Modifying the Hormone Strategy for Superovulating Donor Cows to Reduce Drug Costs without Decreasing the Number of High Quality Transferable Embryos Recovered Public Deposited

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  • Two research projects comprise this thesis. The first project investigated modifying the hormone dosing strategy traditionally used in superovulating donor cows for an embryo collection to decrease drug costs without decreasing the number of high quality, transferable embryos recovered. The objective of this project was to evaluate the number and quality of embryos recovered from donor cows superovulated with a reduced dose of the pituitary gonadotropic hormone, follicle stimulating hormone (FSH), and a greater dose of the hypothalamic releasing hormone, gonadotropin releasing hormone (GnRH). In Experiment 1, 24 crossbred beef cows from the Oregon State University Beef Cattle Ranch were assigned to one of four superovulation treatment groups according to age and parity: 1) 400 mg FSH and 100 μg GnRH (the traditional doses), 2) 400 mg FSH and 200 μg GnRH, 3) 200 mg FSH and 100 μg GnRH, or 4) 200 mg FSH and 200 μg GnRH. Embryos were collected non-surgically 7 d after estrus onset and scored for stage of development and quality using a four-rank grading scheme. In Experiment 2, 12 crossbred beef cows from the Oregon State University Beef Cattle Ranch were superovulated twice with either 1) 400 mg FSH and 100 μg GnRH or 2) 200 mg FSH and 100 μg GnRH. Embryos were collected non-surgically as described in Experiment 1 and good to excellent quality late morulae to blastocysts were cultured, one embryo per 15-microliter drop, for 8 d. At 24-h intervals, embryos were evaluated for viability and overall development and transfered to a fresh micro-drop and conditioned medium was recovered. Conditioned media were assayed for plasminogen activator (PA), a protease correlated with embryo cell number and development to advanced cell stages. In Experiment 1, cows superovulated with 200 μg GnRH produced more unfertilized ova (P = 0.02) than cows superovulated with 100 μg GnRH. Cows superovulated with 200 mg FSH produced a higher percentage (P = 0.07) of transferable embryos and a lower percentage (P = 0.10) of degenerate embryos than cows superovulated with 400 mg FSH. Superovulating cows with the reduced FSH and GnRH doses (200 mg and 100 μg, respectively) yielded fewer total transferable embryos (1.8 embryos) but a greater percentage of transferable embryos at a reduced cost compared to the traditional dose ($24 vs $31 per embryo, respectively). Increased GnRH dosing had a negative effect on transferable embryos and the reduced FSH and increased GnRH dosing was not cost effective. In Experiment 2, cows superovulated with 400 mg FSH produced more unfertilized ova (P = 0.08) than cows superovulated with 200 mg FSH. More (P=0.04) embryos recovered from cows treated with 200 compared to 400 mg FSH developed to the hatched blastocyst stage in culture. Embryos recovered from cows treated with 200 compared to 400 mg FSH also developed to the expanded blastocyst stage sooner (P = 0.08). Embryos collected from cows receiving 200 mg FSH produced more (P = 0.04) PA in the first round of superovulation compared to 400 mg FSH and both doses in the second round. Similar to Experiment 1, the 200 mg FSH dose yielded fewer total transferable embryos in the first round of superovulation but at a reduced cost compared to the 400 mg dose ($25 vs $37 per embryo, respectively). Collectively, these data suggest higher FSH dosing is likely inducing ovulation of poorer quality ova which either fail to fertilize or, if fertilization occurs, may generate a reduced percentage of competent embryos. The reduced FSH dose not only contributes to a reduced cost but may also provide more embryos with a greater likelihood of pregnancy establishment. The second project in this thesis attempted to develop a dipstick-style enzyme assay to assess embryo viability prior to transfer. The objective of this project was to develop a dipstick that rapidly quantified PA production by an embryo and could be used on the farm for an embryo collection and transfer. Dipsticks were constructed by cutting and mounting 5 X 5 mm squares of cellulose acetate, chromatography paper, glass fiber membrane or nitrocellulose on the end of 5 X 25 mm plastic strips. Five microliters of 1, 10 or 50mM of the tripeptide glutamic acid-glycine-arginine (EGR; C₂₇H₃₆N₈O₇•CH₃COOH), a colorimetric substrate for urokinase (UK), were pipetted onto the 5 X 5 mm squares and dried. Dipsticks were incubated in 25-μL of culture medium containing 0, 1, 10, or 100 IU UK/mL or embryo-conditioned medium (ECM) and visually observed for color development at 30-min intervals for up to 90 min. Color development was scored using the following 3-point system where: 0 = absence of yellow, 1 = light yellow and 2 = bright yellow. Dipsticks were able to detect EGR cleavage in 10 and 100 IU/mL UK after 90 and 30 min of incubation, respectively, but no color was observed in dipsticks incubated in ECM. A second approach was developed using a 96-well plate. Twenty-five microliters of 1, 5, 10, 20 or 50 mM EGR were combined with 25-μL of culture mediumcontaining 0, 1, 10, or 100 IU UK/mL or ECM and observed for color development visually and photometrically at OD 405 using an ELISA plate reader at 30-min intervals for up to 120 min. Color development was visually observed in 1, 5, 10 and 50 mM EGR after 30 min of incubation with 100 mM UK/mL but no color was observed in wells containing ECM. However, when the plates were evaluated photometrically EGR cleavage was detected in wells containing ECM and 50 mM EGR after 30 min of incubation. Although the PA dipstick enabled visual detection of EGR cleavage by 10 and 100 IU/mL UK, it was not sensitive enough to detect PA in ECM. The 96-well plate assay was successful in detecting PA in ECM after 30 min of incubation but only photometrically. To use the plate-based assay would require the embryo transfer practitioner to have an ELISA plate reader on hand and the expense associated with such an instrument may limit its on farm application.
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