JOURNAL OF AGRICULTURE & SOCIAL SCIENCES 1813–2235/2006/02–2–117–119 http://www.fspublishers.org Short Communication Effect of Buffers on Freezing of Buffalo Bull Semen MUHAMMAD SIDDIQUE, RIASAT ALI AND ALI RAZA 1 Army Veterinary School, Sargodha, Pakistan 1 Corresponding author’s e-mail: alikhanbaloch@hotmail.com ABSTRACT Sixteen semen samples (ejaculates) from two Nili-Ravi buffalo bulls (8 from each) were preserved in liquid nitrogen at -196 o C for 24 hours to study the effect of five extenders on post-thaw motility, liveability (hours) and absolute index of liveability of spermatozoa. Immediately after collection and evaluation, the semen samples were diluted with respective extenders viz. A, B, C, D and E having different concentrations of Tris and Sodium citrate fructose, glucose, lactose, egg yolk, glycerol and antibiotic penicillin and dihydrostreptomycin. After an equilibration period of 6 hours, samples were frozen by fast method and stored in liquid nitrogen for 24 hours. Thawing was carried out at 37 o for 30 seconds in water bottle to observe post-thaw motility percentage, liveability (hours) of spermatozoa and absolute index of liveability for comparing the relative efficiency of the five extenders. The average values of post-thaw motility percentage were 45.9 ± 5.2, 38.1 ± 5.2, 50.0 ± 6.4, 41.9 ± 7.9, and 40.9 ± 6.3 for extenders A, B, C, D and, respectively. Corresponding values for liveability at 37 o C were 8.6 ± 0.9, 7.6 ± 0.9, 9.2 ± 1.1, 8.3 ± 0.9 and 7.6 ± 1.0 hours. The absolute index of liveability at 37 o C averaged 178.0 ± 38.4, 130.3 ± 24.2, 207.5 ± 41.4, 153.5 ± 55.3 and 141.0 ± 42.5 for extenders A, B, C, D and E, respectively. Post-thaw motility for two bulls (B 8 and B 9) ) averaged 44.7 ± 6.0 and 42.0 ± 5.5% while the post-thaw liveability (hours) at 37 o C averaged 8.4 ± 1.2 and 8.1 ± 1.0, respectively. The absolute index of liveability at 37 o C for two bulls averaged 168.3 ± 42.3 and 155.7 ± 38.5. The effect of five extenders on post-thaw motility, liveability (hours) at 37 o C and absolute index of liveability was highly significant (P< 0.01). The effect of two bulls on these parameters was non- significant. It was found that the extender C containing Tris and Sodium citrate in 1:1 ratio was the best for successful preservation of buffalo bull semen at - 196 o C. The order of merit for five extenders used was C, A, D, E and B. Key words: Freezing; Buffers; Buffalo bull; Semen INTRODUCTION The buffalo, a great national asset and with its population 26.3 million heads (Anonymous, 2004-05), is the major source of milk in the country. Low productivity of our dairy animals is an obstacle to meet the nutritional requirements of our ever increasing human population. The existing gap of milk and meat supply in the country can be well abridged by exploiting the productive and reproductive potential of the buffalo. Artificial insemination is one of the most important techniques ever devised for the genetic uplift of livestock. Among various factors affecting fertility of frozen semen, composition of extending medium is the most important one. Different extenders have been tried for deep freezing of buffalo bull semen. Among these Tris (hydroxymethyl) aminomethane has successfully been used as an organic buffer for deep freezing of bull semen (Foote, 1970). Another organic buffer, sodium citrate, has also proved very useful for this purpose (Glen et al. 1956). The present study was designed to study the effect of two buffers Tris and sodium citrate dihydrate, combined in different proportions on the freezability of buffalo bull semen. MATERIALS AND METHODS Experimental material. The semen of two buffalo bulls of Nili-Ravi breed, maintained at the Semen Production Unit, Department of Animal Reproduction, University of Agriculture, Faisalabad was used in this study. Semen collection and evaluation. In order to get a complete ejaculate of good quality, each bull was given enough time for sexual stimulation and one false mount was allowed for sexual preparation before the collection of first ejaculate. The second ejaculate was collected 10-15 minutes following the first collection. Ejaculates showing motility estimate of at least 60 % were pooled. A total of 16 pooled samples were available for further processing. Experimental methods. Five experimental extenders were used, the composition of which is given in Table I. Egg yolk was separated by complete removal of albumen. Egg yolk was poured into a cylinder by puncturing the yolk membrane. The required amount of Tris, sodium citrate dihydrate, citric acid, fructose, glucose, lactose, penicillin and streptomycin for each extender were weighed and placed in separate beakers. Distilled water was added to dissolve these ingredients and make the final volume of 73 ml. Then the