Molecular fish sexing on Kohaku Koi (Cyprinus carpio) based on ArS.9-15 gene amplification by PCR method

Abstract

Abstract This study aims to sex determine on Kohaku Koi fish rapidly, precisely, accurately, and as early as possible based on the amplification of the ArS.9-15 gene using the PCR method. This study began with the maintenance of 10 Kohaku Koi fish, obtained from Koi fish farmers in the Sleman area in Yogyakarta. The 10 Kohaku Koi were adapted in a filtered aquarium with O2 aeration and fed ad libitum by fish pellets for 1-3 days. Then, a collection of 0.5 ml peripheral blood samples per fish from the caudalis vein was performed under anesthesia conditions using special anesthetic preparation for Koi fish, namely Koi Anesthesia mixed with fresh water. Those samples were then collected in tubes containing the Ca-EDTA anticoagulant. The DNA then was extracted from peripheral blood samples and used as a template for PCR amplification by specific oligonucleotide primer pairs to amplify the ArS.9-15 gene. PCR products in the form of DNA fragments were visualized with 1.5% agarose gel electrophoresis and SybrSafe staining using Transilluminator-UV in a dark room. The DNA fragments were analyzed descriptively to sex determination between male and female Kohaku Koi. Electrophoresis results of PCR products showed two DNA bands of 850 bp and 1,100 bp in male Kohaku Koi, while in female Kohaku Koi showed only a band DNA in size of 850 bp. The results of the molecular fish sexing of the 10 Kohaku Koi showed that 70% are female and 30% are male.