2016 Midland Bull Test Edition
Page A12
Scientists make progress in embryo preservation, genotyping Genotyping embryos that are produced in vitro, or in an artificial environment, is becoming more of a reality thanks to the work of scientists at California Polytechnic State University in San Luis Obispo, Calif. Researcher Fernando Campos-Chillon says, “Genotyping embryos involves so many different disciplines – embryology, molecular biology and so forth. It’s very complicated.” Not only is producing embryos in a laboratory environment a relatively new technology, he adds that the following steps – including freezing of the embryos – present a problem. In vitro production Campos-Chillon says, “The in vitro production system is very simple. We have simulated an ovary, and we need to incubate the oocytes.” Next, semen is processed through a density gradient to separate the cytoplasm from the sperm and to obtain a rich sperm population. “Sperm go into culture and into an incubator for six or seven days, depending on the application,” he says. “Then we get embryos. The nice-looking embryos have a potential
to create a calf.” The highest rate of fertilization that they see are around 70 percent, and Campos-Chillon adds that they are able to reach pregnancy rates of around 55 percent by implanting in vitro embryos. “We average around 7.5 embryos transferred per session, and we see around 4.5 pregnancies per session,” he says. “The system is getting more consistent.” However, genotyping the embryo isn’t as simple as just creating it. Genotyping Genotyping is based in the technology of single nucleotide polymorphisms (SNPs). “When we have a strand of DNA from one individual and one from another, we can see a single nucleotide difference in the chain,” Campos-Chillon explains. “Scientists have been able to look at these changes and then relate them to specific traits that we are looking at.” Traits, such as calving ease and growth, have been correlated, and producers are interested in genotyping embryos before they are implanted to see if the resulting calves will exhibit desired traits. “To genotype the
embryos, we need to have enough DNA,” Campos-Chillon says. Several techniques can be used to retrieve the DNA, with varying levels of success. Micro-Blade dissection involves cutting a piece of the embryo away, which can result in genetic defects. “We tried to do this with lab-produced embryos, and we managed to kill them all,” he says. “We had to look for alternatives.” Piezo-assisted drilling (PAD) and laser-assisted drilling (LAD) are techniques that allow the blastocyst to be dislodged to extract a sample. In PAD, the piezo is a small jackhammer-like device that creates holes to dislodge cells from the membrane. DNA is extracted from dislodged cells, amplified and then genotyped. For LAD, a laser is used to stimulate production of a tropoblast, which essentially herniates from the blastocyst and provides cell samples without harming the embryo itself. Amplification After samples are obtained, an additional challenge comes in the fact that small DNA samples may be difficult to amplify
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so enough DNA is present to genotype. “We also get allelic dropout when we amplify DNA,” Campos-Chillon says. “Basically, one of the alleles of the genome doesn’t get amplified.” Additionally, low call rates can be a problem. The call rate is defined by the number of correctly identified SNPs over the total number of SNPs for the data set. “This number has to be over 0.9 or we get discrepancies when we fill in the blanks in the genome,” he says. “Those are the technical challenges.” Whole-genomic amplification can also be utilized, if the results must be extremely accurate. Preservation After DNA is collected, amplification and genotyping take time, so Campos-Chillon says the embryos must be frozen, a process called cryopreservation. “Our embryos are good, but we get in trouble with cryopreservation because they aren’t that good,” he says. “They have a lot of lipids.” High lipid content is associated with low cryosurvival, he continues, because embryo membranes lose fluidity, lipid peroxidation occurs and osmotic shock may result. Several cryopreservation strategies may be employed to attempt to mitigate for lipid content. “Some labs try to reduce lipid content, and that has worked okay,” he says. “There are two tech-
“Genotyping embryos involves so many different disciplines– embryology, molecular biology and so forth. It’s very complicated.” – Fernando Campos-Chillon, California Polytechnic State University niques for cryopreservation we can also use.” Slow freezing was developed in 1992 and can be used with ethylene glycol for direct embryo transfer. Embryos may also be preserved through vitrification, which creates a physical state similar to glass. There is a theoretical advantage to vitrification, he says, but it is a challenging procedure with multiple formulations and other downfalls. “Vitrified embryos are not user-friendly because we have to warm the embryo, put it under the microscope and do several steps before we implant it,” he says. “It might take 10 to 15 minutes per embryo, which is a lot of time.” A final approach is to collapse the blastocoele fluid prior to preservation in an attempt to preserve the embryos better. Experiments Campos-Chillon and his team of researchers performed four experiments to determine the best methods of cryopreservation. First, they used a collapsed blastocoele and intact blastocoele. Then, the embryos were frozen using slow freezing with glycerol, a vitrification process and an open-pulled straw and liquid nitrogen. “The main effect was that, on re-expansion, col-
lapsing the blastocoele is superior than leaving the blastocoele intact,” he says. He also notes that vitrification was the superior method of cryopreservation, but the expense and impracticality of the technique disqualified it from future experiments. Next, he looked at the effect of re-expansion of intact embryos versus PAD-biopsied embryos. The PAD-biopsied embryos had better re-expansion. Those embryos were also preserved using slow freezing by both ethylene glycol and glycerol, as well as the open-pulled straw method. “The ethylene glycol was 70 percent compared to glycerol and open pulled straw at 86 and 85 percent successful re-expansion,” Campos-Chillon says. “These two techniques are not user-friendly, though.” Finally, they looked at call rates of PAD compared to LAD biopsied cells, finding greatly improved results from LAD. “The call rates seem sufficient for robust analyses, but we need to look at the calves to corroborate the validity of our genotyping,” he concludes. Saige Albert is managing editor of the Wyoming Livestock Roundup and can be reached at saige@ wylr.net.
The Wyoming Livestock Roundup team would like to thank Bill Angell for all of his hard work in helping put together the Midland Bull Test Edition. His incredible effort selling and laying out ads helps this edition come together each year.
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