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Verification of Caprine Sexed-separated Spermatozoa by Real Time PCR

Somarny, W.W.M.Z¹*, Aslinda¹,K., Md. Tasol¹, S., Mohd. Hifzan², R., Nasir², J.M., Mohd Hafiz³, A.W., Ab Rashid⁴, B. and Musaddin¹, K.

¹Strategic Livestock Research Center, Malaysian Agricultural Research and Development Institute, 43400 Serdang, Selangor, Malaysia.
²MARDI Muadzam Shah, Pahang.
³MARDI Kluang, Johor.
⁴Faculty of Agriculture, Biotechnology and Food Science, Universiti Islam Sultan Zainal Abidin, Kuala Terengganu, Terengganu.

Malaysian J. Anim. Sci. 2014 17(1): 73-83

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Sexed spermatozoa could contribute to increasing profitability of the dairy and beef industries worldwide by producing offspring of predetermined sex. Various methods have been designed to separate sperm population into X- and Y-chromosome bearing spermatozoa. In this study, the caprine semen sample from Savanna (n=2) and Boer (n=4) breeds were sex-separated using free flow electrophoresis. In order to determine the efficiency of sex-separation procedure, a rapid and accurate technique real time PCR (qPCR) was used to validate the X- and Y-chromosome bearing spermatozoa following separation. The primers and probes were developed to detect the X- and Y- chromosome bearing spermatozoa of Capra hircus based on ZFX and SRY genes, respectively, using TaqMan qPCR assay. The amplification of ZFX and SRY genes produced a single fragment of 93 bp and 137 bp, respectively. The primers used in separated spermatozoa by free-flow electrophoresis technique showed that the mean ratio of X- and Y-chromosomes bearing spermatozoa from the cathodic and anodic fractions were 36:64 and 44:56, respectively, compared to unseparated semen was 51:49. Thus, these primers can be used to validate the gene copy of X- and Y-chromosome bearing spermatozoa in caprine semen.