The effect of supplements on digestibility and faecal microbial communities in ponies

Background to the work. Equids have evolved as browsing and grazing herbivores and their digestive tract is well adapted to low-energy, high-fibre forage ingested continuously throughout the day. In domesticated equines, current feeding practices often involve restricted grazing, feeding conserved forage, consumption of large amounts of starchy cereals in often large meals per day, abrupt changes to the diet and management, all of which may lead to microbial disturbances, metabolic and health disorders such as hindgut acidosis, gastric ulceration, colic, and laminitis. The art of feeding the horse is to meet nutrient requirements, while maintaining the health and welfare, which is essential for the success of equine athlete. Dietary supplements may serve as a tool in manipulating the equine gut microbiome to increase forage digestibility, reduce the negative impact of feeding high-starch concentrates and alleviate digestive upsets related to stress caused by abrupt changes in the diet. Aims of the thesis. To investigate the effects of a fibrolytic enzyme preparation and yeast (S. cerevisiae) products on nutrient digestibility in vivo, fermentative parameters in vitro, and faecal pH. To describe alterations in faecal microbial communities of ponies after an abrupt change from pasture to hay and vice versa. To explore the effects of yeast supplement on faecal microbial communities, when ponies were fed high-starch and high-fibre diets and when they were subjected to abrupt dietary changes from grass to hay and vice versa. Methods. An automated in vitro gas production system (ANKOM) was used to assess the fermentation parameters of substrates incubated with an equine faecal inoculum according to technique of Theodorou et al. (1994). Vacuum filtration was used to estimate dry matter loss in vitro. The pH of the resultant suspension was also measured. A total collection digestibility technique followed by proximate analysis of feed and faeces was used to calculate nutrient digestibility in vivo and faecal pH was measured. A NASCO digestive analyser was used to measure faecal particle size. The data were statistically analysed in Minitab 17 (ANOVA GLM according to Latin square design) and Genstat 18.1 (repeated measures ANOVA). Freshly frozen faecal samples were processed to extract bacterial DNA (QIAamp Fast DNA Stool Mini Kit) before creation of 16S rRNA gene amplicon libraries and sequencing using Illumina MiSeq platform. The Illumina output was analysed with 16S metagenomics pipeline using QIIME v1.9.0. LEfSe package on Galaxy was used for microbial biomarkers identification. Results. The addition of a fibrolytic exogenous enzyme product to grass hay prior to feeding resulted in greater gas production and improved fermentation kinetics in vitro. However, enzyme treatment of grass hay prior to feeding it to ponies did not enhance the apparent total tract digestibility of the diets offered or nutrient intakes. Yeast supplementation resulted in improved fermentation parameters in vitro and fibre digestibility in vivo. S. cerevisiae increased the relative abundance (RA) of members of Lachnospiraceae and Dehalobacteriaceae families, associated with healthy core microbiome. Moreover, yeast increased RA of fibrolytic bacteria (Ruminococcus) when added to high-fibre diet and reduced RA of lactate producing bacteria (Streptococcus) when a high-starch diet was fed. An increased RA of acetic, succinic acid producing bacterial taxa (unassigned genera from family Succinivibrionaceae) and butyrate producing bacteria (Roseburia) were seen when yeast was fed with high-starch and high-fibre diets, respectively. Abrupt changes from pasture to hay and vice versa affected faecal microbiome community structure. Moreover, the order of dietary change had a profound affect in the first few days following the transition. The most abundant phylum, Bacteroidetes, decreased after abrupt dietary changes. Yeast increased the RA of Bacteroidetes phylum (P-value=0.009) when ponies were subjected to abrupt dietary changes, thus maintaining a more stable microbial profile. Contribution of the results to the existing field. This thesis provides novel information on the use of feed supplements and their effect on nutrient digestibility in vivo and in vitro, and on faecal microbial communities under stress caused by abrupt dietary changes