Tris-citric Acid Research Articles

71 BOVINE SPERM DEATH KINETICS: CHANGES IN MOTILITY, ACROSOMES, AND PLASMA MEMBRANE

Extent and timing of alterations in structures and functions of sperm after its placement in the female reproductive tract are important for successful fertilization. To our knowledge, the few reports are available on the kinetics of alterations in bovine sperm structures and functions during pathway to their death. Therefore, the present study was conducted to determine the changes in motility, acrosome and plasma membrane asymmetry in fresh and frozen–thawed semen during incubation at 37°C over the period of 24 h. Semen was collected from 3 breeding beef bulls, pooled, and considered as one replicate (total replicates = 5). Each pooled semen sample was diluted in Tris-citric acid egg yolk glycerol extender (pH 6.8), cooled to +4°C over 90 min, and then cryopreserved by a programmable cell freezer. Fresh (pooled semen) and frozen–thawed semen were incubated at 37°C for 24 h. Each semen sample was evaluated for sperm motility with computer-assisted semen analysis and acrosomal integrity and plasma membrane asymmetry using fluorescein isothiocyanate-peanut agglutinin/propidium iodide and Annexin V/propidium iodide assays, respectively, at 0, 2, 4, 6, 12, and 24 h of incubation at 37°C, with a flow cytometer. Statistical analysis was conducted using PROC MIXED model in statistical analysis system as 2 (semen types) × 6 (times) factorial model, using time as repeated measure. Progressive motility was higher (P < 0.05) in fresh than in frozen–thawed semen until 6 h. Progressive motility declined (P < 0.05) below the threshold level (i.e. 30%) much later (12 h) in fresh as compared with frozen–thawed semen (2 h). However, acrosomal integrity and plasma membrane asymmetry deteriorated (P < 0.05) below threshold at the same time interval (2 h) in both fresh and frozen–thawed semen. Viable sperm (AN–/PI–) remained higher (P < 0.05) during the first 6 h in fresh than in frozen–thawed semen and declined (P < 0.05) below the threshold at 12 h in fresh and at 6 h in frozen–thawed semen. In fresh semen, the necrotic sperm (AN–/PI+) population increased (P < 0.05) over time and reached maximum (97%) at 24 h. In frozen–thawed semen, a mixed population of late apoptotic (53%) and necrotic (34%) sperm was found at 24 h. In conclusion, the alterations in sperm motility, acrosomes, plasma membrane integrity, and asymmetry were slower in fresh than in frozen–thawed semen. Fresh sperm followed necrosis and frozen–thawed sperm underwent necrosis and apoptosis-like pathways, respectively. This study was supported by the Canadian Commonwealth Scholarship Program by the Canadian Bureau for International Education (CBIE), and Agriculture and Agri-Food Canada.

Cryopreservation of aoudad (Ammotragus lervia sahariensis) sperm obtained by transrectal ultrasound-guided massage of the accessory sex glands and electroejaculation

This study examines (1) the effectiveness of transrectal, ultrasound-guided massage of the accessory sex glands (TUMASG) combined with electroejaculation for obtaining aoudad (Ammotragus lervia sahariensis) sperm samples for cryopreservation, and (2) the effectiveness of a Tris-citric acid–glucose-based medium (TCG; usually used for freezing ibex sperm) and a TES-Tris–glucose-based medium (TTG; typically used in the cryopreservation of mouflon sperm) as sperm extenders. After TUMASG, just one to three electrical pulses were required for ejaculation to occur in five of the six animals studied; one ejaculated after TUMASG alone. Transrectal, ultrasound-guided massage of the accessory sex glands would therefore appear to be useful in obtaining sperm samples from this species, requiring few subsequent electrical electroejaculation stimuli and sometimes none at all. After thawing, the membrane integrity (assessed by nigrosin-eosin staining) of sperm extended with TTG was greater than that of sperm extended with TCG (P < 0.05). The total percentage of sperm showing an intact acrosome, as assessed by fluorescein isothiocyanate-conjugated peanut (Arachis hypogea) agglutinin, was also higher in the TTG-extended sperm (P < 0.05), and the percentage of dead sperm with a damaged acrosome was lower (P < 0.05). No differences were seen between TCG and TTG in terms of apoptotic manifestations (DNA damage, caspase activity, mitochondrial membrane potential, and plasmalemma stability). Therefore, TTG appears to be a better extender than TCG for cryopreserving aoudad sperm.

Glutathione-supplemented tris-citric acid extender improves the post-thaw quality and in vivo fertility of buffalo (Bubalus bubalis) bull spermatozoa

In this study we evaluated the effects of semen extender supplementation with different concentrations of glutathione (GSH) on buffalo (Bubalus bubalis) bull sperm motility, plasma membrane integrity, viability and DNA integrity as well as in vivo fertility. Semen from three Nili-Ravi buffalo bulls was collected, and qualified semen ejaculates (n=18) were split into five aliquots for dilution (37°C; 50×106spermatozoaml−1) with experimental tris-citric acid extender containing 0, 0.5, 1.0, 1.5 or 2.0mM GSH. Extended semen was cooled to 4°C, equilibrated and filled in French straws. The straws were kept on liquid nitrogen vapors (5cm above the LN2 level) for 10min and plunged in liquid nitrogen for storage. Sperm motility (%), plasma membrane integrity (%), viability (%) and DNA integrity (%) were assessed at 0, 2 and 4h post-thawing (37°C). Extender supplementation with GSH (0.5, 1.0, 1.5 and 2.0mM) increased sperm motility, plasma membrane integrity and viability in a dose dependent manner. Sperm DNA integrity was higher (p<0.05) in all experimental extenders containing GSH when compared to the control extender (0mM GSH). The in vivo fertility rate of cryopreserved buffalo bull (n=2) spermatozoa was higher (p<0.05) in extender containing 2.0mM GSH compared to that of control. In summary, tris-citric acid extender supplemented with glutathione improved the freezability of buffalo bull spermatozoa in a dose dependant manner. Moreover, the addition of 2.0mM GSH to the extender enhanced the in vivo fertility of buffalo (Bubalus bubalis) bull spermatozoa.

Effect of antioxidants on post thaw microscopic, oxidative stress parameter and fertility of Boer goat spermatozoa in Tris egg yolk glycerol extender

This study was conducted to determine the effect of antioxidants on standard semen parameters, lipid peroxidation and fertility of Boer goat semen after cryopreservation. Ejaculates from four bucks were collected, evaluated and pooled at 37°C. The pooled semen was diluted with Tris citric acid fructose for washing. Semen samples, which were diluted with a Tris-based extender containing the antioxidant ascorbic acid (8.5mg/ml), butylated hydroxytoluene (2mM), cysteine (5mM) and hypotaurine (10mM) and an extender without antioxidant supplementation were cooled to 4°C and frozen in 0.25 straws with programmable freezer and finally stored in liquid nitrogen. Data (10 replicates) were analyzed by one-way analysis of variance. Mean (±SEM) progressive motility was significantly higher in ascorbic acid than other supplement groups and control samples (P>0.05). Best values were observed in ascorbic acid followed by BHT, cysteine, and hypotaurine. Antioxidant supplementation in extender showed significant (P<0.05) better values than the control group for sperm membrane integrity, acrosome integrity and viability. The ability of antioxidants to reduce the lipid peroxidation (LPO) after freeze thawing was measured by the formation of malondialdehyde (MDA) using the thiobarbituric acid method. Results showed that addition of antioxidants significantly reduced the rate of LPO in comparison to control (P<0.05). Ascorbic acid exhibited better values (1.27±0.28), than butylated hydroxytoluene, cysteine and hypotaurine 1.32±0.42, 2.27±0.16 and 2.38±0.17 respectively, which are significantly better than control (3.52±0.54). Higher pregnancy rate was observed with ascorbic acid followed by butylated hydroxtolune, hypotaurine and cysteine. However, differences in the fertility rate were non-significant with hypotaurine, cysteine and control groups.

A strategy for improvement of postthaw quality of bison sperm

The objective was to improve the postthaw quality of bison semen using zwitterion (ZI)-based extenders, glycerol addition at a lower temperature (4 °C), adding reduced glutathione (GSH) in extender, or treating bison sperm with cholesterol-loaded cyclodextrin (CLC) before freezing. Postthaw sperm motility and structural characteristics were analyzed using a computer-assisted sperm analyzer and flow cytometer respectively, at 0 and 3 hours postthaw incubation at 37 °C. In experiment 1, each ejaculate (N = 11) was diluted in Triladyl extender (control) or in ZI extenders (Tes-Tris or HEPES-Tris). In addition, glycerol in semen was added either at 37 °C or 4 °C before cryopreservation. Extenders had no significant effect on postthaw sperm motilities at 0 hour. However, sperm velocity parameters were higher (P < 0.05) in ZI extenders than in Triladyl. Sperm population with intact plasma membrane (IPM) and acrosomes (IACR) were higher in Triladyl than in ZI extenders (P < 0.05). Postthaw sperm total and progressive motilities, average path velocity, straight-line velocity, IPM, and IPM-IACR did not improve with the addition of glycerol at 4 °C. In experiment 2, semen was diluted (50 × 106 sperm per mL) in Triladyl extender containing 0 (control), 0.5, 1.0, or 2.0 mM GSH (an antioxidant) at 37 °C. Postthaw sperm motility and structural characteristics at 0 hour and percentage declined after 3 hour incubation, but did not differ because of GSH in the extender (P > 0.05). In experiment 3, fresh bison sperm (100 × 106 sperm in 1 mL) were pretreated with 0, 1, 2, or 3 mg/mL of CLC at 22 °C for 15 minutes and diluted to 50 × 106 sperm per mL in Tris-citric acid–egg yolk–glycerol extender before cryopreservation. The CLC pretreated sperm had higher (P < 0.05) postthaw total and progressive motilities, IPM, and IACR at 0 hour and less percentage of decline in these characteristics after 3 hour postthaw incubation. In conclusion, zwitterion extenders (Tes-Tris and HEPES-Tris), temperatures of glycerol addition, and GSH in extender did not significantly improve postthaw quality of bison sperm. However, pretreatment with CLC significantly improved postthaw quality of bison sperm, which should enhance its use in assisted reproductive technologies.

Quantification of damage at different stages of cryopreservation of endangered North American bison ( Bison bison) semen and the effects of extender and freeze rate on post-thaw sperm quality

Semen cryopreservation is an important technique for the banking of animal germplasm from endangered species and exploitation of genetically superior sires through artificial insemination. Being a member of bovidae family, bison semen has poor freezing ability as compared to dairy and beef bulls’ semen. This study was designed to quantify the damage to bison sperm at different stages of cryopreservation, and to determine the effects of extender (commercial Triladyl ® vs. custom made tris-citric acid [TCA]) and freeze rate (−10, −25 and −40 °C/min) on post-thaw quality of bison semen. Semen was collected from five bison bulls (three woods and two plains) via electroejaculation. In Experiment 1, semen was diluted in Triladyl ® extender and frozen with freeze rate −10 °C/min. Sperm motility characteristics were recorded in fresh, diluted, cooled (4 °C) and freeze-thawed semen using computer-assisted sperm analyzer (CASA). In Experiment 2, semen was diluted in Triladyl ® or TCA extender, and frozen with three different freeze rates, i.e. −10, −25 or −40 °C/min. Thawing was performed at 37 °C for 60 s. Post-thaw sperm motility characteristics were assessed using CASA, and sperm structural characteristics (plasma membrane, mitochondrial membrane potential and acrosomes) were evaluated using flow cytometer, at 0 and 3 h while incubating semen at 37 °C. In Experiment 1, total and progressive motilities did not differ among pre-freeze stages of cryopreservation ( P > 0.05). However, sperm total and progressive motilities declined ( P < 0.001) in freeze-thawed semen by 35% and 42%, respectively, compared to after cooling (pre-freeze) semen. In Experiment 2, Triladyl ®, as compared to TCA, yielded greater ( P < 0.05) post-thaw sperm total motility (41% compared to 36%) and progressive motility (34% compared to 29%) at 0 h, respectively. The percent change in post-thaw sperm total and progressive motilities, VAP, VCL, VSL, IPM-high Δ Ψm and IPM-IACR during 3 h incubation at 37 °C, was less ( P < 0.05) in TCA than in Triladyl ®. There was an effect of freeze rate on post-thaw sperm average path velocity at 0 h, and total motility, progressive motility, VCL, IPM and IPM-IACR at 3 h were the greatest ( P < 0.05) when bison semen was frozen at −40 °C/min. Likewise, the percent change in post-thaw sperm total and progressive motilities, during 3 h incubation at 37 °C, was less ( P < 0.05) in bison semen frozen at −40 °C/min. All post-thaw bison sperm characteristics decreased ( P < 0.05) from 0 h to 3 h, during incubation at 37 °C. In conclusion, the maximum damage to bison sperm occurred during freeze–thaw processes. Post-thaw total and progressive motilities of bison sperm were greater in Triladyl ® at 0 h whereas sperm survival was greater in TCA extender during 3 h post-thaw incubation. Bison sperm had greater survival ( P < 0.05) when frozen at −40 °C/min freeze rate.

Effect of low density lipoproteins in extender on freezability and fertility of buffalo (Bubalus bubalis) bull semen

This study was designed to determine whether low-density lipoporoteins (LDLs) extracted from egg yolk in extender improve the freezability and fertility of buffalo bull semen. Semen from three Nili-Ravi buffalo bulls was diluted at 37 °C with tris-citric acid extender (50 × 106 motile spermatozoa mL−1) containing LDLs 2.5%, 5%, 10%, and 15% extracted from egg yolk and extender containing 20% egg yolk was kept as control. Diluted semen was cooled to 4 °C in 2 h, equilibrated at 4 °C for 4 h, filled in 0.5 mL French straws, and kept on liquid nitrogen vapors for 10 min. Straws were then plunged and stored in liquid nitrogen (–196 °C). Sperm motility (visually; %), plasma membrane integrity (%; with supravital hypo-osmotic swelling test), and viability (%; with dual staining test using Trypan-blue Giemsa) were assessed at post-dilution, pre-freezing and post-thawing. At post-dilution and pre-freezing, sperm progressive motility, plasma membrane integrity and viability was similar (P > 0.05) in extender containing 10% LDLs or the control. However, at post-thaw the aforementioned parameters were higher (P < 0.05) in extender containing 10% LDLs compared with the control and other experimental extenders. The fertility rate of inseminations performed were higher (P < 0.05) with extender containing 10% LDLs than the control. It was concluded that LDLs (10%) in extender improved the freezability and fertility of buffalo bull spermatozoa.

Spermatozoa recovery and post-thawing quality of brown bear ejaculates is affected for centrifugation regimes

Sperm cryopreservation protocols for brown bear (Ursus arctos) require the centrifugation of semen samples to increase sperm concentration and to clean urine in contaminated samples. We evaluated the effect of centrifugation regimes (time and relative centrifugal force—RCF) on the quantity of sperm recovered and the quality of post-thawed sperm. Thirteen brown bears were electroejaculated. The ejaculates were diluted 1:1 in Tris–citric acid–glucose (TCG) extender and centrifuged with different RCF/time combinations: 600×g, 1,200×g and 2,400×g, for 3, 6 or 12 min. After centrifugation, spermatozoa were diluted in TES–Tris–fructose extender with egg yolk and glycerol (final glycerol concentration of 8%) and frozen in 0.25-mL straws. In the post-thawed semen, motility was assessed by CASA, and acrosomal status (PNA-FITC), viability (SYBR-14 with propidium iodide) and chromatin status (SCSA) were determined by flow cytometry. The longest centrifugation time (12 min) significantly decreased some motility parameters. Sperm recovery significantly decreased in brown bear at 600×g. Our results suggest that brown bear spermatozoa are more sensitive to long centrifugation times than to high RCF. Centrifugation regimes showed no effects on the post-thawing chromatin status. We recommend preparing the brown bear semen for freezing by centrifugation 1,200×g or 2,400×g for 6 min, after electroejaculation and dilution 1:1 in TCG extender, since these procedures increase the spermatozoa recovery without harmful effects on the post-thawed quality of brown bear spermatozoa.

Cryopreservation of bull semen shipped overnight and its effect on post-thaw sperm motility, plasma membrane integrity, mitochondrial membrane potential and normal acrosomes

In the Canadian Animal Genetic Resource Program, bull semen is donated in frozen or fresh (diluted) states. This study was designed to assess the cryopreservation of diluted bull semen shipped at 4 °C overnight, and to determine the post-thaw quality of shipped semen using different straw volumes and freezing rates. Semen was collected from four breeding bulls (three ejaculates per bull). Semen was diluted in Tris–citric acid–egg yolk–glycerol (TEYG) extender, cooled to 4 °C and frozen as per routine (control semen). After cooling to 4 °C, a part of semen was removed and shipped overnight to the research laboratory via express courier (shipped semen). Semen was packaged in 0.25 or 0.5 ml straws and frozen in a programmable freezer using three freezing rates, i.e., −10, −25 or −40 °C/min. Control semen was also shipped to the research laboratory. Post-thaw sperm motility characteristics were assessed using CASA, and post-thaw sperm plasma membrane, mitochondrial membrane potential and normal acrosomes were assessed using flow cytometry. Post-thaw sperm quality was greater in shipped semen as compared to control ( P < 0.001). The shipped semen packaged in 0.25 ml straws had better post-thaw sperm quality than in 0.5 ml straws ( P < 0.001). Freezing rate had no effect on post-thaw sperm quality. In conclusion, bull semen can be shipped overnight for subsequent cryopreservation and gene banking. Overnight shipping of semen was found advantageous for bull semen cryopreservation. Semen packaging in 0.25 ml straws yielded better post-thaw quality than 0.5 ml straws.

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 × 10 9 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.

Response of buffalo spermatozoa to low temperatures during cryopreservation

This is the first detailed report on the response of buffalo spermatozoa to low temperatures during freezing. The study determined the critical temperature zone for buffalo spermatozoa and developed a suitable freezing rate for this species. Semen from four Nili-Ravi buffalo bulls diluted in Tris-citric acid was frozen in a programmable freezer. Motion characteristics, plasma membrane integrity and acrosome morphology were determined at +4, 0, -5, -10, -20, -30, -40, -50, -80 and -196 degrees C by removing semen straws from the freezer at exactly these temperatures and rewarming them at 37 degrees C. The first statistical decline in sperm motility and lateral head displacement was observed at -40 degrees C. For all other parameters, there was biphasic decline: for curvilinear velocity, at 0 degrees C and -50 degrees C; and for plasma membrane integrity and acrosome morphology, at -30 degrees C and -50 degrees C. In a second series of experiments, buffalo spermatozoa were frozen using slow (-10 degrees C min(-1)), medium (-20 degrees C min(-1)) or fast (-30 degrees C min(-1)) freezing rates, between -10 degrees C and -80 degrees C. Freezing of buffalo spermatozoa at a rate of -30 degrees C min(-1) yielded higher post-thaw motion characteristics, plasma membrane integrity and normal acrosomes. In conclusion, different sperm characteristics respond differently at low temperatures and the freezing of buffalo spermatozoa at a higher rate ensures higher post-thaw semen quality.

Prediction of electrophoretic enantioseparation of aromatic amino acids/esters through MIA-QSPR

Multivariate image analysis (MIA) descriptors have been applied to predict the enantiomer migration orders of a series of aromatic amino acids and aromatic amino esters. In MIA-QSPR, pixels of chemical structures (2D images) stand for descriptors, and structural changes account for the variance in relative migration times (RMTs). R and S enantiomers were differentiated by drawing up or down stereo bonds at the chiral carbon, and the RMT predictions of the title compounds in specific medium (20 mM Tris–citric acid background electrolyte (pH 2.50) containing 5.0 mM of (+)-18-crown-6-tetracarboxylic acid) were reliably obtained ( r 2 = 0.992, q LOO - CV 2 = 0.926 , and q L - 20 % - O - CV 2 = 0.910 ) after removing two outliers from the dataset. MIA descriptors were capable to recognize the physicochemical information and may be useful to predict enantiomer migration orders of amino acids and amino esters whose pure enantiomers are unavailable.

Cryopreservation of Spanish ibex ( Capra pyrenaica) sperm obtained by electroejaculation outside the rutting season

The effectiveness of electroejaculation for obtaining Spanish ibex sperm samples for freeze preserving outside the rutting season was evaluated—the aim being to optimise biological resources for the establishment of germplasm banks. The effect of different egg yolk concentrations (6% or 12%, v/v) in diluents of different buffer composition (Tris–citric acid buffer or Tes–Tris buffer) on frozen-thawed samples of the above also investigated. Experiments were undertaken with six ibex males in February–May, and involved four different semen samples from each animal with four combination of extender, respectively: Tes–Tris–glucose (TTG)–6% egg yolk, TTG–12% egg yolk, Tris–citric acid–glucose (TCG)–12% egg yolk, TCG–6% egg yolk. The results show that electroejaculation is a useful way of obtaining sperm samples from Spanish ibex outside the rutting season (i.e., at a time coinciding with plasma testosterone levels of <0.4 ng/ml). According to the results of the eosin–nigrosin staining and the hypo-osmotic swelling test, the freezing–thawing process significantly reduced the viability and membrane integrity of the spermatozoa extended with TTG–6% egg yolk, TTG–12% egg yolk, and TCG–12% egg yolk, but did not affect these variables in spermatozoa extended with TCG–6% egg yolk. Therefore, the use of Tris–citric acid-based extenders containing low concentrations of egg yolk is recommended for cryopreserving Spanish ibex spermatozoa obtained by electroejaculation outside the rutting season.

A comparison of the protective action of chicken and quail egg yolk in the cryopreservation of Spanish ibex epididymal spermatozoa

Egg yolk-based diluents provide adequate cryoprotection for the sperm of several mammalian species. Traditionally, chicken egg yolk has been used as additive for the freeze preservation of spermatozoa because of its wide availability. Variations in the chemical composition of the egg yolk of different avian species appear to influence the protection afforded during cooling, freezing, and thawing. The aim of the present study was to assess the use of quail egg yolk as a novel additive for the epididymal spermatozoa of a threatened wild ruminant species—the Spanish ibex—and to compare its efficacy with chicken egg yolk. Epididymal spermatozoa were diluted using a Tris–citric acid–glucose medium (TCG) composed of 3.8% Tris (w v −1), 2.2% citric acid (w v −1), 0.6% glucose (w v −1), 5% glycerol (v v −1), and 6% egg yolk (v v −1). Sperm masses from the right epididymes were diluted with TCG-6% chicken egg yolk medium, while those from the left were diluted with TCG-6% quail egg yolk. The thawed spermatozoa preserved with TCG-6% quail egg yolk extender exhibited lower motility ( P < 0.001), membrane integrity ( P < 0.001), and viability ( P < 0.01) than those diluted with the TCG-6% chicken egg yolk extender. The fertility of spermatozoa frozen in TCG-6% chicken egg yolk tended to be higher than in those frozen with TCG-6% quail egg yolk (63.3% vs 36.4%, P = 0.19). These results show that quail egg yolk offers no advantages over chicken egg yolk in the cryopreservation of Spanish ibex epididymal spermatozoa.

Sperm sex-sorting in the Asian elephant ( Elephas maximus)

In captive Asian elephants, there is a strong need for production of female offspring to enhance reproduction, counter premature aging processes in female animals and reduce challenging management situations derived from husbandry of several bulls in one institution. Artificial insemination of flow cytometrically sex-sorted spermatozoa offers the possibility to predetermine the sex of offspring with high accuracy. The aims of this study were to determine a suitable semen extender and basic parameters for flow cytometrical sex-sorting of Asian elephant spermatozoa. In total 18 semen samples were collected by manual rectal stimulation from one bull. Sperm quality parameters and sex sortability of spermatozoa were evaluated after dilution in three semen extenders (MES-HEPES-skim milk, MES-HEPES, TRIS–citric acid) and DNA staining. MES-HEPES-skim milk was the only semen extender found suitable to sex Asian elephant spermatozoa. From 18 ejaculates collected, 12 were successfully sorted with a purity of 94.5 ± 0.7% at an average sort rate of 1945.5 ± 187.5 spermatozoa per second. Sperm integrity, progressive and total motility were 42.6 ± 3.9%, 48.1 ± 3.3%, 59.4 ± 3.8% after DNA labelling, and 64.8 ± 3.2%, 58.0 ± 5.0%, 70.8 ± 4.4% after sorting, respectively. After liquid storage of sorted spermatozoa for 12 h at 4 °C, sperm integrity, progressive and total motility were 46.4 ± 5.2%, 32.2 ± 4.2% and 58.2 ± 3.9%, respectively. The obtained results provide a promising base to inseminate Asian elephants with sexed semen.

Antagonist effect of DMSO on the cryoprotection ability of glycerol during cryopreservation of buffalo sperm

The objective of the present study was to investigate the synergistic effect of DMSO and glycerol added at various temperatures on the post-thaw quality of buffalo sperm. Pooled ejaculates from four Nili-Ravi buffalo bulls were divided into 18 aliquots and extended (1:10) in Tris–citric acid extender differing in glycerol:DMSO ratios (0:0, 0:1.5, 0:3; 3:0, 3:1.5, 3:3; and 6:0, 6:1.5, 6:3, respectively; %, v:v) either at 37 or 4 °C. Semen was packaged in 0.5 mL French straws and frozen in a programmable cell freezer. Thawing was performed at 37 °C for 50 s. Post-thaw motion characteristics, plasma membrane integrity and acrosome morphology of buffalo sperm were determined using computer-assisted semen analyzer (CASA), hypoosmotic swelling (HOS) assay and phase-contrast microscopy, respectively. Glycerol (6%) in extender yielded better post-thaw sperm motility, velocities (straight-line and average path), plasma membrane integrity, and normal acrosomes ( P < 0.05). Post-thaw sperm motility and plasma membrane integrity declined in the presence of DMSO ( P < 0.01). The addition of glycerol (6%) at 37 °C yielded better post-thaw sperm motility, plasma membrane integrity and velocities than addition at 4 °C ( P < 0.05). In conclusion, glycerol is still an essential cryoprotectant for buffalo sperm. The addition of DMSO antagonized the cryoprotection ability of glycerol and reduced the post-thaw quality of buffalo sperm. Furthermore, 6% glycerol added at 37 °C, provided better cryoprotection to the motility apparatus and plasma membrane integrity of buffalo sperm.

Duck egg yolk in extender improves the freezability of buffalo bull spermatozoa

We investigated the use of duck egg yolk (DEY), Guinea fowl egg yolk (GFEY) and Indian indigenous hen (Desi) egg yolk (IDEY) in extender for improving the post-thaw quality of buffalo ( Bubalus bubalis) bull spermatozoa, and compared it with commercial hen egg yolk (CHEY; control). For this purpose, two consecutive ejaculates of semen from each of two Nili–Ravi buffalo bulls were collected on 1 day each week for 5 weeks (replicates; n = 5) with artificial vagina (42 °C). Split pooled ejaculates, were diluted in tris-citric acid glycerol extender containing either DEY or GFEY or IDEY or CHEY at 37 °C. Extended semen was cooled to 4 °C in 2 h and equilibrated for 4 h at 4 °C. Cooled semen was then filled in 0.5 ml straws at 4 °C and frozen in programmable cell freezer. Thawing of semen was performed at 37 °C for 30 s. Sperm motility, plasma membrane integrity and sperm morphology (acrosome integrity, head, mid-piece and tail abnormalities) of each semen sample were assessed at 0, 3 and 6 h after thawing and incubation at 37 °C. Visual motility (%) and percentage of intact plasma membranes assessed at 6 h post-thaw of buffalo bull spermatozoa were highest ( P < 0.05) due to DEY as compared to GFEY, IDEY and control. The percentage of spermatozoa with normal acrosomes at 0, 3 and 6 h post-thaw was highest ( P < 0.05) in DEY extender than GFEY, IDEY and CHEY. Sperm tail abnormalities (%) observed at 0, 3 and 6 h post-thaw in samples cryopreserved with freezing extender having DEY were lower ( P < 0.05) as compared to extender containing GFEY, IDEY and CHEY. In conclusion, DEY compared to other avian yolks in extender improves the frozen-thawed quality of buffalo bull spermatozoa.

77 EFFECT OF SUGAR SUPPLEMENTATION ON THE CRYOPRESERVATION OF BOAR SPERMATOZOA

The optimization of cryopreservation extenders is an essential issue for the successful establishment of boar sperm cryobanks. The aim of the present study was to investigate the importance of sugar supplementation to the freezing extender and/or to the thawing diluents, and the interactions between these treatments on post-thaw survival of boar spermatozoa. Pooled sperm-rich fractions from 5 mature hybrid boars (5 ejaculates per boar) were divided into 7 aliquots, centrifuged at 2300g 3 min, and diluted to a final sperm concentration of approximately 1000 � 106 spermatozoa mL–1 with the freezing extender prior to freezing in 0.5-mL plastic straws; a standard freeze–thaw procedure with computer-controlled freezing equipment was utilized. All of the freezing extenders were based on Tris-citric acid buffer supplemented with 20% egg yolk and 0 mm (TC, no glucose), 0.05 mm, 2 mm, 4 mm, 10 mm, 50 mm, or 185 mm glucose (all media adjusted to 310 mOsm kg–1; pH 6.8). After thawing at 37�C/20 s, semen was immediately diluted 1:2 (v/v) with either TC (no glucose) or BTS (205 mm glucose, 20.39 mm NaCl, 5.4 mm KCl, 15 mm NaHCO3, and 3.35 mm EDTA). Post-thaw sperm motility (assessed with a computer-assisted semen analysis system) and plasma membrane and acrosome integrity (viability, assessed simultaneously by flow cytometry using triple fluorescent staining) were evaluated at 0, 30, and 150 min post-thaw and used to estimate the success of cryopreservation. Data were analyzed as a split plot design using a mixed model ANOVA including cryopreservation extender, thawing diluent, incubation time, and interactions as fixed effects and replicates as a random effect. The freezing extender did not have any significant effect on the percentage of motile or viable spermatozoa at either thawing or after 150 min (P &gt; 0.05). There was a significant effect of incubation time (P &lt; 0.0001) and thawing diluent (P &lt; 0.0001) on motility and viability, with a significant interaction between them on motility (P = 0.018). Motility was similar (P &gt; 0.05) at time 0 in both thawing diluents (mean � SEM: 49.4 � 3.7 v. 46.2 � 3.9% for BTS and TC, respectively), but decreased in Tris-diluted samples in a time-dependent manner (54.6 � 1.9 v. 42.5 � 2.6% at 30 min, and 40 � 3.4 v. 27.1 � 3.7% at 150 min). In contrast, viability was significantly higher (P &lt; 0.05) in BTS-diluted samples at 0 (53.9 � 3.7 v. 49.7 � 3.8%), 30 (55 � 3.1 v. 47.7 � 3.31), and 150 min (51 � 4.2 v. 43.7 � 4.4%). These results indicate that, despite common beliefs, sugars are not necessary for cryopreservation but are beneficial for maintaining boar sperm metabolism for a longer time after thawing.

Effect of egg yolk concentration on cryopreserving Spanish ibex ( Capra pyrenaica) epididymal spermatozoa

Tris-egg yolk based diluents provide adequate cryoprotection for the sperm of most wild species in which they have been tested. The objective of the current study was to evaluate various Tris-based diluents containing different concentrations of egg yolk, for the fertilizing ability of epididymal spermatozoa of the Spanish ibex ( Capra pyrenaica) after freezing and thawing. For this purpose, we used heterologous in vivo fertilization by intrauterine insemination of domestic goats ( Capra hircus). In Experiment 1, a Tris–citric acid–glucose (TCG) diluent containing 6% (v/v) egg yolk and a TCG extender containing 20% egg yolk were compared. In Experiment 2, a TCG-6% egg yolk extender was compared with Triladyl-20% egg yolk. Diluted samples were cooled slowly to 5 °C over 1 h and equilibrated at that temperature for 2 h. At that point, aliquots of samples were loaded into 0.25 ml straws, and frozen in nitrogen vapor for 10 min. The fertility of spermatozoa frozen in TCG-6% egg yolk was higher ( P < 0.05) than for those extended with TCG-20% egg yolk, and tended to be higher than for those frozen with Triladyl-20% egg yolk. From the results of this study, the use of Tris-based extenders containing low concentrations of egg yolk (6%) is recommended for cryopreserving Spanish ibex epididymal spermatozoa.

Effect of holding and washing on the quality of goat semen

A total of 18 pooled semen samples collected from 4 healthy crossbred (Beetal × Assam Local) bucks, twice weekly for a period of 12 weeks, were processed via three processing methods, viz. Method I (holding after washing: semen sample was washed twice, extended, held for 0, 1, 3 and 5 h and preserved), Method II (washing after holding: semen sample held for 0, 1, 3 and 5 h, washed twice, extended and preserved) and Method III (holding of undiluted semen: semen sample held for 0, 1, 3 and 5 h, extended and preserved). An isotonic Tris buffer was used to wash the spermatozoa with the aid of centrifugation at 3000 rotations per minute (rpm) for 10 min. A Tris–citric acid–fructose–egg yolk extender was used to extend the semen processed with all three processing methods and preserved at 5 °C up to 72 h. Holding of semen at 24 °C in each processing method caused deterioration of the semen quality during preservation, as the holding time increased from 0 (without holding), 1, 3 and 5 h. The initial sperm motility declined from 85.8 to 71.7% in Method I, from 85.8 to 21.7% in Method II and from 84.2 to 45.0% in Method III, as the holding time increased from 0 to 5 h. The corresponding values were 66.7 and 49.2% for Method I, 66.7 and 8.0% for Method II and 42.5 and 25.8% for Method III, following 72 h of preservation. The percentage of live sperm and intact acrosomes also showed a similar decreasing trend as the holding time increased from 0 to 5 h in all processing methods as revealed in percentage of sperm motility. Washing of spermatozoa without a holding period maintained significantly higher sperm motility (66.7%), live sperm counts (83.0%) and incidence of intact acrosomes (79.3%) following 72 h at preservation, compared to the corresponding values (42.5, 65.2 and 73.3% for sperm motility, live sperm counts and intact acrosomes, respectively) recorded in unwashed semen without a holding period. The values recorded for washed semen without holding were also significantly higher than for semen held for 1, 3 and 5 h in all the three processing methods following 72 h of preservation. This study revealed that the holding of semen caused deterioration of the spermatozoa, whereas, washing without holding had a beneficial effect on the viability of spermatozoa in maintaining its storage ability for up to 72 h of preservation at refrigeration temperature (5 °C).