Preimplantation genetic diagnosis of equine embryos
Date
2010
Authors
Cullingford, Erika L., author
Seidel, George Jr., advisor
McCue, Patrick, advisor
Ahola, Jason K., committee member
Bouma, Gerrit, committee member
Journal Title
Journal ISSN
Volume Title
Abstract
In horses, determination of certain genetic traits/alleles in embryos before embryo transfer would be advantageous due to the costs of resulting pregnancies. An attractive option is preimplantation genetic diagnosis (PGD), but to date few biopsied equine embryos have resulted in pregnancies. In the current experiment, 37 embryos ranging from 160 - 575 μm in diameter were biopsied. To obtain embryos, donor mares were monitored using transrectal ultrasonography. When a follicle > 35 mm in diameter was observed, 2,500 IU hCG or 1.5 mg deslorelin acetate was administered, and mares were inseminated daily until ovulation was detected. Embryos were recovered nonsurgically on days 6.5 – 7 (day 0 = ovulation). Trophoblast biopsies were collected in a 30 μl droplet of Syngro Holding Medium (Bioniche, Belleville, ON) using a piezo drill and beveled injection pipette. After removal of the embryo, the droplet containing the biopsied cells was moved into an Eppendorf tube and centrifuged. Supernatant was removed leaving ~5 μl sample, which was snap frozen for later genetic testing. Fifteen biopsied embryos were immediately transferred nonsurgically into uteri of synchronized recipients. Day 16 pregnancy rate for embryos ≤ 300 μm was 75.0% (6 of 8; 175 – 240 μm), which was not significantly different from control embryos of the same size (77.3%; 17 of 22). For embryos > 300 μm, day 16 pregnancy rate was 28.6% (2 of 7; 320 and 400 μm), which was not significantly different from control embryos of the same size (62.5%; 10 of 16). Additionally, 22 embryos (150 - 440 μm) were vitrified by standard procedures after biopsying and later warmed and transferred directly. No embryos > 300 μm (n = 3) became pregnancies after vitrification. Day 16 pregnancy rate for ≤ 300 μm was 47.4% (9 of 19; 150 – 225 μm), which was significantly different (p < 0.05) from direct transfer and control embryos of the same size (75.0% and 77.3%, respectively). Three of these pregnancies (150 - 200 μm) resulted in the formation of empty trophoblastic vesicles by 25 d. All pregnancies were terminated on or after 25 d to collect embryos for further genetic testing. For preimplantation genetic testing, a duplex nested polymerase chain reaction (PCR) was developed for amplification of the DNA from the biopsied cells using primers for sex chromosome-linked zinc finger protein genes (ZFx/ZFy; 445 bp), and 2 pairs of primers for equine-specific sex-determining region on the Y-chromosome (SRY; 217 bp, 121 bp). Experiments on XX and XY genomic DNA from white blood cells revealed accurate genetic testing on as little as ~9 pg DNA, which equals ~1 cell. Sex determination on biopsied material occurred for 30% of samples, one of which was confirmed from a placental sample. Low PGD results indicate either lack of sensitivity of the test, or more likely the loss of cells during the steps of transfering the biopsied cells to Eppendorf tubes. We concluded that biopsy collection, preimplantation genetic diagnosis, and direct transfer can be performed on equine embryos without compromising pregnancy rates when performed on embryos ≤ 300 μm. Vitrification lowered pregnancy rates of biopsied embryos (p < 0.05). Continued effort in improving genetic tests and in vitrifying equine embryos, especially those > 300 μm, is warranted.
Description
Department Head: Barbara Sanborn.
Rights Access
Subject
Preimplantation genetic diagnosis
Horses -- Embryos