© 2016 PP House Research on Freezability of Spermatozoa by Use of Various Concentrations of Glycerol in Mithun (Bos frontalis) Semen Cryopreservation K. K. Baruah 1 , M. Mondal 2 , A. Dhali 3 , B. Bora 1 , A. Mech 3 , A. Baruah 4 , P. Perumal 1* and K. C. Das 5 1 ICAR- National Research Centre on Mithun, Jharnapani, Nagaland (797 106), India 2 ICAR- National Dairy Research Institute, Kalyani, West Bengal (741 235), India 3 ICAR-National Institute of Animal Nutrition and Physiology, Bengaluru, Karnataka (560 030), India 4 AAU- College of Veterinary Science, Khanapara, Guwahati, Assam (781 022), India 5 ICAR- Central Institute of Fresh water Aquaculture, Bhubaneswar, Odisha (751 002), India Abstract A study was conducted to investigate the various glycerol concentrations on freezability of mithun semen. The present study was conducted in semen production laboratory, ICAR-NRC on Mithun, Medziphema, Nagaland, India. A total of 20 ejaculates were collected from apparently healthy mithun bulls (n=6) by trans-rectal massage method. These ejaculates were extended with tris–egg yolk-citrate-glycerol diluent (TECG) and 5, 6 and 7% (v/v) glycerol, respectively were used as a cryoprotectant. Extended semen was equilibrated, cryopreserved and stored in liquid nitrogen with standard protocols. Semen quality parameters (SQPs) such as percentage of forward progressive motility, viability (Eosin Nigrosin staining), acrosomal integrity (Giemsa staining) and total morphological abnormality (Eosin Nigrosin staining) were estimated with standard procedure at different stage of preservation and with different concentration of the glycerol. The result revealed that these semen quality parameters were differed non- signifcantly among the three experimental groups at immediately after fnal dilution (Stage: 1) and differed signifcantly (p<0.05) at 4 hrs of equilibration (Stage: 2) and at post thawing stage (Stage: 3) of semen cryopreservation. Further, 5% glycerol treated group has shown signifcantly (p<0.05) higher values of motility and live with acrosomal integrity and lower value of total morphological abnormality than in 6% or 7% glycerol treated groups. Based on the result, it was concluded that 5% glycerol was best for the TECG diluent for preserving mithun semen in liquid nitrogen. Article History Correspondence to Keywords Manuscript No. AR1454 Received in 30 th August, 2015 Received in revised form 16 th August, 2016 Accepted in fnal form 20 th August, 2016 * E-mail: perumalponraj@gmail.com Mithun semen, glycerol, cryopreservation DOI: 10.5958/0976-4038.2016.00188.3 1. Introduction The production and reproduction performance of mithun cows can be increased by breeding females with semen of the bulls of superior genetic merit. But due to non-availability of a system to replace bulls at regular interval to maintain the genetic quality, there is a need to focus on some alternative way for breeding in mithun species. This can be achieved easily through the artifcial insemination (AI) with the semen of proven mithun bulls or by multiple ovulation and embryo transfer (MOET). AI has become one of the most important techniques ever devised for the genetic improvement of farm animals like cattle, buffalo etc. Fresh bovine semen can be preserved successfully at 5 °C for 6 days using tris-egg yolk based extender without glycerol (Verberckmoes et al., 2004) and bull spermatozoa can be preserved by using 4% to 8% glycerol (Fennessy et al., 1990). However, there is very limited information in mithun. In this context, it is very essential to standardize a suitable semen preservation protocol in mithun in order to adopt AI. Mithun semen preserved at refrigeration temperature using tris-egg yolk based extender or cryopreserved in liquid nitrogen using tris-egg yolk-glycerol diluents, coupled with artifcial insemination would be a viable option for propagation and conservation of quality germplasm. Considering the importance of it, the present research programme was taken not only for conservation but also for propagation of mithun. 2. Materials and Methods The present study was conducted in semen production laboratory, ICAR-NRC on Mithun, Medziphema, Nagaland, India. A total number of 20 semen samples were collected from the adult healthy mithun bulls (n=6) and the semen International Journal of Bio-resource and Stress Management 2016, 7(5):1173-1176 Short Research Article 1173