Equine Hindgut Research Articles

0812 Investigation of equine hindgut microbiota development in young horses

0812 Investigation of equine hindgut microbiota development in young horses

0820 Changes in equine hindgut fermentation and carbohydrate digestion in response to varying sources of nitrogen

0820 Changes in equine hindgut fermentation and carbohydrate digestion in response to varying sources of nitrogen

Equine colic: a review of the equine hindgut and colic

Colic is the major cause of morbidity and mortality, premature deaths, and is the number one health concern in horses. To increase the quality of life for horses, it is vital to understand the causes, signs, diagnostics, treatment, and prevention of colic. Horses are hindgut fermenters and their cecal microbiome can easily be altered due to environmental and physiological changes. Colic can be induced from starch overloading, which can have detrimental effects on the cecum microbiome, such as decreasing hindgut pH and altering the production of volatile fatty acids. In addition, if the immune system becomes compromised, causing changes in the hindgut microbiome, this will trigger a proliferation of opportunistic and pathogenic bacteria, which can cause numerous gastrointestinal diseases, resulting in colic. However, identifying a variety of microorganisms in the cecum is extremely difficult and understudied because difficulties in sampling and not all bacteria strains can be cultured. Therefore, next generation sequencing has helped in identifying microbes found in the gastrointestinal tract. Using the application of metagenomics, allows scientists to be able to sequence thousands of microorganisms simultaneously. In addition, exploring new techniques to help identify and achieve a clearer picture of the microbiome population and how it may change during bouts of colic could revolutionize the way veterinarians diagnose colic and tailoring a treatment specific to the microoganisms that are proliferating. Therefore, understanding the mechanisms of colic and next technological advances could increase the quality of life for horses and minimize the mortality rates.

Preliminary Investigation of the Changes in Fecal Streptococcal Population due to Diet and Time of Day in Horses

A growing number of equine studies examine management factors that influence the microbial populations in regions of the gastrointestinal tract using culture-independent molecular techniques. We used quantitative polymerase chain reaction (qPCR) to evaluate changes in fecal streptococcal populations in horses fed different maturities of teff hay with fecal samples collected at 7 AM, 12 PM, and 7 PM. The objective of this study was to use qPCR and species-specific probes for 16S ribosomal DNA to quantify the percentage of equine hindgut streptococcal species (EHSS) relative to the total bacterial load in the feces. Feces from horses fed the most mature teff hay had the lowest %EHSS, and feces collected at 12:00 had the highest %EHSS (P < .05). Our interest in investigating %EHSS developed out of research that examined associations between changes in EHSS populations and the onset of laminitis. Although we expected almost no risk of laminitis in these forage fed horses, our hypothesis was that the different carbohydrate fractions in the three maturities of hay would result in differences in %EHSS in horses fed these hay maturities. The supply of carbohydrates, nonstructural or structural, influences the microbial species composition through the gastrointestinal tract. This study highlights the occurrence of measurable changes in %EHSS due to subtle changes in dietary nonstructural carbohydrate and also changes in samples taken at different times of day. This information is useful to others considering investigations of dietary influences on gastrointestinal microbial populations, particularly those that plan to use feces as their main sample medium.

HORSE SPECIES SYMPOSIUM: The microbiome of the horse hindgut: History and current knowledge.

In the early 1990s, the equine hindgut microbial ecosystem looked like a "black box." Its vital role in hydrolyzing and fermenting fiber, thus providing energy to the host, was recognized. Although there was a critical lack of information on the hindgut microbes, their role in preventing intestinal diseases was suggested. Traditionally, the microbes of the horse hindgut were studied using culture-dependent techniques. More recently, culture-independent methods have been used and provided further insight. This review presents the history and updated knowledge regarding the microbes that live inside the different intestinal ecosystems and which collective genomes compose the hindgut microbiome. In the first section, the quantification and diversity are described for each microbial community as well as the implication of plant fiber degradation and their crucial role for an herbivore host. The microbial communities are presented in chronological order of discovery: due to their large size, protozoa were brought to light as early as 1843 in the horse cecum; in 1897, bacteria were described in the horse intestine; as early as 1910, monoflagellated eukaryotic organisms resembling protozoa were observed in the horse cecum; since then, they have been identified to be zoospores of anaerobic fungi; in 1970, bacteriophage-like particles were recognized in the cecum and colon of pony and horse; and finally, in 1996, archaea were identified in the horse cecum. The second section discusses the variations that can occur between digestive segments or between individuals. The representativeness of the fecal microbiota to the hindgut one is debated, especially as the majority of recent studies conducted on the horse hindgut are in fact focused on the feces, rather than the cecum or colon. Also, the representation of microbiota between individuals is questioned. It has long been suggested in the literature that some ponies or horses that were more susceptible to intestinal diseases may harbor a specific intestinal microbiota. Alternatively, some new studies aim at identifying a core microbiome between all individual equine. A deeper knowledge of the microbiome and its core may allow improvement of nutrition and health, understanding of the onset of digestive diseases, and the development tools for health monitoring and disease prevention.

A comparison of microbial profiles of different regions of the equine hindgut

The microbial profiles of the luminal content of five hindgut segments of one healthy horse were compared with rectal sample to elucidate the effect of anatomical region on bacterial and archaeal community structure and to evaluate the use of faeces as a representative model of large intestine. The qualitative and quantitative changes of the microbial community composition of caecum, right ventral colon, left ventral colon, left dorsal colon, right dorsal colon and faeces were monitored by denaturing gradient gel electrophoresis (DGGE) and real-time PCR using universal primers amplifying the V3 region of 16S rDNA. DGGE fingerprints revealed extensive bacterial as well as archaeal diversity in all studied samples and reflected shifts in the community structure among the caecum, the different segments of the colon and the faeces. Archaeal DGGE pattern of the caecum differed from all the other parts of the hindgut. Microbial profile similarities were found between the left and the right dorsal colon and between the left ventral colon and the faeces. The excised DGGE bands were related to uncultured bacteria and methanogens, the dominant archaeal bands of caecum and faeces were related to Methanocorpusculum sp. Diversity indices indicated the higher diversity for bacteria than for archaea and the dominance of some methanogenic species. The real-time PCR revealed the differences in the microbial quantitative composition of each segment, showing the highest number of total bacteria and archaea in the right ventral colon. The analyses of bacterial and archaeal composition along the one equine hindgut indicate that the faecal sample is similar to that of the left ventral colon, but does not represent the microbial community of the caecum and other parts of the colon.

In vitro analysis of the effect of supplementation with activated charcoal on the equine hindgut.

ABSTRACTThe present study uses in vitro analytical techniques to investigate the effect of activated charcoal on the microbial community of the equine hindgut and the metabolites they produce. Incubations were performed in Wheaton bottles using a 50 ml incubation of a high-energy feed or a low-energy feed, plus bottles with no added food source, together with five levels of activated charcoal (0, 10, 25, 50 or 100 mg per bottle) and fecal samples as a bacterial inoculum. Using this method the rate of gas production, volatile fatty acid and ammonia concentrations, and pH values were analyzed and found to vary depending on the addition of feed, but the activated charcoal had no effect (P>0.05) on any of these. It is already believed that the effect of activated charcoal as a control for toxic substances is at its highest in the foregut or midgut of animals, and therefore should have little impact on the hindgut. The data presented here suggest that if any of the activated charcoal does reach the hindgut, then it has no significant impact on the microbial community present, nor on the major metabolites produced, and so should not have a detrimental effect on the principal site of fermentation in the horse.

Effects of Inulin Chain Length on Fermentation by Equine Fecal Bacteria and Streptococcus bovis

Grass fructans can be fermented by Gram-positive bacteria (e.g., Streptococcus bovis) in the equine hindgut, increasing production of lactic acid and decreasing pH. The degree of polymerization (DP) of fructans has been suggested to influence fermentation rates. The objective of the present study was to determine how DP impacts fermentation by equine fecal bacteria and a model S. bovis. Fecal microbes from three mares were harvested by differential centrifugation, washed, and resuspended in anaerobic media containing short-chain (SC; DP ≤ 10) or long-chain (LC) inulin (DP ≥ 23) from 0% to 2% wt/vol. After 24 hours of incubation (37°C), samples were collected for pH determination. Data were analyzed using the general linear models (GLM) procedure testing for the effect of treatment, concentration, and treatment × concentration (SAS v. 9.3). At all concentrations, the pH was lower in SC fermentations than in LC (P < .0001, in all cases). To determine the effect of DP on S. bovis, cultures were grown (39°C, 9 hours) with 0.1%, 0.5%, or 1.3% SC or LC inulin. Optical density (600 nm) was determined by spectrophotometry. Maximum specific growth rates (μ) were determined by linear regression (2–5 hours). Data were analyzed using the one-way analysis of variance procedure (SAS v. 9.3). The final optical density (600 nm), μ, and yield were higher with SC than with LC fermentation (P < .05). These results indicate that SC inulin may be more available for fermentation than LC inulin by equine fecal bacteria and S. bovis, specifically.

Effects of different amounts of Saccharomyces cerevisiae supplementation on apparent digestibility and faecal parameters in horses fed high-roughage and high-concentrate diets

The aim of this study was to evaluate the influence of different amounts of Saccharomyces cerevisiae (SC) supplementation in high-roughage (HR) and high-concentrate (HC) diets on apparent total tract digestibility (ATTD), faecal microbial profile and faecal pH. Eight gelding miniature horses, about 36-months-old with an average weight of 113±12kg were randomly assigned into a double 4×4 Latin Square. Two distinct experiments of 4 periods each were conducted with SC-supplementation of 0 (control), 10, 20 and 30g (5×108cfu/g), per animal per day. Experiment 1 used a HR diet (70% grass hay, 30% concentrate) and experiment 2 used a HC diet (30% grass hay, 70% concentrate). Each experimental period consisted of 23 days: 15 adaptation days, 5 days for data collection and a 3-day-wash-out interval between periods. Nutrient digestibility was evaluated by total faecal collection for each animal. The cellulolytic and lactic acid bacteria populations in faeces were calculated and faecal pH was measured. In the HR diet, S. cerevisiae supplementation was not associated with any changes in ATTD of nutrients, microbial profile in faeces and did not increase faecal pH values. In the HC diet, only the addition of 20g SC reduced crude protein digestibility when compared with the control group and 30g SC. For the other variables of digestibility the amounts of SC supplementation did not differ from control group. Furthermore, the microbial profile in faeces and faecal pH were not affected by S. cerevisiae supplementation. The present study showed that the S. cerevisiae strain used was not able to induce any changes in the equine hindgut and did not improve the fibrolytic activity with high-roughage and high-concentrate diets.

In vitro simulation of the equine hindgut as a tool to study the influence of phytosterol consumption on the excretion of anabolic–androgenic steroids in horses

Traditionally, steroids other than testosterone are considered to be synthetic, anabolic steroids. Nevertheless, in stallions, it has been shown that β-Bol can originate from naturally present testosterone. Other precursors, including phytosterols from feed, have been put forward to explain the prevalence of low levels of steroids (including β-Bol and ADD) in urine of mares and geldings. However, the possible biotransformation and identification of the precursors has thus far not been investigated in horses. To study the possible endogenous digestive transformation, in vitro simulations of the horse hindgut were set up, using fecal inocula obtained from eight different horses. The functionality of the in vitro model was confirmed by monitoring the formation of short-chain fatty acids and the consumption of amino acids and carbohydrates throughout the digestion process. In vitro digestion samples were analyzed with a validated UHPLC–MS/MS method. The addition of β-Bol gave rise to the formation of ADD (androsta-1,4-diene-3,17-dione) or αT. Upon addition of ADD to the in vitro digestions, the transformation of ADD to β-Bol was observed and this for all eight horses’ inocula, in line with previously obtained in vivo results, again confirming the functionality of the in vitro model. The transformation ratio proved to be inoculum and thus horse dependent. The addition of pure phytosterols (50% β-sitosterol) or phytosterol-rich herbal supplements on the other hand, did not induce the detection of β-Bol, only low concentrations of AED, a testosterone precursor, could be found (0.1ng/mL). As such, the digestive transformation of ADD could be linked to the detection of β-Bol, and the consumption of phytosterols to low concentrations of AED, but there is no direct link between phytosterols and β-Bol.

Inhibition of fructan-fermenting equine faecal bacteria and Streptococcus bovis by hops (Humulus lupulus L.) β-acid.

The goals of this study were to determine if β-acid from hops (Humulus lupulus L.) could be used to control fructan fermentation by equine hindgut micro-organisms, and to verify the antimicrobial mode of action on Streptococcus bovis, which has been implicated in fructan fermentation, hindgut acidosis and pasture-associated laminitis (PAL) in the horse. Suspensions of uncultivated equine faecal micro-organisms produced fermentation acids when inulin (model fructan) was the substrate, but β-acid (i.e. lupulone) concentrations ≥9ppm inhibited lactate production and mitigated the decrease in pH. Inulin-fermenting Strep.bovis was isolated from the β-acid-free suspensions after enrichment with inulin. The isolates were sensitive to β-acid, which decreased the viable number of streptococci in faecal suspensions, as well as growth, lactate production and the intracellular potassium of Strep.bovis in pure culture. These results are consistent with the hypothesis that hops β-acid prevented the growth of fructan-fermenting equine faecal bacteria, and that the mechanism of action was dissipation of the intracellular potassium of Strep.bovis. Bacterial hindgut fermentation of grass fructans has been linked to PAL and other metabolic disorders in horses. Hops β-acid is a potential phytochemical intervention to decrease the growth of bacteria responsible for PAL.

The Genome of the Predominant Equine Lactobacillus Species, Lactobacillus equi, Is Reflective of Its Lifestyle Adaptations to an Herbivorous Host

We report the draft genome sequence of Lactobacillus equi strain DPC6820, isolated from equine feces. L. equi is a predominant Lactobacillus species in the horse hindgut microbiota. An examination of the genome identified genes and enzymes highlighting L. equi adaptations to the herbivorous gastrointestinal tract of the horse, including fructan hydrolases. This genome sequence may help us further understand the microbial ecology of the equine hindgut and the influence lactobacilli have on it.

Comparison of the bacterial community structure within the equine hindgut and faeces using Automated Ribosomal Intergenic Spacer Analysis (ARISA)

The horse’s hindgut bacterial ecosystem has often been studied using faecal samples. However few studies compared both bacterial ecosystems and the validity of using faecal samples may be questionable. Hence, the present study aimed to compare the structure of the equine bacterial community in the hindgut (caecum, right ventral colon) and faeces using a fingerprint technique known as Automated Ribosomal Intergenic Spacer Analysis (ARISA). Two DNA extraction methods were also assessed. Intestinal contents and faeces were sampled 3 h after the morning meal on four adult fistulated horses fed meadow hay and pelleted concentrate. Irrespective of the intestinal segment, Principal Component Analysis of ARISA profiles showed a strong individual effect (P<0.0001). However, across the study, faecal bacterial community structure significantly (P<0.001) differed from those of the caecum and colon, while there was no difference between the two hindgut communities. The use of a QIAamp® DNA Stool Mini kit increased the quality of DNA extracted irrespective of sample type. The differences observed between faecal and hindgut bacterial communities challenge the use of faeces as a representative for hindgut activity. Further investigations are necessary to compare bacterial activity between the hindgut and faeces in order to understand the validity of using faecal samples.

In vitrodegradation of grass fructan by equid gastrointestinal digesta

AbstractLaminitis is a debilitating disease of the equid foot and appears to result from altered metabolism due to insulin resistance (IR) or hindgut acidosis. These disorders may be elicited by high intakes of simple sugars or fructans in pasture grasses. The aim was to determinein vitro(i) whether grass fructan is degraded in the equid foregut and (ii) the fermentation kinetics and organic acid production in cultures containing grasses differing in fructan content incubated with an equid hindgut microbial inoculum. When grass fructan was incubated with equid gastric and small intestinal digesta, polymeric fructan was partially degraded to oligo‐fructan. When incubated with small intestinal digesta, but not gastric digesta, there was limited net loss of total fructan (oligomeric + polymeric fructan). WhenLolium perenneof elevated (PRGH) or moderate (PRGL) fructan content was incubated with an equine hindgut microbial inoculum, the rate of fermentation and accumulation of lactate 6 and 9 h post‐inoculation were significantly greater forPRGHthan forPRGL(P &lt; 0·05). This may suggest that grass fructan ingested by grazing horses is incompletely digested in the foregut and passes into the hindgut, resulting in elevated production of lactate, which may indirectly lead to metabolic disorders such as laminitis.

Technical note: Fatty acids and purine profile of cecum and colon bacteria as indicators of equine microbial metabolism1

The potential use of odd- and branched-chain fatty acids (OBCFA) and purine bases (PB) as microbial markers in the equine hindgut was studied. For this purpose, feed particles adherent bacteria [solid associated bacteria (SAB)] and planktonic bacteria [liquid associated bacteria (PAB)] were isolated from total cecum and colon contents of 8 healthy, crossbred horses (9 ± 3 yr). Horses were fasted for 12 to 15 h before slaughter, and the cecum and colon were identified and clamped in their extremities to avoid mixing of digesta contents. The total cecum or colon contents was collected into thermal containers previously filled with CO2, immediately transported to the laboratory, and subjected to separation of solid and liquid phases to obtain bacterial PAB and SAB pellets from each horse. Overall differences observed were mainly between site of bacterial collection (cecum vs. colon) rather than between type of bacterial population (PAB vs. SAB). Cecal bacteria fraction had greater (P < 0.05) OM, PB, and branched-chain fatty acids (BCFA):odd-chain fatty acids (OFA) ratio but less (P < 0.05) BCFA, OFA, BCFA:PB ratio, and adenine:guanine ratio than colon bacterial biomass. Results indicated that the composition of cecal and colon bacteria is very different from that of similar ecosystems (e.g., rumen). These differences can be a reflection of different growth stages or nutrition of particular populations as well as different bacterial metabolic activities. Results presented herein provide evidence that PB and fatty acids can be used as microbial markers in equine studies.

Analysis of stomach bacterial communities in Australian feral horses

We investigated the community structure of bacteria that populate the stomach of the Brumby, a breed of feral horses from the Australian outback. Using a 16S rRNA gene clone library, we identified 155 clones that were assigned to 26 OTUs based on a 99.0 % sequence identity cutoff. Two OTUs represented 73.5 % of clones, while 18 OTUs were each assigned only a single clone. Four major bacterial types were identified in the Brumby stomach: Lactobacillaceae, Streptococcaceae, Veillonellaceae and Pasteurellaceae. The first three groups, which represented 98.1 % of the Brumby stomach library clones, belonged to the bacterial phylum Firmicutes. We found that 49.7 % of clones were related to bacterial species previously identified in the equine hindgut, and that 44.5 % of clones were related to symbiotic bacterial species identified in the mouth or throat of either horses or other mammals. Our results indicated that the composition of mutualistic bacterial communities of feral horses was consistent with other studies on domestic horses. In addition to bacterial sequences, we also identified four plastid 16S rRNA gene sequences, which may help in further characterizing the type of vegetation consumed by Brumby horses in their natural environment.

Fecal pH and Microbial Populations in Thoroughbred Horses During Transition from Pasture to Concentrate Feeding

Abrupt dietary transitions and feeding of rapidly fermentable diets are common practices in the horse industry and have been associated with digestive and metabolic disorders that can impair the performance of horses. The present study investigated the effect of dietary transition from pasture grazing to confinement with concentrate feeding, and back, on fecal pH and bacterial populations of Streptococcus spp and Lactobacillus spp. Six Thoroughbred fillies, previously grazing perennial ryegrass and white clover-based pasture, were housed in individual stalls and fed an increasing ratio of concentrate to conserved forages for 13 days (days 1-13), followed by an abrupt transition back to only pasture-grazing for 3 days (days 14-16). The concentrate was initially offered at 0.83 kg dry matter (DM)/d and increased to 5 kg DM/d, whereas ensiled alfalfa was initially offered at 0.61 kg DM/d, increasing to 1.22 kg DM/d. Meadow hay was initially offered at 6.73 kg DM/d, decreasing to 1.6 kg DM/d. Fecal specimens were collected daily for determination of pH, and every 2 days for quantitative analysis of Streptococcus spp and Lactobacillus spp. Mean fecal pH increased significantly from pasture baseline values (pH 6.18) during the initial confinement and supplementation on day 1 (6.37), day 2 (6.52), day 3 (6.58), and day 4 (6.43) (standard error of mean [SEM]: 0.056; P < .001). By day 5, mean fecal pH values had decreased to, and remained at, baseline values until the horses returned to pasture, when another increase occurred at day 15 (6.45). Fecal colony forming units (cfu) of Streptococcus spp and Lactobacillus spp increased linearly (r = 0.94; P < .001) from 6.0 and 6.1 log10 cfu/g on day -4, to 7.8 log10 cfu/g on day 14 (SEM: 0.2 P < .001), respectively. Fecal cfu decreased on return to a pasture-only diet (P < .001). In this study, the increment of bacterial populations was associated with a relatively stable fecal pH and highlights the difficulty in identifying the effects of dietary transition on the equine hindgut health, without microbial culture.

A comparison of the microbiome and the metabolome of different regions of the equine hindgut

The microbiome and associated metabolome of faecal samples were compared to those from the caecum and right dorsal colon of horses and ponies euthanised for nonresearch purposes by investigating the microbial population community structure as well as their functional metabolic products. Through the use of 16S rRNA gene dendrograms, the caecum microbiome was shown to cluster separately from the other gut regions. 16S rRNA gene-based quantitative PCR (q-PCR) also demonstrated differences between the caecum and the other gut regions. Metabolites as identified by Fourier transform infrared clustered in a similar way and specific metabolic products (volatile fatty acids and ammonia) also varied by region. Protozoal 18S rDNA concentration and archaeal mcrA gene concentration quantified by q-PCR were found in higher numbers in the colon than the other gut regions. Diversity calculations using Simpson and Shannon-Wiener indices demonstrated higher diversity in the right dorsal colon and faeces than in the caecum. All findings of this study suggest that faecal samples are likely to represent the microbial population of the right dorsal colon to some extent but not that of the caecum, indicating careful consideration is required when planning microbial investigations of the hindgut of the horse.

Dynamic host–bacteria interactions during an acidotic state induction

Mainly due to the lack of time-series data, we know very little about the underlying interactions leading to adverse states in the gut. I therefore investigate the host-bacteria dynamic interactions in a recently published acidotic state induction time-series experiment. Too high levels of fermentation in the hindgut may lead to elevated serum lactate levels, which is a characteristic of the acidotic state. The acidotic state was induced through injection of oligofructose directly into the caecum of five horses, with subsequent temporal measurements of host serum lactate, and a range of caecum bacteria and metabolites. Principal component analysis (PCA) showed that equine hindgut streptococcal species (EHSS) showed the main positive correlation to caecum lactate, and negative correlation to acetate. By dynamic modelling I found that serum lactate interacted with several caecum components, with the main interactions being with caecum lactate and Enterobacteriaceae. Enterobacteriaceae showed a positive interaction with serum lactate, while the interaction between serum and caecum lactate was more complex. The main individual differences in horse serum lactate could be explained by the levels of Enterobacteriaceae and caecum lactate. In conclusion, the dynamic models revealed simple host-bacteria interactions that can explain changes and individual differences in serum lactate.

Influence of Total Sulfur Amino Acids on Growth and Nitrogen Retention of Weanling Horses

was 1.11; however, fold change of the LVM, IM, and CM compared to the JM was 0.59, 0.48, and 0.46, respectively. The fold change of CAT-1 in the IM or the CM compared to the JM was 0.03 and 0.0006, respectively; CAT-1 was not detected in the LVM nor the LDM. The fold change of LAT-2 in the IM compared to the JM was 1.34. The fold change of LAT-2 in the LVM, CM, and LDM com- pared with the JM was 0.18, 0.17, 0.06, respectively. The fold change of LAT-3 in the LDM compared to the JM was 0.20; however, the fold change of LAT-3 in the CM, IM, and LVM compared to the JM was 6.25, 3.30, and 3.13, respectively. mRNA abundance of the AA transporters B 0,+ , LAT-2, and LAT-3 was detected in other segments of the hindgut mucosa of the horse. Amino acid transporter B 0,+ showed highest abundance in the LDM, followed by the JM, LVM, IM, and CM. The CAT-1 transporter was highest in the JM, with low abundance in the IM and CM, and no detection in the colon. Amino acid transporter LAT-2 was highest in the IM compared to the JM, followed by the LVM, CM, and LDM. Amino acid transporter LAT-3 showed the highest abundance in the CM, IM, and LVM, with lowest abundance in the JM. The results imply that the large intestine is not capable of Na+-independent cationic AA uptake via the CAT-1 across the colon, but may be capable of utilizing CAT-1 in the CM and IM as well as the JM. In contrast, the equine hindgut may be as equally capable as the small intestine of Na+-dependent uptake of neutral AA through the B 0,+ transporter, with lower capacity for Na+-independent up- take of neutral AA through the LAT-2 transporter and equal capacity for Na+-independent uptake of neutral AA through the LAT-3 transporter. Relative mRNA abun- dance forall genes of interest reported above willbe further determinedintissuesfromthreeadditionalhorsesforstatis- tical inference on fold change values relative to the JM.