368 Field Studies on the Effectiveness of the Ycd Embryo Sexing Technique in Bovine

Abstract

The YCD sexing technique is applied in many commercial breeding programs due to its ease of operation in the field and its reliable and accurate sex determination results. Less equipment requirements and more simplified protocols provide great convenience for field applications of this method, and processing errors and chances of assay contamination are reduced. To increase the effectiveness of this technique in future field applications, field data collected from commercial embryo transfer programs in 63 farms in China during June 2002 to December 2005 were analyzed. Embryos were flushed from superovulated dairy cattle and subjected to a biopsy for sex determination using protocols and YCD sexing kits supplied by AB Technology Ltd. (Pullman, WA, USA). Embryos were implanted fresh on the same day or frozen with AG freeze medium (AB Technology Ltd.) for later transfer. The chi-square test was used for statistic analysis. This study indicates that sampling with a piece of zona pellucida (ZP) during embryo sexing increased the incidence of no response (NR) but did not alter the accuracy of determining sex of offspring. Larger biopsies significantly reduced the incidence of NR after DNA amplification and electrophoresis separation, and increased the accuracy of sex determination of calves (from 88.1%, 111/126 to 98.5%, 129/131; P < 0.01). Biopsies containing dead and/or detached cells did not increase the NR incidence, but decreased the accuracy of sex determination of calves from 93.4% (240/257) to 86.6% (58/67; P < 0.05). There were no relationships between NR incidences and embryo quality, nor between the sex determination accuracy of calves and embryo quality or stages. However, the NR incidence increased for PCR analyses of earlier development stages (5.1% at morula to early blastocyst) compared to blastocyst (3.2%) and expanded blastocyst (2.2%; P < 0.05). The sex determination accuracy decreased to 77.4% (65/84) when embryos with unclear or expanded and faint female electrophoresis bands were transferred compared to those with clear bands (97.1%, 233/240). Field data suggest that larger biopsies during embryo sexing favor the effectiveness and accuracy of the YCD technique; however, increased sample size may affect the embryo survival, resulting in a decrease in the pregnancy success of embryo transfer. Reduced accuracy in YCD analyses using dead or detached blastomeres may be caused by the loss of male genes in dead or degenerated cells. Increased NR incidence in sampling of ZP for analysis may be due to biopsy loss when transferring the biopsy at the sampling step of the YCD method. In addition, the accuracy of sexing will be lower when embryos with unclear electrophoresis results are used. It is concluded that a proper and stage-dependent size of biopsy should be collected and used to ensure the accuracy of sex determination without reducing embryo survival, and the inclusion of a few live cells in a biopsy is important to the success of the sexing determination of this technique.

Conception rate after sex determination and cryopreservation of D7 bovine embryos:

“…In this study, the gender was determined in 91.3% of embryos for fresh transfer and in 87.5% of embryos intended for freezing (Table 1), which accords with our previous work with whole, intact (88.7%; Lopatarova et al, 2007) and splitted embryos (89.4-92%; Lopatarova et al, 2008). Our results are similar to those published by other authors (85-95%) using embryos from superovulated donors (Thibier and Nibart, 1995;Shea, 1999;Li et al, 2007;Yu et al, 2007) as well as IVP embryos (Lopes et al, 2001;Hasler et al, 2002;Tominaga and Hamada, 2004). However, the successful sex detection was inversely related to the number of isolated embryonic cells (≤ 3 cells 85.5%, 4-6 cells 97.4%, ≥ 7 cells 100%; Lacaze et al, 2008).…”

“…Better results were published later using PCR amplification of specific DNA sequences to determine embryonic sex in cattle (Herr et al, 1990;Schroder et al, 1990;Peura et al, 1991), pigs (Pomp et al, 1995), horses (Peippo et al, 1995), humans (Handyside et al, 1990) and mice (Han et al, 1993). Recently published results support the suitability of the PCR method for sex determination in cattle on the basis of its high accuracy and quick results (Thibier and Nibart, 1995;Lopes et al, 2001;Ekici et al, 2006;Yu et al, 2007).…”

Lower quality bovine embryos may be successfully used for sex determination

“…First of all, it was necessary to refine microsurgical intervention to enable the isolation of blastomeres from different developmental stages of embryos with minimal damage. The procedure may differ in details from laboratory to laboratory according to the skill and experience of the staff (Bredbacka et al, 1994;Thibier and Nibart, 1995;Shea, 1999;Yu et al, 2006). As described in more detail in the Material and Methods section we used for this purpose the twinner system (AB Technology, Inc., Pullman, WA), and similar to others we recorded only minimal harm to embryonic morphology after controlled operations under a stereomicroscope.…”

“…From experimental aspects these data are certainly valuable, but field praxis requires exclusively embryos in more advanced developmental stages and Shea (1999) after the evaluation of numerous experiments concluded that the removal of 10-20% of embryonic mass for sex analyses had no detectable harmful effect on the pregnancy rate after transfer. Yu et al (2006) also posits that larger samples are better for sex determination but that the viability of treated embryos may be compromised. This is particularly true when such embryos are subsequently exposed to freezing and thawing procedures (Lee et al, 2004).…”

“…Soon after the sequencing of the Y chromosome, specific DNA probes were used in PCR, and the first, highly promising results were published (Herr et al, 1990;Schroder et al, 1990). Recently, quite a few reports have indicated that under certain circumstances the sex of embryos may be determined with high accuracy and in acceptable time intervals (Thibier and Nibart, 1995;Lopes et al, 2001;Ekici et al, 2006;Yu et al, 2006).…”