Effect of Antioxidants on Refrigeration (5°C) and Cryopreservation (-196°C) of Buffalo Semen
Loading...
Date
2015
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
AAU, Anand
Abstract
This study was undertaken on five sexually mature healthy buffalo bulls of Surti breed during the breeding season at Central Sperm Station of the College. The bulls were maintained in nearly identical nutritional and managemental conditions throughout the period of study and were regularly dewormed and vaccinated against FMD & HS. They were in regular, twice a week, semen collection schedule using AV. Eight ejaculates with >70% initial motility were studied from each of the 5 bulls at weekly interval. Soon after collection, the ejaculates were evaluated for routine physicomorphological attributes, including motility, viability, morphology (eosin-nigrosin), acrosomal integrity (Giemsa stain) and plasma membrane integrity (HOST 150 mOsm/L; an in vitro fertility test) through standard procedures and using phase contrast microscope. Each ejaculate was then divided into five equal aliquots, and extended at the concentration of 100 x10 power 6 spermatozoa ml-1 at 34°C with standard Tris citrate fructose egg yolk glycerol (TFYG) extender as control and TFYG having 2 additives - Cysteine HCl (0.5 & 1.0 mg/ml) and Taurine (4.0 & 6.0 mg/ml)- each at 2 levels to study their comparative efficacy for refrigeration (5°C up to 72 hrs) and cryopreservation (-196°C) of buffalo semen based on above 5 sperm quality parameters, including interrelationships of these parameters in fresh, refrigerated and cryopreserved semen. Small portions of the extended semen samples (2 ml from each aliquot) were transferred to a refrigerator for 5°C preservation and evaluated at 24 hrs interval up to 72 hrs for above quality parameters. The remaining portions of extended semen samples were used for filling the French mini straws on IS4 system (IMV, France). After gradual cooling over 60-90 minutes and equilibration for 4 hrs in cold handling cabinet, the straws were frozen in liquid nitrogen vapour using a programmable bio-freezer (Digitcool 5300, IMV, France). The straws of all 5 extenders were evaluated on dilution, at pre-freezing (after equilibration) and after 18-24 hrs of freezing (post-freeze stage) for the above quality parameters. Post-thaw incubation test (37 °C) was also performed to evaluate sperm survival after 1,2 and 3 hr of incubation. The mean values of ejaculate volume, density (1-4 score), sperm concentration, mass activity (0-5 score), individual sperm motility, live sperm, abnormal sperm, intact acrosome and HOS reactive sperms observed in fresh semen in Surti bulls were 3.04±0.11 ml, 3.06±0.08, 963.05±50.97 million/ml, 2.97±0.06, 75.00±0.69 %, 88.22 ±0.48 %, 4.40±0.26 %, 93.67±0.31 % and 89.17±0.57 %, respectively. The variation between bulls was significant (P<0.05) only for HOS test. With respect to cryopreservation, the overall mean percentages of progressively motile spermatozoa observed initially, after equilibration (pre-freeze stage) and after freezing (post-thaw stage) of buffalo semen split-diluted in TFYG extender, irrespective of additives, were 79.25±0.37, 70.15±0.43 and 41.72±0.53, respectively. The corresponding values for live spermatozoa were 84.80±0.32, 76.61±0.45, 48.05±0.55 %; morphologically abnormal spermatozoa 4.73±0.11, 5.94±0.12 and 8.23±0.13 %; intact acrosomes 92.40±0.18, 89.20±0.21 and 83.06±0.18 % and HOST reactive sperms 84.77±0.35, 75.25±0.46 and 46.42±0.56 %, respectively, all of which differed highly significantly (P<0.01) between stages of freezing process. The mean percentages of progressively motile spermatozoa observed soon on dilution (initial/0 hr) in control TFYG extender and in extender having cysteine 0.5 & 1 mg/ml and taurine 4 & 6 mg/ml were 75.12±0.76, 77.75±0.69, 80.51±0.65, 81.75±0.79 and 81.00±0.82, respectively. The corresponding values for live spermatozoa were 82.75±0.76, 84.27±0.66, 85.95±0.65, 86.17±0.64 and 84.80±0.82 %; intact acrosomes 92.85±0.34, 93.07±0.31, 91.00*2.30, 92.22±0.39 and 90.55±0.43 % and HOS reactive sperms 82.72±0.91, 83.40±0.70, 85.72±0.67, 86.25±0.64 and 85.62±0.86 %, respectively. The mean percentages of progressively motile spermatozoa observed after equilibration (at pre-freeze stage) in control TFYG and TFYG with cysteine 0.5 & 1 mg/ml and taurine 4 & 6 mg/ml were 65.37±0.84, 68.37±0.81, 72.62±0.69, 73.25±0.81 and 71.12±1.02; and after freezing-thawing 38.37±0.95, 39.50±0.80, 46.50±0.72, 50.00±0.62 and 34.25±0.71, respectively. The corresponding values of live spermatozoa observed at pre-freeze stage in control TFYG and in TFYG with cysteine 0.5 & 1 mg/ml and taurine 4 & 6 mg/ml were 72.75±0.99, 70.62±0.81, 78.97±0.93, 79.37±0.88 and 76.32±1.14 %; and after freezing-thawing 44.82±1.02, 46.37±0.99, 52.97±0.79, 55.02±0.80 and 41.07±0.99 %, respectively. The mean percentages of morphologically abnormal spermatozoa observed at pre-freeze stage in control TFYG diluent and in TFYG with cysteine 0.5 & 1 mg/ml and taurine 4 & 6 mg/ml were 6.35±0.27, 5.73±0.28, 5.45±0.25, 5.80±0.28 and 6.38±0.29; and after freezing-thawing 9.10±0.22, 8.15±0.26, 7.10±0.26, 7.90±0.29 and 8.90±0.31 %, respectively. The overall mean values of sperms with head, midpiece and tail abnormalities recorded initially were 1.19±0.04, 1.54±0.04 and 2.01±0.08 %, respectively. The corresponding values noted after equilibration (pre-freeze stage) of extended semen were 1.33±0.04, 1.99±0.05 and 2.70±0.09 %, and after freezing (postthaw stage) 1.62±0.04,2.58±0.06 and 4.03±0.09 %, respectively. The mean percentages of spermatozoa with intact acrosomes found at pre-freeze stage in control TFYG and in TFYG with cysteine 0.5 & 1 mg/ml and taurine 4 & 6 mg/ml were 89.70±0.39, 90.58±0.34, 90.90±0.35, 88.53±0.46 and 86.28±0.45; and after freezing-thawing 82.43±0.32, 84.40±0.18, 85.73±0.18, 82.75±0.30 and 79.98±0.35 %, respectively. The overall mean percentages of sperms with swollen, ruffled, detached and denuded acrosome, and total damaged acrosomes recorded initially were 3.18±0.08, 2.30±0.06, 1.36±0.04, 0.76±0.04 and 7.58±0.18 %, respectively. The corresponding values noted after equilibration (pre-freeze stage) of extended semen were 4.55±0.09, 3.30±0.08, 1.85±0.05, 1.11±0.04 and 10.80±0.21 %, and after freezing (post-thaw stage) were 6.73±0.07, 5.25±0.07, 2.99±0.06, 1.94±0.05 and 16.94±0.18 %, respectively. The values of all the traits differed highly significantly (P < 0.01) between stages/periods of cryopreservation process. The mean percentages of spermatozoa with biochemically active intact plasma membrane recorded at pre-freeze stage in control TFYG extender and in TFYG with cysteine 0.5 & 1 mg/ml and taurine 4 & 6 mg/ml 70.82±0.92, 74.17±0.93, 78.12±0.79, 78.08±0.95 and 75.03±1.08; and after freezing-thawing 42.52±1.04, 45.10±1.04, 51.62±0.82, 53.73±0.69 and 39.10±0.91 %, respectively. The mean percentages of motile spermatozoa observed immediately after thav^ng (0 hr), and after 1, 2 and 3 hrs of post-thaw incubation at 37°C in water bath were 41.52±0.57, 27.88±0.58, 15.05±0.58 and 5.47±0.35 (P<0.01), respectively. The mean percentages of progressively motile spermatozoa observed in control TFYG with cysteine 0.5 mg/ml and 1 mg/ml and taurine 4 mg/ml and 6 mg/ml after 1 hr of post-thaw incubation were 24.13±0.88, 27.18±1.02, 32.75±0.82, 36.13±0.92 and 19.25±0.88; after 2 hrs of incubation 12.38±0.86, 14.38±0.84, 18.88±0.70, 21.50±0.88 and 8.13±0.64 %, and after 3 hrs of incubation 3.25±0.58, 4.63±0.58, 8.88±0.66, 10.00±0.65 and 0.63±0.2 %, respectively. Significantly higher progressive motility, better viability and post-thaw survival, acrosome integrity and HOS reactivity with fewer abnormalities of sperm/acrosome were observed at all stages of cryopreservation of buffalo semen extended with TFYG extender having taurine 4 mg/ml and cysteine 1 mg/ml than cysteine 0.5 mg/ml and control TFYG, while taurine 6 mg/ml in TFYG showed comparatively inferior results towards cryopreservability of buffalo semen may be due to its hypertonicity and osmotoxic effect induced by this higher level. As regards to refrigeration preservation, the overall mean percentages of progressively motile spermatozoa observed on dilution in TFYG based extender at (0- hr), and after 24, 48 and 72 hrs of refrigeration preservation of split-diluted semen samples were 79.22±0.37, 66.75±0.40, 58.02±0.43 and 50.20±0.49 respectively. The corresponding values for live spermatozoa were 84.79±0.31, 72.66±0.45, 63.53±0.48 and 55.99±0.51 %; abnormal sperms 5.17±0.46, 5.79±0.11, 7.05±0.12 and 8.56±0.13 %; intact acrosome 91.94±0.48, 89.85±0.18, 86.80±0.18 and 83.55±0.22 %, and HOST reactive sperm were 84.47±0.35, 72.00±0.46, 62.50±0.48 and 54.95±0.50%, respectively. All these traits differed highly significantly (P<0.01) between refrigeration intervals with progressive decline in sperm quality with increasing storage time. The mean percentages of progressively motile spermatozoa observed after 24 hrs of refrigeration storage in control TFYG extender and in extender having cysteine 0.5 & 1 mg/ml and taurine 4 & 6 mg/ml were 62.37±0.66, 65.37±0.76, 70.00±0.69, 69.00 ±0.82 and 67.00±1.02, respectively. The corresponding live spermatozoa were 82.75 ±0.76, 84.27±0.66, 85.95±0.65, 86.17±0.64 and 84.80±0.82; abnormal sperms 68.60± 0.77, 72.05±0.92, 75.87±0.69, 75.05±0.93 and 71.75±1.17; intact acrosomes 89.75 ±0.31, 90.92±0.25, 91.72±0.32, 89.42±0.39 and 87.45±0.34; and HOS reactive sperms 67.35±0.70, 71.17±0.90, 75.30±0.93,74.37±1.00 and 71.80±1.13 %, respectively. The mean percentages of progressively motile spermatozoa observed after 72 hrs of refrigeration storage in control TFYG extender and in extender having cysteine 0.5 & 1 mg/ml and taurine 4 & 6 mg/ml vy^ere 44.57±0.92, 48.87±0.93, 55.00±0.85, 55.00± 0.82 and 47.62±0.91, respectively. The corresponding values for live spermatozoa were 50.47±1.03, 54.85±1.05, 60.50±0.85, 60.20±0.93 and 53.95±1.01; abnormal sperms 9.28±0.29, 8.30±0.25, 7.78±0.26, 8.23±0.31 and 9.23±0.24; intact acrosomes 83.10± 0.33, 85.10±0.29, 86.32±0.31, 82.50±0.47 and 79.75±0.39; and HOS reactive sperms 49.67±0.94, 53.40±0.94, 59.85±1.01, 59.00±0.82 and 52.77±0.99 %, respectively. Significantly higher progressive sperm motility, better viability, acrosome integrity and HOS reactivity with lesser sperm/acrosome abnormalities of buffalo spermatozoa were observed at all intervals of refrigeration storage (0-72 hrs) in semen diluted with standard TFYG extender having cysteine HCl 1 mg/ml or taurine 4 mg/ml as additive than with 0.5 mg/ml cysteine and 6 mg/ml taurine and both the additives maintained significantly better sperm quality when compared with non-added control TFYG diluents. Taurine 6 mg/ml at times revealed insignificant differences with control TFYG for some sperm parameters, particularly intact acrosome which was significantly depressed. Thus in general, TFYG with cysteine HCl 1.0 mg/ml or taurine 4.0 mg/ml has been considered as a better additive for refrigeration preservation, while cysteine HCl 1 mg/ml and 0.5 mg/ml was better for cryopreservation, while taurine at 6 mg/ml level was found to be detrimental/toxic to buffalo sperm particularly at post-thaw stage. Hence, standard TFYG diluent with cysteine HCl 1.0 mg/ml or taurine 4.0 mg/ml can be recommended as a better option for improved refrigeration and cryopreservation of buffalo semen. However, actual fertility trials are required to substantiate the present findings. Some of the interrelationships between sperm motility, viability, morphology, intact acrosome and plasma membrane integrity in fresh, refrigerated and cryopreserved buffalo semen were observed to be significant (P<0.01), which proved that initial motility and membrane integrity can be used as predicative measures in routine semen evaluation.
Description
Keywords
ANIMAL REPRODUCTION, GYNAECOLOGY AND OBSTETRICS, A STUDY