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Determination of Sperm Sex Ratio in Bovine Semen Using Multiplex Real-time Polymerase Chain Reaction.

Khamlor T, Pongpiachan P, Sangsritavong S, Chokesajjawatee N - Asian-australas. J. Anim. Sci. (2014)

Bottom Line: However, assessment of the sex ratio of the sorted semen is tedious and expensive.In this study, a rapid, cost effective and reliable method for determining the sex ratio was developed using a multiplex real-time polymerase chain reaction (PCR) assay.The measured %X in semen samples were the same within 95% confidence intervals as the expected values, i.e., >90% in X-sorted semen, <10% in Y-sorted semen and close to 50% in the unsorted semen.

View Article: PubMed Central - PubMed

Affiliation: National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathum Thani 12120, Thailand ; Department of Animal and Aquatic Science, Faculty of Agriculture, Chiang Mai University, Muang District, Chiang Mai 50200, Thailand .

ABSTRACT
Gender selection is important in livestock industries; for example, female calves are required in the dairy industry. Sex-sorted semen is commonly used for the production of calves of the desired gender. However, assessment of the sex ratio of the sorted semen is tedious and expensive. In this study, a rapid, cost effective and reliable method for determining the sex ratio was developed using a multiplex real-time polymerase chain reaction (PCR) assay. In this assay, the X and Y chromosome-specific markers, i.e., bovine proteolipid protein (PLP) gene and sex-determining region Y (SRY) were simultaneously quantified in a single tube. The multiplex real-time PCR assay was shown to have high amplification efficiencies (97% to 99%) comparable to the separated-tube simplex real-time PCR assay. The results obtained from both assays were not significantly different (p>0.05). The multiplex assay was validated using reference DNA of known X ratio (10%, 50%, and 90%) as templates. The measured %X in semen samples were the same within 95% confidence intervals as the expected values, i.e., >90% in X-sorted semen, <10% in Y-sorted semen and close to 50% in the unsorted semen. The multiplex real-time PCR assay as shown in this study can thus be used to assess purity of sex-sorted semen.

No MeSH data available.


Related in: MedlinePlus

Standard curves and linear equations obtained from the simplex real-time polymerase chain reaction assay showing relationship between threshold cycle (Ct) and DNA copy number (logC) from triplicate experiments. (A) X chromosome-specific amplification, (B) Y chromosome-specific amplification.
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f1-ajas-27-10-1411: Standard curves and linear equations obtained from the simplex real-time polymerase chain reaction assay showing relationship between threshold cycle (Ct) and DNA copy number (logC) from triplicate experiments. (A) X chromosome-specific amplification, (B) Y chromosome-specific amplification.

Mentions: Standard curves showing relationship between the amount of template (copy number, logC) and Ct for each sex-related DNA from the simplex and multiplex assays are shown in Figure 1 and 2, respectively. All curves fitted to linear regression models with correlation coefficient (r)>0.99. For the simplex PCR, the general linear equations obtained from the three runs can be represented as: Ct_X = 35.40–3.34 logC for X amplification and Ct_Y = 36.12–3.39 logC for Y amplification. For the multiplex PCR, the equations for X and Y were: Ct_X = 40.56.16–3.39 logC and Ct_Y = 41.01–3.35 logC, respectively.


Determination of Sperm Sex Ratio in Bovine Semen Using Multiplex Real-time Polymerase Chain Reaction.

Khamlor T, Pongpiachan P, Sangsritavong S, Chokesajjawatee N - Asian-australas. J. Anim. Sci. (2014)

Standard curves and linear equations obtained from the simplex real-time polymerase chain reaction assay showing relationship between threshold cycle (Ct) and DNA copy number (logC) from triplicate experiments. (A) X chromosome-specific amplification, (B) Y chromosome-specific amplification.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150173&req=5

f1-ajas-27-10-1411: Standard curves and linear equations obtained from the simplex real-time polymerase chain reaction assay showing relationship between threshold cycle (Ct) and DNA copy number (logC) from triplicate experiments. (A) X chromosome-specific amplification, (B) Y chromosome-specific amplification.
Mentions: Standard curves showing relationship between the amount of template (copy number, logC) and Ct for each sex-related DNA from the simplex and multiplex assays are shown in Figure 1 and 2, respectively. All curves fitted to linear regression models with correlation coefficient (r)>0.99. For the simplex PCR, the general linear equations obtained from the three runs can be represented as: Ct_X = 35.40–3.34 logC for X amplification and Ct_Y = 36.12–3.39 logC for Y amplification. For the multiplex PCR, the equations for X and Y were: Ct_X = 40.56.16–3.39 logC and Ct_Y = 41.01–3.35 logC, respectively.

Bottom Line: However, assessment of the sex ratio of the sorted semen is tedious and expensive.In this study, a rapid, cost effective and reliable method for determining the sex ratio was developed using a multiplex real-time polymerase chain reaction (PCR) assay.The measured %X in semen samples were the same within 95% confidence intervals as the expected values, i.e., >90% in X-sorted semen, <10% in Y-sorted semen and close to 50% in the unsorted semen.

View Article: PubMed Central - PubMed

Affiliation: National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathum Thani 12120, Thailand ; Department of Animal and Aquatic Science, Faculty of Agriculture, Chiang Mai University, Muang District, Chiang Mai 50200, Thailand .

ABSTRACT
Gender selection is important in livestock industries; for example, female calves are required in the dairy industry. Sex-sorted semen is commonly used for the production of calves of the desired gender. However, assessment of the sex ratio of the sorted semen is tedious and expensive. In this study, a rapid, cost effective and reliable method for determining the sex ratio was developed using a multiplex real-time polymerase chain reaction (PCR) assay. In this assay, the X and Y chromosome-specific markers, i.e., bovine proteolipid protein (PLP) gene and sex-determining region Y (SRY) were simultaneously quantified in a single tube. The multiplex real-time PCR assay was shown to have high amplification efficiencies (97% to 99%) comparable to the separated-tube simplex real-time PCR assay. The results obtained from both assays were not significantly different (p>0.05). The multiplex assay was validated using reference DNA of known X ratio (10%, 50%, and 90%) as templates. The measured %X in semen samples were the same within 95% confidence intervals as the expected values, i.e., >90% in X-sorted semen, <10% in Y-sorted semen and close to 50% in the unsorted semen. The multiplex real-time PCR assay as shown in this study can thus be used to assess purity of sex-sorted semen.

No MeSH data available.


Related in: MedlinePlus