vs. 3.4% ± 1.7). These results indicate that feline urethral semen is susceptible to high dilution rate, and some sperm characteristics can be artifactually changed by semen dilution. It also suggests the potential role of seminal plasma in maintaining sperm motility and viability in high dilution rates.     

Induced immotility during long-term storage at +5 degrees C does not prolong survival of dog spermatozoa

This study investigated whether the immotility induced by the CLONE chilled semen extender prolongs the lifespan of dog spermatozoa stored at 5 degrees C, compared with a Tris-egg yolk-glucose (TG) extender, which maintains motility. Pooled semen was split in four aliquots, centrifuged, and the four sperm pellets mixed with TG extender; with the CLONE chilled semen (CL) extender; with TG extender mixed with an activator (TG+A(TG)); or with the CLONE extender mixed with the CLONE activator (CL+A(CL)). Samples were stored at 5 degrees C for 23 days and examined 12 times for sperm motility, plasma membrane and acrosome integrity, glucose consumption, and DNA fragmentation index (DFI). The experiment was performed in triplicate. Glucose consumption was not significantly different between extenders until the period 15-23 days, when it was higher in CL and CL+A(CL) than in TG (P=0.0055) and TG+A(TG) (P=0.0010). No breakdown of DNA chromatin (P>0.05) occurred until day 14. Spermatozoa preserved in TG or TG+A(TG) showed better values for all the different parameters throughout the experiment compared with sperm subjected to CL or CL+A(CL). In conclusion, the immotility induced by the CLONE chilled semen extender during long-term cold storage at 5 degrees C did not prolong the lifespan of spermatozoa compared with the lifespan following storage in Tris-egg yolk-glucose. In addition, our results indicate that good quality dog semen may possibly be stored for up to 14 days in TG extender at 5 degrees C, with retained fertilizing capacity. In vivo studies should, however, be performed to further support this conclusion.     

L-carnitine is a survival factor for chilled storage of rooster semen for a long time

Rooster sperm is sensitive to cooling, which restricts procedures to store sperms for extended periods of time for artificial insemination of commercial flocks. This study was conducted to evaluate the suitability of adding L-carnitine (LC) to chilled-storage of rooster sperm and its effects on sperm quality parameters and its fertility potential during storage at 5 °C. Pooled semen from roosters were divided into six equal aliquots and diluted with media supplemented with different concentrations of LC (0, 0.5, 1, 2, 4 and 8 mM LC). Diluted semen samples were cooled to 5 °C and stored over 48 h. Motility, viability, membrane functionality, lipid peroxidation and mitochondria activity of the sperm were assessed at 0, 24 and 48 h of storage. Moreover, fertility potential of chilled stored sperm was considered at 24 h of storage. While sperm quality was not affected by LC at the beginning of storage (0 h), supplementation of extender with 1 and 2 mM of LC significantly improved the percentage of sperm motility, viability, membrane integrity and mitochondria activity at 24 h and 48 h compared to other groups. Lipid peroxidation was significantly reduced in sperm samples diluted with 1 and 2 mM LC at 24 h (2.15 ± 0.52 nmol/ml and 2.21 ± 0.52 nmol/ml) and 48 h (3.42 ± 0.49 nmol/ml and 3.38 ± 0.49 nmol/ml) compared to other groups. Furthermore, fertility rates during artificial insemination using sperms cooled for 24 h in the presence of 1 and 2 mM LC were significantly higher (78%) than in the control group (64%). These findings suggest that optimum doses of LC could protect rooster sperm against cool storage-induced functional and structural damages.     

Effects of different extenders on DNA integrity of boar spermatozoa following freezing–thawing

The sperm-rich fraction, collected from eight mature Yorkshire boars, was frozen in an extender containing 9% LDL (w/v), 100 mM trehalose, or 20% yolk (v/v), respectively. Sperm DNA integrity was assessed using the single-cell gel electrophoresis (SCGE). Other sperm quality characteristics such as motility, acrosome and membrane integrity were also monitored. The results showed that freezing–thawing caused an increase in sperm DNA fragmentation, and extender containing 9% LDL could significantly protect sperm DNA integrity (P < 0.05) from the damage caused by cryopreservation and decrease DNA damages compared with extender containing 100 mM trehalose and 20% yolk (v/v). No significant difference in damaged DNA was detected between frozen and unfrozen semen samples for extender of 9% LDL and 100 mM trehalose, but cryopreservation could increase the degree of DNA damage (P < 0.05), the percentage of damaged DNA degree of grade 2 and 3 was significantly increased. The deterioration in post-thaw sperm DNA integrity was concurrent with reduced sperm characteristics. The data here demonstrated that the cryoprotectant played a fundamental role in reducing boar sperm DNA damage and protecting DNA integrity. It can be suggested that evaluation of sperm DNA integrity, coupled with correlative and basic characteristics such as motility, acrosome integrity and membrane integrity, may aid in determining the quality of frozen boar semen.     

The synergistic effect of trehalose and low concentrations of cryoprotectants can improve post-thaw ram sperm parameters

The objective of this study was to compare the effects of different concentrations of two different cryoprotectants (glycerol, G and ethylene glycol, EG) and trehalose (T), added to the semen extender, on post-thaw ram sperm parameters. Ejaculates, collected from 6 Merino rams, were pooled and evaluated at 37 °C. The pooled samples were divided into six equal aliquots, and diluted in Tris-based extenders containing 5% G, 3% G + 60 Mm T, 1.5% G + 100 Mm T, 5% EG, 3% EG + 60 mM T, and 1.5% EG + 100 Mm T. Subsequently, the samples were cooled to 5 °C, frozen in 0.25-ml French straws, and stored in liquid nitrogen (LN₂). Frozen samples were thawed individually, at 37 °C for 25 s in a water bath, for evaluation. Sperm motility was assessed using a phase-contrast microscope with a warm stage maintained at 37 °C. Acrosome integrity (FITC/PNA-PI), sperm viability (SYBR-14/PI), mitochondrial activity (JC-1/PI), DNA damage (COMET assay) and DNA fragmentation (TUNEL test) were determined. The group of samples diluted in an extender containing 5% of glycerol (Group 5% G) displayed higher percentages of subjective motility, viability and mitochondrial activity of sperm, compared to the other groups (P < 0.05). On the other hand, Group 3% G + 60 mM T yielded the second-best results for subjective motility, viability and mitochondrial activity of sperm, when compared to the other groups. The post-thaw sperm parameters of Group 3% G + 60 Mm T did not show any statistically significant difference from those of Group 5% G. There were no statistically significant differences between the groups for acrosome integrity (P > 0.05).The results of the COMET assay showed that the use of low concentrations of cryoprotectants in combination with trehalose decreased sperm DNA damage. Accordingly, Group 1.5% G + 100 mM T and Group 3% EG + 60 mM T benefited from a significantly stronger cryoprotective effect on DNA integrity, in comparison to Group 5% G (P < 0.05). According to the results of the TUNEL test, the combined use of low concentrations of cryoprotectants with trehalose decreased sperm DNA damage, when compared to the use of 5% glycerol, but this difference was statistically insignificant (P > 0.05).In conclusion, G and EG concentrations can be reduced by adding various amounts of T (60 mM, 100 mM) to the semen extender. The addition of 5% of glycerol and 3% G + 60 mM T to the semen extender did not yield statistically different post-thaw sperm parameters, when compared for protection against cryoinjury. Post-thaw sperm parameters can be improved by the supplementation of the semen extender with 3% G + 60 mM T. Thus, we recommend the use of freezing extenders containing low cryoprotectant concentrations (3% G) combined with trehalose to avoid the high level of toxic and osmotic damage caused by 5% G.     

Effect of antioxidant supplementation in semen extenders on semen quality and reactive oxygen species of chilled canine spermatozoa

The objective of this study was to evaluate quality of chilled dog semen processed with extenders containing various antioxidants. Single ejaculates from five dogs were always pooled and evaluated for concentration, sperm motility, progressive motility (RSF-movement), viability, acrosomal integrity and by the hypo-osmotic swelling (HOS)-test. Also, superoxide (O(2)(-)) production, hydroxyl radicals (OH) and total reactive oxygen species (tROS) were determined. Pooled semen was divided in seven aliquots (for control and test conditions), which were diluted to a final concentration of 67x10(6)spermatozoa/ml with TRIS-glucose-egg yolk extender with or without the following supplements: control (without antioxidants), vitamin C (0.5mM), N-acetyl-l-cysteine (NAC; 0.5mM), taurine (0.2mM), catalase (100u/ml), vitamin E (0.1mM) and 5-(4-dimethylamino-phenyl)-2-phenyl-penta-2,4-dienoic acid (B16; 0.1mM). The semen aliquots were chilled and preserved at 4 degrees C. Portions of chilled semen were removed at 24 and 72h, and semen quality was evaluated after rewarming. At 24h the mean (+/-S.E.M.) sperm motility was higher (p<0.001) when vitamin E, taurine and B16 were added in the extender, whereas more spermatozoa with RSF-movement were observed (p<0.001) in the vitamin E, catalase, B16 and taurine groups. Sperm viability was higher (p=0.040) in B16 and vitamin E groups and the percentage of swollen spermatozoa was higher (p=0.002) only in the B16 group. Acrosomal integrity and OH were not significantly influenced by any of the antioxidants tested. Superoxide production was significantly lower when vitamin C, B16 and vitamin E were added in semen extenders compared with the control (p=0.017). All antioxidant groups, except vitamin C and NAC, contained less tROS compared to the control group, but only the B16 group value differed significantly (p=0.05). At 72h sperm motility was higher (p<0.001) when vitamin E, catalase, B16, taurine and NAC were added in the extender. More spermatozoa with RSF-movement were observed (p<0.001) in the vitamin E, catalase, B16, taurine and NAC treatment groups. Sperm viability was higher (p=0.001) when vitamin E, B16, taurine and vitamin C were added in semen extenders. HOS-test percentages were higher (p=0.016) in the B16, vitamin E, catalase and NAC groups. Acrosomal integrity was not influenced in any case. Production of O(2)(-) was significantly higher using catalase compared to all the other groups (p=0.006), while OH was not significantly influenced by any of the antioxidants tested. The addition of vitamin E, catalase and B16 in semen extenders resulted in significantly lower tROS values compared with the controls (p<0.0005). The results suggest that vitamin E and B16 had the most pronounced effect in preserving semen quality of chilled dog spermatozoa.     

Impact of Eurycoma longifolia extract on DNA integrity,lipid peroxidation,and functional parameters in chilled and cryopreserved bull sperm

This study aims to assess the effect of Eurycoma longifolia aqueous extract on chilled and cryopreserved quality of bull sperm. Semen samples were obtained from four Simmental–Brangus. Each sample was divided into two fractions: the first fraction was used for chilling the semen, and the second fraction was used for the freezing process. Both fractions were extended with Tris–egg yolk extender supplemented with 0.0, 0.25, 0.5, 1.0, 2.5, 5.0, and 7.5 mg/ml Eurycoma longifolia aqueous extract. The diluted chilled fraction was chilled at 5 °C for 6 days, whereas the frozen–thawed fraction was frozen in liquid nitrogen. Data revealed that 1 mg/ml E. longifolia aqueous extract yielded significantly (p < .05) higher sperm motility, morphology, viability, and sperm membrane integrity compared with the control group and other treated groups in chilled semen evaluation. For cryopreserved sperm, a significant difference (p < .05) in sperm motility, viability, sperm membrane integrity, DNA integrity, and lipid peroxidation was observed between 5 mg/ml E. longifolia aqueous extract and other treated and control groups. However, no significant difference in the percentage of sperm exhibiting normal sperm morphology was observed among the groups. In conclusion, the addition of 0.25 and 1 mg/ml E. langifolia extract to chilled semen and 5 mg/ml E. longifolia aqueous extract to cryopreserved sperm into Tris–egg yolk extender helps in maintaining superior quality of bull spermatozoa during chilling and freezing.     

Effect of different calcium concentration on sperm motility and fertilisation capacity of rainbow trout (Oncorhynchus mykiss)

Calcium is a prerequisite factor for triggering the sperm activation in salmonids. The aim of this study was to test different concentrations of Ca²⁺ on sperm motility activation and fertility of rainbow trout (Oncorhynchus mykiss). Semen samples activated with calcium free activator (0.0 mM) and increasing concentrations of CaCl₂ (6.3, 7.9, 9.5 and 11 Mm) were evaluated by computer-assisted sperm analysis (CASA). To assess fertility, eggs from three females were fertilized with semen and activated with each of the five solutions and incubated until four-cell stage when the rate fertility and the symmetry of the first blastomeres were evaluated. The results show that eliminating calcium from the activator solution significantly reduced the motility rate and fertility in comparison with the other treatments, while increasing the CaCl₂ from 6.3 to 11 mM generated high sperm motility (78.5 ± 5.8–95.4 ± 4.0%) and fertility (85.0 ± 9.3–96.0 ± 6.51%) rates, and low symmetry rates in the first blastomeres (<20%). No significant differences were found between solutions for the latter two variables, suggesting that the calcium concentrations were in balance with the other components of the medium and within the requirements for the fertilization of rainbow trout.     

Comparison of two commercial extenders for cryopreservation of goat semen without sperm washing

The objective of this study was to evaluate the effects of two commercially available semen extenders on the motility of cryopreserved goat sperm and to simplify the cryopreservation protocol. Individual goat ejaculates were split and processed in parallel for freezing in either commercially available soy-based extender (Bioxcell®) or egg yolk-based extender (Irvine TYB). Sperm quality was assessed using total and progressive sperm motility, measured by computer-assisted sperm analysis (CASA). Total motility was higher for samples processed in soy-based extender, both at pre-freeze (P = 0.002) and at post-thaw (P < 0.0001). Progressive motility was higher for semen processed in soy extender at post-thaw (P < 0.0001). Approximately 10% of samples processed in egg yolk-based extender had a large (> 50%) reduction in total motility prior to freezing. However, this type of extreme reduction in pre-freeze motility did not occur in semen samples processed in soy extender. In addition, the use of soy-based extender eliminated the need for a time-consuming sperm washing protocol. We concluded that a commercially available soy-based extender was superior to an egg yolk-based extender in preserving motility of cryopreserved goat sperm, using a two-step method.     

Protective effect of taurine,glutathione and trehalose on the liquid storage of ram semen

Oxidative damage to sperm resulting from reactive oxygen species generated by the cellular components of semen during liquid storage is possibly one of the main causes for the decline in motility and fertility during storage—the other detrimental cause is low temperature on the destabilisation of sperm membrane structure. The aim of this study was to determine the effects of the addition of the anti-oxidants taurine and glutathione (GSH), and the membrane structure stabiliser, trehalose, on sperm viability during low temperature liquid storage. A total number of 36 ejaculates were collected using the artificial vagina from four Chios rams and nine replicates of the ejaculates were diluted with a Tris-based extender containing additives as the control. The sperm motility, percentage abnormal sperm, plasma membrane intact sperm and the hypo-osmotic swelling test (HOST) were determined during storage of semen at 5 °C for a period of 0, 6, 24 and 30 h of liquid storage, respectively. Trehalose at a level of 50 mM provided the best maintenance of motility at 6 and 30 h (P < 0.05), and gave the highest percentage (69.0 ± 2.0% and 64.6 ± 1.8%, respectively) of viable sperm at 24 and 30 h (P < 0.01). Trehalose treatment at a concentration of 50 mM also resulted in the highest percentage of membrane-intact sperm (53.7 ± 2.9%) after performing HOST at 30 h. The anti-oxidant treatments GSH 5–10 mM and taurine at 50 mM provided a significant improvement in sperm survival during the 6 h of liquid storage at 5 °C (P < 0.05). In conclusion, many aspects of sperm protection, e.g. sperm motility, viability and membrane stabilisation of the sperm cells during relative low temperature storage, are the key factors determining the preservation of sperm function. Future efforts toward improving function of ram sperm kept in low temperature storage should concentrate on anti-oxidant additives. The results of this study provide a new approach to the preservation of sperm from rams of the Chios and related breeds, and so contribute to the improvement of these breeds for the world sheep industry.     

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.     

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.     

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.     

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.