SYBR Green quantitative PCR for sex determination of bovine spermatozoa

Tan Y. J., . and Mahanem Mat Noor, . and Wan Somarny Wan Md. Zain, . SYBR Green quantitative PCR for sex determination of bovine spermatozoa. pp. 29-39. ISSN 1394-9829

Spermatozoa sexing technology in cattle breeding is being done by separation of X- and Y- chromosome bearing spermatozoa using flow-sorting technology. However the sexing technique needed to be validated to ensure the accuracy of the technology. A technique to determine the sex of bovine spermatozoa using SYBR Green real-time quantitative PCR (qPCR) was developed. Two sets of primers ZFX and SRY were designed specifically to X- and Y- chromosome bovine genes respectively. Plasmid was inserted with ZFX and SRY gene fragment separately to create standard curves that ranged from 3.0 x 102 to 3.0 x 106 copies. The standards generated linear relationship with regression coefficient r2 0.984 for ZFX and r2 0.996 for SRY. Both standards of ZFX and SRY showed melting peak at temperature of 83 C and 85 C respectively. Real-time qPCR of bovine spermatozoa DNA samples and cloned plasmid ZFX and SRY genes for creating standard samples were performed simultaneously. The percentages of unsexed X- and Y- chromosome-bearing spermatozoa did not differ much from the 1:1 as reported in unsexed spermatozoa population. Therefore the highly sensitive and fast method of real time PCR is a suitable technique for quantitating X- and Y- chromosome-bearing spermatozoa in semen samples.

Full text available from: Official URL: http://jtafs.mardi.gov.my/jtafs/43-1/SYBR.pdf

Abstract

Spermatozoa sexing technology in cattle breeding is being done by separation of X- and Y- chromosome bearing spermatozoa using flow-sorting technology. However the sexing technique needed to be validated to ensure the accuracy of the technology. A technique to determine the sex of bovine spermatozoa using SYBR Green real-time quantitative PCR (qPCR) was developed. Two sets of primers ZFX and SRY were designed specifically to X- and Y- chromosome bovine genes respectively. Plasmid was inserted with ZFX and SRY gene fragment separately to create standard curves that ranged from 3.0 x 102 to 3.0 x 106 copies. The standards generated linear relationship with regression coefficient r2 0.984 for ZFX and r2 0.996 for SRY. Both standards of ZFX and SRY showed melting peak at temperature of 83 C and 85 C respectively. Real-time qPCR of bovine spermatozoa DNA samples and cloned plasmid ZFX and SRY genes for creating standard samples were performed simultaneously. The percentages of unsexed X- and Y- chromosome-bearing spermatozoa did not differ much from the 1:1 as reported in unsexed spermatozoa population. Therefore the highly sensitive and fast method of real time PCR is a suitable technique for quantitating X- and Y- chromosome-bearing spermatozoa in semen samples.

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Item Type:	Article
AGROVOC Term:	Bovines
AGROVOC Term:	Spermatozoa
AGROVOC Term:	PCR
AGROVOC Term:	Sex determination
AGROVOC Term:	Sexing
AGROVOC Term:	Cattle
AGROVOC Term:	Animal breeding
AGROVOC Term:	Chromosomes
AGROVOC Term:	Separation
AGROVOC Term:	High temperature
Depositing User:	Ms. Suzila Mohamad Kasim
Last Modified:	24 Apr 2025 06:29
URI:	http://webagris.upm.edu.my/id/eprint/24422

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