The influence of a probiotic/prebiotic supplement on microbial and metabolic parameters of equine cecal fluid or fecal slurry in vitro

Jennifer L MacNicol 1,✉, Simone Renwick 2, Caroline M Ganobis 3, Emma Allen-Vercoe 4, Jeffery S Weese 5, Wendy Pearson 6

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PMCID: PMC9994591  PMID: 36715114

Abstract

The microbes that reside within the equine hindgut create a complex and dynamic ecosystem. The equine hindgut microbiota is intimately associated with health and, as such, represents an area which can be beneficially modified. Synbiotics, supplements that combine probiotic micro-organisms with prebiotic ingredients, are a potential means of influencing the hindgut microbiota to promote health and prevent disease. The objective of the current study was to evaluate the influence of an equine probiotic/prebiotic supplement on characteristics of the microbiota and metabolite production in vitro. Equine cecal fluid and fecal material were collected from an abattoir in QC, CAN. Five hundred milliliters of cecal fluid was used to inoculate chemostat vessels maintained as batch fermenters (chemostat cecal, N = 11) with either 0 g (control) or 0.44 g of supplement added at 12 h intervals. One hundred milliliters of cecal fluid (anaerobic cecal, N = 15) or 5% fecal slurry (anaerobic fecal, N = 6) were maintained in an anaerobic chamber with either 0 g (control) or 0.356 g of supplement added at the time of vessel establishment. Samples were taken from vessels at vessel establishment (0), 24, or 48 h of incubation. Illumina sequencing of the V4 region of the 16S rRNA gene and bioinformatics were performed for microbiome analysis. Metabolite data was obtained via NMR spectroscopy. All statistical analyses were run in SAS 9.4. There was no effect of treatment at 24 or 48h on alpha or beta diversity indices and limited taxonomic differences were noted. Acetate, propionate, and butyrate were higher in treated compared to untreated vessels in all methods. A consistent effect of supplementation on the metabolic profile with no discernable impact on the microbiota of these in vitro systems indicates inoculum microbe viability and a utilization of the provided fermentable substrate within the systems. Although no changes within the microbiome were apparent, the consistent changes in metabolites indicates a potential prebiotic effect of the added supplement and merits further exploration.

Keywords: cecal, equine, in vitro, metabolome, microbiome, synbiotic

Using in vitro systems to capture the response of the equine hindgut to synbiotic supplements.

Introduction

The equine hindgut contains a diverse microbial ecosystem which is necessary for horses to derive nutrients from their highly fibrous diet. The gastrointestinal (GI) microbiota has numerous important roles with regards to host health. The GI microbiota is involved in the maintenance of mucosal integrity and protection against GI pathogens (Thursby and Juge, 2017), has been linked to the proper functioning of the immune system and is pivotal for the prevention of inflammation (Yoo et al., 2020). Furthermore, microbiota derived signaling molecules communicate with various organs and influence systemic function (Schroeder and Bäckhed, 2016). It is clear that the health of the GI microbiota is related to the health and function of the host on multiple levels. As such, dysbiosis (a state of imbalance within a microbial community) (DeGruttola et al., 2016) can have a severe impact on host health. In horses, several severe diseases including colitis (Costa et al., 2012; Arroyo et al., 2020), laminitis (Milinovich et al., 2008, 2010), postpartum colic (Weese et al., 2015), and equine grass sickness (Garrett et al., 2002) have been related to hindgut dysbiosis. Due to its strong influence on health, manipulation of the GI microbiota is of clinical and practical relevance.

The challenges associated with in vivo animal research make in vitro studies a useful means of evaluating different stimuli within the context of the hindgut microbial ecosystem. Measurement of metabolites within these systems offers a context for microbiota data and can provide insight into potential microbial responses to feed additives of interest. Horses derive approximately 70% of their energy from volatile fatty acids (VFA) synthesized by microbial fermentation (Frape, 2004). Therefore, the impact of dietary additives on microbial VFA production is of particular interest in equine nutrition.

Probiotics are defined as “live micro-organisms that, when administered orally at adequate concentrations, provide a beneficial effect to the host beyond that of their nutritional value”. Prebiotics, on the other hand, are “nondigestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of one or more of a limited number of bacterial species already resident” (Gibson and Roberfroid, 1995). Synbiotics, supplements that blend both prebiotic ingredients and probiotic bacteria, can result in a combined effect (Mohanty et al., 2018). Probiotic, prebiotic, and synbiotics are popular supplements in human and animal nutrition. In theory, probiotics represent a powerful tool to support optimal health through an enhancement of the host immune system, competitive exclusion of potential pathogens, stimulation of the intestinal barrier, production of antimicrobial metabolites, and the inactivation of toxins (Schoster, 2018). Significant challenges exist regarding the development of probiotics. A good probiotic must be robust against manufacturing and storage conditions, survive passage through the GIT to reach its site of action, ideally adhere to the GIT epithelial surface and persist within the host, and importantly not harm or induce disease the host (Saarela et al., 2000). Due to the vital role of the equine hindgut microbiota, there is significant potential for these types of supplements to aid in modulating and maintaining equine health and nutritional status. However, substantial challenges in probiotic development remain regarding the selection of appropriate organisms, as well as the maintenance of organism viability during processing, storage, and survival through the GIT (Weese, 2002). Prebiotics, on the other hand, are not viable organisms and therefore not vulnerable to the manufacturing process in the same manner as probiotics. However, according to a consensus statement set out by the International Scientific Association of Probiotics and Prebiotics, a prebiotic compound must meet several criteria including selective impact on microbial growth/activity and health benefits confirmed within the species the prebiotic is intended for (Gibson et al., 2017). Synbiotics are somewhat less defined and more challenging to assess. A synbiotic implies a combined effect, suggesting that the probiotic in the supplement should be selectively stimulated by the particular prebiotic ingredient (Olveira and González-Molero, 2016). Synbiotics were initially conceptualized as a means of assisting probiotic organism survival (Pandey et al., 2015). However, the distinct prebiotic and probiotic effects that are elicited in vivo from the consumption of the combination could also be considered a synergistic effect (Schrezenmeir and De Vrese, 2001; Pandey et al., 2015).

The use of in vitro systems to screen probiotic, prebiotic or synbiotic products is an interesting concept. Probiotics are generally considered safe. However, certain strains of a species generally thought to be innocuous can induce disease (Lancefield and Hare, 1935; Tonzetich and McBride, 1981; Lorenz et al., 2020). The significantly different characteristics of bacterial strains of the same species makes the selection and implementation of a safe product more challenging than often assumed. Furthermore, the potential for the unintentional use of antibiotic resistant strains in probiotics or antibiotic resistance transfer through the use of probiotics (Sharma et al., 2014) stresses the significance of ensuring, at the very least, an efficacious product. Purposeful application of in vitro evaluations of probiotics could offer insight into products that are most suitable for further investigation.

The objectives of this study were to evaluate the impact of a commercial equine synbiotic dietary supplement on the microbial and metabolic profiles of cecal inoculum maintained in either a chemostat batch fermenter or anaerobic chamber, and fecal inoculum maintained in an anaerobic chamber.

Materials and Methods

This research did not require IACUC approval or CCAC approval as it used samples from animals from a commercial abattoir. Details of sample collection and microbiome and metabolite sample preparation can be found in MacNicol et al. (2022).

In brief, sample material was collected directly from the GIT with 10 min of death from mature horses deemed suitable for consumption at Viande Richileu (QC, CAN). Cecal and fecal material were collected from the same animal directly into sterile containers. All sample materials were maintained separately. Cecal and fecal material were transported in anaerobic conditions on ice to the University of Guelph where experimental vessels were established within 24 h of collection. Cecal material was collected from a total of 17 horses. Solely cecal fluid was collected from 11 horses. Fecal material was collected in addition to cecal fluid from six horses. Cecal fluid from two horses was used solely in chemostat experiments, cecal fluid from nine horses was used in both chemostat and anaerobic chamber experiments, cecal fluid and fecal material from six horses was used solely in anaerobic chamber experiments.

Chemostat vessel establishment and sampling (chemostat cecal, N = 11)

At the time of vessel establishment and every 12 h for the 48 h experimental period, 6 ml of content was removed directly from the vessel through the sampling port via sterile syringe. At this time, the volume was replaced by the addition of either 0.44 g of probiotic + 6 ml sterile water (or 6 ml sterile water) through the sampling port. The supplement used in these experiments was a commercially manufactured equine synbiotic supplement (Probioplus, Herbs for Horses, Selected Bioproducts INC, LOT 04332320) containing Enterococcus faecium, Lactobacillus plantarum, and Pediococcus acidilactici at a stated level of 2 × 1010 CFU/g total micro-organisms, amino acids, amylase, beneficial fiber, chicory, fenugreek, flaxseed, glucanase, inulin, nutritional yeast, and protease. The supplement was weighed, aliquoted, and stored at −80 °C until use. The amount of supplement added to the vessel was calculated to reflect a total of a 32g/d dose (as per the manufacturer’s recommendation) in a cecal volume of approximately 18 Liter (Ross and Hanson, 1972; Miyaji et al., 2008) scaled to a 500 mL volume and added over four additions (0, 12, 24, 36 h). Samples taken at the time of vessel establishment (0), 24, and 48h were aliquoted into subsamples. Subsamples for microbiome analysis were stabilized in anhydrous EtOH (Hale et al., 2015; Song et al., 2016; Vogtmann et al., 2017). Subsamples for metabolome analysis were prepared as previously stated. Subsamples were stored at −80 °C until further analysis.

Anaerobe vessel establishment and sampling (anaerobic cecal, N = 15; anaerobic fecal, N = 6)

At the time of vessel establishment 0.356 g of the equine synbiotic supplement (Probioplus, Herbs for Horses, Selected Bioproducts INC, LOT 04332320), was added to fecal probiotic vessels (anaerobic fecal) and cecal probiotic vessels (anaerobic cecal). The amount of probiotic added to the vessels was calculated in the same manner as for the chemostat vessels and scaled to a 100 ml volume. No additions were made to fecal or cecal control vessels at the time of establishment. Every 12 h for the 48 h experiment 4 ml of content was removed directly from the vessel via sterile syringe and replaced with 4 ml sterile H2O. At the time of sampling, vessel pH was also measured with a portable pH meter (OHAUS ST20, NJ, USA) which was cleaned with 70% isopropyl alcohol prior to use.

Statistical analysis

The purpose of the present analysis was to evaluate the impact of Probioplus additions on the microbial and metabolic parameters within each of the fermentation methods separately at 24 and 48 h. All statistical analyses were run in SAS 9.4 (SAS Inc, NC, USA) using a RM ANOVA in PROC GLIMMIX. For all models, residuals were analyzed to identify the most appropriate structure, lsmeans of time and treatment within time were evaluated. The interaction was sliced by time and treatment and a Tukey adjustment was applied. P < 0.05 was considered significant.

pH

Statistical analysis of pH (γ) was run according to the following model:

𝛾=𝜇+ a⁢n⁢ii+t⁢i⁢m⁢ej+t⁢r⁢t⁢(t⁢i⁢m⁢e)k⁢j+𝜀

where µ = the overall mean, anii = the random effect of animal (i = 1 to n), timej = fixed effect of time (j = 0-48h), trt (time)kj = the fixed effect of treatment (k = pbp, co) nested within sampling time, ε = the residual error.

Microbiome

The model used to evaluate the difference between control and probiotic vessels for alpha diversity indices including richness (Chao1), evenness (Shannon evenness), and diversity (Inverse Simpson index) and taxonomic relative abundance data for all phyla, order, and genera greater than 5% (γ) included:

𝛾i⁢j⁢k=𝜇+ a⁢n⁢ii+t⁢i⁢m⁢ej+t⁢r⁢tk+t⁢r⁢t∗t⁢i⁢m⁢ek⁢j+𝜀

where µ = the overall mean, anii = the random effect of animal (i = 1 to n), timej = fixed effect of time (j = 24h, 48h), trtk = the fixed effect of treatment (k = pbp, co), the interaction of time and treatment, ε = the residual error. For taxonomic data, False Discovery Rate was applied with a Benjamini–Hochberg adjustment (Benjamini and Hochberg, 1995) to the P-values for each taxonomic level within a comparison using the PROC MULTTEST procedure in SAS 9.4. An adjusted P-value < 0.05 was considered significant.

Beta diversity measures of community membership (Jaccard index) and community structure (Bray-Curtis index) within fermentation method between control and probiotic vessels at 24 or 48h were evaluated using AMOVA and HOMOVA.

Metabolite analysis

Individual metabolites and the ratio of acetate+butyrate/propionate (γ) were assessed according to the following model:

𝛾i⁢j⁢k=𝜇+𝛽⁢x⁢i⁢n⁢i⁢t⁢i⁢a⁢l+a⁢n⁢ii+t⁢i⁢m⁢ej+t⁢r⁢tk+t⁢r⁢t∗t⁢i⁢m⁢ek⁢j+𝜀

where µ = the overall mean, β = the covariate slope, initial = metabolite concentration measured at the time of sample collection, anii = the random effect of animal (i = 1 to n), timej = fixed effect of time (j = 24,48 h), trtk = the fixed effect of treatment (k = pbp, co), the interaction of time and treatment, ε = the residual error.

Results

pH

The pH of supplement treated vessels was lower than that of control vessels for both anaerobic cecal (N = 15; means: 6.95 ± 0.05, 7.01 ± 0.05, respectively) and anaerobic fecal (N = 6; means: 6.13 ± 0.02, 6.61 ± 0.02, respectively) at all times (P < 0.05), with the exception of 36h in anaerobic cecal vessels (Figure 1).

Figure 1.

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Asterisk (*) represents a significant difference between control and supplement treated vessels within a fermentation method at a particular time point (P < 0.05). Comparison of pH in control and supplement treated vessels maintained in an anaerobic chamber. Comparison of pH between anaerobic chamber control (black) and supplement (Probioplus; green) treated vessels at 12 h intervals following vessel establishment (0 h) within a fermentation method. One hundred milliliters of cecal fluid (N = 15; solid) or 5% fecal slurry (N = 6; dashed) were used to inoculate 100 mL vessels maintained in an anaerobic chamber with either 0 g (control) or 0.356 g of supplement added at the time of vessel establishment.

Microbiome

Sequence analysis

The total number of raw sequences was 31,776,710. Following quality control filtering the total number of sequences was 24,331,235 (average per sample 137,465; SD 18,625; median 134,786; range 96,744–192,151). Sequences were subsampled at a sequencing depth of 90,000 sequences per sample to adjust for the uneven depth across samples. This resulted in a coverage of >99% for all samples.

Alpha and beta diversity indices

There was no effect of supplement treatment on any alpha diversity indices in anaerobic cecal or chemostat cecal vessels (N = 11). In samples from anaerobic fecal vessels, richness at 24 h was lower in samples from supplement treated vessels than controls. (Table 1). There was no effect of supplement treatment in any fermentation method on measures of beta diversity at either 24 or 48h.

Table 1.

Comparison of Alpha diversity indices of richness (Chao1), evenness (Shannon Evenness), and diversity (Inverse Simpson) between control and supplement treated vessels at 24 and 48 h of fermentation ± SEM

TreatmentIndiceTime, hControlSupplementP-valueChao1 Anaerobic cecal242210 ± 1202174 ± 1200.5408482242 ± 1202207 ± 1200.5644 Anaerobic fecal243427 ± 2702967 ± 2700.0172483172 ± 2702969 ± 2700.2562 Chemostat cecal242267 ± 1302182 ± 1300.5191482339 ± 1302229 ± 1300.4102Inverse Simpson Anaerobic cecal2437.9 ± 739.5 ± 70.72804854.2 ± 756.8 ± 70.5570 Anaerobic fecal2458.9 ± 1243.4 ± 120.39354894.8 ± 1258.6 ± 120.0582 Chemostat cecal2447.5 ± 852.7 ± 80.36864851.9 ± 846.4 ± 80.3427Shannon Evenness Anaerobic Cecal240.634 ± 0.020.634 ± 0.020.6271480.664 ± 0.020.662 ± 0.020.8578 Anaerobic Fecal240.686 ± 0.020.647 ± 0.020.1557480.730 ± 0.020.684 ± 0.020.1012 Chemostat Cecal240.664 ± 0.020.670 ± 0.020.6050480.667 ± 0.020.656 ± 0.020.3303

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One hundred milliters of cecal fluid (anaerobic cecal, N = 15) or 5% fecal slurry (anaerobic fecal, N = 6) were used to inoculate 100 mL vessels maintained in an anaerobic chamber with either 0 g (control) or 0.356 g of supplement added at the time of vessel establishment. Five hundred mL of cecal fluid was used to inoculate chemostat vessels maintained as batch fermenters (chemostat cecal, N = 11) with either 0 g (control) or 0.44 g of supplement added at 12 h intervals.

Taxonomy

None of the evaluated taxonomic classifications were different between treatment and control in anaerobic cecal or chemostat cecal vessels at either 24 or 48h. Table S1 provides the full scope of taxonomic relative abundances.

In anaerobic fecal treated vessels compared to controls at 48 h, the relative abundances of Bacteroidales (adj P = 0.0024) and Bacteroides (adj P = 0.0165) were higher, whereas unclassified Bacteroidetes (adj P = 0.0064) was lower. Fig. 2.

Figure 2.

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Lines attach taxa from vessels that significantly differ (P < .05). Percentage relative abundances of taxa from 5% fecal slurry maintained in an anaerobic chamber after 48 h of fermentation. Relative abundance (%) of Bacteroidales (green), Bacteroidetes_unclassified (yellow) and Bacteroides(blue) at 48 h in vessels containing 5% fecal slurry (anaerobic fecal, N=6) with either 0 g (co) or 0.356 g of supplement (pbp) added at the time of vessel establishment.

Metabolites

The main byproducts of microbial metabolism and major metabolites present in all samples were acetate, propionate, and butyrate (Figure 3). The concentration of both acetate and propionate in all methods was greater in the supplement treated vessels than controls at both 24 h (P < 0.05) and 48 h (P < 0.001; Table 2). This was also the case for butyrate in anaerobic fecal (P < 0.05) and anaerobic cecal (P < 0.0001) supplement treated vessels compared to the controls. In chemostat cecal supplement treated vessels, an increase butyrate occurred by 48 h (P = 0.0188). The acetate+butyrate/propionate ratio was significantly lower at both 24 and 48h in supplement treated vessels compared to control vessels across all methods, with the exception of 24 h in chemostat cecal at which time no difference was noted.

Figure 3.

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Asterisk (*) represents a significant difference between control and supplement treated vessels within fermentation method at a particular time point (P < 0.05). Comparison of acetate (A,B,C), propionate (D,E,F), and butyrate (G,H,I), mM between control and supplement treated vessels at 24h and 48h of fermentation. A,D,G) One hundred ml of cecal fluid (N = 15; solid) was used to inoculate 100 mL vessels maintained in an anaerobic chamber with either 0 g (control) or 0.356 g of supplement added at the time of vessel establishment. B,E,H) One hundred milliliters of 5% fecal slurry (N = 6; dashed) were used to inoculate 100 mL vessels maintained in an anaerobic chamber with either 0 g (control) or 0.356 g of supplement added at the time of vessel establishment. C,F,I) Five hundred milliliters of cecal fluid was used to inoculate chemostat vessels maintained as batch fermenters (N = 11; dotted) with either 0 g (control) or 0.44 g of supplement added at 12 h intervals.

Table 2.

Comparison of acetate+butyrate/propionate ratio between control and supplement treated vessels at 24 and 48 h of fermentation ± SEM

TreatmentMethodTime, hControlSupplementP-valueAnaerobic cecal243.63 ± 0.1342.93 ± 0.108<0.0001483.60 0.1332.94 ± 0.109<0.0001Anaerobic fecal243.30 ± 0.2421.97 ± 0.2420.0015483.18 ± 0.2421.85 ± 0.2420.0015Chemostat cecal243.46 ± 0.1883.21 ± 0.1750.2384483.50 ± 0.1913.05 ± 0.1660.0354

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One hundred mL of cecal fluid (anaerobic cecal, N = 15) or 5% fecal slurry (anaerobic fecal, N = 6) were used to inoculate 100 mL vessels maintained in an anaerobic chamber with either 0 g (co) or 0.356 g of supplement added at the time of vessel establishment. Five hundred mL of cecal fluid was used to inoculate chemostat vessels maintained as batch fermenters (chemostat cecal, N = 11) with either 0 g (control) or 0.44 g of supplement added at 12 h intervals.

Discussion

Prebiotic, probiotic, and more recently synbiotic supplements have been explored as potential means of improving the GI microbial ecosystem and preventing diseases associated with dysbiosis (Gotić et al.; Quigley, 2019). Probiotics and synbiotics require the addition of live bacteria which must reach the site of action within the GIT alive such that they can benefit the health of the host. However, there are substantial challenges in maintaining microbe viability during supplement manufacturing, storage, and even throughout the harsh conditions of other regions of the GIT prior to the site of action, which is generally the colon. At this time there is limited evidence of the benefits of probiotic type supplements within the veterinary industry (Schoster et al., 2014; Schoster, 2018). However, select in vivo equine studies have demonstrated microbial shifts following synbiotic supplement administration. Grimm et al. (2020) identified an increase in the cecal and fecal relative abundance of XIII Clostridiales, as well as colonic and fecal Veillonellaceae in a study that utilized six fistulated geldings fed a yeast/microalgae supplement. The authors speculated these changes represented a potential for the improvement of fibrinolytic function and modulation of dysbiosis under dietary change. A study by Ishizaka et al. (2014), that included only 10 horses observed a reduction in the presence of enteropathogenic bacteria. Furthermore, they noted a consistency in fecal pH in horses fed a fermented supplement whereas fecal pH increased in the control horses over time. These results led the authors to conclude that the administration of probiotics could modulate changes within the GIT. However, in both studies, a limited number of horses were enrolled, and the majority of parameters investigated demonstrated no change with probiotic administration. Furthermore, the changes that were observed demonstrate only a tangential correlation to the benefits of probiotic administration but are far from definitive cause and effect evidence. Any resultant beneficial health effects of the enhanced VFA production are also unclear. Although VFAs produced by colonic microbes are often associated with positive impacts on health, particularly butyrate (Canani et al., 2011), no measures of health could be accounted for in this particular study and therefore remain unknown. In vitro studies assessing the alterations associated with probiotic administration specifically within the microbial communities of different areas within the equine GIT may aide in identifying changes that are lost in vivo when only feces are assessed. In vitro studies may also identify more minor microbial shifts that may be overwhelmed by physiological processes in vivo.

The microbial communities within all three methods evaluated in this study demonstrated viability and the capacity to metabolize the provided substrate. However, the consistent lack of any major or minor microbial alterations between supplement treated and nontreated vessels indicates that the probiotic organisms did not substantially impact the established communities present within the inoculum. Certain probiotic organisms may have more difficulty in colonizing adult, healthy horses that have a well-established and complex GI microbiota. This is supported by the longer colonization time when the probiotic are administered to foals as compared to adult horses (Schoster et al., 2016). This reasoning is, in part, why equine probiotic studies have focused on foals (Weese et al., 2003), as their GI microbiota is not fully developed; or horses exposed to antibiotic treatment (Collinet et al., 2021), as antibiotic treatment induces a state of microbial dysbiosis which may be more susceptible to influence by probiotic organisms.

In this study, unaltered supplement was added directly to the in vitro system. Although this design lacked a simulation of the substantial prehindgut digestion that occurs in vivo, the lack of probiotic influence on the microbial community composition or structure, despite the direct use of probiotic organisms, provides strong evidence that in vivo microbial shifts due to the probiotic organisms within the supplement would be unlikely in healthy horses.

Nevertheless, there were consistent effects of the supplement across all three methods on metabolite production. The addition of the supplement into all systems increased concentrations of acetate, propionate, and butyrate, clearly demonstrating that the microbial communities present utilized components of the supplement. The stable microbial communities present, despite increased VFA production, likely indicates that the incubation time was not prolonged to the point of a microbial ecosystem crash. A crash within the microbial communities would eventually occur within these systems due to the build up of VFAs and reduced pH. It is possible that the reduction in Bacteroidetes and increase in Bacteroidales and Bacteroides identified in Probioplus treated anaerobic fecal vessels at 48 h represents the beginning of destabilization, particularly considering these vessels had the lowest pH.

Prebiotics were initially defined in 1995 as “nondigestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria already resident in the colon” (Gibson and Roberfroid, 1995). Since the concept of prebiotics were first introduced, the definition of what constitutes a prebiotic has undergone many iterations. However, a selective effect on the microbiota and resultant health benefits conferred to the host are core principles of the prebiotic effect. Initial culture based studies of feces and GI contents identified increased counts of Bifidobacteriumfollowing the consumption of prebiotics (Okazaki et al., 1990; Hsu et al., 2004). High throughput sequencing has also been used successfully to characterize changes in the microbiome associated with the consumption of prebiotics. Consumptions of prebiotics frequently results in an increased relative abundance of Bifidobacterium which is often accompanied by changes in various other taxa (Swanson et al., 2020), although the changes in taxa other than Bifidobacterium are not particularly consistent across studies. However, the selectivity criteria of prebiotics has been challenged based on the current lack of definitive ability to delineate “beneficial” from “detrimental” microbes within the context of the gut ecosystem and the metabolic benefits do not necessitate discriminate fermentation (Bindels et al., 2015; Verspreet et al., 2016). Dietary fiber on the other hand is a more lenient definition which refers to carbohydrates that resist hydrolysis via endogenous enzymes but can be fermented by some colonic microbes (Olveira and González-Molero, 2016). Dietary fibers provided fermentable substrate for the colonic ecosystem and there are documented health benefits attributed to these compounds (Verspreet et al., 2016; Makki et al., 2018). The definition of what constitutes a prebiotic is still being adjusted and the line between prebiotics and dietary fiber is somewhat vague. Nevertheless, a key feature of both is the beneficial impact on host physiology and as such in vivo trials are a critical component to assess potential health benefits.

The high fiber content and prebiotic ingredients of this supplement are likely the key contributors to the observed increase in microbial VFA production. The acetate+butyrate/propionate ratio is considered an applicable marker of microbial fibrolytic activity within the equine hindgut (Grimm et al., 2017). However, no change was observed in this ratio when horses were switched from a high fiber to high starch diet (Grimm et al., 2020), and it did not correspond to microbiological fibrolytic activity despite a numeric increase for 2 days following trimethoprim sulfadiazine administration in horses (Collinet et al., 2021). Similarly, in the present study this ratio decreased in all systems fed the high-fiber supplement. Therefore, this particular ratio may be mainly reflective of in vivo fibrolytic activity by indicating an alteration in VFA metabolism or absorption and not directly corresponding to microbial activity per say. Several of ingredients within the Probioplus supplement demonstrate prebiotic potential in various contexts. Chicory fructans are fermented in the large colon (Roberfroid, 1998) and fenugreek seed gum has demonstrated in vitro resistance to enzymatic digestion and can be completely fermented by the cecal microbes in rabbits (Zemzmi et al., 2020). Inulin is an added ingredient in the supplement, as well as a component of several other ingredients. The prebiotic characteristics of inulin are well-documented, and inulin-type prebiotics are considered bifidogenic (they selectively stimulate the growth of bifidobacteria) (Kelly, 2008). Interestingly, in this study, no influence of supplementation on bifidobacteria or other bacterial taxa was observed. Although the study was not designed in such a way as to ascertain why the addition of inulin, inulin containing ingredients, and other ingredients with prebiotic potential would not stimulate Bifidobacteria or other generally associated with prebiotic administration, it is possible that a 48 h exposure is insufficient to generate substantial changes in the relative abundance of microbes present within a complex community to a degree that can be captured by 16S rRNA gene sequencing. The lack of a predigestion process or assessment of the individual supplement ingredients prevented us from determining exactly which components or ingredients were responsible for the VFA changes. Gut microbes are able to utilize dietary protein (Zhao et al., 2018) and carbohydrates as sources of energy. However, equine hindgut microbes are particularly sensitive to carbohydrates. Carbohydrate overload is used as a reliable and repeatable means of provoking hindgut dysbiosis and inducing laminitis in horses (Milinovich et al., 2008). The stability of the microbiota during supplementation with Probioplus indicates that any soluble carbohydrate faction of the supplement was likely playing only a minor role in providing a substrate for microbial fermentation. Further exploration of this would be ideal and could be undertaken through experimentation with the probiotic strains removed from the product or a sterilization procedure prior to use.

As with any in vitro study, caution must be exercised when interpreting these results as significant differences exist between the artificial environment generated within this study and that which occurs in a live animal. As previously mentioned, the lack of a pre-digestion and exposure to upper GI tract conditions (e.g. low gastric pH) process limits our ability to interpret which fraction of the supplement is most likely leading to the observed changes in VFA concentration between treated and control vessels. Furthermore, these in vitro systems were used as batch fermenters and thus had no continuous influx or outflow. However, even in continuous flow systems, absorption and host metabolism of microbial biproducts can, as of yet, not be mimicked. Conversely, the lack of host metabolism can be beneficial when strictly interested in the outcome of a particular factor on the microbial community and its activity. Cecal contents were employed in this study as the cecum sits at the beginning of the hindgut and is the initial site of hindgut microbial fermentation. The use of cecal content from a variety of animals strengthens the applicability of these results. The microbiome is highly individualized and can significantly vary even between a homogenous group of individuals (Garber et al., 2020). Therefore, results that are apparent across a broad range of subjects are more highly generalizable. Additionally, the use of inoculum from the specific GI site of interest is advantageous, despite representing a challenge in terms of collection, as it has been reliably demonstrated that the microbiome across the equine GIT varies significantly (Costa et al., 2015; Ericsson et al., 2016). A consideration of all these factors must be applied when interpreting the results from this study.

Conclusions

From the in vitro conditions applied within this study, the addition of Probioplus, an equine synbiotic supplement composed of probiotic organisms, as well as select high-fiber and prebiotic ingredients, did not alter the microbial composition of equine cecal or fecal inoculum. However, the supplement did consistently increase the microbial activity as assessed through the measures of VFA production within all systems, likely due to the high-fiber ingredients. The prebiotic potential of this supplement requires further exploration to determine whether microbial shifts would occur following a longer exposure and confirming in vivo health benefits following administration. Additional in vitro assessment following a predigestion protocol to evaluate whether prececal GI conditions might alter the ability of hindgut microbial communities to utilize the fiber factions of the supplement would also be particularly useful.

Supplementary Material

skad034_suppl_Supplementary_Table

Click here for additional data file. (17KB, docx)

Acknowledgments

We would like to gratefully acknowledge Viande Richelieu and Luc Bailgeron (Massueville, QC) for their assistance in collection of samples for this research. We would also like to acknowledge the Allen-Vercoe lab for their guidance as well as the use of equipment and lab space. Funding was provided by Selected BioProducts INC.

Glossary

Abbreviations

AMOVA

analysis of molecular variance

CFU

colony forming units

GI

gastrointestinal

GIT

gastrointestinal tract

HOMOVA

homogeneity of molecular variance

VFA

volatile fatty acids

Contributor Information

Jennifer L MacNicol, Department of Animal Biosciences, Ontario Agricultural College, University of Guelph, Guelph, ON N1G2W1, Canada.

Simone Renwick, Department of Molecular and Cellular Biology, College of Biological Science, University of Guelph, Guelph, ON N1G2W1, Canada.

Caroline M Ganobis, Department of Molecular and Cellular Biology, College of Biological Science, University of Guelph, Guelph, ON N1G2W1, Canada.

Emma Allen-Vercoe, Department of Molecular and Cellular Biology, College of Biological Science, University of Guelph, Guelph, ON N1G2W1, Canada.

Jeffery S Weese, Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, ON N1G2W1, Canada.

Wendy Pearson, Department of Animal Biosciences, Ontario Agricultural College, University of Guelph, Guelph, ON N1G2W1, Canada.

Conflict of Interest Statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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Maria Sempe