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.

Data collected from commercial embryo transfer programs in 63 farms in China during June 2002 to December 2005 was analyzed to examine the effects of various factors (biopsy, freezing, sample size, embryo development and quality, in vitro culture, and recipient quality) on pregnancy rates of in vivo-biopsied embryos. Embryos were flushed from superovulated dairy cattle and subjected to a biopsy for sexing determination using protocols and sexing kits supplied by AB Technology Ltd. Fresh embryos were implanted on the same day or frozen with AG freeze medium (AB Technology Ltd., Pullman, WA, USA) for later transfer. Recipients were synchronized with CIDA + PG protocols. Embryos were cultured in 6-well dishes containing 1.3 mL of holding medium (AB Technology Ltd.) in each well at room temperature (20–25�C) for examination of embryo survival in vitro. The chi-square test was used in statistic analysis. The implantation of fresh embryos after biopsy did not affect pregnancy rates (49.6%, 257/518) compared to that of non-biopsied fresh and frozen–thawed embryo groups (52.9%, 47/140 and 46.6%, 177/380, respectively). However, for biopsied embryos subjected to frozen and thawed procedures before implantation, particularly for those subjected to the removal of a larger biopsy, a reduced pregnancy rate was observed (41.8%, 297/710; P < 0.01). Pregnancy rates among biopsied embryos at 3 different development stages (morula-early blastocyst, blastocyst, and expanded blastocyst) were not different. Similar results were found between embryo groups of grade 1 and 2. A significant decrease in pregnancy rate (0/10) was observed with embryos held in vitro for a longer period of time (>5 h), suggesting detrimental effects of in vitro conditions on embryo survival. The highest pregnancy rate (68.0%) was observed in recipients synchronized for the first time before being implanted with biopsied embryos. Significant decreases in such rates were found in recipients synchronized for the second or third times or those with an abortion history at the first or second synchronization-implantation treatment (P < 0.01). Better pregnancy rates (45.6%, 41/90; 46.1%, 76/165; and 45.5%, 5/11) were obtained for recipients implanted with biopsied embryos at Days 7.5, 8.0, and 8.5 post-heat detection, respectively, compared to 16% at Day 7 (3/18, P < 0.05). It is concluded that mechanical treatment (cutting) does not reduce the survival of biopsied embryos; however, cryopreservation reduces their ability to survive in vivo. The analyses also suggest that holding embryos in vitro should not be longer than 5 h unless more favorable in vitro conditions can be provided. To achieve better results of implantation of biopsied embryos, embryo transfer should be performed during 7.5–8.5 days post-estrus, and the healthy recipients synchronized for the first time should be used.