AGRICULTURAL RESEARCH COMMUNICATION CENTRE www.arccjournals.com/www.ijaronline.in *Corresponding author’s e-mail: bhakat.mukesh@gmail.com Indian J. Anim. Res., 52 (4) 2018 : 527-529 Print ISSN:0367-6722 / Online ISSN:0976-0555 1 Dairy Microbiology Division, ICAR-National Dairy Research Institute, Karnal, 132001, India. 2 Animal Nutrition Division, ICAR-National Dairy Research Institute, Karnal, 132001, India. INTRODUCTION Good quality disease free semen production and its propagation is one of the major goals of semen laboratories in India and throughout the world (Meena et al., 2015). High microbial loads not only affect semen quality (Diemer, et al., 1996) but also impact fertility status of spermatozoa. Bacteria compete with spermatozoa for nutrients and oxygen necessary for growth and normal functioning. Semen acts as a vehicle for a wide range of pathogens (Vinodh et al., 2007). Microbes enter into semen through number of routes from diseased animals, preputial sheath, extenders, as well as faulty procedure followed during collection, processing and packaging of semen. Microorganisms affect the male reproductive function directly by reducing the ability of acrosome reaction (Morrell, 2006) and agglutination of motile sperm and indirectly, by formation of reactive oxygen species generated by inflammatory response to the infection. Regular microbial evaluation of semen samples is a prerequisite to ensure the quality of semen. The rising issue of bio-security and cross boundary disease transmission has unveiled new concerns over the use of egg yolk based extenders, as they have been often incriminated for facilitating the transmission of some diseases. Various pathogens such as E. coli, Staphylococcus, Streptococcus, Pseudomonus, Haemophilus, Salmonella, Avian influenza, Campylobacter, Listeria and Mycoplasma are transmitted through egg yolk (Thibier and Guerin, 2000). Besides the risk of disease transmission, egg yolk globules interfere in the microscopic examination of the spermatozoa (Bousseau et al., 1998). Further, composition of egg yolk also varies upon their source, which prevents implementation of strict quality control of the extender. On the other side egg yolk inflicts damages to the chromatin integrity (Stradaioli et al., 2007). All these lead to an extensive search for an alternate semen extender, which is free from the risk of introducing exotic diseases. Now-a-days soya lecithin and liposome based extenders are commercially available but till date no proper validation is available regarding the microbial load status of soya lecithin and liposome based extenders. As per the World Organization for Animal Health Microbial load of frozen thawed Sahiwal semen extended in egg yolk, soya lecithin and liposome based extender A. Singh, M. Bhakat*, S. Mondal, T.K. Mohanty, P. Behare 1 , G. Mondal 2 , A. Rahim, S.K. Yadav, A.K. Gupta, M.D. Gupta and M. Abdullah Artificial Breeding Research Centre, ICAR-National Dairy Research Institute, Karnal-132 001, Haryana, India. Received: 20-09-2016 Accepted: 17-11-2016 DOI: 10.18805/ijar.v0iOF.8455 ABSTRACT Present investigation was carried out to study the bacterial load of different semen extender in frozen thawed semen samples of Sahiwal bulls maintained at Artificial Breeding Research Centre, ICAR-NDRI, Karnal, Haryana. Twenty four ejaculates from four bulls were collected during morning hours using sterilized artificial vagina and the ejaculates were evaluated. Semen samples showing more than 70 per cent progressive motility and having more than 800 million sperms per ml were used for further processing and freezing. The samples were split into six aliquots and extended in six different extenders [Conventional egg yolk (CEY), egg yolk of hen supplemented with omega enriched diet (group I), Egg yolk of hen supplemented with herbal feed (group II), 1% soya lecithin (SY1), commercially available Bioxcell (SY2) and Optixcell (LP)]. One ml of post thaw semen (four straws) was serially diluted (ten-fold) with normal saline, loaded in sterilized nutrient agar plate and incubated at 37°C for 48 hours. Number of colonies were counted in duplicate and multiplied by dilution factor. The Standard plate counts (CFU/ml) of frozen thawed semen samples extended in different extenders were analyzed by one way analysis of variance. The results revealed that bacterial load of SY1, SY2 and LP was significantly (p<0.05) different from egg yolk based extenders. The highest microbial load of frozen thawed semen samples was recorded in group II (646.30±71.65) egg yolk extender and lowest in optixcell (91.67±6.72) extender. Microbial load in all the extenders are well within the standard limit (5000CFU/ml) of frozen thawed semen sample. Lower bacterial load in present finding may be due to maintenance of better HACCP protocol for hygiene during preparation, semen collection and semen processing. Key words: Bacterial load, Egg yolk, Liposome, Soya-lecithin, Sahiwal bull semen.