Effects of Different Equilibration Times on Post-Thaw Cryopreserved Semen Quality of Cattle and Buffalo Bulls

Nine mature bulls were selected in three (n=9) different groups (Holstein Fresian, Jersey and Nili Ravi) to find out equilibration time (T1, 2 h; T2, 6 h and T3, 6 h) effects on different semen quality parameters. Highest sperm progressive motility, sperm plasma membrane integrity were observed in T2. It reflects high damages at T3 and T1 due to cryo shock and oxidative stress respectively. Sperm DNA integrity and pH were increased linearly from T1 to T3. The dead sperm percentage was significantly (P<0.01) low in groups with semen sample treated with T2 compared to other groups. Significantly (P<0.01) high sperm individual motility percentage was observed for T2 than T3 and T1. Sperm acrosomal integrity percentage showed significantly ((P<0.01) high value in T2. It is concluded that 4 h equilibration time improved semen qualitative characteristics. Among the three breeds, Jersey bull semen samples showed


INTRODUCTION
T he successful cryopreservation enables spermatozoa to maintain its fertilizing ability at post thawing.During cryopreservation, the sperm should be able to maintain normal plasma membrane integrity, motility, and enzymes activity like acrosin to allow the penetration of pserm into the ovum.Fertilization ability of the spermatozoa will not be achieved in case of any disturbance in any of these functions.The major menace in maintaining these properties is due to ice crystals formation and the osmotic gradients taken up by water movement during cryopreservation process (Bushra et al., 2013).
The survivability of sperms is enhanced by equilibration time during freezing.Water within the cell is replaced with glycerol, which prevents ice crystal

Selection of bulls
Nine andrologically and physically normal adult bulls of each breed including Friesian, Jersey and Nili Ravi bulls were selected for assessment of their semen quality for 4 weeks at the interval of twice a week (Monday and Thursday).

Semen collection
Semen samples were collected early in the morning from all the selected bulls kept at Government Cattle Breeding and Dairy Farm Harichand Charsadda using artificial vagina from each bull (n=9) maintaining all hygienic measures (Qureshi, 2011).Collection of two semen aliquots (total=6) were made from each bull of each breed on the same day.Semen ejaculates with motile sperm (>60%), volume (>1 mL) and concentration (>0.5 billion/ml) from each bull were used (Ansari et al., 2011).Semen was shifted to hot water bath at 34 o C immediately after collection and was evaluated for initial qualitative and quantitative tests.Sperm concentration in semen aliquot was assessed by bovine photometer ACCUCELL (IMV, France) at 539nm wave length.Each ejaculate was supplemented in single step with Tris-egg yolk-glycerol (TRIS) at 34 o C, getting the final concentration of 50×10 6 sperm/ml.The semen was extended (Tris-citric egg yolk extender) soon after assessing sperm mass motility and concentration, 5-10 min after collection (Leite et al., 2010).

Semen processing
Semen samples were incubated in a water bath at 34 o C for 10 min.after that, it was cooled to 25 o C (room temperature), which took about 20 min and then cooled down to 4 o C in 2 h.After cooling, semen was exposed to different equilibration times (2, 4, and 6 h) at 4 o C (Leite et al., 2010).At the end of each equilibration time, 0.5 mL semen was transferred to liquid nitrogen with the help of French straw.To assess post thaw semen quality after 24 h of cryopreservation, the frozen semen straws were thawed in water bath at 37 o C for 30 seconds and incubated for 6 h for qualitative tests (Ansari et al., 2011).

Determination of sperm quality parameters
Semen was assessed for individual motility, progressive motility and dead sperm percentage were determined according to Qureshi (2011).

Determination of sperm plasma membrane integrity
This test was performed as directed by Qureshi (2011).Briefly, 0.1 ml of the thawed test whole semen was mixed with 1 ml test solution [0.5 ml of D-fructose (1.47%) and sodium citrate (2.7%)] and was incubated for 40 minutes at 37 o C.After incubation in hypo-osmotic solution, semen drop (5 µl) was placed on a prewarmed slide.The slide was then covered with cover slip and was examined under phase contrast microscope (400X; LABOMED LX400).

Determination of semen pH
A thawed semen drop was spread evenly on the pH paper, waited for <30 seconds to get uniform colour of the impregnated zone.The semen color of impregnated zone comparable with >7.0 were considered as of basic nature and that of with <7.0 were considered as acidic in nature (Haugen and Grotmol, 1998).

Determination of acrosomal integrity
The dual staining technique comprising of trypan-blue and Giemsa stain was performed following protocol directed by Kovacs and Foote (1992).In this procedure, trypan-blue was used for distinguishing live and dead spermatozoa while for evaluating acrosomal membrane integrity Giemsa stain was used.For this intention, one drop of both trypan-blue (2.0%) and semen was placed on a clean slide and mixed, air dried and fixed (formaldehyde-neutral red solution, MP Biomedicals, Eschwege, Germany)] for 5 min.The slides were then rinsed with distill water and for 4 h and then dipped in Giemsa stain (MP Biomedicals, Eschwege, Germany).The Giemsa stained slides were washed with distill water, air dried and cover slips were placed on it before covering with Balsam of Canada (Merck, Darmstadt, Germany) and observed under phase contrast microscope at 1000X (LABOMED LX400).

Determination of sperm DNA integrity
Semen straw was thawed in water bath at 37 o C. 20 μl semen was mixed in 200 μl TNE buffer (Tris = 50mM, NaCl =140mM, EDTA =5mM) to final concentration of approximately 2×10 6 spermatozoa/ml.From each semen sample of 20 μl, three smears were prepared on prewarmed clean glass slides, smeared and air dried.Carnoy's solution [methanol and glacial acetic acid (Merck, Darmstadt, Germany) in 3:1 ratio] was applied over night for the fixation of all smears.Subsequently, slides were washed with distill water, made air dried and dipped in tampon solution [(80mM citric acid (Merck, Darmstadt, Germany) and 15mM Sodium phosphate (Sigma, NY, USA), pH 2.5)] in water bath at 75 o C for 5 minutes.After the incubation in tampon solution, acridine orange (Sigma, NY, USA), pH 2.5) (0.2 mg/ml) was applied for 2 minutes on processed slides.Slides were then washed with phosphate buffer saline (PBS), covered with cover slip whilst they were still soaked and evaluated under fluorescent microscope (LABOMED LX400).

Statistical analysis
Effects of equilibration times on sperm progressive motility, individual motility, acrosomal integrity, plasma membrane integrity, dead sperm, semen pH and DNA integrity were analyzed with the help of Statistix (8.1 version) for Windows using 2×3×2 factorial design as described by Steel and Torrie (1997).To find the significant difference, Duncan multiple range test was used (Duncan, 1955).

RESULTS
The results of membrane integrity, progressive motility, individual motility and acrosomal integrity in different breeds at different equiliberation times is given in Table I.The results indicated that membrane integrity, progressive motility, individual motility, acrosomal integrity increased significantly (P<0.01) at 4 h equilibration time and decreased at 2 h.Also, Jersey has significantly (P<0.01)high values followed Holstein Friesian and Nili Ravi.
The result of different equilibration times on semen pH, DNA integrity, and dead sperm of Jersey, Holstein Fresien and Nili Ravi bulls is given in Table II.The effect of Semen pH was significantly (P<0.01)high at 2 h equilibration time and was significantly low at 6 h.Breed has no significant effect on seminal pH.Sperm DNA integrity was significantly (P<0.01)high at 2 h equilibration time and was significantly low at 6 h.Sperm DNA integrity value was significantly (P<0.01)high in Holstein Friesian followed by Jersey and Nili Ravi.Dead spermatozoa were significantly (P<0.01)high at 2 h equilibration time and was significantly high at 4 h.Dead sperm percentage was significantly (P<0.01)high in Nili Ravi followed by Holstein Friesian and Jersey.

DISCUSSION
In this study, the effect of different equilibration times on post thaw cryo preserved semen quality of cattle and buffalo bulls were evaluated.The result of the present study indicated that 4 h equilibration time produced optimum results regarding semen traits.In the present study, sperm individual motility, progressive motility, membrane integrity and acrosomal integrity were significantly high for 4 hr equilibration time which is in line with other works (Herold et al., 2006;Liete et al., 2010).Spermatozoa faced partial injuries during equilibration step of semen processing, became high post freezing and thawing.During cryopreservation, low temperature and high salt concentration are mainly responsible for destabilization of sperm plasma membranes (Holt and North, 1994).The present results which are in line with above research findings presented that sperm membrane integrity percentage of the tested semen samples was significantly (P<0.01)high for 4 h equilibration time followed by 6 h and the low value of membrane integrity percentage was recorded for 2 h equilibration time and this has been also supported in other studies (Herold et al., 2006;Liete et al., 2010).
The current research results illustrated that sperm progressive motility percentage of the tested samples was significantly highest for 4 h equilibration time followed by 6 h and the lowest value of progressive motility percentage was noted for 2 h equilibration time and this has been supported by many scientist (Herold et al., 2006;Liete et al., 2010).Breed effect showed that plasma membrane of buffalo sperm constitutes higher percentage of polyunsaturated fatty acids than sperm of cattle bull (Parks et al., 1992).Due to this, buffalo sperm is more vulnerable to cryo damages and oxidative stress induced during freezing and cryopreservation process than cattle spermatozoa (Kumar et al., 2011).As a result there is decrease in progressive motility percentage of buffalo spermatozoa.This is supported by the findings of present research work as progressive motility percentage was high in cattle bulls (Jersey, Holstein Friesian) than buffalo bull (Nili Ravi) spermatozoa.
The presented results in term of sperm motility was supported by the above findings as individual motility percentage of the studied semen samples was significantly highest for 4 h equilibration time followed by 6 h and the least value of individual motility percentage was recorded for 2 h equilibration time and this has also been supported by other works (Herold et al., 2006;Liete et al., 2010).Buffalo spermatozoa lose their motility following equilibration, freezing and thawing processes (Kakar and Anand, 1981).Moreover, a significant loss in buffalo sperm motility occurs following equilibration time than cattle sperms (Tuli et al., 1981).Similar pattern was supported by our presented data as cattle sperm (Jersey and Holstein Friesian) had high individual motility than Buffalo sperm motility (Nili Ravi).
The presented results showed the symmetrical relation between different equilibration times and acrosomal integrity percentage of the studied semen samples as it was significantly high for 4 h equilibration time followed by 6 h and the least value of sperm membrane integrity percentage was recorded for 2 h equilibration time and this is in line with other works (Herold et al., 2006;Andrabi, 2009).Acrosomal damages were high for 2 h equilibration time than 4 h (Niasari-Naslaji et al., 2006;Liete et al., 2010).Breed effect showed that plasma membrane of buffalo sperm constitutes higher percentage of polyunsaturated fatty acids than sperm of cattle bull (Parks et al., 1992).Due to this buffalo sperm is more vulnerable to cryo damages and oxidative stress induced during the process of freezing and cryopreservation than cattle spermatozoa (Kumar et al., 2011).As a result, there is a decrease in acrosomal integrity percentage of buffalo spermatozoa.These relations were supported by our presented data having high acrosomal integrity percentage for cattle (Jersey, Holstein Friesian) than buffalo (Nili Ravi) sperms.Semen storage for long equilibration time may decrease post freezing seminal pH because of lactic acid production from fructose and post freezing semen pH may increase, when it is stored for short equilibration time as production of lactic acid formation will be low (Qureshi, 2011;Andrabi et al., 2009).This is supported by present research findings as seminal of the studied semen samples was significantly high for 2 h equilibration time followed by 4 h and the least value of pH was recorded for 6 h equilibration time.
Because of lipid peroxidation of the plasma membrane (Kankofer et al., 2005), the process of semen dilution, cooling, equilibration, freezing and thawing speed up the production of reactive oxygen species molecules (Mehr et al., 2013).These unnecessary reactive oxygen species molecules can damage the sperm DNA integrity of cattle and buffalo semen (Kumaresan et al., 2006(Kumaresan et al., , 2005;;Garg et al., 2008).In the presented data, our findings are in line with the results for above studies on bovine semen.Buffalo semen protects spermatozoa from oxidative stress due to its both enzymatic and non-enzymatic antioxidant mechanism (Andrabi, 2009;Khan, 2011).Plasma Effects of Different Equilibration Times on Cryopreserved Semen Quality 617 membrane of buffalo sperm constitutes higher percentage of polyunsaturated fatty acids than sperm of cattle bull (Parks et al., 1992).Due to this buffalo sperm is more vulnerable to and oxidative stress induced during freezing and cryopreservation than cattle spermatozoa (Kumar et al., 2011), results in high DNA damage for buffalo after thawing (Parks et al., 1992).
Effective freezing procedure requires determination of proper equilibration time which suggests that if equilibration time is low then huge amount of water is left inside the cell resulting in cell death due to ice crystal formation which damages the membrane may lead to sperm death.If the equilibration time is too high then the cells may undergo excessive shrinkage and extra solute contact.For that reason the ideal equilibration time is that which is as quick as possible to elude effects of solution, but slow enough to avoid intracellular ice formation to maintain sperm viability (Andrabi, 2009).

CONCLUSION
Equilibration time of 4 h showed best post thaw cryopreserved semen quality characteristics in all breeds.Jersey bulls showed the best quality attributes at 2, 4, and 6 h of equilibration.Sperm membrane integrity showed positive correlation with acrosomal integrity, progressive motility, individual motility and was inversely related the dead sperm percentage, DNA integrity and seminal pH.

Statement of conflict of interest
The author have declared no conflict of interset.

Table I . Mean± SE of membrane integrity, progressive motility, individual motility and acrosomal integrity of Jersey, Holstein, Friesian and Nilli Ravi bulls at different equilibration times.
Effects of Different Equilibration Times on Post-Thaw Cryopreserved Semen Quality