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

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.

Exposure to male-dominated environments during development influences sperm sex ratios at sexual maturity

“…; Khamlor et al. ). Here, we modified and optimized an absolute quantification qPCR protocol to measure the proportion of Y‐CBS in the sperm samples of house mice.…”

The enriched Y-bearing sperm combined with delayed fixed-time artificial insemination for obtaining male Simmental crossbred offspring

“…Unfortunately, the sex ratio of spermatozoa was not examined before and after the sexing process due to the limited time because this project must be completed in 1 year. Further research is needed to determine sperm sex ratio using polymerase chain reaction as reported by Khamlor et al [42] and its application in a larger recipient population.…”

Technical note: Droplet digital PCR precisely and accurately quantifies sex skew in bovine semen

“…Semen-sexing cytometers quantify sex skew using Technical note: Droplet digital PCR precisely and accurately quantifies sex skew in bovine semen the same DNA-binding dye and detection scheme that originally generated the sexed semen, rather than an independent test; the skew data are therefore subject to the same variations or biases as manufacturing the sexskewed product and are limited by the lack of unique, positive identifiers for the X and Y chromosomes. The FISH and qPCR approaches both specifically identify X and Y chromosomes (Kawarasaki et al, 1998;Rens et al, 2001;Habermann et al, 2005;Parati et al, 2006;Puglisi et al, 2006;Khamlor et al, 2014), but lack quantitative sensitivity and statistical rigor due to sampling (FISH) and sensitivity (qPCR) limitations. In an effort to overcome these challenges, we present methods for a droplet digital PCR (ddPCR) sex skew assay that quantifies total X and Y chromosome copies, reporting sex skew as a percentage with a variance of ±0.5 percentage point.…”