Undergraduate Thesis Or Project
 

Endometrial Inflammatory Cytokine Expression in Postpartum Beef Heifers Following Platelet Rich Plasma Treatment

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  • Background: Uterine inflammation (e.g., endometritis) causes negative economic effects in the beef industry by reducing reproductive performance. Current treatments for endometritis in beef cattle involve hormones and antibiotics, which can be expensive and are often ineffective. The endometrial immune response regulates the release of cytokines and chemokines in response to inflammation. The objective of this research was to measure endometrial pro-inflammatory cytokines (IL-6, TNFα) and chemokine (IL-8) expression in postpartum beef heifers following treatment with intrauterine platelet rich plasma (PRP), platelet poor plasma (PPP), or saline. Methods: Twelve Angus-crossbred heifers calving under supervision were used for this study. Nine heifers calved without assistance (eutocia) and were divided equally into groups: PRP, PPP, and saline-treated eutocia (EUT; this group served as the control for all other groups). Three heifers needed assistance during calving (dystocia) and these heifers were put into a separate saline-treated group (DYS). Endometrial cells were collected at 2- and 4-weeks post-calving using a double-guarded endometrial swab. Intrauterine treatment was administered once after samples were collected 2-weeks post-calving. Total RNA was isolated from endometrial cells using a standard Trizol-chloroform protocol. The concentration and integrity of the RNA (RIN) was determined. Complementary DNA (cDNA) was prepared from the total RNA using a commercial kit. Forward and reverse primer sequences for IL-6, IL-8, and TNFα as well as the housekeeping gene (β-actin) were used as they had been previously validated for use in an endometrial gene expression study in dairy cattle. These primers were used in the current study for real-time polymerase chain reactions (PCR) to determine relative gene expression. Expression of each gene of interest (IL-6, IL-8, TNFα, DYS) relative to the expression of saline-treated controls (EUT) was calculated using the delta delta CT method. Differences between time points (2- and 4-weeks post-calving) were determined using a one-way analysis of variance where significance was defined as p<0.05. Results: RNA samples were partially degraded based upon RIN analysis, but cDNA creation resulted in good yields and PCR amplification from all samples. Relative gene expression of IL-8 and IL-6 did not differ between time points in any treatment group. Relative gene expression of TNFα was reduced (p=0.002) at the second time point in the DYS group but not in the other groups. Conclusion: Difficult calving increases the risk for developing endometritis but in healthy cattle, the increase in cytokine expression related to dystocia will resolve spontaneously. This explains why the relative expression of TNFα was higher 2-weeks post-calving compared to 4-weeks post-calving. This study also shows that intrauterine administration of platelet-rich plasma to normal calving heifers does not alter endometrial cytokine gene expression. Future studies will focus on investigating the effect of PRP treatment in cattle that experienced dystocia or have clinical evidence of endometritis.
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