Cholesterol is an important constituent in the membrane fluidity regulation. This role becomes
more important in the cryopreservation of sperm cells where destabilization of plasma membrane
leads to intracellular ice formation causing death of the cell. The objective of the present study
was to verify if addition of cholesterol in semen extender has a beneficial effect on post-thaw
semen quality in buffalo bulls. This study was carried out at Al-Haiwan Sires, Sahiwal, Pakistan.
Cholesterol was added to Tris-citric acid semen extender in the form of cholesterol-loaded
cyclodextrin (CLC). Semen was collected by using artificial vagina (420 C) from four buffalo
bulls. Split pooled ejaculate (n = 7), having more than 60 % visual sperm motility, were diluted
at 370 C in extender containing CLC either 3mg Iml (LOW), 4 mgl ml (MED), 5 mg Iml (HIGH)
or without (CON). Motility of semen samples before freezing was checked and cryopreservation
was done through routine procedures. Beneficial effect of CLC in cryopreserved semen was .
assessed by post-thaw motility (%), live spermatozoa (%), morphological abnormalities (%),
normal acrosornes (%) and plasma membrane integrity (%) in four groups. Analysis of variance
revealed that motility before freezing was significantly (P<0.05) more in MED (68.93 ± 0.51 %)
as compared to CON and HIGH but non-significantly higher than LOW. Percent post thaw
motility (PTM) from semen samples containing MED concentration of CLC was the highest
(61.43 ± 0.74 %; P < 0.05) followed by LOW (50.36 ± l.15 %; P < 0.05) or HIGH (38.57 ± 2.66
%; P < 0.05) (Table 1). Percent PTM was the lowest in CON (32.86 ± 2.07 %; P < 0.05) semen
samples. Mean plasma membrane integrity (PM I) from semen samples containing MED
concentration of CLC was the highest (59.36 ± 1.32 %; P < 0.05) followed by LOW (51.93 ±
1.32 %; P < 0.05) or HIGH (44.00 ± 1.65 %; P < 0.05) and was least in CON (41.14 ± 1.71 %; P

< 0.05) semen samples. Mean normal acrosomes from semen samples containing MED
concentration of CLC was the highest (52.93 ± 1.78 %; P < 0.05) followed by HIGH (40.57 ±
2.01 %; P < 0.05) and CON (34.93 ± 2.89 %; P < 0.05) semen samples. Buffalo bull semen in
MED & LOW had significantly (P < 0.05) higher live sperm percentage (69.64 ± 1.84 & 61.43 ±
1.62, respectively) as compared to HIGH and CON samples. CON samples had significantly
(P<0.05) higher morphological abnormalities (9.86 ± 0.14 %) as compared to LOW & MED but
non-significantly higher as compared to HIGH samples. All the variables under study had strong
positive correlation (P < 0.01) with each other except morphological abnormalities having strong
negative correlation (P < 0.01) with all others. It is concluded that addition of cholesterol in
MED concentration (4 mg CLCI ml of semen extender) to buffalo bull semen can improve post-
thaw semen quality.
Conception rate of AI in buffaloes is generally lowered than in cows. Cholesterol is an
important constituent in the membrane fluidity regulation. This role becomes more important in
the cryopreservation of sperm cells where destabilization of plasma membrane leads to
intracellular ice formation causing death of the cell. The objective of the present study was to
verify if addition of cholesterol in semen extender has a beneficial effect on semen post-thaw
quality in buffalo bulls. This study was carried out at Al-Haiwan Sires, Sahiwal, Pakistan.
Cholesterol was added to Tris-citric acid semen extender in the form of cholesterol-loaded
cyclodextrin (CLC). Semen was collected by using artificial vagina (42° C) from four buffalo
bulls. Split pooled ejaculate (n = 7), having more than 60 % visual sperm motility, were diluted
at 37° C in extender containing CLC either 3mg Iml (LOW), 4 mgl ml (MED), 5 mg Iml (HIGH)
or without (CON). Motility of semen samples before freezing was checked and cryopreservation
was done through routine procedures. Further examination was done to check post-thaw motility
(%), live spermatozoa (%), morphological abnormalities (%), normal acrosomes (%) and plasma
membrane integrity (%) using phase contrast microscope and hypo osmotic swelling assays.
Analysis of variance revealed that motility before freezing was significantly (P<0.05) more in
MED (68.93 ± 0.5 I %) as compared to CON and HIGH but non-significantly higher than LOW.
Percent post thaw motility (PTM) from semen samples containing MED concentration of CLC
was the highest (61.43 ± 0.74 %; P < 0.05) followed by LOW (50.36 ± 1.15 %; P < 0.05) or
HIGH (38.57 ± 2.66 %; P < 0.05) (Table 1). Percent PTM was the lowest in CON (32.86 ± 2.07
%; P < 0.05) semen samples. Mean plasma membrane integrity (PMI) from semen samples
containing MED concentration of CLC was the highest (59.36 ± 1.32 %; P < 0.05) followed by
LOW (51.93 ± 1.32 %; P < 0.05) or HIGH (44.00 ± 1.65 %; P < 0.05) and was least in CON
(41. I 4 ± 1.71 %; P < 0.05) semen samples. Mean normal acrosomes from semen samples
containing MED concentration of CLC was the highest (52.93 ± 1.78 %; P < 0.05) followed by
HIGH (40.57 ± 2.01 %; P < 0.05) and CON (34.93 ± 2.89 %; P < 0.05) semen samples. Buffalo
bull semen in MED & LOW had significantly (P < 0.05) higher live sperm percentage (69.64 ±
1.84 & 6 1.43 ± 1.62, respectively) as compared to HIGH and CON samples. CON samples had
significantly (P<0.05) higher morphological abnormalities (9.86 ± 0.14 %) as compared to LOW
& MED but non-significantly higher as compared to HIGH samples. All the variables under
study had strong positive correlation (P < 0.01) with each other except morphological
abnormalities having strong negative correlation (P < 0.01) with all others. It is concluded that
addition of cholesterol in MED concentration (4 mg CLCf ml of semen extender) to buffalo bull
semen can improve post-thaw semen quality.