PRODUCTION OF SEXED SPERM AND CALVES BORN AFTER ARTIFICIAL INSEMINATION USING SEXED SPERM IN BALI PROVINCE

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1 PRODUCTION OF SEXED SPERM AND CALVES BORN AFTER ARTIFICIAL INSEMINATION USING SEXED SPERM IN BALI PROVINCE SYAHRUDDIN SAID and F. AFIATI Research Center for Biotechnology, Indonesian Institute of Sciences Jl. Raya Bogor km 46, Cibinong ABSTRACT The objective of this research is to apply the innovation of sperm sexing biotechnology to obtain calf of Bali catle with certain sexes. Production of sexed sperm was condacted in Balai Inseminasi Buatan Daerah (BIBD) Baturiti, Bali on December 18, Insemination was done in Negara District during September 18, untill October 28, Data collected covers number of acceptors, sperm type to be inseminated, birth, conception rate (CR), service per conception (S/C), successfull rate of sperm insemination as according to expectation. Quality of sperm was analized using sperm vision TM (Mini Tube Germany), obtained data were analyzed using SPSS 12s test. Sperm motility post thawing sperm X (82%) and sperm Y (86%) did not significantly different (P < 0.05) from fresh sperm (87%), after sperm diluted X (89%) sperm Y (90%), and after equilibrasi sperm X (92%) sperm Y (90%). Sperm motility X post thawing (82%) significantly (P < 0,05) lower than Sperma Y after thinning (90%), equilibrated sperm X ( 92%) and Sperm Y (90%). While for progressive of sperm post thawing (sperm X and Y each 52%) significantly (P < 0,05) lower than progressive of fresh sperm (78%). After sexed sperm Y were inseminated to 43 acceptor cows in Jembrana District, 26 cows were pregnant (S/C = 1.65). Out of 26 pregnant cows, calf born 21male calf dan 5 female calf. These indicated that level accordance of sperm sexing up to 87,77%. Result of this research indicated that quality of sperm sexing did not different (P < 0.01) from sperm without sexed (control). After insemination to acceptor, shown result of S/C = 1.65, CR = and level of concordance of calf gender was 80.77%. Key words: Sperm Sexing, Bali Cattle, Quality of Sperm, Sperm Vision TM INTRODUCTION Livestock sub-sector in agricultural development is now increasingly important. Without the presence of cattle, a high efficiency in farming is difficult to achieve. Farms have also to be used as part of resource conservation programs and environmental protection cycle. In addition to the orientation of livestock products (meat, milk and eggs), the function of livestock is used as a savings for the farmer or as an income, fertilizer, agrotourism and alternative energy resources. Jembrana is one district of Bali Province in whose territory borders the Buleleng district in the north, the Indian Ocean to the south, the Strait of Bali in the west, and the Tabanan regency in the east. Jambrana District administratively divided into 4 districts, with an area of 841,80 km 2. Livestock products that are the most widely produced in Jembrana is beef, pork, and poultry meat. Beef cattle is raised mainly in Negara and Melaya. Bali cattle is the native beef catle type of Indonesia whose population continues to decline each year. Bali cattle is a breed of beef cattle Bos Sondaicus that successfully domesticated and have evolved considerably in Bali island. The original Bali cattle have the same characteristics as banteng, but its size is relatively small due to the influence of domestication. The government currently has attempted to increase the population and productivity of cattle, so that meat production in the country increased, including through artificial insemination program (AI). Although meat production increased, but the cattle population is relatively fixed or stagnant, that it could be imported calves are still high and accompanied by depletion or over-slaughtering of local livestock. When viewed from the existing population structure, there are still opportunities to improve productivity and 61

2 production of cattle through the application of innovative technologies in the AI program, which was followed by improvement of feed, health and housing system management (Diwyanto and Herliantien, 2006). Artificial insemination can increase the added value when supported by the development of reproductive biotechnology to get the calf with the expected sex. Sex is determined by the X and Y chromosomes in sperm. Bovine sperm separation has been widely used abroad using flow cytometry that can accurately measure the DNA content of sperm so that sperm can be distinguished female sex (X) carrier or male sex (Y) carrier. In addition there are other sperm separation methods such as sedimentation, electrophoresis, Sephadex G-50 filtration, centrifugation, column albumin (BSA) and other methods (Garner, 2001). Accuracy of sperm sexing using flow cytometry results is estimated to reach 90% (Seidel and Garner, 2002), whereas Johnson and Saidel (1999) states that by sexing sperm using flow cytometry can obtain 85 95% of births according to sex. X chromosome spermatozoa will produce a female embryo, while the Y spermatozoa will produce a male embryo on fertilization (Susilawati et al., 1999). Each X and Y spermatozoa is different in size and shape, weight, density, motility and biochemical content of the charge on its surface (Hafez, 1993). These differences cause the X and Y spermatozoa allows to be separated (Susilawati, 2004). The objective of this research was to apply biotechnology innovations (sperm sexing) that was produced in the location, followed by insemination to the acceptor (heifer) to obtain the birth of bali calves certain sex. MATERIAL AND METHODS Place and time of research Sperm sexing was conducted in the Central Regional Artificial Insemination (BIBD) Baturiti, Bali on December 18, 2006, inseminations was performed in the State District from September18 October 28, The number of acceptors, the type of sperm in the AI, birth, Conception rate (CR), Service per conception (S/C) and the success rate of AI as expected data was collected. Collection and production of sperm sexing Collection and sexing of sperm was done at BIBD Baturiti, Bali. Semen was collected by an artificial vagina method at the temperature of 42 25ºC. Sperm were observed in macroscopic reservoirs (volume, color, smell, viscosity and ph) and microscopic (mass movement, motility, concentration, live cells, abnormalities and membrane integrity) using sperm vision TM (Mini Tube, Germany). Separation was carried out with albumin column method (Kaiin, et.al., 2003). Sperm with a concentration of cells/ml were stored in the albumin-story columns. Each fraction was collected and the collected sperm in the centrifuge tube, then washed using BO medium by centrifugation at a speed of 2500 rpm for 10 minutes. Media Tris egg yolk cryoprotectant containing 20% (v/v) was used as a diluent for spermatozoa separation. The results which was diluted spermatozoa incorporated into 0.25 ml straw (filling), then sealed. Before placing in liquid nitrogen -196 C, spermatozoa was equilibrated for 3 4 hours at a temperature of 5 C. Artificial insemination, pregnant and birth The owner of the cow herds to be used as acceptors was recorded. Acceptor cows that showed signs of estrus, such as swollen vulva condition, wet, red and mucus out with anxious temperament recto vaginal checked to verify the condition of livestock in the circumstances is not pregnant. After the insemination, the date of AI, the acceptors number, the number of times AI, type of sperm (X/Y) and the males used were recorded. Cows that do not estrus after breeding was suspected to pregnant possibilities. Cow pregnancy examinations were performed three to four months after the last insemination. Inspection was done by rectal palpation, touch inspection can be done in the fetal membranes (branching uteri) which was enlarged. Recording of pregnant aceptors was conducted. 62

3 The cow showed signs of giving birth, if showed the loosened of the vulva and the presence of yellowish hanging mucus. Cows that had given birth then shortly be given the vitamins and antibiotics in order not to prevent the weaken condition after giving birth. Statistical analysis Data obtained using sperm vison TM (Mini Tube, Germany), then analyzed using SPSS 12 program. RESULTS AND DISCUSSION Bali cattle sperm sexing quality Production of sperm sexing was held in the Central Regional Artificial Insemination (BIBD) Baturiti, Bali on December 18, The quality of frozen sperm separation results can be seen in Table 1 and males that used is showed in Figure 1. The above data shown that motility of postthawing sperm X (82%) and sperm Y (86%) were not significantly different (P > 0.05) with fresh sperm (87%), after dilution of sperm X (89%), sperm Y (90%), and after equilibration sperm X (92%) and sperm Y (90%). After thawing sperm X motility (82%) was significantly smaller (P < 0.05) than sperm Y after dilution (90%), sperm X after equilibration (92%) and sperm Y after equilibration (90%). As for the progressive sperm after thawing (X and Y sperm respectively 52%) was significantly lower (P < 0.05) than fresh sperm progressive (78%). However, sperm quality was still above the recommended standard of the post-thawing motility (> 40%). Birth results of IB sexing program Frozen sperm sexing results after insemination in cattle acceptor in the Negara District, data resulted by two inseminator is showed in Table 2. Table 1. Sperm sexing quality of Bali cattle sperm Description Fresh sperm quality Post dilution Post equilibration Post thawing Sperm X Sperm Y Sperm X Sperm Y Sperm X Sperm Y Motility (%) 87 a 89 a 90 ab 92 ab 90 ab 82 ac 86 ab Progressive (%) 78 a 57 b 65 ab 77 ac 79 ac 52 b 52 b a, b, c differ in the same row, significantly differences (P < 0.05) * Sperm were analyzed by using Vision TM (Mini Tube, Germany) Figure 1. One of the Bull in Baturiti BIBD - Bali 63

4 Table 2. Birth of Bali cattle resulted by sperm sexing artificial insemination Inseminator Acceptor (head) Sperm Pregnant (head) Birth (sex) Desten 24 sperm Y head (12, 2 ) Made Swarnawa 19 sperm Y head (9, 3 ) S/C C/R (%) Concordance (%) Mean , The results show that from 43 acceptors, 26 head was pregnant or services per conception (S/C) by an average of The results of sexing AI was lower than the previous practice of S/C = 1.37 (Said et al., 2005) but from other research as reported in accordance Diwyanto and Herliantien (2006), that S/C AI program in beef cattle and dairy cattle ranged from 1.5 to over 4.0 because technically the success of AI at least influenced by four factors: (1) the quality of sperm after freezing and thawing, including the maintenance and handling of frozen semen; (2) the quality or condition of the recipient, related with the management, feed and health; (3) accuracy of detection of estrus and (4) inseminator skill which are related to the overall success of an AI program. Another factor that needs to be concerned is the possibility of a genetic disorder as a result of a cross, as well as the possibility of genotype environment interaction (GEI) or the interaction of environmental factors with the offspring of cattle genotype. The data in Table 2 shows that of 26 calves born by using the AI sperm sexing Y or male type, 21 head male and 5 head female were born (Figure 2a and 2b). This suggests that the frozen sperm sexing produced by 80.77% compliance rate, and there was no differentce from previous results where the success rate of births to the expectations of 81% (Said et al., 2005). CONCLUSION It is concluded that sexed sperm straw can be produced with good quality and is not significantly different from the unsexed sperm. When acceptor were inseminated using sexed sperm, the results shown that S/C = 1.65, CR = and concordance rate of birth was 80.77%. ACKNOWLEDGEMENT Thank you to the Ministry of Research and Technology of Republic of Indonesia, that funded this research through Agroteknopark program (ATP) in Bali. Our gratitude also to Figure 2a. Two months calves sexed sperm resulted from AI Figure 2b. Ten months calf resulted from sexed sperm AI (arrow). 64

5 the entire team of Bali ATP and Animal Research Group in Research Center for Biotechnology LIPI that has helped so that research can be finished. REFERENCES Diwyanto, K. dan Herliantien Aplikasi teknologi inovatif sexing dalam program inseminasi buatan dan usaha cow-calf operation. Wartazoa 16(4): Garner, D.L Sex-sorting mammalian sperm: Concept to application in animals. J. Androl. 22: Hafez, E.S.E Reproduction in Animals. 6 th Ed. Lea and Febiger, Philadelphia. Johnson, L.A. and G.E. Seidel Jr Current status of sexing mammalian spermatozoa. Theriogenology 52: Kaiin, E.M., B. Tappa, F. Afiati, M. Gunawan dan N.D. Yanthi Aplikasi Bioteknologi Untuk Produksi Bibit Yang Sudah Diketahui Jenis Kelaminnya. Laporan Teknik Proyek Penelitian Bioteknologi. Puslit Bioteknologi- LIPI. pp Said, S., E.M. Kaiin dan B. Tappa Produksi anak sapi potong dan sapi perah berjenis kelamin sesuai harapan. Pros. Seminar Nasional Industri Peternakan Modern II. Mataram, Juli pp Seidel, G.E. Jr. and D.L. Garner Current status of sexing mammalian spermatozoa. Reproduction 124: Susilawati, T Keberhasilan IB menggunakan semen sexing setelah dibekukan. Pros. Seminar Nasional Teknologi Peternakan dan Veteriner. Bogor, 4 5 Agustus Puslitbang Peternakan. Bogor. pp Susilawati, T., S.B. Sumitro, Harjopronjoto, Y. Mantara dan Nuryadi Pola kapasitasi spermatozoa X dan Y sapi hasil pemisahan menggunakan filtrasi sephadex dan sentrifugasi gradient densitas percoll. J. Penelitian Ilmu-ilmu Hayati 11: DISCUSSION Questions: 1. How many percentage of sperm X and Y that you got from collecting? 2. What % of animal that male and female calf? 3. How much the price of sexed sperm and unsexed sperm? Answers: 1. 80% Y sperm, 80% X sperm % sperm x (female). 3. 2x the cost. Rp 3000 (sexed) and Rp 1500 (unsexed). 65