Tris-citric Egg Yolk Research Articles

Effect of black cumin (Nigella sativa) seeds extract in tris egg yolk citrate extender on sperm motility of cryopreserved Surti buck semen

Effect of black cumin (Nigella sativa) seeds extract in tris egg yolk citrate extender on sperm motility of cryopreserved Surti buck semen

Effect of black cumin (Nigella sativa) seeds extract in tris egg yolk citrate extender on viability of cryopreserved Surti buck semen

Effect of black cumin (Nigella sativa) seeds extract in tris egg yolk citrate extender on viability of cryopreserved Surti buck semen

Morphometric features, seminal profile and diluters effect on post-thaw semen quality of Munshiganj cattle in Bangladesh

The study investigates morphometric features, seminal profile and post-thaw semen quality of Munshiganj cattle. Morphometric features were measured using measuring tape from 20 Munshiganj cattle while coat color was measured by observing in naked eye. Fresh and post thaw semen quality parameters were analyzed using Computer Assisted semen analyzer (CASA). Coat color of Munshiganj male cattle were creamy white to dull pinkish and female were white to creamy. The mean body weight, body length, hearth girth, height at wither, head length, head width, ear length, ear width, fore leg length, hind leg length, tail length, tail doc circumference, horn length, horn diameter and mouth circumference were 362.80 kg, 137.31 cm, 160.66 cm, 135.21 cm, 50.97 cm, 20.58 cm, 19.75 cm, 9.88 cm, 73.02 cm, 74.84 cm, 106.10 cm, 20.75 cm, 13.60 cm, 16.12 cm and 43.00 cm, respectively. There was significant difference (p < 0.05) between male and female in terms of body weight (418.00 vs 307.60 kg), heart girth (173.74 vs 147.57 cm), head width (22.50 vs 18.67 cm), horn diameter (18.58 vs 13.66 cm) and mouth circumference (46.60 vs 39.40 cm). Average scrotal length was 16.76 cm while scrotal circumference was 32.70 cm. Age had significant effect (p < 0.05) on morphometric characteristics of Munshiganj male and female cattle. On the other hand, season had no significant effect on semen quality. Mean semen volume, sperm concentration, total motile, progressive, static, slow and live spermatozoa were 5.83 ± 0.88 ml, 1510.27 ± 844.07 million/ml, 91.9 ± 2.17 %, 63.80 ± 12.53 %, 8.10 ± 2.17 %, 0.10 ± 0.10 %, 91.38 ± 0.25 %, respectively. On the other hand, sperm head length and width, sperm tail length of Munshiganj cattle were 10.39 ± 0.16 μm, 4.26 ± 0.07 μm, 21.5 ± 0.52 μm, respectively. Individual breeding bull had a significant (p < 0.05) effect on post-thawed motile sperm percentage. Four different diluters (Triladyl, Steridyl, Tris-egg yolk-Citrate and Andromed) were used to compare the effects of diluter on post-thaw semen quality of Munshiganj cattle and found that diluter had no significant effect (p > 0.05) on post thaw semen quality except slow motility and proximal droplet percentages. Munshiganj cattle had a distinctive phenotypic feature with standard quality semen and had no effect of egg yolk free and egg yolk based diluters on post thaw semen quality.

Effect of clarified tris egg yolk citrate diluter and seminal plasma removal on plasma membrane integrity in cryopreserved Surti buck semen

Effect of clarified tris egg yolk citrate diluter and seminal plasma removal on plasma membrane integrity in cryopreserved Surti buck semen

Improving Capacities of Chilled- and Cryo-Preserved Buck Spermatozoa by Enriching Preservation Medium with Moringa oleifera leaves extract

In the present work, two experiments were conducted to investigate the efficiency of supplementing buck sperm medium with Moringa oleifera leaves extract on liquid-chilled storage and cryopreservation capacities of spermatozoa. Twenty ejaculates were collected by an artificial vagina from 5 Damascus bucks, 4 ejaculates each, during September-October. The pooled specimens of each collection session were diluted (1:10) with Tris-citric egg yolk extender, and were split into 6 equal aliquots. The first aliquot served as control (moringa-free), whereas the other five aliquots were supplemented with 0.05, 0.1, 0.2, 0.3 or 0.4 mL/ml (v/v) moringa leaves extract, respectively. The samples were then stored and maintained at 4 oC for the subsequent 48 h during which sperm traits were assessed alongside malondialdehyde (MDA) production and glutathione peroxidase (GPX) activity. The optimum concentration of moringa leaves extract that showed potential for maintaining sperm traits throughout the chilled storage period was further investigated for preserving the lifespan of spermatozoa after exposure to the cryopreservation stress against control. The results showed that moringa leaves extract supplementation reduced (P&lt;0.05) the oxidative stress on spermatozoa and recorded a positive correlation (P&lt;0.01) with sperm motility (%), viability (%), normal sperm (%), acrosome integrity and GPX activity (r= 0.36, 0.42, 0.61, 0.79 and 0.35, respectively) while recording a negative correlation (P&lt;0.01) with production of MDA (r= - 0.56). Furthermore, supplementing buck sperm medium with 0.4 mL/ml moringa leaves extract efficiently improved (P&lt;0.05) all sperm characteristics and decreased (P&lt;0.05) DNA fragmentation index (DFI) post thawing compared to those of control. These results imply the powerful potential of moringa leaves extract as an exogenous antioxidant supplement in buck sperm medium particularly when spermatozoa are used for AI and IVFM applications.

Supplementation of Alpha-lipoic acid-loaded nanoliposomes in semen extender improves freezability of buffalo spermatozoa

This research was designed to explore the protective effect of alpha-lipoic acid–loaded nanoliposomes (ALAN) during cryopreservation of buffalo sperm. Buffalo semen was cryopreserved in a tris-citrate egg yolk extender without any supplement (ALAN0, control group) or with ALAN at levels of 25, 50, 75 or 150 µg (ALAN25, ALAN50, ALAN75 and ALAN150, respectively). The ALAN had a size of 171.80 nm and a negative zeta potential (− 43.40 mV). The progressive motility, vitality and membrane integrity significantly improved in all ALAN groups (except ALAN25 for membrane integrity). ALAN150 group exhibited the best values of progressive sperm motility, vitality and membrane integrity after thawing at 37 °C for 30 s or incubated for 2 h at 37 °C and 5% CO2 compared with those in other groups. Both ALAN75 and ALAN150 groups significantly improved the TAC, GR and catalase, while lipid peroxidation and early apoptotic spermatozoa significantly decreased in ALAN150 group followed by ALAN75 group. Collectively, the adding ALAN to buffalo semen freezing extender plays a substantial shielding function against cryodamage by preserving the sperm functional parameters.

Motion characteristics and plasma integrity evaluation of Murrah buffalo semen

Buffalo semen collected from Murrah bull were cryopreserved and evaluated for different motility parameter, kinematics and plasma membrane integrity. Buffalo bulls were maintained uniform standard management and nutritional practices. Semen was collected regularly twice a week semen collection schedule from four (04) Murrah bull. Collected semen was immediately transported to laboratory and evaluated for different macroscopic parameter (color, volume and thickness). Fresh semen was then diluted with saline solution and evaluated for sperm concentration, motility, sperm kinematics and morphology. Semen samples that fill all the standard were selected for freezing and diluted with Tris-egg yolk citrate diluter. Diluted semen was equilibrated, cryopreserved and finally evaluated for post thaw sperm quality. Different motility parameter (total, progressive, static and slow motility) varied significantly (p&lt;0.01) irrespective of different freezing stages. Significantly higher progressive sperm motility and viability of buffalo spermatozoa were observed at fresh semen whereas lower progressive sperm motility and viability was found at post thaw stage. Total and progressive motility reduced by 2.5 and 2.12% following equilibration, whereas following cryopreservation, total and progressive motility reduced by 35.7 and 28.51% and static motility increases accordingly (35.4%). Significantly higher plasma membrane integrity of sperm was observed at fresh semen followed by pre freeze and post thaw semen. Following freezing, integrity of plasma membrane reduces at the rate of 10.81% and 26.7% at pre freezing and post thaw stages. Significantly higher average path velocity (VAP), straight line velocity (VSL), curvilinear velocity (VCL), straightness (STR), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF) were found for fresh semen followed by pre-freeze and post-thaw semen. Frozen buffalo semen with higher progressive motility and motion characteristics may be produced if motility losses can be reduced during freezing stage as this stage results higher motility losses. Asian Australas. J. Biosci. Biotechnol. 2022, 7(2), 75-81

The effects of green synthesized anionic cupric oxide nanoparticles on Zaraibi goat spermatozoa during cryopreservation with and without removal of seminal plasma

Sperm motility, normal morphology, viability, spermatozoa DNA damage, and lipid peroxidation are all affected by semen cryopreservation. The goal of this study was to see how effective cupric oxide nanoparticles (CuONPs) are as a cryo-extender additive on post-thawed sperm parameters. An artificial vagina was used to collect semen samples from five mature Zaraibi bucks (2–3 years). Ejaculates were pooled and separated into two fractions (A&B), a fraction (A) was left without being centrifuged and a fraction (B) was centrifuged to remove seminal plasma. Both fractions were diluted with tris egg yolk citrate extender (TECE) and then divided into five equal aliquots, each supplemented with (0, 10, 20, 40, and 60 ppm/ml) CuONPs. The findings revealed that removing seminal plasma before cryopreservation harms sperm parameters. Sperm motility, viability index, membrane integrity, biochemical antioxidant marker, DNA integrity, and MDA level improved after supplementation with CuONPs up to 60 ppm/ml, the most prominent significant positive effect was obtained with the highest dose (60 ppm/ml) without removal of the seminal plasm compared to control group. In conclusion: The presence of seminal plasma with a high concentration of CuONPs (up to 60 ppm/ml) may help to mitigate the negative effects of cryo-preservation.

​Evaluation of Antioxidant Potential of Taurine in Tris Egg Yolk Citrate Extender in Surti Buck Semen Preserved at Refrigerated Temperature

Background: Taurine supplementation in tris egg yolk citrate (TEYC) extender can improve antioxidant defense and reduce motility degeneration rate in semen of Surti buck preserved at refrigeration temperature. Present study has evaluated antioxidant effect of different concentrations of taurine in TEYC extender on oxidative stress, antioxidant defense and motility degeneration rate in Surti buck semen preserved at refrigerated temperature. Methods: Four Surti bucks (age greater than 2years) were selected. Total 64 ejaculates (16 per buck) were collected during a period of 8 week. Post-collection they were pooled and stored at refrigeration temperature after dividing into 5 groups based on different taurine concentrations in TEYC extender viz. 0 mM (control T1), 25 mM (T2), 50 mM (T3), 75 mM (T4) and 100 mM (T5) taurine maintaining final concentration of 200 x 106 sperm/ml (pH 6.5-6.8). Evaluation of motility degeneration rate at 24, 36 and 48 hours and glutathione as well as lipid peroxidation (MDA) at 0 and 48 hours was done. Result: Supplementation of taurine @50 mM in TEYC extender caused post-chilling significant increase in reduced glutathione (GSH) levels, lowering of lipid peroxidation (in terms of MDA production) and reduction of motility degeneration rate (MDR)% in semen of Surti bucks. It improved antioxidant defense thereby maintaining good quality of semen.

Gamma-oryzanol supplemented in extender enhances the quality of semen cryopreservation and alters proteomic profile in Thai swamp buffalo

Reactive oxygen species (ROS) exert an adverse effect on sperm quality during the freezing process. Gamma-oryzanol is an effective antioxidant and has the ability to inhibit lipoperoxidation in various cells. Therefore, this study aims to investigate the effect of gamma-oryzanol supplementation in extender on post-thawed motility and proteomic profiles of swamp buffalo spermatozoa. Each ejaculate of an individual bull was divided into four equal aliquots. Gamma-oryzanol was supplemented at 0 (control), 0.1, 0.25, and 0.5 mM in tris-citrate egg yolk extender. The parameters of sperm motility were evaluated using computer assisted semen analyzer (CASA). The results showed that the progressive motility was significantly higher in 0.5 mM of gamma-oryzanol supplementation group when compared with the control group (p < 0.05), but no significant differences were observed among the treatments. In addition, a proteomic approach was applied to analyze the differentially expressed proteins in post-thawed sperm with or without gamma-oryzanol supplementation in extender. We confirmed that 2-phospho-d-glycerate hydro-lyase (ENO1), glutathione s-transferase mu 1 (GSTM1), phospholipid hydroperoxide glutathione peroxidase (GPX4), outer dense fiber protein 2 (ODF2), tektin-4 (TEKT4), tubulin beta-4B chain (TUBB4B), and ATP synthase subunit beta (ATP5B) were up-regulated in 0.5 mM of gamma-oryzanol supplementation group, which might be associated with the improved post-thawed motility observed in this treatment group. These results demonstrate the beneficial effect of gamma-oryzanol on post-thawed survival of swamp buffalo spermatozoa and help advance the understanding about molecular metabolism of sperm in this species.

Effect of taurine in tris egg yolk citrate extender on quality of surti buck semen at refrigerated temperature

Semen was collected from four Surti bucks (>24 months-old) twice a week for 8 weeks (16 ejaculates per buck) during November, 2020 to March, 2021. Ejaculates of all four bucks were pooled and divided into five equal aliquots. The aliquots were diluted with tris-egg yolk-citrate (TEYC) extender containing 0 (control T1), 25 (T2), 50 (T3), 75 (T4) and 100 mM (T5) taurine with final concentration of 200x106 sperm/ml (pH 6.5 to 6.8) in each. At 24, 36 and 48 h of refrigerated storage, individual sperm motility, per cent live sperm and HOST reacted sperm were significantly (p<0.01) higher in T3 (50 mM) and abnormal sperm % was significantly (p<0.01) lower in T3 (50 mM) and T4 (75 mM) as compared to other groups. With increasing duration of storage, significant (p<0.01) increasing trend for abnormal sperm per cent and decreasing trend for rest of the characters were observed. Motility showed significant (p<0.01) positive correlation with live spermatozoa, abnormal spermatozoa and HOST-reacted spermatozoa. Live count showed significant (p<0.01) negative correlation with abnormal spermatozoa and significant (p<0.01) positive correlation with HOST-reacted spermatozoa. Live spermatozoa showed significant (p<0.01) negative correlation with abnormal spermatozoa and HOST non-reacted spermatozoa. Abnormal spermatozoa showed significant (p<0.01) negative correlation with HOST-reacted spermatozoa. It was concluded that supplementation of 50 mM taurine in TEYC extender maintained optimum quality of buck semen at refrigerated temperature up to 48 h.

Seminal quality attributes and their interrelationships in gaddi and chegu bucks

A study was conducted to compare the seminal quality attributes in Gaddi and Chegu goats. A total of 286 ejaculates (Gaddi -106, Chegu -180) from adult healthy post-pubertal bucks (11 Gaddi and 8 Chegu) were collected twice weekly using artificial vagina. The ejaculates were extended in tris citrate egg yolk (TEY) extender to maintain a concentration of 150x10 6 spermatozoa/straw. Randomly selected straws from each ejaculate were thawed at 37ºC for 30 sec, 24 h post incubation to compare seminal quality characters between fresh and post-thaw semen along with the changes due to processing. The results showed significantly (P<0.01) lower volume (0.66±0.04 vs 0.77±0.04 ml) with higher sperm concentration (3401.0±247.2 vs 1968.76±134.97x10 6 sperms/ml) in Gaddi as compared to Chegu bucks. Interrelationships among gross semen characters showed significant (P<0.01) correlation between sperm concentration and mass motility in Gaddi (r=0.506) and Chegu (r=0.715) bucks. Progressive motility was significantly (P<0.01/0.05) correlated with viability (r=0.326 and r=0.692) and morphological abnormalities (r=-0.393 and r=-0.257) in fresh diluted and post-thaw Gaddi semen. Likewise, in Chegu goats progressive motility was significantly (P<0.05/0.01) correlated with viability (r=0.236 and r=0.641). Morphological abnormalities were significantly (P<0.05) correlated with hypoosmotic swelling test (HOST) reactivity (r=-0.231) in fresh and with progress motility and viability (r=-0.248 and r=-0.323) in post-thaw semen in Chegu only. Significantly (P<0.01) higher progressive motile (35.18±0.87 vs 30.86± 0.78 %) and HOST reactive sperm (52.48±1.43 vs 37.57±1.19%) were observed in Gaddi than Chegu bucks. In conclusion, higher progressive motility and HOS Treactivity along with lower per cent changes in these characters due to processing in Gaddi bucks suggest better resilience of this breed to cryopreservation compared to Chegubucks.

Determination of the optimal concentration of Minitube Equex paste for boar semen cryopreservation based on sperm motility characteristics

Equex paste is a non-permeating cryoprotective agent (CPA) that improved post-thaw survival of spermatozoa during boar semen cryopreservation. However, Equex paste produced by Nova Chemical Sales Inc. (MA, USA) is not currently available. The aim of the present study was to determine the optimal concentration of Minitube Equex paste (Minitube, Tiefenbach, Germany) for boar semen cryopreservation in comparison with Nova Equex STM paste (control). Fifteen ejaculates from 12 mature boars were collected by the glove-hand method. Each ejaculate was aliquoted and cryopreserved in base freezing extender III as Tris-citrate egg yolk (TEY) extender plus 9.0% glycerol classified into four groups. Group I was the control and included only 1.5% Nova Equex STM paste. Groups II, III and IV were the experiment groups, and contained different concentrations of Minitube Equex paste (Group II: 1.5%; Group III: 1.7%; and Group IV: 1.9%) added to the freezing extender III. After freezing and thawing, sperm motility characteristics were evaluated by Sperm Class Analyzer® incubated at 37 °C for 0 (10 min), 1 and 2 h post-thawing. In Group IV after thawing at 0 h, rapid velocity and the velocity curved line were significantly higher than in Groups II and III (P &amp;lt; 0.05) but did not differ from Group I. Moreover, after thawing at 1 h, LIN (linearity) in Group IV was higher than in Group II (P &amp;lt; 0.05), but did not differ from the other groups. In conclusion, the most suitable concentration of Minitube Equex paste in the current protocol was 1.9% supplemented with 9.0% glycerol in TEY-based freezing extender III, based on the conformity between data from manual guides and the observed sperm motility characteristics results.

Melatonin supplementation improved cryopreserved Thai swamp buffalo semen.

The production of reactive oxygen species (ROS) during cryopreservation process impairs the sperm characteristics and fertilizing ability. However, melatonin, an antioxidant, could protect spermatozoa against this cell damage during cryopreservation. Therefore, we attempted to evaluate whether the melatonin supplementing in the semen extender could improve the sperm quality of swamp buffalo during cryopreservation. The semen collected from six swamp buffalo bulls were diluted with tris-citrate egg yolk extender supplementing with 0, 0.1, 0.5, 1.0, 2.0 and 3.0mM of melatonin. The parameters of sperm viability and motility were evaluated using computer-assisted semen analyser (CASA) after cryopreservation on days 1, 7, 15 and 30. The group supplemented with 1.0mM melatonin exhibited the higher viability after cryopreservation on days 1, 7, 15 and 30 with 58.346±2.1a , 57.586±2.0a , 55.082±1.8a and 55.714±1.8a , respectively, and showed the best results of motility parameters. However, higher concentration of melatonin at 3.0mM impaired all the parameters. In conclusion, the addition of melatonin at 1mM to semen extender could exert the best protection against sperm damage in swamp buffalo bull during cryopreservation.

Effect of coconut water in tris egg yolk citrate extender on cauda epididymal buck spermatozoa motility preserved at refrigeration temperature

Effect of coconut water in tris egg yolk citrate extender on cauda epididymal buck spermatozoa motility preserved at refrigeration temperature

Anti-Oxidant Effect of Quercetin in Tris Egg Yolk Citrate Extender on Surti Buck Semen Preserved at Refrigerated Temperature

Anti-Oxidant Effect of Quercetin in Tris Egg Yolk Citrate Extender on Surti Buck Semen Preserved at Refrigerated Temperature

Evaluation of Bull Semen Quality and Sperm DNA Integrity during Cryopreservation with Different Cryoprotectants

The fertility rate in dairy cattle with cryopreserved semen significantly depends on the reduction of the toxic effects on the sperms induced during cryopreservation. DNA integrity in sperm is vital for the precise transmission of genetic information and therefore the production of high yielders and maintenance of good health in future generations. The reliable and consistent assessment of sperm motility can be succeeded by Computer Assisted Semen Analyzer (CASA). Ten ejaculates were collected from Frisian bulls kept at Centre of excellence for bovine genetics (CEBG) Renala Khurd District Okara. The deleterious effects of three cryoprotectants glycerol, dimethylsulfoxide (DMSO) and ethylene glycol as a constituent of tris-egg yolk-citrate extender were compared on diluted post thawed cryopreserved Frisian bull semen. The quality of semen in terms of viability/motility/progressive velocity was determined with computerized external real image optical system (CEROS) and sperm DNA fragmentation was quantified with comet assay using single cell gel electrophoresis (SCGE) technique. Glycerol was the least followed by DMSO and ethylene glycol was the most toxic cryoprotectant both for semen quality and sperm DNA fragmentation. The present study suggests that sperm DNA fragmentation is an associated feature of semen cryopreservation resulting into cell death (non-motile sperms) and glycerol as a cryoprotectant constituent of tris-egg yolk citrate extender offers a better protection for storage of cryopreserved Frisian bull semen in liquid nitrogen.

Assessment of Pomegranate Juice as Antioxidant in Extender on Cauda Epididymis Spermatozoal Quality of Buck at Refrigerated Temperature

The present investigation was carried out to study the refrigeration preservation of the cauda epididymal retrieved spermatozoa of buck in Tris egg yolk citrate (TEYC) dilutor containing pomegranate juice as antioxidant additive. The retrieved cauda epididymal spermatozoa extended in TEYC dilutor were studied in five groups by adding different concentration of pomegranate juice as additive (0% as control T1 group and 5%, 10%, 15% and 20% as treatment T2, T3, T4 and T5 groups, respectively) and storing at refrigerated temperature up to 48 hr. The results showed that the control extender had the least dead, abnormal and HOS non-reacted sperm percent among all treatments tested and that with increasing the pomegranate juice concentration in dilutor, the percentage of the dead, abnormal and HOST non-reacted spermatozoa increased significantly. The same trend was observed at all 12 hourly storage intervals indicating its detrimental effect on epididymal sperms of bucks at refrigeration temperature. The dead, abnormal, and HOST non-reacted sperm were significantly and positively interrelated with each other (r = 0.53-0.83). It was concluded that the inclusion of pomegranate juice in TEYC dilutor did not show any beneficial/antioxidant effect on epididymal sperms of buck in fresh or refrigerated semen and in fact all the levels of pomegranate juice (5% to 20%) were detrimental to cauda epididymal spermatozoa of a buck.

Effect of Tomato Juice in Tris-Yolk-Citrate Extender onRefrigeration Preservation of Cauda Epididymal Spermatozoa of Buck

The study was carried out on the preservation of epididymal spermatozoa of buck at refrigerated temperature without and with tomato juice as a supplement in Tris egg yolk citrate extender. The eight pairs of testicles including epididymis (total 16) from slaughtered bucks were collected within 2–4 hours of their slaughter. Sperms collected from cauda epididymis were preserved at refrigerated temperature up to 48 hours in tris egg yolk citrate extender at 300 million sperm/mL with different concentration of tomato juice (0%, 4%, 6%, 8%, and 10%) and the physical characteristics of spermatozoa were assessed to know the effect of tomato juice (Tj). The mean dead, abnormal and HOST non-reacted spermatozoa increased significantly (p less than0.05) at every 12-hour intervals of preservation in the dilutor without and with different concentration of tomato juice. Tomato juice exerted an adverse effect on physical characteristics of sperm during refrigeration preservation. All the three sperm traits studied however revealed significant (p less thaN 0.01) positive interrelationships with correlations of 0.31 to 0.72.

Effect of Extender and Cooling Rate on the Quality of Frozen Thawed Semen of Bali Bull (Bos Sondaicus)

The purpose of this study was to observe the effect of extender and cooling rate on the quality of frozen thawed semen of Bali bull (Bos sondaicus). The experiment was conducted at Laboratory of Animal Biotechnology, Faculty of Animal Husbandry, Andalas University. A completely randomized design of factorial 2 x 3, 2 different extenders and 3 cooling rates with 3 bulls as the replicate. Data were analyzed using analysis of variance (ANOVA). The first factors was extender such as tris citrate egg yolk (TCEY) and tris citrate soy milk (TCSM). The second factors was the cooling rate such as: 15ºC/min, 10ºC/min and 5ºC/min. Variables were: motility, viability, abnormality, and membrane integrity. The results of treatment of TCEY extender on the average of motility 42.67±5.17%, viability of 53.78±3.79%, abnormality of 18.89±1.07%, and membrane integrity of 35.44±3.01%. The effect of cooling rate of 10°C /min has the highest semen motility of 44.35±5.28%, viability of 57.17±1.18%, abnormality of 17.84 ± 0.23 % and membrane integrity of 36.83±2.12%. The interaction between extender and cooling rate was founded significant different (P&lt;0.05). Otherwise of extender and cooling rate on post-thawed of motility, viability and membrane integrity, had no difference (P&gt;0.05) on semen abnormality. It can be concluded that tris-citrate egg yolk (TCEY) extender with cooling rate of 10ºC / min were the best semen motility, viability, and membrane integrity of spermatozoa.