1. Introduction
Horses thrive on high-forage diets due to the microbial fermentation of fibre and other compounds in the hindgut [
1]. This digestive strategy requires a high food intake level (above 30 g kg
−1 body weight
0.75 day
−1 [
2]) to maintain proper gastrointestinal function, with forage comprising the major component of the diet. Below this threshold level of forage intake, nutrient supply to the hindgut microbiota becomes the major constraint of digestive function and efficiency [
2]. Research has shown that the balance of microbial populations in the hindgut is important to maintain digestive health, immune function, and performance of the animal [
3,
4,
5]. Over the past decade, research has focused on understanding the structure and composition of these microbial communities in the equine hindgut (using caecal and faecal samples) [
4,
6]. Several studies have indicated that the structure of the equine hindgut microbiota is complex and is comprised of a highly diverse community dominated by bacteria, amongst other microbial species such as archaea, protozoa and fungi [
6,
7,
8,
9].
Some studies have indicated that changes observed in the relative abundance of faecal bacteria may be associated with dietary modifications and gastrointestinal disturbances [
10,
11,
12], which theoretically also occur prior to the onset of life-threatening conditions like colic and laminitis [
13,
14,
15]. Colic and laminitis are two of the most common problems that affect horses [
16], so the potential to predict the onset of sub-clinical gastrointestinal disturbances by examining changes in the abundance of faecal bacteria has been suggested as a method that could perhaps prevent the sudden onset of clinical signs of disease [
13].
A previous study conducted by our group [
7] reported that the faecal bacterial community of forage-fed horses was highly diverse, and the profile was diet-specific with significant differences observed in the relative abundance of certain bacterial genera (the most dominant one in both diets was an unclassified genus within the family Ruminococcaceae). Furthermore, when horses housed indoors and maintained on a commercial chopped ensiled forage were moved outside to graze on pasture, alterations in the bacterial community profile were observed within four days. Although the gut microbiota of the horses appeared to adapt quickly to the new (pasture) diet, some fluctuation (both an increase and/or decrease) in the beta diversity of bacterial communities were observed over the 3-week study period, which was hypothesised to be due to changes in pasture composition [
7].
While many horses around the world are stabled and fed formulated compound diets, the major feed source for New Zealand horses is pasture, mostly consumed directly from the paddock, but also after conservation as ensiled forages and hay [
17,
18,
19]. A temperate climate enables continuous growth of pasture in New Zealand, and many horses graze on pasture all year round [
17,
18,
19,
20]. While there is vegetative grass-leaf throughout the year, the proportion of dead-leaf increases during late-summer (February) and autumn (March–April), and during plant stress due to drought or freezing temperatures [
21,
22]. Thus, there are seasonal changes in the dry matter (DM) and macronutrient composition of the pasture. While one study used conventional microscopic enumeration to compare the faecal microbiota of horses kept on summer versus winter pastures in Japan [
23], there have been very few studies that have used metagenomic techniques to examine the dynamics of bacterial communities in the faeces of horses maintained on pasture [
24].
Given the expected seasonal fluctuations in pasture composition, we hypothesised that there is an effect of time (month or season) on the profile of the faecal bacterial community of pasture-fed horses. The aims of the current study were to investigate changes in the structure and composition of faecal bacterial communities over a 12-month period, by collecting cross-sectional snapshot data at monthly intervals and to correlate the changes in microbiota populations with the macronutrient composition of pasture over the study period.
4. Discussion
The present study investigated the effects of seasonal changes in the nutrient composition of pasture on the diversity of faecal microbiota in horses grazing on typical New Zealand pasture. Currently, there are limited data on the hindgut or faecal microbiota profile of pasture-fed horses, and this study is the third (after our previous New Zealand work [
6] and one UK study [
24]) to examine the effects of dietary change on the diversity of faecal microbiota in pasture-fed horses. The results of this study showed significant effects of diet, horse, and season on the diversity within the faecal bacterial community, which was dominated by four genera within two phyla—Firmicutes and Bacteroidetes. The faecal bacterial community profile identified in this study was similar to the profile reported in our previous work on pasture-fed horses in New Zealand [
7] and in the UK [
24], although there was a lack of bacteria belonging to the Fibrobacteres phylum in our study, compared with the UK study which reported up to 18% of the population belonging to this phylum. This may be due to methodological differences between the two studies or reflect differences in pasture composition.
The faecal samples collected, and bacterial sequences obtained in the present study were representative of the year-round population of faecal microbiota, which comprised a rich and diverse bacterial community. The dominant bacterial phyla detected in the present study were consistent with reports from previous studies that used the Illumina next-generation sequencing technique to examine caecal and faecal microbiota in horses [
14,
58,
59]. Some of the bacterial phyla detected were similar to those identified in other studies of horses using various molecular techniques, but the relative abundances reported in those studies varied considerably [
8,
9,
10,
23]. This was perhaps due to the variation in the type of horse selected, the type and composition of the diet fed to the horses, the geographical location, inconsistencies in the management practices, and the variation in molecular and bioinformatics analyses used across the studies [
4]. Although similarities in results were apparent, caution is warranted in comparing some of the conflicting findings reported in previous studies to the results obtained in the present study.
Kobayashi and co-workers [
23] were the first to report a seasonal variation in the diversity of faecal microbiota in horses fed summer versus winter pasture, using conventional microscopy to enumerate the population of the microbiota. However, given that the faecal bacterial community in forage-fed horses is diet specific [
7], the underlying differences in diet (grassland pasture comprising of timothy grass offered during summer and woodland pasture comprising of bamboo grass offered during winter), and perhaps its nutrient composition, may have confounded the effect of season on the microbial diversity reported in that study [
23].
The significant effects of diet reported in the present study, were in agreement with the findings of our previous work in New Zealand Thoroughbred horses that were also fed forage-only diets [
7]. The bacterial diversity was higher when horses were fed exclusively on pasture in both studies when compared to the period when pasture was supplemented with hay or when the horses were fed on a chopped ensiled forage. While there was a temporal effect on the diversity of faecal bacteria within the two diet periods in the present study, the supplementation of hay appeared to dampen the effects of variations in pasture composition. This dampening effect could be associated with the relatively consistent nutrient composition of hay usually harvested at a fixed time during the year when compared to the variation observed in pasture grazed over several months. However, again caution is warranted in extrapolating this finding to other sources and types of hay, because there may be substantial variation in the nutrient composition depending on the type of grass/legume, stage of growth, climate factors, time of harvest, method of processing and storage, which may vary considerably between batches of hay.
Furthermore,
Table 1 in the present study showed decreases in %CP, %NSC, %HWSC and DE of hay that was fed over five months after open/barn storage, even though the hay was harvested and processed as a single batch. This finding indicates that storage conditions have a negative effect on the nutritive value of hay, and perhaps, this may be true for other fermented forages preserved under field conditions over extended periods [
60,
61]. However, a shelf-life study conducted on a batch of chopped ensiled forage (prepared commercially using forages harvested as a single batch, processed via a controlled fermentation technique, and stored anaerobically in polythene-wrapped packaging) showed no change in nutritive value over a storage period of 12 months [
62]. This type of an ensiled forage appears to have a more stable nutrient composition when compared to hay or pasture (present study), and also supports a high diversity of faecal microbiota similar to horses grazing on pasture [
7].
The variation in pasture composition and the fluctuations in the diversity of faecal microbiota observed in the present study may have implications for grazing management and the preparation of conserved forages for horses susceptible to perturbations of the hindgut microbiota. It may be hypothesised that good quality hay or ensiled forages of a similar nutrient composition could support a relatively consistent population of faecal microbiota by minimising the fluctuations in microbial diversity observed in horses when fed on pasture. Further investigations on the effects of different types of conserved forages on the diversity of the faecal microbiota in horses fed over an extended period are required.
The temporal effects of season and the more subtle effect of the month on the diversity of the bacterial community in faeces reported in the present study appeared to be driven by the variation in the nutrient composition of pasture. When the pasture was growing (vegetative phase, high %CP and low %CHO), Firmicutes dominated the bacterial community, whereas when the pasture was dry (drought-stressed, low %CP and high %CHO), the abundance of Bacteroidetes increased, at the expense of Firmicutes. In particular, the trend for increasing DM content during the months from January to March (drought period), was associated with low pasture growth, which significantly affected the nutrient composition. This spike observed in the %DM was in agreement with previous reports from the region [
21,
63] and was closely associated with climatic factors (low rainfall and high temperature). The minimal variation in HWSCs reported in the present study may have been biased by the time at which the pasture samples were collected (around mid-day), when the HWSC content is potentially at its lowest in comparison to the early-morning or late-afternoon hours [
22,
64]. Subtle differences were observed in the bacterial diversity between the months when horses were grazing exclusively on pasture (April, May—winter/limited pasture; November, December—spring/lush pasture and January, February, March—summer/dry pasture) or when limited pasture was supplemented with hay (June, July, August, September, October). This finding was unique and provided evidence in support of the sensitivity of the next-generation sequencing technique to identify these small changes in the diversity of faecal microbial populations [
59].
While the main findings of the present study supported the hypothesis that pasture composition influences the diversity of the faecal microbiota of horses, variation was observed between horses within diet periods. Nevertheless, only a few less abundant phyla differed between horses, whereas the most dominant phyla (Firmicutes and Bacteroidetes) were not significantly different. This finding indicated that the group of horses in the study shared a core microbiome, with minimal variation between the horses, and is similar to previous reports on other groups of horses where the diet and management were relatively consistent [
8,
9,
12]. However, the subtle differences in the less abundant bacterial taxa detected between individual horses, even though they were managed as a single cohort under similar conditions, may be associated with the feed intake of individual horses and the retention times of digesta through the gastrointestinal tract. Some horses consume more feed than others, a behaviour that appears to affect the transit time of digesta through the gastrointestinal tract [
65,
66], and consequently, affects the time available for microbial digestion. This digestive strategy may explain one of the reasons why some horses (due to their inherent feeding behaviour and individual hindgut microbiota) may adapt to dietary changes better than other horses or why some horses may be predisposed to hindgut dysbiosis that induces acute colic or laminitis [
13,
14]. This further suggests that the population of hindgut/faecal microbiota in some horses is more resilient than in other horses, i.e., the microbial populations of some horses bounce back or adapt better than other horses, although this requires further investigation.
When the %DM of pasture increased, the relative abundance of the phylum Bacteroidetes increased and that of Firmicutes decreased, whilst the remaining phyla of lower relative abundances remained fairly constant. This finding suggests that major shifts in bacterial diversity and abundance can occur in these two phyla, and they should be considered important when monitoring changes in microbial populations. Similar shifts in the Firmicutes:Bacteroidetes ratio have been reported in other studies on the gut microbiome of horses [
10,
12,
59,
67] and humans [
68,
69,
70,
71] in relation to dietary manipulation. Furthermore, within the phylum Firmicutes, three of the most dominant genera were classified within the order Clostridiales, indicating the importance of this group of bacteria and its association with dietary changes and the development of intestinal dysbiosis [
13]. However, the functional roles of these unclassified genera within the order Clostridiales are still unknown and require further investigation.