Abstract
The majority of heifer calves born as hetero–sexual twin births are sterile freemartins. It is important that this condition be diagnosed at an early age because freemartin heifers can’t be used as replacement stock. Different methods are available for the diagnosis of freemartinism, however molecular methods are preferred due to their accuracy and shorter duration of process. In the present study, freemartinism status of 40 female calves of heterosexual multiple births was investigated by multiplex polymerase chain reaction (multiplex PCR). The reaction was carried out using two primer sets for the Sry and K–casein genes of domestic cattle. The PCR product was analyzed by agarose gel (3%) electrophoresis, which allowed to identify the genotypes of the animals studied: normal males (453 bp and 163 bp), normal females (453 bp), and 39 (97.50%) study cases that showed chromosome chimerism (freemartin, 453 bp and 163 bp). Compared to the polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) technique, this method (i.e., multiplex PCR) proved a far cheaper and quicker (approximately 27 minutes) way to diagnose freemartinism.
Highlights
IntroductionIn over 90% of such cases, chorionic vascular anastomoses connecting the placentas of the twin fetuses occur at 30 to 40 days of gestation, i.e., before sexual dimorphism takes place
The term freemartin is used to refer a sterile heifer, and is by extension, used to refer to a genetic female from a heterosexual multiple birth
The optimal annealing temperature of the oligo–nucleotide primer used in this study (Sry and K–casein) was determined by using the gradient polymerase chain reaction (PCR) program (Techne gradient thermal cycler, UK)
Summary
In over 90% of such cases, chorionic vascular anastomoses connecting the placentas of the twin fetuses occur at 30 to 40 days of gestation, i.e., before sexual dimorphism takes place. This results in an interchange of cells (hematopoietic chimerism) and plasma substances such as hormones between the twin embryos, leading to an intersexual state in the female (Harvey, 1976; Gustavsson and Johansson, 1980; Long, 1990). Compared to the polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analysis, the multiplex polymerase chain reaction (multiplex PCR) method proved a far cheaper (not using restriction enzyme) and quicker (no time restriction) way to diagnose freemartinism (Olsaker et al.,. 1993; Ennis et al.,. 1999; Padula, 2005)
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