Lipid-soluble compounds in late-cutting alfalfa maximize host immune responses and beneficially modulate the colon microbiota of mice during Citrobacter rodentium infection

Fries-Craft, Krysten
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Bioactive, plant-sourced feed additives for use in animal diets appeal to consumer demand for ‘naturally-raised’ animal products. Alfalfa offers a source of potential nutraceuticals attributed to its membership in the legume (Fabaceae) family, which has been associated with benefits in human and animal health. As a forage, alfalfa contains a high amount of indigestible, insoluble fiber which typically limits its use in non-ruminant livestock diets, as these animals lack the fermentative capacity to extract adequate energy from high-fiber diets. This has created a niche for examining feeding strategies to maximize alfalfa-associated benefits without the limitation of fiber. Extracts provide a simple approach to this issue and have been shown to have health-promoting effects in swine and poultry.

Phytochemical literature is characterized by broad examination into extracts from a multitude of plant sources, contributing to limited insight into functional benefits. In alfalfa literature, focus on one supplementation form (whole plant or extract) is combined with a lack of reports that address changes to phytochemical and nutritional profiles between cuttings within a growing season, despite later cuttings being anecdotally linked to improved health. Livestock studies are limited to general descriptions of performance and overall health but rarely address underlying changes to the immune system and intestinal microbiota. Thus, the objectives of this thesis were to use a well-characterized mouse model to descriptively assess changes to overall health, immunity, and composition of the intestinal microbiota in response to supplementing ground hay, water-, and lipid-soluble extracts from early (1st) and late (5th) cutting alfalfa at both a healthy and challenged state.

The first step in the process of accomplishing this objective was to implement and optimize methods for preparing water- and lipid-soluble extracts (Chapter 2). Once methods for extraction were selected, these compounds were supplemented in mouse diets and fed over the course of a 35d trial, which was divided into a 14d feeding enrichment period to understand baselines in healthy animals, followed by challenge with Citrobacter rodentium and a 21d recovery period to understand the effects of alfalfa during pathogen challenge. The results of this trial are split into chapters examining overall health outcomes including body weight (BW), feed intake (FI), and colon histomorphology (Chapter 3), the immune response (Chapter 4), and alterations to the intestinal microbiota (Chapter 5).

The results of this study suggested that changes to the host immune system and intestinal microbiota due to alfalfa supplementation did not alter BW and FI in healthy mice but contributed to variations in these parameters following infection with C. rodentium. In early timepoints of the infection period (2dpi), 5th cutting alfalfa had a protective effect on BW, while chloroform extracts from both cuttings increased BW over the control in the final timepoints of the study (Chapter 3). Underlying these responses, chloroform extracts contributed to a more pro-inflammatory immune environment prior to inoculation by increasing the percentage of IFN-+ cells, while general alfalfa supplementation had a proliferative effect on splenic lymphocyte populations. In early timepoints of infection, chloroform extracts increased the response time of IFN--producing innate immune cells, while 5th cutting alfalfa maintained elevated lymphocyte populations before recruiting these cells to peripheral tissues at 14dpi (Chapter 4). In addition to immunological changes, alfalfa modulated the intestinal microbiota prior to inoculation without impacting BW and FI. Following inoculation (4dpi), only 5th cutting chloroform extracts reduced abundance of Citrobacter rodentium while chloroform extracts from both cuttings increased the relative abundance of beneficial genera such as Akkermansia at later timepoints (21dpi; Chapter 5). Early responses by innate immune cells in 5th cutting chloroform extracts combined with decreased pathogen abundance may have been underlying protective effects on BW at early stages of infection, while increases in beneficial members of the microbiota by chloroform extracts may have contributed to improvements in final BW.

The work presented in this thesis utilized different cuttings and supplementation forms as a comparative approach to dissecting the beneficial impacts of alfalfa in healthy and pathogen-challenged mice. In healthy animals, BW and FI did not differ between treatments despite varying changes to the splenic immune cell profile and colon microbiota attributed to alfalfa forms and cutting. During physiological stress (i.e. pathogen challenge), lipid-soluble extracts of late-cutting alfalfa enhanced the immune response and intestinal microbiota in a way that could be linked to improved BW during infection with a host-specific enteric pathogen (C. rodentium). These outcomes in a well-characterized animal model (C57BL/6J mice) provide preliminary results to guide future livestock research to focus on lipid-soluble compounds enriched in later cuttings of alfalfa to maximize its health-promoting effects on the immune system and intestinal microbiota.