Tris-citric Egg Yolk Extender 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

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<0.05) the oxidative stress on spermatozoa and recorded a positive correlation (P<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<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<0.05) all sperm characteristics and decreased (P<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.

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.

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.

Comparative efficacy of different concentrations of egg yolk for cryopreservation of goat semen

Gaddi goats are important livestock species of Himachal Pradesh, India. The sensitivity to cryopreservation varies among different species as also between animals of same species. Ejaculates (180) from 11 adult Gaddi bucks aged between 1.1 to 4.5 years (2.16±0.36 years), weighing 31–57 kg (39.1±2.82 kg) were collected using artificial vagina and selected on basis of standard quality parameters. The ejaculates were extended in Tris citrate egg yolk extender containing 6% Glycerol with varying concentrations of egg yolk (EY; 5, 10, 15 and 20%) to maintain a concentration of 150 × 106 sperms/straws. Filled and sealed straws were equilibrated at 5°C for 4 h followed by vapour freezing of straws for 7 min at 4 cm above the liquid nitrogen and finally plunged into liquid nitrogen. The representative straws from each ejaculates were thawed at 37°C for 30 sec, 24 h post incubation to compare the progressive motility, viability, morphological abnormalities and HOST reactive sperms in between different EY concentrations along with per cent change due to the processing. The data was analyzed using package R version 3.4.3. Post thaw progressive motility (35.18±0.87) and viability (45.26±1.32) was higher with least per cent change due to processing (52.03 and 40.12) in 10% EY than other EY concentrations. Absolute average values of morphological abnormalities, were least in 10% EY (7.93±0.28) than 20, 15 and 5% EY (11.42±0.67, 10.84±0.53 and 8.39±0.35), respectively. The absolute average values of HOST did not differ between 15, 10 and 5% (59.96±1.93, 52.48±1.43 and 59.07±2.18) EY, all of which were higher than 20% (42.57±4.20) EY concentrations. In conclusion, extender containing 10% EY was best with respect to progressive motility and viability for Gaddi goat semen 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.

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.

Cryopreservation and fertility of frozen thawed Chegu goat semen

Goats are important livestock species of India. Chegu is a pashmina producing goat native to the cold arid region of Himachal Pradesh (H.P.), India. Semen cryopreservation from six Chegu elite males (aged 2.05±0.40 years; weighed 29.16±2.02 kg) was practiced using Tris Citrate Egg Yolk extender containing 10% EY and 6% Glycerol. Gross semen parameters includes volume (0.80.85±0.07 ml), color (Creamy white to yellowish), concentration (2238.5±231.0 x106 spermatozoa/ml) and mass motility (3.92±0.03).The significant changes (P less than 0.01) in post thaw seminal parameters (75.48±0.69 v/s 37.38±0.90; progressive motility), viability (75.79±0.95 v/s 48.25±1.78), morphological abnormalities (5.64±0.29 v/s 7.02±0.32) and HOST reactive spermatozoa (64.07±1.75 v/s 43.35±1.79) were observed in present study. Artificial insemination using frozen thawed semen having concentration (150 x106 spermatozoa/straw) from three different bucks was practiced in 40 synchronized goats with conception rate of 42.5 per cent. Non-significant variations amongst different bucks were observed with birth of 1.12 kids per doe and twinning rate of 11.8 per cent. It was concluded that semen cryopreservation along with artificial insemination can be practiced in Chegu goats to improve the population of this endangered species.

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.

Effect of adding different levels of caffeine in the extender on some biochemical constituents, enzymatic activities and physical characteristics of chilled and frozen ram semen.

Two experiments were carried out to determine the efficiency of supplementation of ram semen extender with caffeine on chilled storage and frozen capacity of spermatozoa. In the first experiment, eighty ejaculates were collected by an artificial vagina from five adult Barki rams, aged 2-3years and weighted 45.0±2.0kg throughout the experimental period (January to February 2017). The ejaculates were pooled and diluted (1:10) with tris-citric egg yolk extender and were split into five groups. Group 1 served as control, whereas groups 2-5 were supplemented with 0.1, 0.2, 0.3 and 0.4mM caffeine. All diluted semen specimens were evaluated for physical characteristics immediately after dilution (T0 ) and throughout preservation period of 48hr at 4°C. Simultaneously, oxidative stress and indices such as total antioxidant capacity (TAC), malondialdehyde concentrations (MDA) and alkaline transaminase (AKP) concentrations and value of resazurin reduction test (RRT) were determined. In the second experiment, the raw pooled ejaculates were diluted (1:10) with glycerolated tris-citric egg yolk extender, receiving the previously mentioned caffeine levels. The post-thaw assessment of cryopreserved spermatozoa, in all groups, was conducted by a computer-assisted sperm analysis (CASA) system. The results revealed that adding caffeine to ram semen extender at low (0.1mM) or medium (0.2mM) levels had positive impact on both physical characteristics of ram sperm and the enzymatic activities compared to the other semen groups. Caffeine supplementation also enhanced post-thaw sperm dynamics, which implies its potential as an exogenous antioxidant supplement.

Effect of supplementing ram semen extender with melatonin on oxidative stress indices and physical properties of chilled spermatozoa

The current study aimed to evaluate the efficiency of supplementing ram semen extender with melatonin on chilled storage capacity of spermatozoa. Eighty ejaculates were collected from 5 Barki rams, 16 ejaculates each, using an artificial vagina throughout the period from January – February, 2017. The ejaculates of each collection session were pooled and diluted (1:10) with Tris-citric egg yolk extender, and were split into 4 aliquots. The first portion served as control (melatonin-free), whereas the other 3 portions were supplemented with 0.1, 0.2 or 0.3 mM melatonin. Thereafter, the samples were stored at 4 oC for 48 hrs, during which sperm physical and morphological properties were evaluated at 24 hr interval. Simultaneously, oxidative stress indices in terms of total antioxidant capacity (TAC), malondialdehyde concentration (MDA), resazurin reduction test, in addition to enzymatic activities of AST, ALT and ALP were determined. The results revealed that, over the 48 hr preservation period, level of melatonin in the diluent was positively correlated (P<0.01) with sperm motility, viability, percent of normal sperm, intact acrosome, sperm cell membrane integrity and total antioxidant capacity (r= 0.75, 0.96, 0.82, 0.95, 0.96 and 0.74, respectively). Contrarily, a negative correlation (P<0.01) was observed between melatonin level and each of primary and secondary sperm abnormalities, MDA, AST, ALT and ALP over time of storage (r= – 0.71, – 0.85, – 0.46, – 0.71, – 0.95 and – 0.84, respectively). These results elucidate that supplementing the diluent with 0.3 mM melatonin efficiently reduced oxidative stress and improved chilled storage of ram spermatozoa.

Improving cryopreservation capacity of ram spermatozoa by supplementing the diluent with melatonin

The aim of this investigation was to evaluate the efficiency of supplementing ram semen extender with melatonin on cryopreservation capacity of spermatozoa. A total of 80 ejaculates were collected from 5 Barki rams, 16 ejaculates each, by an artificial vagina throughout the period from January – February, 2017. Ejaculates of each collection session were pooled and diluted (1:10) with glycerolated Tris-citric egg yolk extender, and were split into 4 portions. The first portion served as control (melatonin-free), whereas the other 3 portions were supplemented with 0.1, 0.2 or 0.3 mM melatonin. The post-thaw objective assessment of cryopreserved spermatozoa, in all groups, was conducted by a computer-assisted sperm analysis (CASA) system. The results revealed that melatonin supplementation was positively correlated (P<0.01) with post-thaw total motility, progressive motility, viability, acrosome integrity, sperm cell membrane integrity, VSL, VAP and WOB (r= 0.88, 0.71, 0.78, 0.85, 0.96, 0.95, 0.68, 0.57 and 0.53, respectively. However, it was negatively correlated (P<0.01) with primary and secondary sperm abnormalities, as well as non-progressive motility and immotile spermatozoa (r= -0.73, -0.85, -0.49 and -0.76, respectively. These results elucidate that adding melatonin to ram semen extender substantially enhanced post-thaw sperm physical properties, which implies its powerful potential as an exogenous antioxidant supplement.