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Effect of Supplementation of Butylated Hydroxytoluene on Post-Thaw Viability, Motility and Membrane Integrity of Crossbred Bovine Spermatozoa

Anoop Singh Mridula Sharma Shiv Prasad Yaqoob Bhat Anil Kumar Dinesh Pandey Santosh Shukla
Vol 7(4), 93-101

This study was conducted to evaluate effect of butylated hydroxytoluene (BHT) on post thaw semen quality of cattle bull. Thirty six ejaculates from two bulls were collected and extended with glycerolated egg yolk tris diluter. Samples were divided into three groups according to BHT concentration as 0 mM (C) 0.5 mM (T1) and 1.0 mM (T2). The samples were analyzed for progressive motility, sperm viability and membrane integrity at three stages i.e. post dilution (stage I), post equilibration (stage II) and post thaw (stage III). Progressive motility, sperm viability and sperm membrane integrity were significantly (p<0.05) higher in T1 & T2 compared to the control. The concentration 1mM of BHT showed better results as compared to 0.5 mM. BHT addition improved the sperm quality probably due to its antioxidant property as it reduces the lipid peroxidation of the sperms.

Keywords : Sperm Viability Butylated hydroxytoluene Membrane Integrity Antioxidant Crossbred Bovine Spermatozoa


Cryopreservation is detrimental to sperm function and fertility even with the most up to date technique. Sperm viability and fertility is decreased by about 50% after cryopreservation (Lessard et al., 2000). There are many harmful conditions such as cold shock, osmotic stress, ice crystal formation or oxidative damage etc which cause sperm cryoinjury and loss of sperm viability and fertility. The detrimental conditions of sperm are due to mechanical and osmotic phenomenon, oxidative stress, increased membrane permeability, lipid per oxidation and subsequent membrane damage. Physiological levels of reactive oxygen species (ROS) play essential roles in sperm capacitation; acrosomal reaction and fertilization process (Bailey et al., 2000). However, higher production of ROS due to oxidative stress during freezing and thawing of bull semen is responsible for the deterioration of semen quality (Bilodeau et al., 2000). The damage due to increased ROS can occur in sperm bio-membrane system due to presence of high content of polyunsaturated fatty acids (Kankofer et al., 2005). Increased ROS affect motility, plasma membrane integrity, viability and acrosomal integrity (Bilodeau et al., 2001 and Lenzi et al., 2002) which ultimately reduce the fertilization potential of the spermatozoa. The ROS are highly reactive in nature and combine readily with other molecules causing oxidation and structural and functional changes which lead to cellular damage.

The sperm plasma membrane is the primary site of damage induced by cryopreservation. Sperm cells lack cytoplasmic component which contain significant antioxidants. Hence, they are highly susceptible to lipid per oxidation by oxygen free radicals and H2O2 (Baumber et al., 2003 and Ogretmena, F. and Inanan, B.E. 2014). Free oxygen radicals in the sperm cells play a major role in lipid per oxidation as well as in protein damages that lead to cell death (Shoae, A. and Zamiri, M.J. 2008) or altered characteristics (DNA and sperm membrane damages). Concentration of naturally present antioxidant is usually decreased during the cryopreservation process by dilution of semen with extender and excessive generation of ROS molecules (Roca et al., 2004) and its harmful effect to sperms is not nullified by naturally present antioxidant. Hence supplementation of natural or synthetic antioxidants in semen extender would be beneficial for the improvement of post-thawed semen characteristics (Jamadia et al., 2016). Addition of vita-E, vita-C, glutathione (GSH), cysteine, taurine, hypotaurine, caffine, bovine serum albumin (BSA), trehalose and hyaluronan etc. to semen extender has been reported to have a positive influence in control of cryodamages of spermatozoa (Patel et al., 2015).

Butylated hydroxytoluene (BHT) has an excellent antioxidant capacity. It behaves as a synthetic analogue of vitamin E, primarily acting as terminating agent that suppresses autoxidation by converting peroxy radicals to hydroperoxides (Fujisawa et al., 2004). Being lipid soluble, BHT functions as an antioxidant within and outside the sperm membrane, hence it is preferred over other antioxidants. BHT easily penetrates the sperm membrane increasing their fluidity (influencing the membrane phase transition) to decrease ice-crystal formation within the cell thereby protecting the sperm (Naijian et al., 2013). Hence, the present study was planned to evaluate the effect of butylated hydroxytoluene (BHT) on post thaw bovine semen characteristics of bovine crossbred spermatozoa.

Material and Methods

Two crossbred bulls (1/2 HF x 1/2 J) of aged 4-6 years weighing 450-500 Kg, reared at Semen Production Centre, Department of Veterinary Gynaecology & Obstetrics, College of Veterinary and Animal Sciences, G. B. Pant University of Agriculture and Technology, Pantnagar, Uttarakhand-263145 were used for experiment. The bulls were kept under identical feeding and management conditions during entire course of this study.

Semen Collection

Total eighteen ejaculates from each bull were collected by artificial vagina method during morning hours. Eighteen ejaculates from each bull were frozen separately after dilution in Tris extender with or without BHT.

Fresh Semen Evaluation

The fresh semen samples were evaluated for volume, pH, mass motility and sperm concentration. Ejaculates were selected on the basis of mass activity and individual motility. Ejaculates that had mass activity of 3.0 + (0-5.0 scale) and a progressive motility of 70% or more were selected for further extension and freezing (Salisbury and Vandemark 1978).

Preparation of 0.5 and 1.0 mM BHT Solution and Extension of Semen

BHT (275.44 mg) (SRL, India) was dissolved in 25 ml of ethanol making a BHT concentration of 0.05 mole/ml. 0.05 and 0.1ml of 0.05 mole/ml BHT was taken in the two glass test tubes and kept at 370C. Ethanol containing BHT was evaporated at resulting in sticking of BHT in the inner wall of the test tubes. The semen was extended with glycerolated egg yolk tris (EYT). In each BHT containing test tubes 5 ml of extended semen was added to make concentration @ 0.5 mM BHT (T1) and 1.0 mM BHT (T2). Tubes were kept at 370C for 5 min to allow uptake of BHT by spermatozoa. Simultaneously, the same amount of extended semen was kept in another test tube (without BHT) and considered as control.

Processing of Semen for Freezing

Semen was filled in the 0.25 ml French mini straws at room temperature (370C) with the help of straw filling nozzle immediately after dilution. Polyvinyl alcohol powder was used to seal after creating air space with the help of air bubbler comb in each straw. Straws were wiped, dried, sealed and transferred to a refrigerator for equilibration at 4-50C for 4 hours. The samples were evaluated before deep freezing. After equilibration, semen samples were frozen using horizontal liquid nitrogen vapor freezing technique in a thermo cool box. The straws were maintained at a distance of 4 cm above the liquid nitrogen for 15 minutes to allow vapor freezing and transferred into goblets placed in the thermo cool box placed in the cryopreservation containers. After 24 hours of cryopreservation, the straws were thawed at 370C for 1 minute and evaluated for acrosomal integrity.

Evaluation of Seminal Attributes

Semen was evaluated for progressive motility, viability and membrane integrity at three stage i.e. after dilution and addition of BHT (0, 0.5 and 1.0 mM) (Stage I), after equilibration at 4-50C for 4h (Stage II) and after 24 h of cryopreservation or post thaw (Stage III). After 24 h of cryopreservation, the straws were thawed at 37°C for 45 s. After thawing the semen was evaluated for progressive motility, sperm viability and sperm membrane integrity (HOST) in the control and test samples (T1 and T2). Progressive motility was observed on a thermostatically regulated stage, whereas, the sperm membrane integrity was evaluated by exposing the sperms to hypo-osmotic solution (150 mOsmol/L) (Jeyendran et al., 1984). Sperm viability was observed by Eosin-Nigrosin staining.

Statistical Analysis

The data generated from the experiment was analysed statistically (Snedecor and Cochran, 1989) by using SPSS computer package. Effect of BHT (antioxidant) on semen quality was analyzed using two-way analysis of variance and significance was tested at 5% level (p<0.05).


Mean per cent progressive motility was significantly (p<0.05) higher at all three stages in T2(supplemented with 1mM BHT) compared to control. At stage III, Tshowed significantly (p<0.05) higher progressive motility compared to other groups. The per cent viability was also significantly (p<0.05) higher in T2 at all three stages (stage I stage II and stage III) compared to T1 and control. The mean per cent membrane stable spermatozoa were also significant (p<0.05) higher in T2 at all three stages (stage I stage II and stage III) as compared to T1 and control.


Sperm membrane integrity is the index of sperm function. Loss in the membrane permeability is a common limitation during freezing and thawing due to the per-oxidative damage caused by free radicals and dilution of antioxidant defense (Khalifa et al., 2008). The membrane intactness can be retained by increasing the antioxidant defense and subsequent reduction in free radical mediated oxidative damage to the cells (Andreea, A. and Stela, Z. 2010). Progressive motility serves as the index of fertilization (Ball et al., 2010). Post thaw viability of the sperms is always a challenging step because 30 to 40% viability of the sperms are decreased at the time of freezing and thawing, associated due to changes in the temperature, and ultra-low temperature maintained during cryopreservation (Watson 2000).

The present study was planned to evaluate the role of BHT as an antioxidant additive of semen to minimize the cryodamage. Semen was evaluated at three stages i.e. post dilution, post equilibration and post thawing (After 24 hr of deep freezing). The results of study are presented in graphs 1-3.


Graph 1 shows the effect of butylated hydroxytoluene (BHT) supplementation on post dilution, post equilibration and post thaw per cent progressive motility of crossbred bull spermatozoa. The percent progressive motility at all 3 stages showed higher values in extender containing 1.0 mM BHT. Post dilution, the percentage of progressive motility was 77.33±2.24 in 1 mM as compared to the 0 (73.92±2.15) and 0.5 mM (75.22±2.15) BHT. The post equilibration percentage of progressive motility was 68.47±2.58 in 1 mM as compared to the 0 (64.28±2.96) and 0.5 mM (66.42±2.89) BHT. The Post thaw percentage of progressive motility was 56.58±1.38 in 1 mM as compared to the 0 (49.53±2.00) and 0.5 mM (52.78±1.63) BHT.


Graph 2 shows the effect of butylated hydroxytoluene (BHT) supplementation on post dilution, post equilibration and post thaw percent viability of crossbred bovine bull spermatozoa. The percent viability at all 3 stages showed higher values in extender containing 1.0 mM BHT. Post dilution, the percentage of viability was (86.58±2.34) in 1 mM as compared to the 0 (81.56±2.30) and 0.5 mM (82.69±4.47) BHT. The post-equilibration percentage of progressive viability was (81.33±2.77) in 1 mM as compared to the 0 (72.50±2.63) and 0.5 (77.06±2.59) mM BHT. The post thaw percentage of viability was (69.22±2.99aC) in 1 mM as compared to the 0 (59.14±2.18) and 0.5 mM (64.31±2.19) BHT.


Graph 3 shows the effect of butylated hydroxytoluene (BHT) supplementation on post dilution, post equilibration and post thaw percent membrane integrity of crossbred bovine bull spermatozoa. The percent membrane integrity at all 3 stages showed higher values in extender containing 1.0 mM BHT. Post dilution, the percentage of membrane integrity was (83.36±1.95) in 1 mM as compared to the 0 (77.67±1.81) and 0.5 mM (80.28±1.79) BHT. The post equilibration percentage of membrane integrity was in 1 mM as (76.61±2.20) compared to the 0 (69.92±2.30) and 0.5 mM (5 72.53±2.26) BHT. The post thaw percentage of membrane integrity was (70.31±3.27) in 1 mM as compared to the 0 (60.56±2.93) and 0.5 mM (65.31±3.35) BHT.

Sperm motility is nearly reduced by 50% after freezing and thawing in most of the species of the animal and may be a probable factor behind the reduction in the sperm quality (Watson 2000). Thus, it is essential to maintain the sperm motility at a higher level after cryopreservation to achieve optimal fertility. To retain the motility and to minimize the free radical mediated oxidative stress, it is essential to improve the antioxidant defense of sperms opted for cryopreservation. It can be achieved by supplementing the extender with antioxidant additives (Ijaz et al., 2009).

Our results are in agreement with the study in which 1.0mM BHT improved post thaw motility of Holstein-Frisian bull spermatozoa in sodium citrate extender. It is believed that species and type of extender affect the sperm protection by BHT (Ball et al., 2001; Roca et al., 2004). It is important to mention that exotic bull semen has higher level of ROS as compared to zebu bull semen (Nichi et al., 2006). It is suggested that increase in sperm motility after BHT supplementation is due to a decrease in oxidative stress and ROS production (Alvarez and Storey, 1983). It is well established that viability of sperm and membrane integrity of the sperm have critical role in the process of fertilization. Therefore, assessment of sperm viability and plasma membrane integrity is of paramount importance due to its interaction with surrounding medium for metabolic exchange. In addition, capacitation, acrosome reaction and the oocyte penetration require a biochemically active sperm plasma membrane. In this study sperm viability and plasma membrane integrity was higher in human semen containing 0.5mM BHT in extender (Jeyendran et al., 1984).

Our results are contradictory with the study on Holstein-Friesian bull semen, where percentage of sperm with intact plasma membrane was higher in sodium citrate extender containing 0.5mM BHT (Shoae and Zamiri, 2008). Our results were also not same with the study that concluded that the supplementation of 0.5mM BHT in tris-citric acid extender improved the motility, plasma membrane integrity and viability of Sahiwal bull spermatozoa (Ansari et al., 2011).

In another study conducted on Hariyana bull semen, addition of 1.0 mM BHT was found better in cryopreservation of compared to 0.5 mM BHT and control samples. The addition of BHT has improved the sperm quality by acting as an antioxidant thereby reducing the lipid peroxidation of the sperms (Patel et al., 2015). The highest (P < 0.05) motility, acrosomal integrity and hypo-osmotic swelling response of spermatozoa were achieved by addition of 1.0 and 2.0 mM BHT to semen extender to improve the semen quality of buffalo bulls (Ijaz et al., 2008). Beneficial role of BHT varies with species, BHT concentration, sperm membrane composition, time for incubation and the type of semen extender used. The excessive amount of antioxidants caused increase fluidity of plasma membrane above the desired point making sperm more prone to acrosomal damages (Shoae and Zamiri, 2008). Supplementation of BHT with higher concentrations has shown detrimental effects on the sperms processed for cryopreservation.

In the present study, a significantly higher post-thaw motility in BHT added semen samples was probably due to its preventing role against ROS and reduction in membrane lipid peroxidation. Furthermore, reduction in motility from dilution to equilibration and post thaw was less in BHT added samples compared to control. Post equilibration and post thaw higher sperm motility in supplemented groups is also indicative of protective action of BHT in cryopreserved sperms.


The maximum beneficial effects were observed by addition of 1.0mM BHT. Addition of BHT significantly improved post thaw semen quality as revealed by higher progressive motility, viability and HOS response of spermatozoa. Hence BHT can be used as additive in semen dilutor for routine cryopreservation to improve the fertility of frozen- thawed spermatozoa.


The authors are highly thankful to Vice Chancellor, Dean, College of Veterinary and Animal Sciences and Director Experiment Station of G.B Pant University of Agriculture and Technology, Pantnagar-263145, Uttarakhand, India for providing necessary facilities and financial support for carrying out this research.


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