Molecular Confirmation Test of Sexing Method on Limousin Cattle Sperm with Swim Up Technique

Budi Utomo, Rimayanti, Widya Paramita Lokapirnasari

Abstract

The purpose of this study was to determine the quality of the sperm and confirm the results of the separation of the sperm carrying the X and Y chromosomes using the molecular swim up method. Determining the calves' sex is a strategic step in the development of artificial insemination technology. One of the efforts to separate an X chromosome spermatozoa from a Y gene was carried out using the swim-up method, in the isotonic medium. This method was a preparation procedure, which allowed motile spermatozoa to migrate or swim from the lower to the upper layer of semen. Furthermore, this research aimed to test for the molecular confirmation of sexing method, through the use of the swim up technique. The motility and the viability parameters of the separated spermatozoa, were also microscopically tested for the quality. The separated sperm DNA was extracted by using the spin-column method, which was amplified by Polymerase Chain Reaction (PCR) at Sex-determining Region Y (SRY) in the Y chromosome area, and the GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) in the HMGB (High Mobility Group Box) location. Also, the sperm separation results in the upper layer and control, showed that there were 2 bands, namely the SRY gene (318 bp) and GAPDH (415 bp). This result further showed that after the swim up method, more Y genes were detected in the lower layer of the technique process. Moreover, the results showed that there was only 1 band of GAPDH (415 bp), which indicated the presence of X genes in the lower layer. Also, these results showed that sexing with the swim-up method was verified molecularly, in order to separate the spermatozoa carrying X and Y chromosomes, which were then tested using the duplex PCR method.

 

 

 

Keywords: XY chromosome, swim up, Polymerase Chain Reaction, Sex-Determining Region Y, glyceraldehyde 3-phosphate dehydrogenase.

 

 

 


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MARI G., CASTAGNETTI C., RIZZATO G., MISLEI B., IACONO E., and MERLO B. Density gradient centrifugation of sperm from a subfertile stallion and effect of seminal plasma addition on fertility. Animal reproduction science, 2011, 126(1-2): 96-100. https://pubmed.ncbi.nlm.nih.gov/21616614/

MOROTTI F., SANCHES B.V., PONTES J.H.F., BASSO A.C., SIQUEIRA E.R., and LISBOA L.A. Pregnancy rate and birth rate of calves from a large-scale IVF program using reverse-sorted semen in Bos indicus, Bos indicus-taurus, and Bos taurus cattle. Theriogenology, 2014, 81(5):696-701. https://pubmed.ncbi.nlm.nih.gov/24412681/

PELLEGRINO C.A.G., MOROTTI. F., UNTURA, R.M., PONTES, J.H.F., PELLEGRINO, M.F.O., CAMPOLINA J.P., SENEDA, M.M., BARSOSA F.A. and HENRY M. Use of sexed sorted semen for fixed-time artificial insemination or fixed-time embryo transfer of in vitro-produced embryos in cattle. Theriogenology, 2016, 86(3): 888-93. https://pubmed.ncbi.nlm.nih.gov/27068357/

YAN J., FENG H.L., CHEN Z.J., JINGMEI H., XUAN G. and YINGYING Q. Influence of swim-up time on the ratio of X-and Y-bearing spermatozoa. European Journal of Obstetrics & Gynecology and Reproductive Biology. 2006, 129(2): 150-154. https://www.sciencedirect.com/science/article/abs/pii/S0301211506001916

ONDHO Y. S., SUTIYONO, KURNIANTO E., SURYAWIJAYA A., and LESTARI D. A. Effect of electrode length on the separation and quality of X and Y sperm in sheep. Jurnal Sain Peternakan Indonesia, 2019, 14: 324-331. https://ejournal.unib.ac.id/index.php/jspi/article/download/9239/4917

GRANT V.J., & CHAMLEY L.W. Sex-sorted sperm and fertility: an alternative view. Biology of Reproduction, 2007, 76(2): 184-188. https://academic.oup.com/biolreprod/article/76/2/184/2629645

BLONDIN P., BEAULIEU M., FOURNIER V., MORIN M., CRAWFORD L., MADAN P., and KING W.A. Analysis of bovine sexed sperm for IVF from sorting to the embryo. Theriogenology, 2009, 71(1): 30-38. https://pubmed.ncbi.nlm.nih.gov/19004490/

MACHADO G.M., CARVALHO J.O., FILHO E.S., CAIXETA E.S., FRANCO M.M., RUMPF R., and DODE M.A.N. Effect of percoll volume, duration and force of centrifugation, on in vitro production and sex ratio of bovine embryos. Theriogenology, 2009, 71: 1289-1297. https://pubmed.ncbi.nlm.nih.gov/19230963/

HIKMAWAN S.K., CIPTADI G., and WAHYUNINGSIH S. Quality of swim up spermatozoa from Etawah grade buck that frozen with vitrification method with glycerol addition in different percentage. J. Ternak Tropika, 2016, 17 : 42-48. https://ternaktropika.ub.ac.id/index.php/tropika/article/view/248

HARRIS S.J., MILLIGAN M.P., MASSON G.M., and DENNIS K.J. Improved separation of motile sperm in asthenospermia and its application to artificial insemination homologous (AIH). Fertility and Sterility, 1981, 36(2): 219-221. https://www.sciencedirect.com/science/article/abs/pii/S0015028216456827

PARRISH J.J., SUSKO-PARRISH J.L., LEIBFRIED-RUTLEDGE M.L., CRITSER E.S., EYESTONE W.H., and FIRST N.L. Bovine in vitro fertilization with frozen-thawed semen. Theriogenology, 1986, 25(4): 591-600. https://pubmed.ncbi.nlm.nih.gov/16726150/

DE JONGE C.J., FLAHERTY S.P., BARNES A.M., SWANN N.J., and MATTHEWS C.D. Failure of multitube sperm swim-up for sex preselection. Fertility and sterility, 1997, 67(6): 1109-1114. https://pubmed.ncbi.nlm.nih.gov/9176452/

CORREA J.R., ZARMAKOUPIS-ZAVOS P.N., and ZAVOS P.M. Quantitative and qualitative characteristics of frozen-thawed bovine spermatozoa recovered via a conventional and a standardized swim-up technique. The Tohoku journal of experimental medicine, 1997, 181(2): 267-274. https://pubmed.ncbi.nlm.nih.gov/9163843/

GOUR D.S., DUBEY P.P., JAIN A., GUPTA S.C., JOSHI B.K., and KUMAR D. Sex determination in 6 bovid species by duplex PCR. Journal of applied genetics, 2015, 49 (4): 379-381. https://link.springer.com/article/10.1007/BF03195637

TAYLOR T.M. Comparing calf sex ratio and semen sex ratio determined by conventional PCR. [Thesis]. The Interdepartmental Program In Animal and Dairy Sciences. Southern Arkansas University, Arkansas, 2005.

OGUZ Y., GULER I., ERDEM A., MUTLU M.F., and GUMUSLU S. Effect of swim-up and gradient Sperm preparation techniques on deoxyribonucleic acid (DNA) fragmentation in subfertile patients. Journal of assisted reproduction and genetics, 2018, 35(6): 1083–1089. https://pubmed.ncbi.nlm.nih.gov/29572695/

POMP D., GOOD B.A., GEISERT R.D., CORBIN C.J., and CONLEY A.J. Sex identification in mammals with polymerase chain reaction and its use to examine sex effects on diameter of day-10 or-11 pig embryos. Journal of animal science, 1995, 73(5): 1408-1415. https://pubmed.ncbi.nlm.nih.gov/7665371/

SHI L., YUE W., REN Y., LEI F., and ZHAO J. Sex determination in goat by amplification of the HMG box using duplex PCR. Animal reproduction science, 2008, 105(3-4): 398-403. https://pubmed.ncbi.nlm.nih.gov/18096334/

INDRIAWATI, VOLKANDARI S.D., and MARGAWATI E.T. The Application of UTY and SRY Molecular Markers for Determination of Unknown Sex Samples in Bali Cattle. Jurnal Ilmu Dasar. 2020, 21(1): 55-60. https://jurnal.unej.ac.id/index.php/JID/article/view/9333

GOUR D.S., DUBEY P.P., JAIN A., GUPTA S.C., JOSHI B.K., and KUMAR D. Sex determination in 6 bovid species by duplex PCR. Journal of applied genetics, 2008, 49(4) :379-381. https://link.springer.com/article/10.1007/BF03195637

SHENDE A.M.,. KUTTY V.H.M., RAMTEKE S.S., GHOSH S.K., PRASAD J.K., and BHURE S.K. Molecular cloning and expression of buffalo SRY gene: A mammalian sex determination protein. Indian Journal of Biotechnology, 2014, 13: 364-369. http://nopr.niscair.res.in/bitstream/123456789/29617/1/IJBT%2013(3)%20364-369.pdf

MALIK A. Reproductive Efficiency Through Estrus Resynchronization, Timed Artificial Insemination and Spermatozoa Separation in Beef Cattle Universiti Putra Malaysia, Serdang, 2012.


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