Comparative study of PCR-sexing procedures using bovine embryos fertilized with sex-sorted spermatozoa

Abstract

Sex determination in bovine embryos is a useful tool in reproductive biotechnology. This work compares two techniques of embryo sexing by polymerase chain reaction (PCR). Embryos were produced in vitro with sex-sorted sperm and two techniques of DNA lysis were tested (proteinase K versus heat shock). Subsequently, halves of each lysed sample was amplified both by amelogenin and BRY4a/SAT1 primers. Male embryos treated by both digestion methods and amplified by BRY4a/SAT1 gave higher rates of false negatives. Amelogenin amplification failed with embryos previously digested by proteinase K. In contrast, the lysis method allowed obtaining the best accuracy in terms of sex verification when using amelogenin amplification.Additional key words: amelogenin; blastocyst; BRY4a/SAT1; sex technique. ResumenEstudio comparativo de dos métodos de sexaje por PCR en embriones bovinos producidos con semen sexado La determinación del sexo en embriones bovinos es una herramienta útil en las biotecnologías reproductivas. Este trabajo compara dos técnicas de determinación del sexo en embriones mediante la reacción en cadena de la polimerasa (PCR). Los embriones fueron producidos in vitro con semen sexado y se analizaron dos técnicas de lisis de ADN (proteinasa K y choque térmico). Posteriormente, la mitad de cada muestra lisada fue amplificada por la amelogenina y por los cebadores BRY4a/SAT1. Los embriones machos tratados por ambos métodos de digestión y amplificados por BRY4a/SAT1 dieron mayores tasas de falsos negativos. La amplificación de la amelogenina no dio resultados en embriones previamente digeridos por proteinasa K; sin embargo, el método de lisis por choque térmico junto con la amplificación por amelogenina permitió obtener la mayor precisión en términos de verificación de sexo.Palabras clave adicionales: amelogenina; blastocistos; BRY4a/SAT1; técnica de sexaje.

In vitro and in vivo quality of bovine embryos in vitro produced with sex-sorted sperm

“…Our sexing method [33] consists of an accurate built-in control PCR, which is particularly appropriate when an accurate embryonic sex diagnosis is necessary [64]. Commercially available, sex-sorted sperm usually allow embryos of the desired sex at rates of 90% or higher, which has been confirmed by PCR in the present work.…”

“…On the other hand, the use of Vero cells shows an embryotrophic effect by producing growth factors and also removing toxicants or inhibitory substances derived from the embryo [59]. However, in a previous work [33] we demonstrated that embryos produced without serum had better survival and hatching rates after vitrification (open pulled straw), pointing toward different qualities among the embryos. To our knowledge, there are no data about how culture systems might improve the diminished developmental ability of embryos produced with sexed sperm, and we hypothesize that in our case the developmental ability of the embryos fertilized with sexed sperm does not depend on the culture system, but on the bull and the damage inflicted to the sperm by the sorting process.…”

“…The efficiency of the sex-sorting sperm was inferred by determining the embryonic sex by a built-in control PCR technique using transferable stage embryos [33]. Briefly, fresh and vitrified/warmed blastocysts were incubated for 2 min in pronase (5 mg/mL in PBS) to remove the zona pellucida.…”

Embryonic Sex Induces Differential Expression of Proteins in Bovine Uterine Fluid

“…6 Embryos were in vitro produced (IVP) with frozen male or female sex-sorted sperm (Sexing Technologies; Navasota, TX, USA) as previously reported. 31 Early morulae were transferred to the ipsilateral horn of estrus-synchronized, cross-bred beef heifers on day 5 after in vitro fertilization (IVF) and were recovered on day 8 (see below) or sustained in vitro up to day 8.…”