Technical note: Droplet digital PCR as a new molecular method for a simple and reliable diagnosis of freemartinism in cattle

Freemartinism is the most common type of disorder of sex development in cattle. It leads to sterility in the female co-twin in heterosexual twin pregnancy, and is thus a serious problem in cattle production. The incidence of freemartin syndrome is directly dependent on the prevalence of twinning, wh...

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Bibliographic Details
Published in:Journal of dairy science 2019-11, Vol.102 (11), p.10100-10104
Main Authors: Szczerbal, I., Nowacka-Woszuk, J., Albarella, S., Switonski, M.
Format: Article
Language:English
Subjects:
Amelogenin - genetics
amelogenin gene
Animals
Cattle
Chimerism - veterinary
disorder of sex development (DSD)
Female
freemartinism
Freemartinism - diagnosis
Freemartinism - genetics
intersexuality
Leukocytes
Male
Polymerase Chain Reaction - methods
Polymerase Chain Reaction - veterinary
Pregnancy
Sensitivity and Specificity
sex chromosomes
Sex Chromosomes - genetics
X Chromosome - genetics
Y Chromosome - genetics
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Summary:Freemartinism is the most common type of disorder of sex development in cattle. It leads to sterility in the female co-twin in heterosexual twin pregnancy, and is thus a serious problem in cattle production. The incidence of freemartin syndrome is directly dependent on the prevalence of twinning, which has increased in dairy cattle populations in recent years. Thus, early and rapid identification of freemartins is needed to reduce economic loss. Of the various methods used to diagnose this condition, identifying the XX and XY cell lines in blood samples using cytogenetic techniques is the gold standard; however, this technique is time consuming. Faster and more reliable techniques are thus being sought. Droplet digital PCR (ddPCR) is a third-generation PCR method and it has not previously been used to detect XX/XY leukocyte chimerism in cattle. The aim of the present study was to verify the usefulness of ddPCR to detect and quantify leukocyte chimerism in this species. The X and Y copy numbers were estimated by identifying the copy numbers of 2 genes located on the sex chromosomes: amelogenin X-linked (AMELX) on the X chromosome and amelogenin Y-linked (AMELY) on the Y chromosome. In the first step, we performed ddPCR on samples prepared from female DNA mixed with male DNA in serially diluted proportions. We determined that the sensitivity of this method was sufficient to detect a low-frequency (
ISSN:0022-0302
1525-3198
DOI:10.3168/jds.2019-17021