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1. Background The Artificial Insemination (AI) technology has been proven to improve the genetic quality of livestock through animal reproduction engineering. The one of factors that could affect in AI implementation is sperm quality. To maintain of sperm quality during storage and freezing can be done by adding of sperm diluents solution. The Tris buffer mix with egg yolk is one of the diluents that commonly used in dilution of spermatozoa. The Bovine Serum Albumin (BSA) is one of protein source diluent that widely used since has abundant protein content in plasma with concentration of 5 gr 500 ml-1 by contains 20 kinds of amino acid. Also, the acting of extracellular cryo-protectants of BSA can also supply reserve energy during the preservation and cryopreservation of semen. But, BSA is still imported product and the price is expensive and difficult to obtain as well. Various studies reported that BSA can substitution with other compounds, like Snakehead fish (Channa striata) extract (Chasanah, 2015) that rich on minerals, essential amino acids and non-essential amino acids that closed to BSA contents. Hence, this study was aim to assay the using of Snakehead fish (Chana striata) extract as an supplement component of the extender compounds on the spermatozoa freezing processes toward the motility and mortality of Limousin bull sperms. 2. Materials and Methods This study was using Limousin bull sperms that was get from ejaculated sperms. Then, the sperms was diluted on Tris egg-yolk and supplemented with Snakehead fish extract (SFE). The supplementation of Snakehead Fish Extract (SFE) was applied for the treatments. The treatments were consist of four treatments; i.e. G0 = 0% SFE; G1 = 2% SFE; G2 = 4% SFE; G3 = 6% SFE and G4 = 8% SSFE. Each treatment was repeated by three times. Observation of mortality and motility of sperms were done by macroscopic observations (volume, pH, consistency, color and smell) and microscopic observations (mortality and motility before and after freezing). Data analysis was done by analysis of variance (ANOVA) and followed by Least Significant Difference (LSD) test if the ANOVA has any significance or very significance effect. 3. Results Table 1 showed the ANOVA results of spermatozoa mortality treatments with SFE. Table 1. The ANOVA of SFE treatments on sperms mortality Source Degree of Freedom Sum of Squares Mean Sum of Squares F-ststistic P-Value 0,05 0,01 Treatments 4 191,60 47,9 1,12ns 3,48 5,99 Error 10 425,33 42,53 Total 14 616,93 Notes: ns = not significance effect (P>0,05) of the treatments The ANOVA showed that supplementation of SFE in egg yolk tris diluent had no significant effect (P>0.05) on the spermatozoa mortality of frozen semen in the post thawing test. This was suspected that the supplementation of SFE to Tris-egg yolk diluents until 8% was not able to protect the plasma membrane of spermatozoa in the cryo-preservation process. Table 2