Browsing by Author "Seidel, George E., Jr., advisor"
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Item Open Access Applying in vitro-produced embryos and sexed sperm to dairy cattle reproduction(Colorado State University. Libraries, 2011) Rasmussen, Sara-Lesley, author; Seidel, George E., Jr., advisor; Graham, James K., committee member; McCue, Patrick M., committee memberThis study compared the pregnancy rates between embryo transfer of bovine embryos produced in vitro with sexed vs control sperm and artificial insemination (AI) using sexed and unsexed sperm. Cleavage rates for oocytes fertilized with sexed vs control sperm were not different for two of the three bulls used, but were lower (p < 0.05) for the third bull sexed (44%) vs control sperm (70%). There were fewer transferable blastocysts produced per oocyte with sexed sperm (9-19%) than for unsexed sperm (18-26%); (p < 0.05). All cows were on an Ovsynch program to synchronize ovulation. Respective 60 d pregnancy rates at two Colorado dairies were as follows: control AI (43%, n=88; 43%, n=44); AI with X-sorted sperm (34%, n=82; 34%, n=62); embryo transfer (ET) with in vitro-produced (IVP) embryos using unsexed sperm (22%, n=68; 21%, n=39); and ET with IVP embryos using sexed sperm (7%, n=72; 37%, n=40). The pregnancy rate (day 60) for AI using sexed sperm was 78% of that of control sperm. ET pregnancy rates were generally lower than AI rates. At one dairy, abortions between days 32 and term were higher for X-sort ET pregnancies (79% n=14) than for AI control pregnancies (20% n=40); (P < 0.001). However, the other dairy experienced only a 12%, (n=17) abortion rate for transferred embryos produced from X-sorted sperm. The sex ratio of calves was similar to previous studies for AI with control sperm (52% bull calves, n=50), AI with X-sorted sperm (12% bull calves, n=40); ET with IVP embryos using unsexed sperm (50% bull calves, n=18); and ET with IVP embryos using sexed sperm (11% bull calves, n=18). Findings from this experiment indicate that embryo production with sexed sperm is not successful enough to be applied to large-scale dairies that already have successful breeding programs in place.Item Open Access Energy substrates, metabolic regulators, and lipid accumulation during culture of in vitro-produced bovine embryos(Colorado State University. Libraries, 2007) Barceló-Fimbres, Moisés, author; Seidel, George E., Jr., advisorThe main objective of this experiment was to optimize in vitro culture conditions for bovine embryonic development, using alternative energy sources and metabolic regulators. Replacing glucose with fructose in culture medium consistently increased blastocyst production per oocyte and decreased lipid content in bovine embryos. The use of phenazine ethosulphate (PES) or fetal calf serum (FCS) supplementation did not affect embryonic development; however, PES consistently decreased, and FCS increased lipid content of embryos compared to the control. There was no effect of glucose or fructose on survival of embryos after cryopreservation by slow freezing or vitrification; however, embryos-treated with PES to reduce lipid content resulted in improved cryotolerance, and FCS decreased cryotolerance compared to the control. Transfer of embryos treated with PES during in vitro culture did not affect pregnancy rates, conceptus losses, or fetal or post-natal development in calves born normally. The sex ratio of calves born was skewed toward males. This effect likely was due to a toxic effect of glucose to female embryos cultured in vitro. Therefore, the more expanded day 7 blastocysts were mostly male embryos.Item Open Access Luteinizing hormone induced oocyte maturation initiates mRNA decay in cattle(Colorado State University. Libraries, 2010) Walker, David Joshua, author; Seidel, George E., Jr., advisor; Clay, Colin M., committee member; Bruemmer, Jason E., committee member; Anthony, Russell V., committee memberOocyte maturation is a complex process consisting of signal transduction, ultrastructural changes, and mRNA transcription, translation, storage, and degradation. In vitro-matured oocytes initiate maturation in response to removal from an inhibitory follicular environment while in vivo-matured oocytes mature in response to the LH surge. Oocytes matured in vivo lead to more successful embryo production than those matured in vitro. This research concerned study of expression levels and action of selected transcripts involved in RNA processing that occur in in vivo oocyte maturation. The first experiment focused on the inability of GnRH to induce oocyte maturation in superstimulated cows during the luteal stage of the estrous cycle. Superstimulated cows were treated with PGF2a and GnRH to induce in vivo maturation or were treated with GnRH without PGF2a to induce an LH surge at 0, 3, 12, or 24 h before aspiration. While treatment with GnRH caused an increase in LH over no GnRH treatment, it was a smaller increase than that observed in cows treated with PGF2a before GnRH treatment (P<0.001) (No GnRH; 0.84 ng/mL, GnRH: 9.45 ng/mL, PGF2a: 93.86 ng/mL). Thus, increases in LH were sufficient to initiate epiregulin mRNA transcription in granulosa cells (P<0.06) with the greatest expression levels after 6h. However, germinal vesicle breakdown did not occur as reliably in cows with an intact corpus luteum 23 h after GnRH (treated with PGF2a 36 h prior to GnRH injection) (GV stage oocytes; Oh; 79%, 6h: 58%, 12h: 83%, 24h: 60%, vs. controls 6%)(P<0.001). In addition, cAMP levels remained stable in oocytes from cows treated with GnRH in the presence of progesterone regardless of post injection time, while control oocytes had a slightly elevated level of cAMP (Oh: 4.95, 6h: 3.98, 24h: 4.12, control: 7.41 fmol) (P>0.1). Phosphodiesterase 3A mRNA levels were unaffected by any treatment (P>0.10). These data suggest that although the stimulatory signaling of LH and epiregulin occur, cAMP levels are unaffected by GnRH treatment in the presence of progesterone. The second experiment evaluated mRNA concentrations in bovine oocytes of four transcripts involved in RNA regulation in mammalian cells; CUG-BP, PARK, eIF-4E, and PAP-1. In vivo- and in vitro-matured oocytes were collected 0, 3, or 6 hours after initiation of maturation via GnRH injection for in vivo-maturation and aspiration from follicles for in vitro-maturation. eIF-4E and PARN mRNA concentrations increased over time in both in vitro- and in vivo-matured bovine oocytes (P<0.05). In vivo-matured oocytes contained more eIF-4E mRNA molecules than in vitro-matured bovine oocytes (P<0.10). CUG-BP and PAP-1 concentrations remained stable over the first 6 h of maturation and were similar in the in vivo- and in vitro-matured oocytes. The final experiment concerned deadenylation patterns for the cyclin B1 3’ untranslated region (UTR) and GDF-9 3’UTR with a poly(A) tail of 60 adenosines. Bovine oocytes were injected with radiolabeled constructs after 0, 5, or 19 h of maturation and then cultured for 0, 1, or 3 h. Radiolabeled RNA was recovered from oocytes after culture and analyzed for changes in construct size, reflective of deadenylation or general degradation. Cyclin B1 underwent deadenylation before being degraded regardless of the stage of oocyte maturation. Analysis of gels showed an intermediate with 0 adenosines (AO) in the Cyclin B1 injected oocytes, while those injected with GDF-9 displayed no such intermediate. These findings indicate that injected GDF-9 transcript remains stable with a poly(A) tail of 60 adenosines or is simply degraded randomly in bovine oocytes matured for 0, 5, or 19 h. Deadenylation of Cyclin B1 mRNA begins immediately in bovine oocytes resulting in degradation of the Cyclin Bl.Item Open Access Removing seminal plasma improves sex-sorting of bovine sperm(Colorado State University. Libraries, 2011) Burroughs, Chelsie Ann, author; Seidel, George E., Jr., advisor; Graham, James K., advisor; Curthoys, Norman P., committee memberTo view the abstract, please see the full text of the document.Item Open Access 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 memberThe 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.