Frozen semen of 26 breeding cattle bulls (Pure HF and HF crosses) were procured from Semen Freezing Laboratory, GADVASU, Ludhiana, Semen Bank Bhattian, Khanna and Government Semen Bank, Ropar, Punjab, India during 2013-2014. Freshly ejaculated semen and blood of these bulls were also collected to harvest seminal plasma and serum, respectively from the respective farms. All the tests were performed in duplicate.
Sperm concentration was taken from the records of respective semen freezing laboratories.
A drop of frozen-thawed semen was placed on micro slide, covered with cover slip and progressively motile spermatozoa were observed under bright field microscope (400×) at 37˚C. A total of 200 spermatozoa were counted in different fields and percentage motility was calculated.
Frozen thawed semen was evaluated for viability and abnormalities through Eosin Nigrosin staining method. A total of 150 sperms were counted in different fields under oil immersion at 1000 X and per cent live sperm was calculated. Spermatozoa with various abnormalities of head, tail and mid piece were also observed in eosin-ni- grosin stained slides.
A total of 150 spermatoazoa were counted at 400X in different fields and total number of coiled tailed sperms was calculated. The number of coiled tailed spermatozoa in phosphate buffered saline (PBS) was deducted from the number in hypo osmotic solution and the resultant figure was taken as the HOS reactive spermatozoa.
Cervical mucus was collected from a normal cycling cow in estrus and was filled in a capillary by capillary action. Capillary was sealed from one side with PVA powder and pre heated at 37˚C for 10 min. Approximately 100 μl of frozen semen was placed at the bottom of an eppendorf tube and a capillary tube was placed with its open end in the semen. After 30 min of incubation at 37˚C, the capillary tube was fixed on scaled glass slide and viewed under microscope at 400×. The length of the tube was then scanned to establish the distance furthest from the semen reservoir attained by spermatozoa. The maximum distance of migration of spermatozoa after 30 min of incubation was defined as the migration distance. Number of migrated spermatozoa was counted in the peak 0.5 cm.
Sperm smears stained with giemesa were examined microscopically under oil immersion at 1000 X. About 200 spermatozoa with intact acrosomes (stained dark purple) and damaged acrosome (without stain/stained light purple) was counted in different fields and percentage of spermatozoa with intact acrosomes was calculated.
About 200 × 106 spermatozoa/ml were incubated in Tyrode albumin lactate pyruvate (TALP) medium (100 mM NaCL, 3.1 mM KCl, 25 mM NaHCO3, 0.3 mM Na2HPO4, 21.6 mM Na lactate, 2 mM CaCl2, 0.4 mM MgCl2 4H2O, 10 mM Hepes, 1 mM Na pyruvate, 0.6% bovine serum albumin (BSA), 5 mM glucose and heparin 10 µg/ml) at 37˚C in an incubator for 4 - 6 hrs. Motility was checked every hour and sperm smears were prepared at 0, 4 and 6 hrs of incubation. Sperm smears were stained with giemsa to observe different stages of acrosome reaction. Giemsa stained slides were observed under bright field microscope at 1000X and about 200 spermatozoa with swollen heads, vesiculated and shedded acrosomes were counted.
Percentage of motile, viable, HOS reactive and acrosome reacted spermatozoa was calculated by the following formula:
Blood serum and seminal plasma were obtained by centrifugation of clotted blood and fresh semen at 3000 rpm for 10 min, respectively. Blood serum and seminal plasma were heated at 56˚C for 30 min to inactivate complements.
Sperm smears on clean slides were incubated with 1% bovine sperm albumin for 2 hours at 4˚C. Slides were washed thrice with PBS, pH 7.4, incubated with 1:200 diluted serum/1:10 diluted seminal plasma for 1 hour at 37˚C, again washed thrice with PBS. Smears were incubated with rabbit anti-bovine IgG (Sigma) for 45 minutes at 37˚C and washed thrice with PBS. Colour was developed with 3, 3’-Diaminobenzindine tetrahydrochloride in Tris buffer (0.05M, pH 7.6 at 25˚C) and 27 μl of 3% hydrogen peroxide for 5 minutes at room temperature. Washed with distilled water, slides were mounted in 10% glycerol in PBS, covered with coverslip and examined under the microscope at 10 × 100 X for dark brownish colouration of the sperm. About 200 sperms with browning on acrosome, post acrosomal cap or whole head were counted in different fields and percentage of IPA positive sperms were calculated (
Diluted inactivated serum/cervical mucus 1/4 with TALP medium, pH, 7.4 and incubated at 37˚C for 30 min. Collected the motile sperms by centrifugation through Histopaque, suspended the sperm pellet in TALP and adjusted the sperm conc to 20 × 106. Incubated 100 µl of the sperm suspension of motile spermatozoa with 100 µl of inactivated ¼ diluted serum or seminal plasma, incubated for 1 hour at 37˚C. Added 2 ml of TALP, mixed well and centrifuged for 10 minutes at 400 g. Resuspended the pellet with 50 µl of TALP. On a slide, mixed 10 µl of sperm suspension and 5 µl of sperm Mar latex particles bound to IgG/IgA, covered with cover slip, kept in humid chamber for 5 min and observed under microscope at 400 X. Attachment of latex particles to the head/tail or whole sperm was observed (
Sperm antigen/extracts were prepared by suspending washed spermatozoa in 62.5 mM Tris-HCl, pH 6.8 containing 2% SDS, 1 mM PMSF, 25 mM benzidine, 10 mM aprotinin, 10 mM pepstatin and 5 mM soyabean trypsin inhibitor, sonicated (3 bursts of 20 sec each) and centrifuged at 15,000 rpm for 30 minutes. ELISA plates were coated with 5 µg protein (sperm antigen) per well by incubating at 37˚C for three hrs. After washing thrice with PBS, antigen coating was blocked by incubating with 300 µl of 2% BSA per well for overnight at 4˚C. Again washed thrice with PBS pH 7.4 and added serial dilutions of serum/cervical mucus into the wells and incubated at 37˚C for three hours. Washed again with PBS and incubated with 100 µl/well of HRP conjugated anti bovine IgG for three hours at 37˚C. Washed the plate twice with PBS and incubated with 100 µl of o-phenyl- diamine + 0.06% H2O2 as a substrate for 20 min at room temperature. Stopped the reaction with 5 N H2SO4 and measured the absorbance at 492 nm using ELISA reader.
The data obtained was analyzed statistically according to Independent Sample T-Test and One-Way ANOVA using difference between means of two groups and means of different group application at 5 per cent level of significance (SPSS, Version 16.0).