1. Sri Pantja Madyawati Departemen of Reproduction Veteriner, Faculty of Veterinay Medicine Airlangga University e-mail : spantja@unair.ac.id

2.  Freezing is a temporary cessation process of cell metabolism without suspending cell function and metabolism process can be continued after freezing is ended (Susilawati, 2000)  In bovine semen freezing, the count of motile spermatozoa after freezing (post thawing motility) as much as 40% (Tanaka, et al. 2000)  This proportion of post-thawing spermatozoa motility as much as 40% remained eligible to use in artificial insemination program.

3.  Pablo, et al. (1995) spermatozoa plasma membrane protein has a function as receptors of enzyme and antigen involved in the recognition of spermatozoa head membrane, such as the adhesion of zona pellucida with spermatozoa, the induction of acrosome reaction and the fusion of spermatozoa and ovum (Herrero et al., 1999)  A preliminary study by Madyawati et al. (2004) showed that the addition of 7% crude tyrosine kinase into Tissue Culture Medium (TCM) -199 could enhance spermatozoa-ovum fusion and in vitro dispersion of granulose cells.

4.  Therefore, it is important to test frozen semen that has been supplemented with tyrosine kinase from FH spermatozoa isolation through in vitro fertilization

5.  Stage 1. Oocyte collection comprosing the collection of cow ovary obtained from slaughterhouse and kept within 0.89% NaCL added with 50 µg/ml gentamycin sulfate in a temperature of 30 - 35°C. Subsequently, oocyte aspiration was carried out using G-18 needle connected to 5 ml syringe containing 1 ml Phosphat Buffer Saline (PBS) that had been added with 3 % Bovine Serum Albumine (BSA) and 50 µg/ml gentamycin. Oocyte collection was done using dissecting microscope. Oocytes used for maturation were those belonged to group A and B.

6.  Stage 2. Oocyte maturation. In the process of oocyte maturation we used the medium TCM -199 added with 0.01 µg/ml Follicle Stimulating Hormone (FSH), 0.01 µg/ml Luteinizing Hormone (LH), 3% BSA and 50 µg/ml gentamycin sulfate. Subsequently, we made microdrop of 50 µl maturation medium (TCM-199) into petri dish and covered with mineral oil, each drop contained 10 oocytes. Oocyte maturation was performed in the temperature of 38.5°C within 5% CO 2 incubator for 20 - 22 hours.

7.  Stage 3. The preparation of in vitro fertilization. For the preparation of in vitro fertilization, we used FH frozen semen based on treatment groups (PO, P1 and P2). The semen was washed within 6 ml Brickket Oliphant (BO), added with 20 µg/ml heparin, 3% BSA and 50 µg/ml gentamycin. Spermatozoa was then diluted within fertilization medium and incubated in 5% CO 2 incubator for one hour for optimizing spermatozoa motility. Incubated spermatozoa were injected into maturated oocyte in a dose of 1 x 10 6 /drop. Oocytes that have been mixed with spermatozoa were reincubated into 5% CO 2 incubator in a temperature of 38.5°C for 24 hours. Fertilization rate was observed for 24 hours by noticing the developing zygote count (Yanagimachi, 1994).

8. TreatmentRepetitionOocyte count Result of in vitro fertilization for 24 hours (zygote) P061914 (73.68%) P162317 (73.91%) P262217 (77.27%) Table 1. Embryo Development in In Vitro Fertilization Process using Tyrosine Kinase- Supplemented Frozen Semen Note: PO = FH frozen semen without TK supplementation P1 = FH frozen semen with supplementation of 100 µg/ml TK P2 = FH frozen semen with supplementation of 200 µg/ml TK

9. Figure 1. The Histogram of Zygote Count Obtained from In Vitro Fertilization After Incubation within CO 2 Incubator for 24 Hours.

10.  Laboratory application of FH frozen semen sample without tyrosine kinase supplementation (P0) and with tyrosine kinase supplementation in a dose of 100 ug/ml (P1) and 200 µg/ml (P2) in in vitro fertilization (FIV) after being incubated within 5% CO 2 incubator for 24 hours revealed the highest mean percentage of zygote count was found in in vitro fertilization using frozen semen in P2 as much as 77.27%, as compared to P2 and control.  In bovine embryo, there is a phenomenon of cell block or impeded cell development that occurs in the stage of two- to four-cell embryo development (Miyoshi, 1994).

11.  In conclusion, frozen semen supplemented with tyrosine kinase isolated from FH spermatozoa can increase fertilization rate in in vitro fertilization. The highest zygote count resulting from in vitro fertilization was found in treatment 2 (P2), which used FH frozen semen supplemented with tyrosine kinase of 200 µg/ml.

12.  Anonimous, 2005. Semen, Processing, Storage, Thawing and Handling. File.//F ;chapter 16 htm.download 9/27/2005  Breitbart H. and Z. Naor, 1999. Protein Kinases in Mammalian Sperm Capacitation and The Acrosomal Reaction. Revisi Reproduction. J. Soc. Reprod. and Fert. 4.151- 159  Brewis, A. Ian, and H. Wong Chi. 1999. Protein Kinase in Mammalia Sperm Capacitation and The Acrosome Reaction. Rev. Reprod. 4th. Pp. 151-159  Hafez,E.S.E., 2000. Reproduction in Farm Animals. 7th.Edition. Lea & Febiger. Philadelphia.  Herrero MB., E. de Lamirande and C.Gagnon, 1999. Nitrit Oxide Regulates Human Sperm Capacitation and Protein Tyrosine. Biol. of Reprod.61:575-581  Kim, J.H., H. Funahashi, K. Niwa and K. Okuda. 1993. Glucosa Requirement of Different Developmental Stages of In Vitro Maturated, In Vitro Fertilized Bovine Oocytes in Medium. J. Theriogenology. 39: 875-886  Madyawati,SP., H.A.Hermadi dan T.Sardjito, 2004. Produksi Protein Tyrosin Kinase hasil Isolasi Spermatozoa Sapi Potong; Altematif Meningkatkan Produksi Semen Beku. Penelitian proyek Due-Like Batch III. Fakultas Kedokteran Hewan. Universitas Airlangga, Surabaya.

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