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Byline: F. Wadood M. Aleem A. Ijaz N. Ahmad M. S. Yousaf D. H. Mughal A. Javid and S. A. Mahmood


This study was designed to determine effect of various concentrations of L- cysteine on frozen-thaw semen quality of Nili Ravi buffalo. Single ejaculate of four buffalo bulls (Nili-Ravi) was collected at weekly intervals for 5 weeks. Ejaculates of acceptable motility (=70%) were pooled and semen was diluted by tris citric acid extender (TCAE; 300 mosm/kg osmotic pressure and ~7.0 pH) having 4 levels of L-cysteine (0.0 2.0 5.0 and 8.0 mM). Semen was equilibrated at 4C and packaged in 0.5 mL French straws with 20A-106 spermatozoa/ straw and then frozen in liquid nitrogen. L-cysteine inclusion levels had non-significant (Pgreater than 0.05) effect on spermatozoa motility viability NAR PMI and on lipid peroxidation. However DNA damage in spermatozoa was significantly higher (Pless than 0.05) in 8.0 mM L- cysteine. It was concluded that L-cysteine (2.0 and 5.0 mM) addition to cryopreserved semen had no significant effect on post thaw semen characteristics except at 8 mM which adversely affected DNA integrity.

This may be due to its toxicity at this concentration.

Key words: cryopreservation semen spermatozoa antioxidants buffalo L-cysteine


L-Cysteine is an amino acid (propionic acid) of thiols group of non-enzymatic antioxidants. It is a precursor of glutathione which plays an important role in hydrogen peroxide oxidation and can prevents DNA damage during cryopreservation. Addition of L-cysteine to semen extender prevents apoptosis spermatozoa motility loss and lipid peroxidation during cryopreservation (Bilodeau et al. 2001). Reactive oxygen species (ROS) affects semen quality (Thuwanut 2007) however certain concentration of ROS is useful for spermatozoa acrosome reaction and capacitation (de Lamirande and Gagnon 1993). Imbalance of oxidants and antioxidants (Oxidative stress) adversely affects spermatozoa motility proteins DNA and membrane integrity which leads to cell apoptosis (Filho et al. 2009).

Various reports indicate that addition of antioxidants to semen reduces oxidative stress and improves post thaw semen quality as antioxidants neutralize the oxidants present in the semen (Bucak et al. 2008). Semen cryopreservation decrease natural antioxidants to an extent that spermatozoa are exposed to oxidative stress. Moreover buffalo have higher lipid oxidation rate due to higher membrane contents of poly unsaturated fatty acids (Chatterjee and Gagnon 2001) which further reduces activity of natural antioxidants. Therefore antioxidants addition to semen extender helps preserving spermatozoa integrity during the process of cryopreservation (Andrabi et al. 2008).

Therefore the present study was designed to determine effect of various concentrations of L- cysteine on frozen-thaw semen quality of Nili Ravi buffalo.


Experimental Animals and Semen Collection: During this study four Nili-Ravi buffalo bulls of Semen Production Unit Qadirabad Sahiwal Pakistan were used. Conditions of feeding and management were same as of Mughal et al. (2013). Single ejaculate was collected at weekly intervals for 5 weeks during spring months (March - April). Collected semen was provided 15 minutes holding time at 37 C in water bath. Preliminary semen assessment e.g. color motility and volume etc. were carried out.

Preparation of Extender and Semen Processing: Composition of TCA extender is given in Table 1. Suitable ejaculates (=70% motility) of four bulls were pooled and then calculated semen quantity was added to the graduated tubes having specific amount of TCAE and L-cysteine (C7352 Sigma Aldrich USA) concentrations of 0.0 2.0 5.0 and 8.0 mM to obtain 40x106 spermatozoa/mL. Semen was equilibrated at 4C and filled in 0.5 mL polyvinyl straws (IMV France) in cold cabinet. These straws were cooled from 4C to -15C @ 3C/min then from -15C to -80C @ 10C/min. Subsequently these straws were placed 4 cm above the liquid nitrogen for 10 minutes in straw grill and finally dipped into liquid nitrogen.

Semen Assessment: Procedure adopted by Ijaz et al. (2009) was used to assess spermatozoa motility after thawing semen at 37 C for 30 seconds. Spermatozoa viability and PMI was determined as per Khan and Ijaz 2008 while NAR by Rasul et al. 2000. Acridine orange staining technique following the procedure of Tejada et al. (1984) was used to determine spermatozoa DNA integrity. Two hundred spermatozoa were examined to assess their viability PMI NAR and DNA integrity. The mean of three observations was considered a single data point. For lipid peroxidation thiobarbituric acid assay was used to determine Malondialdehyde (MDA: stable lipid peroxidation product) through a spectrophotometer (UV 2800 BMS Canada) at 532 nm as per procedure of Ohkawa et al. (1979). The results were expressed in nm of MDA.

Statistical analysis: Statistical Package for Social Science (SPSS for Windows version 12 SPSS Inc. Chicago IL USA) was used for statistical analysis. Data were presented as mean S.E. One way analysis of variance was used. Duncan's Multiple Range Test was employed to compare group differences. Difference in results was considered significant at P less than 0.05.


Present study was designed to determine effect of various concentrations of L- cysteine on frozen-thaw semen quality of Nili Ravi buffalo. The results regarding the effect of varying concentrations of L-cysteine on seminal parameters are presented in Table 2. It was revealed that L-cysteine had significant (Pless than 0.05) effect on spermatozoa DNA damage at 8.0 mM L-cysteine compared to 2.0 5.0 and 0.0 mM however effect on all other parameters was non-significant (Pgreater than 0.05). Non- significant results of percentage spermatozoa motility viability acrosomal integrity PMI and lipid peroxidation may be correlated to the fact that L- cysteine is a water soluble antioxidant and due to this characteristic do not bind properly to plasma membrane (Khalifa et al. 2008).

Moreover in this study a suitable semen extender and optimized osmotic pressure based on previous experiments (data not shown) was selected and it could be another reason for the differences of our findings from other investigators (extender osmotic pressure not mentioned in their studies) as semen extender with optimized osmotic pressure decreases the oxidation rate (Aitken and Krausz 2001).

Over compaction of spermatozoa chromatin occurs due to oxidative stress that break DNA strands (Cordova et al. 2002). These spermatozoa do not attach to ova as they do not decondence during fertilization and transmit defective genome (Sakkas and Alvarez 2010). Higher (Pless than 0.05) DNA damage at 8.0 mM of L-cysteine might be due to toxic effects of antioxidant as excessive antioxidant decreases physiological level of oxidants which is considered essential for normal spermatozoa function (Roca et al. 2004) moreover higher concentrations of antioxidant make spermatozoa vulnerable to cryo-injury by increasing the plasma membrane fluidity (Shoe and Zamiri 2008). As higher concentrations of antioxidant decrease the oxidation rate of PUFA (in spermatozoa membrane) and consequently enhance the membrane fluidity (Sikka 2004). Enhanced fluidity of spermatozoa membrane weakens the function of ionic channels/ATPases in membrane that ultimately affects nutrients availability to the spermatozoa. Higher

Table 1. Composition of TCA Extender

Solution A

Tris (Hydroxymethyl)

###16.78 g


citric acid monohydrate###9.32 g

D-Fructose###7.53 g

Bi-distilled Water###500 mL

Solution B

Solution A###73 mL

Egg Yolk###20 mL

Glycerol###7 mL

Pencillin###1000 I.U./mL

Streptomycin###1.0 mg/mL

Solution A###73 mL

Table 2. Effect of Different Concentrations of L-Cysteine on Post Thaw Spermatozoa Characteristics of Buffalo Bull (n=5).

###L-###Motility (%)###Viability (%)###NAR (%)###PMI (%)###Damaged###Lipid

Cysteine###DNA (%)###Peroxidation






oxidative stress during buffalo semen cryopreservation due to its higher PUFA contents results into DNA damage (Aitken and Krausz 2001). Our results were contrary to Thuwanut (2007) work which may be primarily due to difference in extender composition osmotic pressure and species. Tuncer et al. (2010) noted higher cattle spermatozoa DNA damage at 10.0 mM cysteine concentration compared to control as reported in this study.

In conclusion L-cysteine 2.0 and 5.0 mM inclusion to semen cryopreserved in TCAE had non- significant influence on post thaw semen characteristics except at 8 mM which adversely affected DNA integrity which may be due to its toxic effect.


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Publication:Journal of Animal and Plant Sciences
Article Type:Report
Date:Feb 28, 2015

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