Year of Publication

2017

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Agriculture, Food and Environment

Department

Veterinary Science

First Advisor

Dr. Mats H.T. Troedsson

Abstract

The mare experiences a transient innate immune response to breeding, the resolution of which is crucial for optimal fertility. The majority of mares are able to modulate this inflammation in a timely fashion, but a subpopulation exists which fail to do so and are considered susceptible to persistent breeding-induced endometritis (PBIE). Seminal plasma has been shown to modulate aspects of this inflammation. Recently, two seminal plasma proteins have garnered interest for their immune modulating properties: cysteine-rich secretory protein-3 (CRISP-3) and lactoferrin. These proteins have been found to alter the binding between sperm and neutrophils based on sperm viability in vitro, but minimal work has evaluated their effect on endometrial mRNA expression of cytokines and inflammation in response to breeding. Experiments were performed to analyze the expression of equine CRISP-3. Found to be primarily synthesized in the ampulla of the vas deferens and to a lesser extent in the vesicular gland, CRISP-3 expression was only seen in the postpubertal stallion. Due to the effect of sperm viability on protein function in vitro, varying sperm populations were analyzed for their effect on gene expression in the uterus. It was determined that viable sperm suppressed the gene expression of the inflammatory modulating cytokine interleukin-6 (IL-6) in comparison to dead sperm. Next, the effect of CRISP-3 and lactoferrin on endometrial gene expression in the normal and susceptible mare was investigated. Neither protein had a significant effect on the mRNA expression of inflammatory cytokines in the normal mares at six hours post-breeding. In contrast, lactoferrin was found to significantly suppress the expression of the pro-inflammatory cytokine tumor necrosis factor (TNF)-α in susceptible mares. Due to this, lactoferrin was further analyzed as an immunomodulant for the treatment of PBIE. Susceptible mares were infused with varying doses of lactoferrin at six hours post-breeding. Although not in a dose-dependent fashion, lactoferrin was found to decrease both fluid retention and neutrophil migration, in addition to suppressing the expression of the pro-inflammatory cytokine interferon gamma (IFNγ) and increasing the gene expression of the anti-inflammatory cytokine interleukin-1 receptor antagonist (IL-1RN). In conclusion, CRISP-3 expression occurs in secretory aspects of the male reproductive tract, and appears to be up regulated after sexual maturation. Viability of spermatozoa affects the immune response to breeding and should be taken into consideration for experimental design and interpretation of data. The seminal plasma proteins CRISP-3 and lactoferrin have minimal effect on endometrial gene expression in normal mares, but lactoferrin suppresses the expression of TNF in susceptible mares. Finally, lactoferrin was found to function as a potent anti-inflammatory for the persistent inflammation seen in susceptible mares when administered post-breeding. This protein should be further investigated as a potential therapeutic for the treatment of persistent breeding-induced endometritis.

Digital Object Identifier (DOI)

https://doi.org/10.13023/ETD.2017.228

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