Colorado State University
Vitrification of in vitro- and in vivo-produced bovine embryos for direct transfer.
Degree: MS(M.S.), Biomedical Sciences, 2012, Colorado State University
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…
Advisors/Committee Members: Seidel, George, Jr. E. (advisor), Ahola, Jason K. (committee member), Bruemmer, Jason E. (committee member).
Subjects/Keywords: bovine; direct transfer; embryo; in vitro; in vivo; vitrification
to Zotero / EndNote / Reference
APA (6th Edition):
Kruse, S. (2012). Vitrification of in vitro- and in vivo-produced bovine embryos for direct transfer. (Masters Thesis). Colorado State University. Retrieved from http://hdl.handle.net/10217/65329
Chicago Manual of Style (16th Edition):
Kruse, Shantille. “Vitrification of in vitro- and in vivo-produced bovine embryos for direct transfer.” 2012. Masters Thesis, Colorado State University. Accessed April 11, 2021.
MLA Handbook (7th Edition):
Kruse, Shantille. “Vitrification of in vitro- and in vivo-produced bovine embryos for direct transfer.” 2012. Web. 11 Apr 2021.
Kruse S. Vitrification of in vitro- and in vivo-produced bovine embryos for direct transfer. [Internet] [Masters thesis]. Colorado State University; 2012. [cited 2021 Apr 11].
Available from: http://hdl.handle.net/10217/65329.
Council of Science Editors:
Kruse S. Vitrification of in vitro- and in vivo-produced bovine embryos for direct transfer. [Masters Thesis]. Colorado State University; 2012. Available from: http://hdl.handle.net/10217/65329