Sexing of in vitro produced ovine embryos by duplex PCR

Abstract

The aim of this article was to develop a fast and easy duplex polymerase chain reaction (PCR) method, for sex determination of ovine in vitro produced embryos prior to implantation. We tested the approach with 107 samples of autosomal cells (oviductal sheep cells and male lamb fibroblasts), divided into three groups for each sex according to the number of cells employed (30, 5, 2, respectively). We then used the test on 21 embryos at blastocyst stage. On the same day the embryos were transferred in pairs into 11 recipient synchronized ewes. The PCR utilized two different sets of primers: the first pair recognized a bovine Y-chromosome-specific sequence (SRY), that showed 100% homology with the corresponding sequence of the ovine Y-chromosome and is amplified in males only. The second pair recognized the bovine 1.715 satellite DNA (SAT) which was amplified in all ovine samples but, when submitted to the GenBank database did not show homology with any of the reported ovine sequences. However, after sequencing, ovine amplification product showed 98% homology with the bovine specific satellite sequence. The autosomal samples were amplified with 85.0% efficiency and 91.2% accuracy, while amplification was successful with all 21 embryos (100% efficiency). Eight lambs were born and the sex as determined by PCR corresponded to the anatomical sex in seven (87.5% accuracy). These results confirm that this method can be applied in ovine breeding programs to manipulate sex ratio of offspring.

A fast and simple method for the polymerase chain reaction-based sexing of livestock embryos

“…Moreover, the procedure for sperm cell sorting can alter the semen IVF performance of certain bulls (Blondin et al, 2009), reducing the options for genetic diversity in breeding programs. In turn, embryo sexing by the PCR-based detection of X-and/or Y-specific regions in retrieved blastomeres is an alternative means of overcoming some of the limitations of the sexed semen for in vivo-or in vitro-produced embryos, allowing the producer to choose which embryo should be transferred to synchronous recipients based on sex determination (Mara et al, 2004;Carneiro et al, 2011;Rattanasuk et al, 2011). The molecular method is efficient for field applications, resulting in pregnancy rates comparable to nonbiopsied embryos, even after cryopreservation (Tominaga, 2004;Tominaga and Hamada, 2004).…”

Genetic sex determination assays in 53 mammalian species: Literature analysis and guidelines for reporting standardization

“…Genes that were most often used in the sexing assays are SRY , AMELY with its homologue AMELX and ZFY and homologue ZFX . SRY was used for sexing humans (Esteve Codina et al., 2009), bears (Pages et al., 2009), cattle (Gokulakrishnan, Kumar, Sharma, Mendiratta, & Sharma, 2012), dogs (Prugnard et al., 2016), mouse (Prantner et al., 2016), sheep (Mara et al., 2004), buffalo (Fu et al., 2007), minks, ermines, badgers, otters and pine martens (Statham et al., 2007), elephants (Gorrell et al., 2012), koalas (Wedrowicz, Karsa, Mosse, & Hogan, 2013), and sea mammals (McHale, Broderick, Ovenden, & Lanyon, 2008). Sex determination was also performed on the following genes: DDX3Y and homologue DDX3X, EIF2S3Y and homologue EIF2S3X , Jarid1d and homologue Jarid1c , Sly and SMCY (Table S1).…”

“…An example of such a clear overview was presented in Mara et al. (2004). This enables the reader to compare different sexing assays without laborious search for relevant information in the text, especially when there are multiple validation tests in one article.…”

Comparison between embryonic stem cell versus adult stem cell to restore cartilage repair: an experimental study

“…In group-A, the defects were then treated with Nanofracture: each cartilage lesion was treated with 5 channels having a 9 mm depth using a cannulated awl and a 1mm thick Nitinol needle with a 3 mm distance between each channel; the surgical technique was previously described [6] . In group-B, liquid phase duplex Polymerase Chain Reaction (PCR) was performed on approximately 90 in vitro produced and vitrified blastocysts at day 6 -7 of culture according to the technique of Mara et al [7] as previously described [8] . About 500.000 -700.000 cells were embedded in 60 µl of fibrin glue and engrafted into the cartilage defects.…”