Freezing and thawing of semen leads to production of reactive oxygen species (ROS)
due to plasma membrane lipid peroxidation. Because of this semen quality can be
compromised. To overcome this problem, antioxidants have been used in cryopreservation
medium. Glutathione and cysteine have thiol groups which penetrate into the cell and protect
it from oxidative stress. In this study, effect of different concentrations of cysteine and
glutathione on post thaw quality of Sahiwal bull spermatozoa was determined.
Semen was collected with artificial vagina from five mature regular donor Sahiwal
bulls kept at the Semen Production Unit Qadirabad, Sahiwal. Semen samples possessing
>60% motility and >500x10
6
sperm/ml were included in study. After collection, semen
samples from five bulls were pooled, divided into seven equal aliquots and kept at 37 ºC in
water bath. After that dilution was done with Tris citric egg yolk extender having different
concentrations of cysteine and glutathione as Con (0.0 mM), C1 (1.0 mM cystein), G1 (1.0
mM glutathione), CG0.5/1(0.5 mM Cysteine+1.0 mM glutathione), CG1/0.5 (1.0 mM
cysteine+0.5 mM Glutathione), CG0.5/0.5 (0.5 mM cysteine+0.5 mM glutathione) and
CG1/1 (1.0 mM cysteine+1.0 mM glutathione). Diluted samples were cooled to 4ºC in two
hours and equilibrated for 4 hours at 4
o
C. After that they were packaged into 0.5 ml French
semen straws (20x10
6
sperm/straw). All semen straws were placed 4cm above liquid nitrogen
surface in vapors for 10 minutes. Then, semen straws were plunged into liquid nitrogen for
freezing and stored until post thaw analysis. The experiment was repeated for five times
(replicates = 5). Four semen straws/treatment were thawed for 30 seconds in water bath at
37ºC and evaluated for visual motility, plasma membrane integrity (PMI), acrosome integrity,
mitochondrial trans membrane potential and CASA motility parameters and kinematics.
42
Summary
PMI in group CG0.5/0.5 was significantly higher (40.00±1.42 %) as compared to Con
26.67±0.80 (P<0.5). Plasma membrane integrity in groups CG1/1, CG0.5/1, G1 and C1 was
significantly higher (36.00±1.88 %, 36.20±1.07 %, 33.60±1.21 % and 32.80±0.80 %
respectively) as compared to Con (26.67±0.80 %) (P<0.05). There was no significant
difference in C1 (32.80±0.80 %) and G1 (33.60±1.21 %) (P>0.05). In case of acrosome
integrity, NAR value of group CG0.5/0.5 was significantly higher (71.40±1.08 %) as
compared to Con (59.67±0.37 %) (P<0.05). All other groups also showed significant
differences as compared to Con (P<0.05). CG0.5/0.5 also showed significantly higher NAR
value (71.40±1.08 %) as compared to C1 (64.40±1.40 %) and G1 (67.60±2.07 %) (P<0.05).
CG0.5/0.5 had significantly higher value (71.40±1.08 %) as compared to CG1/0.5 and CG1/1
(65.60±0.81 % and 68.80±0.97 % respectively) (P<0.05). CG0.5/0.5 had significantly higher
subjective motility (54.00±1.88) as compared to Con (36.66±0.92)
Mitochondrial transmembrane potential of CG0.5/0.5 was significantly higher
(37.00±0.71 %) as compared to Con (25.33±1.28 %) (P<0.05). All the other treatment groups
also had higher mitochondrial transmembrane potential as compared to Con (P<0.05). In
groups of combination of cysteine and glutathione, CG0.5/0.5 showed significant difference
(37.00±0.71 %) as compared to CG1/1 and CG1/0.5 (29.00±1.00 % and 33.80±0.86 %)
respectively (P<0.05).
CASA results showed that CG1/1 had significantly higher motility as compared to the
control. But the percentage of progressive spermatozoa was significantly higher in
CG0.5/0.5. VSL of group CG0.5/0.5 was significantly higher (53.33±2.90 %) as compared to
Con (45.10±0.50 %). However, VSL, VCL, ALH and BCF did not vary significantly among
groups. STR and LIN of group CG0.5/0.5 were significantly higher as compared to the
control group.
43
Summary
In conclusion, addition of cysteine and glutathione in tris citric egg yolk extender
improved the post thaw quality of Sahiwal bull spermatozoa. In case of additive effect of
cysteine and glutathione, CG0.5/0.5 showed higher plasma membrane integrity, acrosome
integrity, mitochondrial transmembrane potential, progressive and rapid spermatozoa as
compared to CG0.5/1, CG1/0.5 and CG1/1.
44