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Evaluation of Aerobic Culture Results and the Expression of Embryonic Transcripts From In Vivo Derived Embryos Exposed to Media With and Without Antibiotics




Cowley, Jessica

Type of Degree

Master's Thesis


General Veterinary Medicine

Restriction Status


Restriction Type

Auburn University Users

Date Available



More than 2.8 million antibiotic-resistant infections occur in the United States each year, with a resultant 35,000 deaths. The United States Department of Agriculture is investigating ways to judiciously use antibiotics and stop unnecessary use to minimize resistance. Multiple ovulation embryo transfer (MOET) in cattle has allowed for improvement of genetics worldwide. However, the dissemination of germplasm internationally has raised concerns for pathogen transfer between countries. The addition of antibiotics to embryo flush media is standard in the embryo production industry to limit bacterial contamination in the recovered embryos. Usage of antibiotics in this manner may be unnecessary and potentially detrimental to embryos. A wide variety of types and concentrations of antibiotics used in embryo production systems has resulted in toxic changes to embryos. The aim of this study was to further examine the effects of embryo collection media with antibiotics (AB+ treatment) or without antibiotics (AB- treatment) on the impact of the number of in vivo derived (IVD) embryos collected, bacterial growth of the flush media, and abundance of embryonic transcripts related to apoptosis and free radical scavenging. We hypothesized that, compared to the AB+ treatment, AB- treatment would not significantly affect the number of embryos recovered nor aerobic culture growth but would significantly impact embryonic expression of apoptotic and free radical scavenging genes. In a crossover study design, six superovulated Angus cross cows underwent two conventional (day 7) uterine body embryo collections 77 days apart. Commercial embryo flush media with gentamicin sulphate (ABT Complete Flush, ABT 360, Pullman, WA) was utilized for embryo collections when cows were assigned to the AB+ treatment. Commercial flush media without antibiotics (ABT Complete Flush without antibiotics, ABT 360, Pullman, WA) was utilized when cows were assigned to the AB- treatment. During each embryo collection procedure four samples of flush fluid and an embryo were submitted to the TBS Alabama State Diagnostic Lab for aerobic culture. Stage 6, grade 1 embryos, as outlined by the International Embryo Technology Society (IETS) were used for mRNA RT-qPCR analysis. RNA was isolated utilizing the PicoPure™ RNA Isolation Kit (Thermo Fisher Scientific, Waltham, MA) and quality was confirmed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). The RNA samples, that had passed quality checks, were used to synthesize cDNA using a qScript cDNA synthesis kit (QuantaBio, Beverly, MA). Subsequent molecular analysis using SYBR based Real-time RT PCR was completed (PerfeCTa SYBR, QuantaBio). The relative fold change was determined for abundance of Superoxide dismutase 1 (SOD1), BCL2 associated X (BAX) and Tumor Protein P53 (TP53) normalized to Histone 2A (H2A). Treatment differences for the number of embryos recovered was determined using a paired t-test. Differences between gene expressions were determined by using an unpaired t-test with significance set at p < 0.05. There was no significant difference found between treatments for total number of embryos recovered (p = 0.09) nor the relative fold change of expression of SOD1, BAX, and TP53 (p > 0.05). Bacterial growth was not detected in any sample except for one sample from a single cow. This sample from the unopened bag of embryo flush media grew Microbacterium testaceum, which was suspected to be an environmental contaminant. Based on study findings, utilization of embryo flush media without added antibiotics may be a viable option for reducing antibiotic use in food producing species.