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Department of Biomedical Sciences

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https://hdl.handle.net/10217/100360
These digital collections include theses, dissertations, faculty publications, departmental publications, and datasets from the Department of Biomedical Sciences. Due to departmental name changes, materials from the following historical department are also included here: Physiology.

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Browsing Department of Biomedical Sciences by Author "Ahola, Jason K., committee member"

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    Preimplantation genetic diagnosis of equine embryos
    (Colorado State University. Libraries, 2010) Cullingford, Erika L., author; Seidel, George Jr., advisor; McCue, Patrick, advisor; Ahola, Jason K., committee member; Bouma, Gerrit, committee member
    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.
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    Vitrification of in vitro- and in vivo-produced bovine embryos for direct transfer
    (Colorado State University. Libraries, 2012) Kruse, Shantille, author; Seidel, George E., Jr., advisor; Ahola, Jason K., committee member; Bruemmer, Jason E., committee member
    The overall objective of my thesis research was to improve procedures for vitrifying bovine blastocysts so as to enable direct embryo transfer to the uterus. Blastocysts were produced using standard in vitro procedures in Experiments 1, 2, and 3. Procedures were done at room temperature, 22 ± 2 °C. Unless otherwise mentioned, all media were made in SynGro®. In Experiment 1, base media contained either 1) normal concentrations of sodium (120 mM) and calcium (2 mM);(CON) or 2) 60 mM sodium + 60 mM choline chloride and 0.5 mM calcium (LOW). Blastocysts were exposed to 5 M ethylene glycol (V1) for 3 min and moved to 6.5 M ethylene glycol + 0.5 M galactose + 18% Ficoll (V2). Straws (0.25 mL) were loaded with a column of 120 μl 1 M galactose followed by an air bubble, then V2 containing embryos followed by an air bubble, and 60 μl 1 M galactose followed by sealing with a plastic plug. After 35 s, embryos were vitrified by either 1) standard cooling in liquid nitrogen cooled air (AIR) for 1 min or 2) cooling via contact of straw walls with columns drilled into an aluminum block immersed in liquid nitrogen (BLK) for 2 min and then directly plunged into liquid nitrogen. These combinations resulted in 4 treatments (AIR x CON; n = 61, AIR x LOW; n = 58, BLK x CON; n = 73, BLK x LOW; n = 54). BLK Embryos were warmed by holding straws in air for 10 s, placing them in a water bath at 37 °C for 20 s, mixing embryos with galactose diluent in the straw for 2 min and expelling. Embryos were recovered, rinsed through holding medium, and cultured in chemically defined medium (similar to synthetic oviduct fluid (SOF)) for 24 h before being evaluated for survival. Post warming survival did not differ (P > 0.10) between treatments (AIR x CON = 42.0%; AIR x LOW = 26.8%; BLK x CON = 21.8%, BLK x LOW = 24.5%). Despite lack of statistical significance, we recommend use of LOW base media because both sodium and calcium levels are reduced. Use of this media should therefore have less chance of sodium and calcium toxicity, and could deter apoptosis. The BLK vitrification method is both easier to use and more consistent. In Experiment 2, we sought to identify the most efficacious cryopreservation method for in vitro-produced bovine blastocysts that would enable direct embryo transfer from 0.25 mL straws used as containers for cryopreservation. Although not a method for direct transfer, Cryotops were chosen to serve as positive controls (CON), as they are the industry standard for vitrification of human embryos. Embryos were cryopreserved by vitrification with a Cryotop (CON; n = 118), using an aluminum block (BLK; n = 128), or by slow freezing (SLF; n = 131). Vitrification procedures were as described above for BLK with the exception that CON embryos were placed in < 1 μl V2 onto Cryotops, and after 35 s, vitrified by plunging directly into liquid nitrogen. Embryos cryopreserved via SLF were exposed to 1.36 M glycerol in modified Dulbecco's PBS + 0.4% BSA (PBS) for 10 min, loaded into 0.25 mL straws, and placed into a freezing machine. Straws were cooled to -6 °C at 4 °C per min, held at -6 °C for 5 min, seeded, held at -6 °C for an additional 10 min, and then cooled to -30 °C at 0.5 °C per min and plunged into liquid nitrogen. After storage for at least 24 h in liquid nitrogen, embryos were warmed/thawed. Embryos cryopreserved via CON were removed from Cryotops by direct placement into a 200 μl drop of 1 M galactose for 2 min, whereas BLK/SLF embryos were warmed/thawed as described above with the exception that glycerol was removed in three 6 min steps from SLF embryos: 0.8 M glycerol + 0.3 M sucrose; 0.4 M glycerol + 0.3 M sucrose; and 0.3 M sucrose followed by PBS for 2 min. After recovery, embryos were rinsed through holding medium and cultured as described above. Post warming survival was greater (P < 0.01) for CON than BLK (85.9% and 70.6%, respectively); BLK was greater (P < 0.01) than SLF (56.1%). Although BLK resulted in lower post-warming survival than CON, it may be an acceptable method for direct transfer, which yielded greater post-warming survival than SLF, the current method used for cryopreservation of bovine embryos. In Experiments 3 and 4, the objective was to compare pregnancy rates of recipients of in vitro-(Exp 3) or in vivo-produced bovine blastocysts (Exp 4) cryopreserved via VIT versus SLF. In vitro-produced embryos were produced by standard procedures. In vivo-produced embryos were recovered 7 d post estrus from crossbred, nonlactating superovulated beef cows. Embryos were cryopreserved via BLK vitrification (VIT; Exp 3, n = 78; Exp 4, n = 46 ) or slow freezing (SLF; Exp 3, n = 78; Exp 4, n = 44). Embryos were cryopreserved and warmed/thawed as described above followed by nonsurgical transfer into non-pregnant cows culled for unknown reasons, but with normal-appearing reproductive tracts. Recipients were d 7 ± 0.5 of the estrous cycle, and each received 2 embryos into the uterine horn ipsilateral to the corpus luteum. Pregnancy diagnosis was performed at d 37 ± 2 via ultrasonography. Survival rate per embryo (normal fetus with heartbeat) did not differ (P > 0.10) between methods (Exp 3, VIT = 14.1%; SLF = 16.7%; 9 of 15 pregnant cows carried twins; Exp 4, VIT = 45.7%; SLF = 38.6%; 17 of the 21 pregnant cows carried twins). Therefore, VIT was similarly efficacious to SLF for cryopreservation of bovine embryos, and simpler, requiring less equipment, time, and expense.