STUDIES ON PHYSICO-BIOCHEMICAL ATTRIBUTES AND PRESERVABILITY (AT 5°C AND -196°C) OF SEMEN OF TRIPLEBRED (HF X JERSEY X KANKREJ) BULLS
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Date
2006
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AAU, Anand
Abstract
This study was undertaken in 2 phases on semen of 4 mature triplebred bulls at Livestock Research Station, AAU, Anand. The study covered evaluation of seminal characteristics, seminal plasma biochemical profiles, acrosomal morphology, and effect of extender-additives, viz. cysteine HCl (0.1 %) and EDTA (0.1 %) in Tris fructose yolk glycerol (TFYG) diluent on cryo-freezing as well as refrigeration preservation (5°C till 48-hr) of semen in terms of motility, viability, morphology and acrosomal integrity of spermatozoa. In phase-I, physico-biochemical attributes and their interrelationships were studied, while in phase-II, the effect of additives was studied using split-sample technique (1:10 dilution) on 36 ejaculates for cryo-freezing and refrigeration storage. Sperm motility was examined at 24 hourly intervals till 96- hr in refrigerated semen. The data on various traits of cryopreserved and refrigerated semen were analysed using 3-factors' Factorial CRD. The ejaculate, volume, mass activity (score 0-5), individual sperm motility, sperm concentration, live sperm and abnormal sperm recorded in triplebred bulls' semen during phase-I (winter) were 3.88 ± 0.16 ml, 3.73 ± 0.06, 999.06 ± 16.14 million/ml, 84.00 ± 1.02 %, 86.28 ± 0.97 % and 8.28 ± 0.44 %, respectively. The average seminal plasma content of GOT, GPT, AKP, total protein, total cholesterol, calcium, inorganic phosphorus and magnesium was 189.11 ± 6.29 lU/L, 82.53 ± 4.29 lU/L, 718.94 ± 24.27 lU/L, 6.95 ± 0.34 g/dl, 42.77 ± 2.87 mg/dl, 21.26 ± 0.56 mg/dl, 44.16 ± 1.30 mg/dl and 2.89 ± 0.06 mEq/L, respectively. The bulls varied significantly (P < 0.05) in their ejaculate volume, sperm concentration, abnormal sperm per cent, seminal plasma total protein, total cholesterol, GPT, calcium and magnesium levels. Semen quality of all 4 triplebred bulls was of optimum type and its' biochemical profile was within normal physiological limit. Moreover, the ejaculate volume had significant positive correlation (P < 0.01) with abnormal spermatozoa (r = 0.41); mass activity with the initial motility (r = 0.81), abnormal sperm (r = -0.46) and plasma total protein (r = 0.50); live sperm percentage with initial motility (r = 0.54), abnormal sperm (r = -0.60) and plasma cholesterol (r = 0.36); initial motility with abnormal sperm (r = -0.59) and plasma GPT (r = 0.34) and sperm concentration with seminal plasma GPT, total protein and magnesium levels (r = -0.43, -0.46, -0.39). Seminal plasma GOT activity was significantly (P < 0.01) correlated with plasma GPT, total protein, total cholesterol and magnesium concentrations (r = 0.46, 0.39, 0.36, 0.39, resp), while GPT activity had significant correlations only with plasma protein and magnesium contents (r = 0.64, 0.60), and magnesium with total protein and calcium levels (r = 0.65, 0.39). Plasma AKP and inorganic phosphorus levels did not show significant correlations with any of the physico-biochemical attributes studied. The mean percentages of motile, live and abnormal sperms and intact acrosome observed in freshly extended semen in standard TFYG diluent were 77.92 ± 0.73, 88.89 ± 0.57, 7.44 ± 0.28 and 92.33 ± 0.33, respectively. There was insignificant change in most of these values at prefreeze level, but the post-thaw values differed highly significantly (P < 0.01) from the initial as well as prefreeze values, and so also was the case for the effect of 24-hr and 48-hr of refrigeration storage. The relative % decline in motile and live sperms and intact acrosome at postthaw stage over the initial values was 40.46, 35.63 and 11.82, respectively, while the incidence of abnormal sperm and damaged acrosome increased relatively by 87.10 and 142.24 % at post-thaw stage over the initial values. The values of segment-wise sperm abnormalities and acrosome abnormalities were found to be more than double at post-thaw stage over the initial values. The 48-hr refrigeration storage of extended semen caused significant change in these parameters, but the magnitude of change was relatively small (12 to 18%). Statistically, that there were significant (P < 0.01) differences in percentages of motile, live and abnormal sperms and intact/damaged acrosomes between bulls (n=4), between stages (n=3, initial, prefreeze & post-thaw or 0, 24 & 48 hr) and between additives (n=3, EDTA, cysteine & control) both in cryopreservation and refrigeration storage of semen. Among all the two- and three-way interactions of bulls, stages and additives studied, only bull x stage and/or bull x additive interaction was found significant (P < 0.05) for some of these traits during cryo-freezing and/or refrigeration preservation. The overall pooled mean values of progressively motile sperms (irrespective of diluent additives) at initial, post-thaw and 48-hr post-refrigeration of semen were 81.07 ± 0.48, 50.60 ± 0.77 and 72.41 ± 0.61 %, respectively. The corresponding values for live sperm were 88.89 ± 0.32, 58.63 ± 0.63 and 73.22 ± 0.37 %; abnormal sperm 7.44 ± 0.16, 13.57 ± 0.20 and 11.02 ± 0.15 %; intact acrosome 92.33 ± 0.19, 83.65 ± 0.27 and 86.82 ± 0.21 %, and damaged acrosome 7.67 ± .19, 16.30 ± 0.28 and 13.18 ± 0.21 %, respectively. The sperm motility sustained in the extended semen till 96-hr of refrigeration was 63.47 ± 0.69 %, indicated acceptable preservability of crossbred bulls' semen at 5°C for 3-4 days. The mean percentages of progressively motile spermatozoa at initial, postthaw and 48-hr of refrigeration of semen in control Tris diluent were 77.92 ± 0.73, 46.39 ± 1.27 and 70.00 ± 0.91, respectively. The corresponding values for the diluent containing EDTA were 83.20 ± 0.67, 53.19 ± 1.26 and 74.31 ± 0.96, and that containing cysteine were 82.08 ± 0.85, 52.22 ± 1.20 and 72.92 ± 1.17, respectively. The corresponding values for live sperm per cent in control Tris diluent were 88.89 ± 0.57, 57.22 ± 1.09 and 72.28 ± 0.62, respectively; in EDTA containing diluent 88.89 ± 0.57, 61.50 ± 0.76 and 74.50 ± 0.68, and that in cysteine containing diluent 88.89 ± 0.57, 57.17 ± 1.25 and 72.89 ± 0.57, respectively. The mean percentages of sperms with intact acrosome at initial, post-thaw and 48-hr of refrigeration of semen in plain Tris diluent were 92.33 ± 0.33, 81.42 ± 0.44 and 85.31 ± 0.26, respectively. The corresponding values for the diluent containing EDTA were 92.33 ± 0.33, 85.08 ± 0.39 and 88.08 ± 0.27, and that containing cysteine were 92.33 ± 0.33, 84.44 ± 0.35 and 87.08 ± 0.39, respectively. The trend observed for the effect of freezing steps, storage intervals and additives was identical in the semen of all 4 individual bulls for motile and live sperm and intact acrosome. In general, the values of all three traits were significantly higher at all stages of cryo-freezing and refrigeration preservation of semen in the presence of EDTA and cysteine hydrochloride (EDTA being superior than cysteine) as compared to control Tris diluent. The mean percentages of total sperm abnormalities at initial, post-thaw and 48-hr of refrigeration of semen in control Tris diluent were 7.44 ± 0.28, 13.92 ± 0.26 and 11.42 ± 0.23, respectively. The corresponding values for the diluent containing EDTA were 7.44 ± 0.28, 13.03 ± 0.32 and 10.53 ± 0.28, and that containing cysteine were 7.44 ± 0.28, 13.78 ± 0.41 and 11.11 ± 0.23, respectively. The overall mean percentages of sperms with head, midpiece and tail abnormalities recorded initially in fresh semen of triplebred bulls were 2.33 ± 0.06, 1.37 ± 0.06 and 3.78 ± 0.12, respectively. The corresponding values after freezing thawing of semen were 4.35 ± 0.13, 2.78 ± 0.09, 6.45 ± .018, respectively, and after 48-h of refrigeration storage 3.60 ± 0.11, 2.07 ± 0.09, 4.61 ± 0.13 per cent, respectively. The differences due to freezing stages and storage intervals were significant (P < 0.01) for all the three traits. However, there was no significant effect of diluent-additives on any of these segmental defects in either of the protocols, except tail defects. The mean percentages of sperms with damaged acrosome at initial, post-thaw and 48-hr of refrigeration of semen in control Tris diluent were 7.67 ± 0.33, 18.42 ± 0.50 and 14.69 ± 0.26, respectively. The corresponding values for the diluent containing EDTA were 7.67 ± 0.33, 14.92 ± 0.39 and 11.92 ± 0.27, and that containing cysteine were 7.67 ± 0.33, 15.56 ± 0.35 and 12.92 ± 0.39, respectively. The mean percentages of sperms with swollen, ruffled, denuded and detached acrosome recorded initially in fresh semen were 2.25 ± 0.06, 2.03 ± 0.08, 2.00 ± 0.09 and 2.55 ± 0.10, respectively. The corresponding values at post-thaw stage were 3.69 ± 0.12, 3.79 ± 0.13, 3.63 ± .012 and 5.32 ± 0.16, respectively. The values after 48-h of refrigeration were 3.05 ± 0.10, 4.47 ± 0.18, 2.27 ± 0.11 and 3.28 ± 0.13 per cent, respectively. All types of acrosomal defects were significantly lower (P < 0.01) in presence of EDTA and cysteine than the control diluent, and increased with freezing or storage time. Highly significant (P < 0.01) interrelationships observed for the percentages of motile, live and abnormal sperms and intact/damaged acrosome in fresh, post-thawed and refrigerated semen of triplebred bulls (r = ± 0.19 to 0.88) proved that the assessment of initial motility can be taken as a fairly good indicator of semen quality after freezing and/or refrigeration in terms of above traits.
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Keywords
VETERINARY OBSTETRICS AND GYNAECOLOGY, A Study