Anti-oxidant supplementation improves boar sperm characteristics and fertility after cryopreservation: Comparison between cysteine and rosemary (Rosmarinus officinalis)

Anti-oxidants partially ameliorated the detrimental effects of reactive oxidative substances produced during cryopreservation. The objective of the study was to determine the effect of anti-oxidant addition to the freezing extender on boar semen qualities and fertility capacity. Ejaculates were collected from a previously selected boar and semen samples were processed using the straw freezing procedure. In experiment 1, semen samples were cryopreserved in lactose-egg yolk solution supplemented with various concentrations of cysteine (0, 5 and 10 mM) to determinate a cysteine concentration capable of producing a protective effect during cryopreservation. Semen quality (total motility, progressive motility, viability, acrosome integrity and hypoosmotic swelling test) was evaluated after freezing and thawing and then every hour for 3 h. In experiment 2, ejaculates were cryopreserved with lactose-egg yolk extender with or without the following anti-oxidants: cysteine, rosemary (Rosmarinus officinalis) and cysteine plus rosemary. Semen quality was evaluated. In the experiment 3, fertility capacity of semen frozen in anti-oxidant supplementation extenders was examined in vitro. A total of 2232 oocytes were in vitro matured and inseminated with frozen-thawed sperm. In summary: (i) the effective concentration of cysteine in freezing extender was 10 mM; (ii) the addition of exogenous rosemary or cysteine to the freezing extender positively affected post-thawed viability and acrosome integrity. Only rosemary supplementation improved total motility at 3 h and progressive motility at any time; (iii) the inclusion of rosemary into the extender was effective in penetration and cleavage rate and also in the efficiency of the fertilization system.     

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.     

Alpha-tocopherol improves biochemical and dynamic parameters in cryopreserved boar semen

Cryopreservation is associated with the production of reactive oxygen species which lead to lipid peroxidation of sperm membranes. The objective was to determine an alpha-tocopherol concentration capable of improving the quality of cryopreserved porcine semen. Boar spermatozoa frozen with 200, 500 or 1000 microg/mL alpha-tocopherol were thawed and incubated at 37 degrees C for 4 h. Routine parameters of semen quality, susceptibility to lipid peroxidation 2-thiobarbituric acid (TBARS) and oxygen uptake were evaluated. Motility was higher (P<0.05) in samples treated with different concentrations of alpha-tocopherol up to 2 h of incubation. Viability and acrosome integrity significantly decreased during incubation (no significant differences between treatments). Two hundred micrograms per milliliter alpha-tocopherol protected spermatozoa against lipid peroxidation during incubation, but 1000 microg/mL failed to protect after 2 h of incubation. There was a negative association between TBARS and motility, suggesting that lipid peroxidation affected sperm motility. Both control and 200 microg/mL alpha-tocopherol samples preserved the capacity to generate oxidative energy up to 1 h of incubation. The addition of 200 microg/mL alpha-tocopherol in the semen extender could be useful to preserve boar spermatozoa against the oxidative stress generated by cryopreservation.     

Comparing sugar type supplementation for cryopreservation of boar semen in egg yolk based extender

Cryopreservation of boar semen is still considered suboptimal due to lower fertility when compared to fresh semen. The aim of this study was to evaluate the effects of the addition of different sugars (lactose, trehalose and glucose) on boar spermatozoa cryopreserved in an egg yolk based extender. Ejaculates were collected from a boar previously selected and semen samples were processed using the straw freezing procedure. In experiment 1, subsamples of semen were frozen in three different extenders: recommended lactose egg yolk extender (LEY); trehalose egg yolk extender (TEY) and glucose egg yolk extender (GEY). Sperm quality was assessed for motility, viability, acrosome integrity and hypoosmotic swelling test response upon collection, after freezing and thawing and then every hour for 3 h. Results showed that total motility at 1 and 3 h, progressive motility at 3 h, positive hypoosmotic response at 2 and 3 h and acrosome integrity at all times were significantly improved when trehalose was added to the extender. In experiment 2, sugar influence was also demonstrated in vitro fertilization. A total of 1691 oocytes were in vitro matured and inseminated with frozen-thawed sperm at 2000:1 sperm:oocyte ratio and coincubated for 6 h. Presumptive zygotes were cultured in NCSU-23 medium to assess fertilization parameters and embryo development. Both penetration and monospermy rates were significantly higher for trehalose frozen semen. A significant increase was observed in efficiency and blastocyst formation rates from TEY to the other groups. Our results demonstrated that trehalose extender enhances spermatozoa viability and its in vitro fertilization parameters in boar ejaculates with good sperm freezability. Further studies are necessary to assess the impact of sugars on the entire population.     

Viability and acrosome integrity of rabbit spermatozoa processed in a gelatin-supplemented extender

The effect of gelatin addition to the semen extender on the viability and acrosome integrity of rabbit spermatozoa was studied. Pooled semen samples were processed in a boar semen extender with or without gelatin addition. Semen samples were stored at 5 °C for 72 h. Viability and acrosome integrity was evaluated by light microscope. Results showed that gelatin addition had a significant positive effect on the quality of the stored semen.     

Effect of buffalo seminal plasma heparin binding protein (HBP) on freezability and in vitro fertility of buffalo cauda spermatozoa

The study was conducted to assess the effect of heparin binding seminal plasma proteins (HBP) on freezability and in vitro fertilizing ability of buffalo cauda epididymal spermatozoa. Spermatozoal motility, viability and acrosomal integrity at prefreeze and post-thaw stages were studied. The in vitro fertilizing ability of spermatozoa was assessed by the application of two tests, i.e. bovine cervical mucus penetration test (BCMPT) and hypo-osmotic sperm swelling test (HOST). HBP isolated from buffalo seminal plasma and maintained in the laboratory were used for the study. Twelve pairs of epididymis from adult buffaloes slaughtered at the local abattoir were used for the study. The results indicated that HBP addition improved the progressive motility, BCMPT and HOST response at prefreeze level. HBP at a concentration of 40 microg/ml showed better results than HBP at a concentration of 80 microg/ml. However, subjecting the HBP treated spermatozoa to cryopreservation resulted in significant reduction of motility, viability, acrosomal integrity and response to BCMPT and HOST in the HBP treated groups when compared to those in control group. The deleterious effect of HBP was found to be concentration dependent with the higher concentration causing higher post-thaw damage.     

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.     

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.     

Effect of solid storage on caprine semen conservation at 5 degrees C

In this work, we investigated the effect of storage in solid-phase extender on buck semen conserved at 5 degrees C. Furthermore, we studied the effect of addition of cysteine to the extender and the washing of seminal plasma on sperm survival. In Experiment 1, milk-based extender (M) was used as a control to study the effect of solid media storage (G) and cysteine supplementation (C), and the combination of both (GC), on in vitro sperm survival of washed and non-washed semen, conserved up to 72 h at 5 degrees C. Motility, acrosome integrity (NAR) and hypo-osmotic swelling tests (HOST) were evaluated to assess in vitro sperm survival. In Experiment 2, an artificial insemination (AI) field trial was performed to compare G versus M. Solid media (G) maintained motility of spermatozoa during storage higher than any other extender (67% G versus 62% GC; 61% M and 59% C; P<0.05), but there was no difference in NAR or HOST between extenders (P>0.05). No improvement in sperm viability was obtained by addition of cysteine to the media. Washing of semen improved motility (65% versus 60%; P<0.05), NAR (70% versus 64%; P<0.05) and HOST (37% versus 28%; P<0.05). No significant differences in fertility were obtained between G and M extenders (47% versus 41%; P>0.05). In conclusion, washing of semen and dilution in gelatin-supplemented milk extender (solid storage) appears to be a successful method for goat semen storage at 5 degrees C.     

Idebenone improves quality of ram sperm by mitigating oxidative stress during cryopreservation

The present study was designed to test the effect of different levels of idebenone, a potent antioxidant on the quality of ram semen at post thaw. Eighteen (18) ejaculates were collected and extended with tris extender supplemented with no antioxidant (CON), with 2 μM idebenone (Id2), 5 μM idebenone (Id5), 7.5 μM idebenone (Id7.5) and 10 μM idebenone (Id10). The sperm quality was determined in terms of percent sperm motility, live sperm percentage, percent hypoosmotic swelling test (HOST) positive spermatozoa and percent intact acrosome (PIA). Moreover, malondialdehyde (MDA) level, an end product of lipid peroxidation (LPO) was also measured at post thaw both in seminal plasma and sperm cell. At post thaw, the percent sperm motility was significantly higher (p < 0.05) for Id10 as compared to Id2, Id5, Id7.5 and control. The live sperm percentage was non-significantly (p > 0.05) higher for Id10 as compared to control, Id5 and Id7.5 but significantly higher than Id2. The percent HOST positive spermatozoa was significantly higher (p < 0.05) for Id10 than control, Id2 and Id5. The MDA level in seminal plasma was significantly lower (p < 0.05) for Id10 than control and Id2. The MDA level in spermatozoa did show similar trend as in seminal plasma. Further, all the sperm parameters at all idebenone levels declined significantly from pre freeze to post thaw. In conclusion, idebenone at 10 μM level improved post thaw sperm quality by mitigating peroxidative stress, hence could be considered as a promising antioxidant additive for cryopreservation of ram semen.     

Undiluted or extended storage of ram epididymal spermatozoa as alternatives to refrigerating the whole epididymes

The effect of storage procedure at 5°C on the quality of ram spermatozoa from the cauda epididymis was analyzed. Two strategies were tested at 0, 24, 48 and 72h post-mortem: (1) spermatozoa held in the epididymal fluid and stored either in the cauda epididymis (In-EPID) or in vitro (Ex-EPID), (2) epididymal spermatozoa extended in three media at 320, 370 and 420 mOsm/kg (D320, D370, D420). Analyzed parameters were: osmolality, pH, motility, acrosomal status and viability. In experiment 1, osmolality of the In-EPID samples, but not in Ex-EPID, increased with post-mortem time. Motility of In-EPID spermatozoa in samples, after 24h post-mortem, was higher compared to the Ex-EPID samples, although differences decreased at 48 and 72h. In experiment 2, total (TM) and progressive motility (PM) were not significantly affected by storage time for D320 and In-EPID samples. However, the motility of D370 and D420 samples significantly decreased with time. TM and PM of D320 were significantly higher than D370 and D420 at 72h. At 24h, sperm viability was higher for In-EPID (80.7±3.4%) than for the extended samples (44.8±2.9%, 37.7±3.9% and 48.6±6.0% for D320, D370 and D420, respectively), which also decreased faster with time. At 24h, the percentage of damaged acrosomes was low and similar for the four methods of storage, but damaged acrosomes increased with time for D320 and D370. Storing the spermatozoa in the epididymis is a good strategy for maintaining sperm quality in ram, at least for 48h. The D320 extender preserve motility of epididymal spermatozoa but does not protect the status of the acrosome.     

Influence of porcine spermadhesins on the susceptibility of boar spermatozoa to high dilution

The effect of heparin-binding and non-heparin-binding spermadhesins on the viability, motility, and mitochondrial activity of boar spermatozoa at the high dilution (300,000 sperm/ml) to which sperm are exposed during the process of sex sorting by flow cytometry was investigated. Incubation of spermatozoa with heparin-binding spermadhesins caused a time- and dose-dependent decrease in the percentage of functional spermatozoa. The percentage of viable spermatozoa incubated at 38 degrees C with heparin-binding spermadhesins diluted in PBS (1 mg/ml) dropped from 75% (0.5 h) to 4% (5 h), whereas the percentage of viable spermatozoa incubated in PBS without proteins (control) decreased from 85% (0.5 h) to 19% (5 h). Addition of non-heparin-binding PSP-I/PSP-II spermadhesin to the PBS resulted in a concentration-dependent increment of the percentage of viable cells (65% after 5-h incubation), with maximum effect at 1.5 mg/ml. The heparin-binding spermadhesins totally suppressed sperm motility and mitochondrial activity after 5 h of incubation. The same parameters of sperm incubated in the presence of 1.5 mg/ml of PSP-I/PSP-II were 50% and 58%, respectively, and the percentages of control sperm displaying motility and mitochondrial activity were 21% and 26%, respectively. Moreover, the viability, motility, and mitochondrial activity all decreased on incubation of spermatozoa with mixtures of PSP-I/PSP-II and heparin-binding spermadhesins as the concentration of the latter increased. We conclude that PSP-I/PSP-II and the heparin-binding spermadhesins exert antagonistic effects on the functionality of highly diluted boar spermatozoa. The finding that PSP-I/PSP-II contributes to maintaining sperm with high viability, motility, and mitochondrial activity for at least 5 h at physiological temperature points to its potential use as an additive for sperm preservation, specifically of highly diluted, flow-sorted spermatozoa for sex preselection.     

The ideal holding time for boar semen is 24 h at 17 °C prior to short-cryopreservation protocols

Boar semen cannot be immediately cryopreserved, it need be hold at 17 °C prior to cryopreservation, holding time has been used to improve cryopreserved boar semen, since holding time allows a prolonged interaction between spermatozoa and seminal plasma components. However, until now only few periods of holding time have been studied, and boar semen had been held at 17 °C for 24 h to facilitate its manufacture. Thus, this experiment aims to study the effect several holding time (0, 4, 8, 12, 24, 28 and 32 h) on boar spermatozoa post-thawed (PT) characteristics. Fifteen sperm-rich fractions of ejaculate were extended in Beltsville Thawing Solution and storage at 17 °C. After each holding time (0, 4, 8, 12, 24, 28 and 32 h), a sample was centrifuged, and sperm pellet was diluted in an extender composed of sugars, amino acids, buffers, 20% egg yolk (v/v), antibiotics, 2% glycerol as a cryoprotectant, and 2% methylformamide (v/v). Cryopreservation was performed with an automatic cryopreservation system. Cryopreserved boar semen was evaluated to spermatozoa kinetics, plasma and acrosomal membranes integrity, mitochondrial membrane potential, detection of superoxide anion, plasma membrane fluidity, and peroxidation. Twenty-four hours of holding increase total and progressive motility, rapid spermatozoa, and integrity of plasma and acrosome membranes. To mitochondrial membrane potential, 32 h is needed. However, holding time was not able to control the superoxide anion amount neither membrane lipid peroxidation, and had no effects on membrane fluidity. Thus, to reach the best results of PT boar semen the ideal holding time is 24 h.     

The effect of antioxidants on motility, viability, acrosome integrity and DNA integrity of frozen-thawed epididymal cat spermatozoa

Antioxidants partially ameliorated the negative effects of reactive oxygen species (ROS) produced during cryopreservation. The objective of the present study was to investigate the effect of cysteine and a water-soluble vitamin E analogue on the quality of frozen-thawed epididymal cat spermatozoa. Epididymal spermatozoa were collected from eight male cats and divided into three aliquots; these were resuspended with a tris egg yolk extender I (EE-I), or the same extender supplemented with 5mM dl-cysteine (EE-C) or with 5mM of a water-soluble vitamin E analogue (EE-Ve). Prior to the freezing step, sperm suspensions were added to the extender with Equex STM paste (EE-II). Sperm motility, progressive motility, membrane integrity, and acrosome status were evaluated at collection, after cooling, and at 0, 2, 4, and 6h post-thaw. Sperm DNA integrity was evaluated at 0 and 6h post-thaw. Relative to the control group, supplementation with vitamin E improved (P<0.05) post-thaw motility (69.4+/-5.6%), progressive motility (3.9+/-0.3), and membrane integrity (65.1+/-8.1%) immediately after thawing, whereas cysteine supplementation improved (P<0.05) post-thaw motility after 2h of incubation (53.8+/-12.2%) and DNA integrity after 6h (84.1+/-4.4%). However, neither antioxidant significantly increased the acrosome integrity of frozen-thawed spermatozoa. In conclusion, cysteine or vitamin E supplementation of tris egg yolk extender improved motility, progressive motility and integrity of the sperm membrane and DNA of frozen-thawed epididymal cat spermatozoa.     

Post-mortem semen cryopreservation and characterization in two different endangered gazelle species (Gazella gazella and Gazella dorcas) and one subspecies (Gazella gazelle acaiae)

Both Gazella gazella and Gazella dorcas are endangered species with continually dwindling population size, yet basic knowledge on their spermatozoa is missing. Semen collected post-mortem (PM) from the cauda epididymis of five adult gazelles (three Gazella gazella gazella, one Gazella gazella acaiae and one G. dorcas) was cryopreserved using directional freezing of large volumes (8 mL) with egg-yolk-free extender. Sperm size measurements and SYBR-14/propodium iodide (PI) viability stain validation for use in gazelles were conducted. Post-thaw characterization included motility, viability, acrosome damage evaluation, computerized motility characterization and morphology and sperm motility index (SMI) was calculated. Extracted sperm motility was 71.67+/-11.67% (mean+/-S.E.M.). Post-thaw motility ranged between 15% and 63%, viability was 57.49+/-3.24%, intact acrosome was detected in 63.74+/-2.6% (median 64.8%, upper/lower quartiles 71.79%, 61.82%), and normal morphology ranged between 41% and 63%. Motility characterization showed two sub-groups-highly active and progressively motile spermatozoa with SMI of 62.75+/-0.38 and low activity and poorly progressive with SMI of 46.16+/-1.53. Our results indicate that PM preservation of gazelle spermatozoa with satisfactory post-thaw viability is possible and cryobanking is achievable.     

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.     

Cryopreservation of goat spermatozoa: comparison of two freezing extenders based on post-thaw sperm quality and fertility rates after artificial insemination

TRIS-glucose or skim milk extenders are most commonly used for cryopreserving goat sperm. The aim of this study was to compare the ability of two extenders based on TRIS and skimmed milk buffer to maintain sperm viability after cryopreservation. Goat semen samples (n=110) were frozen with TRIS and with milk extender and thaw. Sperm motion parameters, morphology and acrosomal integrity were assessed in fresh and frozen-thawed samples by Sperm Class Analyzer (SCA) and Diff-Quik and Spermac staining techniques. Pregnancy rates were obtained after cervical insemination with frozen semen doses. The cryopreservation process had a significant effect on acrosome and kinematic parameters. TRIS extender provided more effective preservation of total motility, velocity parameters and amplitude of lateral head displacement after freezing. The percentage of acrosome intact spermatozoa was significantly higher in samples diluted with milk extender. In the insemination doses, mean values of velocity parameters and lateral head displacement were higher in doses processed in TRIS. Spermatozoa frozen in milk extender was mathematically greater than for those frozen with TRIS extenders, though no significant difference exists. We conclude that post-thaw kinematic parameters and acrosome integrity assessed after 1h of incubation was acceptable in both extenders which indicated the feasibility of cryopreserving goat spermatozoa. TRIS extender results in better in vitro performance compared to milk, though these improvements were not reflected in fertility results. Semen doses cryopreserved in milk extender provided greater pregnancy rates after intra-cervical insemination compared to those in TRIS extender (52.4% versus 42.9%).     

Vitamin E Analogue Improves Rabbit Sperm Quality during the Process of Cryopreservation through Its Antioxidative Action

The process of cryopreservation results in high concentration of reactive oxygen species which is detrimental to spermatozoa. The aim of this study was to investigate whether addition of vitamin E analogue to freezing extender can facilitate the cryosurvival of spermatozoa in rabbits, and how vitamin E protects spermatozoa against damages during the process of preservation. Freshly ejaculated semen was diluted with Tris-citrate-glucose extender supplemented with different concentrations of Trolox (a vitamin E analogue). The level of radical oxygen species (ROS) in spermatozoa that was exposed to Trolox was significantly lower than that of the control during each step of the process of preservation. The percentage of frozen-thawed spermatozoa with lipid peroxidation in the Trolox treatments was significantly lower than that of the control. The motility, intact acrosome, membrane integrity and mitochondrial potentials of the frozen-thawed spermatozoa in the treatment of 200 μM Trolox were significantly higher than those of the control. These observations suggest that addition of vitamin E to a freezing extender leads to higher integrity of acrosome, plasma membrane and mitochondrial membrane potential as well as higher motility. Vitamin E protects spermatozoa through its capacity to quench ROS accumulation and lipid peroxidation during the process of preservation. Addition of Trolox is recommended to facilitate the improvement of semen preservation for the rabbit breeding industry.     

Boar sperm velocity and motility patterns under capacitating and non-capacitating incubation conditions

This study compares the velocity and motility of boar sperm under capacitating and non-capacitating incubation conditions. Aliquots of pooled, washed boar sperm were incubated in either Tyrode's complete medium (TCM; a capacitating medium), Ca2+-free TCM (TCM-Ca2+), or Ca2+ and NaHCO3-free TCM (Tyrode's basal medium [TBM]; a non-capacitating medium). Motility patterns were determined every hour over a 3h period of incubation at 38 degrees C. Capacitation status was assessed by the chlortetracycline assay after 1 and 3h of incubation. Experiments were repeated five times. Compared to the TBM control, a significant increase was seen in the percentage of capacitated sperm after 1h of incubation in TCM: the kinematics of these sperm cells were favorably modified. However, the motility patterns of sperm cells incubated in TCM and TCM-Ca2+ were very similar. Under capacitating conditions (TCM), the coefficients of linearity (LIN) and straightness (STR) significantly increased over time (LIN values were significantly different after 3h of incubation, while STR values were significantly different after only 2 h). Significant correlations were seen between LIN and the percentage of cells showing the B pattern (r = 0.334, P < 0.05) and the number of acrosome reacted spermatozoa (r = 0.301, P < 0.05). This suggests that capacitated boar spermatozoa may have a species-specific motility pattern.     

Influence of sugar supplementation of the extender on motility, viability and acrosomal integrity of dog spermatozoa during freezing

Influence of different sugars supplemented to the extender on the motility, viability and intact acrosome rates of dog spermatozoa during dilution, equilibration and freezing was studied. The ejaculate was divided into 10 aliquots, which were diluted 1:3 with TRIS-citric acid extender containing 240 mMTRIS, 63 mM citric acid, 8% (v/v) glycerol, 20% (v/v) egg yolk and 70 mM sugar, which was either fructose, galactose, glucose, xylose (monosaccharide), lactose, trehalose, maltose, sucrose (disaccharide) or raffinose (trisaccharide). No sugar was added to the extender in the control group. Extended semen samples were cooled to 5 degrees C over 45 min, packaged in 0.25-mL straws, equilibrated for 2 h at 5 degrees C and frozen in liquid nitrogen vapor. Samples were thawed by placing straws into 37 degrees C water for 30 sec. Motility, viable sperm and intact acrosome rates decreased gradually in all groups after equilibration and consecutively freezing (P<0.001). The type of sugar significantly effected motility, viability and acrosomal integrity during equilibration and freezing (P<0.05). Galactose, lactose, trehalose, maltose and sucrose reduced damaged acrosome percentages in equilibrated samples (P<0.05). Sugar supplementation did not enhance motility and viability during equilibration. The disaccharides, except lactose, reduced post-thaw dead sperm and/or damaged acrosome percentages without promoting post-thaw motility (P<0.01), whereas monosaccharides, especially fructose and xylose, improved motility (P<0.05) along with viability and intact acrosome rates (P<0.05). Trehalose, xylose and fructose significantly increased total active sperm rates (motility x live sperm rate x normal acrosome rate) compared to other sugars (P<0.01) and control (P<0.0001) in frozen thawed samples. Therefore, sugar supplementation of the extender influenced post-equilibration and post-thaw sperm quality, and the type or locality of protective impact of the sugar on dog spermatozoa vary according to type of the sugar.