Effect of Antioxidant Fortification on Preservability of Buffalo Semen

: During the process of freezing, spermatozoa suffer cold shock which increases their susceptibility to lipid peroxidation which plays an important role in ageing of spermatozoa, shortening their life span and affecting the preservation of semen. An experiment was therefore conducted to study the effect of addition of natural antioxidants into semen diluents on the preservability of buffalo semen. Split semen samples were extended in milk egg yolk diluents fortified with vitamin E (MYE), vitamin C (MYC) and control group (MYO); Tris-egg yolk diluents fortified with vitamin E (TYE), vitamin C (TYC) and control group (TYO) and evaluated for their preservabilities at 4-7 ° C and 37 ° C. Overall least squares mean of percent motility observed after 0, 24, 48, 72 and 96 h of preservation at 4-7 ° C were 66.70, 54.00, 36.80, 21.90 and 12.50, respectively while the estimates for semen extended in MYE, MYC, MYO, TYE, TYC and TYO were 44.80, 42.70, 38.70, 36.00, 35.20 and 33.00 percent, respectively. The results showed that motility was significantly (p<0.01) affected by extender (extender-antioxidant combination) and preservation interval. Overall least squares mean percent motility observed after 0, 4, 8, 12 and 24 h of preservation at 37 ° C were 68.50, 58.90, 45.00, 38.10 and 18.10 percent, respectively, while the estimates for semen extended in MYE, MYC, MYO, TYE, TYC and TYO were 48.20, 49.30, 46.80, 45.30, 42.30 and 42.50 percent, respectively. Extender and storage interval were found to be significantly (p<0.01) affecting spermatozoa motility on room temperature preservation. The results indicated that the incorporation of antioxidants, especially vitamin E, had beneficial effect on preservability of buffalo semen. (Asian-Aust. J. Anim. Sci. 2002. Vol 15, No. 1 : 16-18)


INTRODUCTION
To meet the objective of augmenting milk production, special attention is required to make available sufficient number of semen doses from superior buffalo bulls. The present scenario of semen production is beset with a number of problems such as low sperm harvest, seasonal variation in semen quality, freezability and inconsistencies in frozen semen production technologies. During the process of freezing, spermatozoa are exposed to cold shock, which increases their susceptibility to lipid peroxidation. Lipid peroxidation induces ageing of spermatozoa, reducing their life span and affecting the preservation of semen for AI (Alvarez and Storey, 1982). Slow cooling of semen from 30 to 5°C has been observed to improve fertility performance of cattle. However, slow cooling may lead to increase in respiration, which is subsequently detrimental to sperm viability. Addition of antioxidant might be useful in preventing the damage under such condition.
With an overall objective of improving upon the existing methodologies of cryopreservation of buffalo semen and developing a suitable package, the present experiment was conducted to study the effect of addition of natural antioxidants on preservability of buffalo semen.

MATERIALS AND METHODS
Semen ejaculates from Murrah buffalo bulls maintained at Artificial Breeding Complex, National Dairy Research Institute, Karnal, were used for the present study. Split semen samples extended in milk egg yolk diluents fortified with vitamin E (MYE), vitamin C (MYC) and control group (MYO); Tris-egg yolk diluents fortified with vitamin E (TYE), vitamin C (TYC) and control group (TYO) were evaluated for their preservabilities at refrigerator temperature (4-7°C) and room temperature (37°C). Antioxidant fortification was done by addition of antioxidant to the diluent at the rate of 1 mg/ml of vitamin E (alpha-tocopherol acetate, Loba Chemie, India) for MYE and TYE and 5 mM of Vitamin C (Sodium ascorbate, Fluka, Biochemica) for MYC & TYC extenders. Percent motility (%) was recorded after 0, 24, 48, 72 and 96 h of preservation at 4-7°C, and after 0, 4, 8, 12 and 24 h of preservation at 37°C. Observations on percent motility were made independently by three persons and the average of the observations was taken as percent motility. To study the effect of antioxidant (extender-antioxidant combination) and stage/interval of preservation on preservability of semen, the data were analysed by least squares technique (Harvey, 1975) using the following model: Y ijk =µ+E i +I j +(E I) ij +e ijk Where, Y ijk =kth observation of j th stage of/interval of preservation.
µ=Overall mean E i =the effect of i th antioxidant (Extender-antioxidant combination) I j =the effect of j th stage / interval of preservation (E I) ij =the effect of (ij) th antioxidant (Extenderantioxidant combination) -stage/interval of preservation interaction e ijk =Random error associated with (ijk) th observation, NID( 0, σ 2 e)

RESULTS AND DISCUSSION
The results of percent motility (%) of semen preserved at 4-7°C are depicted in  (table 3). However, percent motility was not significantly affected by extender-interval interaction. With the increase in storage interval, there was deterioration in semen quality. Incorporation of antioxidants improved the performance of extenders. At all the stages of preservation, semen diluted in Milk-egg yolk extender fortified with vitamin E showed maximum percent motility. From the results it could be concluded that Milk-egg yolk extender fortified with vitamin E was most suitable for preserving motility of buffalo bull spermatozoa upon storage at 4-7°C.
The overall results indicated that the incorporation of antioxidants, especially vitamin E, had beneficial effect on preservation of semen. In conformity with the present findings, several investigations using antioxidants for preservation of semen in various species such as ram (Srivastava et al., 1987;Nauk and Boronchuk, 1992), boar (Nishimura and Morri, 1992), bull (AI-Khanak and AI-Hanak, 1989;Beconi et al., 1993) indicated that natural antioxidants exert a protective effect on the plasma membrane, preserving both metabolic activity and cellular viability. The present results were in agreement with other reports on different species, such as bull, boar and ram (Stolbov and Rimanova, 1983;Golyshev, 1985;Beconi et al., 1993;Salmon and Maxwell, 1995).
Positive results in favour of milk-egg yolk extender fortified with antioxidant, especially vitamin E suggested that the addition of antioxidant may lead to achieving better preservability of buffalo semen, thus increasing the number of semen doses available from a buffalo bull.