Early fetal sexing in the rhinoceros by detection of male‐specific genes in maternal serum

Abstract

Genetic sexing of animals with long gestation time benefits the management of captive populations. Here, X and Y chromosome-specific primers, based on equine gene sequencing data, were developed and tested on captive rhinoceroses (10 males, 20 females) representing four species (Diceros bicornis, Certaotherium simum simum, Rhinoceros unicornis, and Dicerorhinus sumatrensis). The Y chromosome-specific primer set targeted SRY (Sex-determining region Y), and amplified a 177-bp product following PCR of DNA extracted from males, but not females, of all species. A primer set based on the equine AMEL (Amelogenin) gene resulted in a 232-bp product following PCR of all rhinoceros species. These gene-specific primer sets were then evaluated for their ability to determine gender in cell-free DNA from rhinoceros serum. Modifications to the original extraction and PCR protocols were required to obtain sufficient DNA quantities from serum, and both DNA yield and PCR amplification were substantially reduced or absent following multiple freeze-thaw cycles of serum. When fresh serum from 14 pregnant rhinoceroses (ultimately bearing seven male and seven female calves), representing four species at different stages of gestation (Days 61-490), were probed in a PCR-based assay, an accuracy of 71% was achieved for male-specific gene detection of SRY, which improved to 100% by including a reamplification step into the protocol. Such early sex determination should be a valuable tool for current management practices as well as future assisted reproduction of rhinoceroses.

Sex determination using circulating cell-free fetal DNA in small volume of maternal plasma in elephants

“…In investigations of ccffDNA, the extraction method used plays a crucial role, therefore a highly-effective protocol with an increased digestion capacity was used for the purpose of avoiding protein inhibition. Concentration of total ccffDNA was 1–1.2 ng/μL for each plasma sample investigated, which was able to be increased by a larger initial volume of maternal plasma 16,25 . Following PCR amplification with fluorescently-labelled primers, an accurate and highly-sensitive capillary electrophoresis method was performed to detect male and female-specific amplicons.…”

“…Molecular sexing methods in elephants using short amplicons (<200 bp) also have a substantial relevance in the areas of population ecology, conservation genetics 11,12 as well as in wildlife forensics 13 . Early and reliable diagnosis of sex in the fetuses of domestic animals has wide-spread and significant commercial and research applications in the livestock industry 10,14,15 , but there is similarly an increasing need for the early determination of fetal sex during pregnancy in zoo species using non-invasive methods and comparable genetic techniques 16 . Ultrasonographic fetal sexing techniques are of limited practical use in larger zoo mammals, particularly in elephants, as the fetus is lying between the huge-sized internal organs of the cow/dam during gestation and is not reachable with ultrasound scanning 17 .…”

Biotechnology for Wildlife

“…The sex-determination envisaged to be a valuable tool for managing assisted reproduction of the managed in captivity (Stoops et al 2018) Sika deer (Cervus nipon)…”

Innervation of internal female genital organs in the pig during prenatal development

“…; Roly et al. ; Stoops et al ). The lack of a gene causes the undifferentiated gonad to transform into an ovary.…”

“…It is known that the genes determining sex, as well as molecular mechanisms have an influence on the further development of gonads and other parts of the genital system (Capel et al 1999;Hashimoto, 2003;Guioli et al 2007;Orvis & Behringer, 2007;Mullen & Behringer, 2014). An important factor in the differentiation of gonads is the SRY gene, located on the short arm of the Y chromosome (Saberivand & Ahsan, 2016;Katsura et al 2018;Roly et al 2018;Stoops et al 2018). The lack of a gene causes the undifferentiated gonad to transform into an ovary.…”