Bovine foetal sex determination—Different DNA extraction and amplification approaches for efficient livestock production

Abstract

Foetal sex determination using polymerase chain reaction (PCR) in mammals is based on the amplification of gender-specific foetal DNA sequences circulating in maternal blood. The bovine synepitheliochorial placenta does not allow a direct contact between the trophoblast and the maternal blood, resulting in difficult passage of foetal DNA and, consequently, its very small amounts in maternal bloodstream. Circulating cell-free foetal DNA (ccffDNA) encompasses short nucleotide fragments (300-600 bp) in maternal circulation. The aim of this study was to assess this non-invasive method in accurate prenatal sexing in early and late gestational periods in comparison with ultrasound diagnostics. As various DNA isolation and amplification methods were tested, their success in obtaining reliable results was evaluated. Two groups were tested, each consisting of 20 pregnant cows. Blood of a bull and a non-pregnant heifer was the controls. Extraction of foetal DNA was accomplished by three different methods: using tubes with silicone membranes, a single-tube extraction without silicone membranes and phenol-chloroform extraction. Following each extraction method, foetal DNA was amplified using PCR and real-time PCR with both bAML and TSPY primers in a separate reaction. Positive results were obtained only after amplification of foetal DNA extracted with a single-tube extraction kit. In comparison with ultrasound examination results and foetal gender recorded at birth, the sensitivity of the PCR test was 90% in Group I, but the technique failed to detect male foetuses in Group II. The real-time PCR test sensitivity in Group I was 90% and in Group II 91.6%.

Beta-Casein Gene Polymorphism in Serbian Holstein-Friesian Cows and Its Relationship with Milk Production Traits

“…For the past few decades, many researchers have been dealing with the problem of rendering agricultural and livestock production more effective. Therefore, different methods are used to make animal breeding and animal selection programs more effective [35]. In this study, we investigated the genotype and allelic frequencies of beta-casein gene in 106 Serbian HF cows.…”

Insight in Leptin Gene Polymorphism and Impact on Milk Traits in Autochtonous Busha Cattle

“…During past few decades, molecular markers proved to be an important instrument for selection and preservation of animal populations [1][2][3][4]. Association between DNA polymorphism and milk production traits have been studied for a number of genes, and among them, leptin (LEP) gene proved to be a powerful biomolecule that can increase the productivity of farm animals [5].…”

Efficient, fast and low-cost strategies for DNA extraction from different nucleated sheep cells

“…It was observed in the present study that the behavior of this protocol as control treatment showed better results when compared to the extraction of DNA from blood cells from the buffalo and cervid species. Variations in the concentration of DNA extracted from the same protocol, in different species, may be related to the divergent leukocyte profile between them (Araújo et al, 2009;Ristanic et al, 2018), since the protocol acts on nucleated cells isolated from the whole blood, which may suffer variations due to external factors, such as stress, causing profile differences among individuals of the same species, albeit in an abnormal way. Caldart et al (2011) evaluated two commercial kits (DNAzol ® -Invitrogen TM , FTA ® -Whatman TM ) for DNA extraction for goat and sheep leukocyte rings as a function of storage time.…”

“…The advent of DNA discovery and its applications in molecular sciences made possible some advances in health, research and animal production (Ristanic et al, 2018;Shalaby et al, 2019;Yadav, Tomar, Jha, & Singh, 2017;Yamamoto et al, 2017). However, genetic resources access and use in most of developing countries is lower than expected (Food & Agriculture Organization of the United Nations (FAO, 2010).…”