Pelvic Flexure Research Articles

Gene Expression Analysis before and after the Pelvic Flexure in the Epithelium of the Equine Hindgut.

Previous research demonstrated the distribution of distinct microbial communities in the equine hindgut surrounding the pelvic flexure. The current study evaluated gene expression in epithelial tissues surrounding the pelvic flexure to characterize patterns that might correlate with microbial distribution. Gene expression was determined by analyzing RNA sequence data from the pelvic flexure, the left and right ventral colon, and the left and right dorsal colon. An average of 18,330 genes were expressed across the five tissues sampled. Most of the genes showed some level of expression in all five tissues. Tissue-restricted patterns of expression were also observed. Genes with restricted expression in the left ventral and left dorsal colons have communication, signaling, and regulatory functions that correlate with their known physiology. In contrast, genes expressed exclusively in the pelvic flexure have diverse functions. The ontology of genes differentially expressed between the pelvic flexure and the surrounding tissues was associated with immune functions and signaling processes. Despite being non-significant, these enrichment trends were reinforced by the functions of statistically significant expression differences between tissues of the hindgut. These results provide insight into the physiology of the equine hindgut epithelium that might influence the microbiota and its distribution.

Flowmetry and spectrophotometry can detect reduced intestinal microperfusion in nonsurvivors during equine colic surgery for large intestinal strangulation.

To evaluate the use of laser Doppler flowmetry and spectrophotometry (LDFS) for large intestinal viability assessment in horses with naturally occurring large intestinal strangulations. By use of LDFS, intestinal microperfusion was quantified as tissue oxygen saturation (tSo2), hemoglobin (tHB), and blood flow (tBF) in cases with large colon volvulus and small colon strangulations undergoing colic surgery (n = 17). Intestinal biopsies were taken from the pelvic flexure in all large colon cases and in small colon cases that underwent intraoperative euthanasia. Measurements were compared between survivors and nonsurvivors, and the correlation between LDFS and (immuno)histology was tested (P < .05). The tSo2 and tBF were clearly lower and tHB was higher than previously reported in healthy horses. Following correction of the lesion, pelvic flexure tBF was significantly lower than that of the left ventral colon. Prior to correction of the lesion, microperfusion did not differ between survivors and nonsurvivors, but following release of the strangulation the survivors had a significantly higher tSo2 and tBF compared to the nonsurvivors. There was a negative correlation between tBF and interstitium-to-crypt ratio and a positive correlation between tHB and the histological hemorrhage score. There were no significant correlations between LDFS measurements and inflammatory cell counts or hypoxia-inducible factor-1α immunoreactivity. Large intestinal microperfusion was decreased in nonsurvivors compared to survivors and was correlated with histological injury, suggesting that LDFS has the potential to predict tissue injury and postoperative survival. The use of LDFS as an ancillary diagnostic aid may improve intraoperative viability assessment during colic surgery.

Unusual left colon displacement and clockwise torsion in a 10‐month‐old Arab filly: A case report

In this case report, we present an unusual clockwise torsion of left colon around mesenteric root in a 10‐month‐old Arab filly, highlighting the clinical presentation, diagnostic approach and successful surgical intervention.A 10‐month‐old Arab filly weighing approximately 250 kg was referred with signs of acute abdominal pain. The history revealed anorexia, restlessness and severe abdominal pain that had begun the previous day. The local practitioner had previously administered flunixin meglumine, an analgesic, but it proved ineffective in relieving the pain. Upon physical examination, the filly exhibited sweating, a body temperature of 38.5°C, tachycardia (65 beats per minute) and tachypnea (25 breaths per minute). Due to the severity of the colic and the lack of response to the conservative treatments, surgical intervention was deemed necessary. An exploratory midline celiotomy was performed to evaluate the abdominal organs. During the examination, no obvious primary lesions were identified in the evaluated organs. However, a restriction in exteriorizing the left colon's length was observed. Further examination revealed an unusual clockwise torsion of the left colon that displaced in left to the right side around the mesenteric root; thereby, pelvic flexure was located in the normal anatomical position with a short length.To the best of our knowledge, this is the first reported case of clockwise torsion and an atypical displacement of the left colon in horses. The surgical correction of the displacement was successfully performed. The filly showed improvement post‐surgery and did not exhibit any complications during the recovery period.

Microbiota characterization throughout the digestive tract of horses fed a high-fiber vs. a high-starch diet.

Diet is one of the main factors influencing the intestinal microbiota in horses, yet a systematic characterization of the microbiota along the length of the digestive tract in clinically healthy horses, homogenous for age and breed and receiving a specific diet is lacking. The study used 16S rRNA amplicon sequencing to characterize the microbiota of the intestinal tracts of 19 healthy Bardigiano horses of 14.3 ± 0.7 months of age fed one of two diets. Nine horses received a high-starch diet (HS), and ten horses received a high-fiber diet (HF). After 129 days, the horses were slaughtered, and samples were collected from the different intestinal tract compartments. The microbiota alpha diversity indices were lower in the caecum, pelvic flexure and right dorsal colon of the horses fed the HS diet (False Discovery Rate, FDR < 0.05). The values of beta diversity indicated significant compositional differences between the studied intestinal tract compartments according to the diet received (FDR < 0.05). At the lower taxonomic level (genus or family), the HS diet was associated with a higher relative frequency of Enterobacteriaceae within the small intestine (jejunum and duodenum) (FDR < 0.05). Within the hindgut (caecum and sternal flexure), the HS diet was associated with lower relative frequencies (i.e., a smaller core community) of bacteria belonging to Fibrobacteraceae and Prevotellaceae (FDR < 0.05). Moreover, horses fed the HS diet displayed a higher relative abundance of Streptococcus in the caecum (FDR < 0.05) and Fusobacterium in the sternal flexure (FDR < 0.05), both of which are pathogenic bacteria responsible for inflammation diseases. Samples collected from the pelvic flexure and rectum of horses fed the HS diet showed significantly higher relative frequencies of Succinivibrionaceae (FDR < 0.05) - amylolytic bacteria associated with acidosis. The relative frequencies of the Lachnospiraceae and Ruminococcaceae were lower in the feces collected from the rectum of horses receiving the HS diet vs. HF diet, indicating smaller core communities of these bacteria (FDR < 0.05). Fibrous diets should be promoted to prevent dysbiosis of the microbiota associated with high-starch diet.

Risk factors associated with development of colitis in horses post-exploratory laparotomy.

Diagnosis of colitis has been shown to impact morbidity and mortality in hospitalised horses. There are no studies to date that describe the incidence of infectious colitis after exploratory laparotomy. To investigate risk factors associated with the development of colitis and infectious colitis post-exploratory laparotomy. Retrospective case-control. Medical records of equids admitted from 2011 to 2020 were reviewed. The primary outcome was a diagnosis of colitis following exploratory laparotomy. Bivariable associations between colitis and risk factors were assessed using the 2-sample t-test and Fisher's exact test. All risk factors were subjected to a backward elimination variable reduction algorithm within a logistic regression framework (p-value set to 0.05). Odds ratios and 95% confidence intervals were computed for the final model. A total of 504 equids were included in the study. Forty-two patients (8.3%) were diagnosed with postoperative colitis. Five patients were diagnosed with Salmonella spp. and two with Clostridioides difficile. The odds of postoperative colitis were higher among patients that had pelvic flexure enterotomy (OR = 3.7, 95% CI = 1.7-7.9, p = 0.001), postoperative leukopenia or leukocytosis (OR = 21.2, 95% CI = 9.7-46.7, p < 0.001), or plasma lactate 2.0-4.0 mmol/L (OR = 3.0, 95% CI = 1.3-6.7, p < 0.008). Patients diagnosed with colitis had a longer median length of hospitalisation (9 days; range 2-21) compared with patients without colitis (7 days; range 2-25). Patients with colitis had a survival to discharge rate similar to patients without colitis (95% compared to 93%). Risk factors for infectious colitis could not be determined due to variation in testing protocols in this retrospective study and the low number of positive cases. Colitis as a postoperative complication does not negatively impact survival to discharge but is associated with longer hospitalisation. Pelvic flexure enterotomy, postoperative leukopenia or leukocytosis, and increased plasma lactate were identified as significant risk factors associated with colitis.

Pelvic flexure enterotomy in the horse: A review of current literature

SummaryEnterotomy of the pelvic flexure is commonly performed during colic surgery in the horse to empty the large colon of its contents. A two‐layer closure with absorbable suture material is recommended, but a consensus has not been established. Many different techniques have been described and tested to evaluate surgical time, luminal diameter reduction, bursting pressure and post‐operative complications including adhesion formation. The results of these studies vary within and between enterotomy techniques. The objective of this narrative review was to evaluate and summarise relevant published literature on pelvic flexure enterotomy techniques in the horse.

Use of a pneumatic device for intraluminal enterolith fragmentation in horses

SummaryHorses with enterolithiasis may develop clinical signs of colic due to partial or complete obstruction of the right dorsal, transverse or small colon. Laparotomy followed by enterotomy at the site of obstruction or mobilisation of enteroliths to intestinal segments deemed more appropriate for incision is indicated in these cases. However, surgical removal of enteroliths lodged in nonexteriorisable intestinal segments and/or not amenable to mobilisation may be labour intensive and dangerous. Pneumatic impact (or ballistic) lithotripsy is a potential tool for surgical resolution of simple and complex cases of enterolithiasis in horses. This study describes the use of a pneumatic lithotripsy device for intraluminal enterolith fragmentation in five cases of enterolithiasis. Seven calculi were successfully fragmented. In four cases, fragmentation was achieved at the site of obstruction and fragments exteriorised through an enterotomy incision created for device introduction or advanced towards the rectum and removed. In two cases, enteroliths were manipulated back to the pelvic flexure, fragmented and removed. Findings of this study suggest that intraluminal enterolith fragmentation can be used to avoid long enterotomy incisions and facilitate removal of calculi lodged in bowel segments that cannot be exteriorised. However, it is important to be aware of the risks of enterolith fragmentation in nonexternalisable bowel segments that cannot be visualised for intestinal viability assessment.

Coccygeal vertebral damage in a multiparous mare following head and tail rope‐assisted recovery from general anaesthesia

SummaryAn 18‐year‐old multiparous Thoroughbred mare with a 2‐month‐old foal at foot presented for investigation of colic. A right dorsal displacement of the large colon with a retroflexion of the pelvic flexure was corrected under general anaesthesia and interventional recovery using head and tail ropes was performed. The mare made multiple attempts to stand, using the ropes for support before achieving and maintaining a standing position. The following day, marked swelling around the perineal and vulval region was noted, combined with reduced tail tone. Urination, but not defaecation, had been observed following surgery. Transrectal ultrasonography indicated formation of a haematoma adjacent and ventral to the caudal sacral and first coccygeal vertebrae. Initial radiographic evaluation was suggestive of an oblique fracture of the caudal endplate of the first coccygeal vertebra, which was confirmed on repeat radiography 7 days later. Treatment included cold compresses to reduce swelling, multimodal analgesia, dietary modification to soften faecal consistency and regular manual evacuation of the rectum to avoid impaction. The mare was discharged after 19 days hospitalisation, gradually recovered the ability to defaecate and produced a viable foal the following year without assistance.

Gut health of horses: effects of high fibre vs high starch diet on histological and morphometrical parameters

BackgroundThe conventional feeding management of horses is still characterized by high starch and low fibre diets, which can negatively affect horse’s gastrointestinal health. Thus, the aim of this study was to compare the effects of a high-starch (HS) vs. a high-fibre (HF) diet on gut health in horses. A total of 19 Bardigiano horses destined for slaughter and aged 14.3 ± 0.7 months were randomly allotted to two dietary groups: HS (5 fillies and 4 colts,) and HF group (7 fillies and 3 colts). They received the same first-cut meadow hay but different complementary feeds for 72 days: HS group was fed 8 kg/animal/day of a starch-rich complementary feed while HF group was fed 3.5 kg/animal/day of a fibre‐rich complementary feed. At slaughter, stomachs were separated and washed for the evaluation of the glandular and squamous regions. Also, duodenum, jejunum, ileum, apex of the caecum, sternal flexure, pelvic flexure, right dorsal colon, rectum and liver were excised and submitted to histomorphometrical evaluation.ResultsThe glandular region of HS group presented more severe gastric mucosa lesions compared to the HF group (P = 0.006). Moreover, a statistical tendency (P = 0.060) was found for the squamous region, presenting a higher score in HS than HF diet. Regarding morphometry, in jejunum villus height to crypt depth (Cd) ratio was influenced by sex, being greater in males than in females (P = 0.037) while in ileum Cd depended on interaction between sex and diet, being greater in males of HS group (P = 0.029). Moreover, in the duodenum and right dorsal colon the severity of the inflammation depended on sex (P = 0.024 and 0.050), being greater in females than in males. On the contrary, in the jejunum and in the pelvic flexure, inflammation was influenced by diet, being more severe in HS than in HF group (P = 0.024 and 0.052).ConclusionsThese results suggested that HS diet provoked more severe mucosa lesions in the glandular region of the stomach and a higher inflammation both in the jejunum and pelvic flexure. The present study can represent a starting point for further investigations on gut health in horses.

Suspected lidocaine toxicity and the use of metoclopramide and neostigmine to treat post‐operative small intestinal ileus in a donkey

AbstractA 17‐year‐old donkey underwent exploratory laparotomy to correct a caecal impaction via typhlotomy in combination with pelvic flexure enterotomy. Intravenous lidocaine (3 mg/kg/h; lidocaine 2% solution, BOVA Specials, London, UK) was administered throughout general anaesthesia following a loading dose (1.3 mg/kg, intravenously [IV]). The donkey recovered uneventfully. One hour after recovery, shortly after returning to the stable and commencement of lidocaine, the donkey showed ataxia, muscle fasciculations and tachycardia with progression to recumbency. Lidocaine toxicity was presumed, so the infusion was stopped and the donkey returned to standing. On Day 2 after surgery, post‐operative reflux was voluminous, so a metoclopramide infusion (0.04 mg/kg/h IV; metoclopramide 5 mg/ml injection, Siegfried Hameln, Hameln, Germany) was commenced. On Day 3, post‐operative reflux persisted, so a neostigmine infusion (0.008 mg/kg/h IV; neostigmine methylsulfate injection 2.5 mg/ml, Siegfried Hameln, Hameln, Germany) was also administered. Small intestinal motility improved by Day 5 and post‐operative reflux resolved by Day 6. Both infusions were stopped and food was re‐introduced. There was no further indication of gastrointestinal hypomotility and the donkey was discharged 11 days later.

Acquired large colon strangulating inguinal herniation in two Arabian foals

SummaryLarge colon inguinal hernias are rare, and successful outcome reports are lacking. The present case report describes the clinical presentation, surgical treatment and outcome of two Arabian foals, presented to the hospital at several weeks of age, with colic signs due to inguinal herniation of the large colon. Case 1 describes a right indirect inguinal hernia and Case 2 describes a left direct inguinal hernia, both involving the pelvic flexure. In both cases, the colts underwent surgical treatment while preserving the ipsilateral testis, recovered well and had good long‐term outcomes.

A high-starch vs. high-fibre diet: effects on the gut environment of the different intestinal compartments of the horse digestive tract

BackgroundHorses are often fed high amounts of starch in their diets despite the well-established benefits of a fibre-based diet to promote gut health and animal welfare. The aim of the present study was to compare the effects of two different diets – one based on high amounts of starch (HS) vs. one base on high amounts of fibre (HF) – on specific parameters of the gut environment across different intestinal compartments of the horse digestive tract. To this end differences in the gastrointestinal environment between HS vs. HF fed horses were assessed in terms of dry matter, organic matter and ash content; the particle size distribution and volatile fatty acid composition were also investigated.ResultsNineteen Bardigiano horses of 14.3 ± 0.7 months of age and destined to slaughter were divided into two group pens – one fed with high amounts of starch (HS; n = 9; 43% hay plus 57% starch-rich pelleted feed); vs. fed with high amounts of fibre (HF; n = 10; 70% hay plus 30% fibre-rich pelleted feed). Horses fed HS diet presented a higher dry matter content in the right dorsal colon. Moreover, they showed a higher organic matter and ash content in the sternal flexure, pelvic flexure, right dorsal colon and rectum. In these latter intestinal compartments, horses fed a HS diet also showed a higher proportion of particles retained on an 8 mm sieve and a higher proportion of particles that washed through the finest sieve (< 1 mm). Moreover, the total amounts of volatile fatty acids as well as valeric acid were found to be significantly higher in horses fed the HS vs. HF diet.ConclusionsA high-starch diet causes significant changes in the horse gut environment. We observed an increase in the dry matter content in the right dorsal colon, as well as reduced particle sizes and an increase in the production of valeric acid in all the gut compartments studied. High-starch diets should be avoided in favour of fibre-based diets with the goal of safeguarding gut health in horses.

Clinical features and treatment response to differentiate idiopathic peritonitis from non-strangulating intestinal infarction of the pelvic flexure associated with Strongylus vulgaris infection in the horse

BackgroundPeritonitis in horses secondary to non-strangulating infarction (NSII) has a guarded prognosis, even after intestinal resection. In contrast, horses with idiopathic peritonitis respond well to medical treatment. Affected horses in both cases often show signs of both colic and systemic inflammation, but early diagnosis is crucial for optimal treatment and an accurate prognosis. One cause of NSII is thrombus formation secondary to Strongylus vulgaris larval migration. There has been a documented increase in S. vulgaris prevalence in Sweden since the implementation of selective anthelmintic treatment in 2007, which subsequently could result in a rise in NSII cases. In a retrospective clinical study, medical records from cases diagnosed with NSII of the pelvic flexure or idiopathic peritonitis from three equine referral hospitals in Sweden during 2017–2020 were reviewed. Information including demographic data, relevant medical history, and clinical- and laboratory parameters were obtained from patient records. To facilitate the differentiation between cases of idiopathic peritonitis and cases with confirmed NSII of the pelvic flexure, the aim of the study was to compare clinical and laboratory parameters, clinical progression and initial response to antimicrobial treatment. A secondary aim was to compare survival-rates.ResultsHorses with NSII (n = 20) were significantly more likely to present during the winter months with a poorer response to medical treatment within 48 h. Cases of idiopathic peritonitis (n = 107) had a 100% survival rate with medical treatment, although one case required surgical correction of a colon displacement. In comparison, all confirmed NSII cases were non-responsive to antimicrobial treatment, with a survival rate to discharge of 50% after colon resection. Specific rectal findings and peripheral blood neutropenia were strongly associated with NSII.ConclusionsIn Sweden, idiopathic peritonitis cases still predominate over S. vulgaris associated NSII cases and have an excellent survival rate with antimicrobial treatment. However, horses presenting with septic peritonitis during the winter months with a palpable rectal mass and displaying fever and colic signs beyond 48 h of medical treatment are likely to suffer from NSII of the pelvic flexure and should be considered for abdominal surgery.

Resolución quirúrgica del fecaloma de Mini Horse: reporte de caso

The presence of fecalomas in horses is related to accidental ingestion of substances such as pieces of rope, bedding, plastic and other materials, taking to an ingestion compaction that can cause obstruction and distension of intestinal loops. It is a common condition that can be found in ponies, mini horses and foals, causing an obstruction, most of the time, in the smaller colon. The present work reports a case of colic syndrome in a 2-year-old Mini Horse, with signs of abdominal discomfort for two days. The physical examination revealed high heart and respiratory rate, hypomotility on auscultation, abdominal distension, pale mucous membranes with the presence of a toxemic halo and pyrexia. Analysis of peritoneal fluid revealed an increase in leukocytes. The blood count identified leukopenia. The animal was subjected an exploratory laparotomy and identified the presence of a fecaloma in the proximal third of the smaller colon and a pelvic flexure retroflexion.

A retrospective study of gastrointestinal disorders in a predominantly Austrian leisure horse referral hospital population

SummaryThe aim of our retrospective study was to produce and analyse epidemiological data on gastrointestinal (GI) disorders of Austrian leisure horses. Case records of horses referred to the Equine Clinic of the Veterinary University of Vienna were collected from an electronic hospital information system using its search options. All cases reported to have suffered a GI disorder between March 2003 and February 2015 were selected. In this period of 4314 days, data of 1833 GI cases, mostly accompanied by colic signs, were analysed. The vast majority of our GI cases appeared in the abdominal parts of the GI tract 96% (1760/1833) while just 1.3% (24/1833) could be definitively attributed to oesophageal disorders, whereas in 2.7% (49/1833) of the cases a definitive diagnosis could not be made. The highest prevalence of GI disorders was seen in July and October/November. A breakdown of figures for cases clustered under the colic syndrome showed that colon impactions in left ventral large and/or pelvic flexure (35%) were the major problem followed by gastric disorders (18%), spasmodic colic (9%), enteritis (7%), caecal tympany (6%); caecal impaction (4%), colitis (3%), abnormal anatomical positions of large colon including complete and partial strangulation/volvulus, nephrosplenic entrapment of the left ventral and dorsal colon and right dorsal displacement of the large colon (3%) and small intestinal volvulus (2.7%). In 12% of the cases, the cause of the abdominal discomfort was likely idiopathic paralytic ileus. The searching tool of the electronic patient data also brought up cases with malabsorbtion/IBD syndrome and GI ruptures, both these contributed less than 1% of the referred GI cases. Results suggested that the causes of colic are diverse, but the major reasons for referred cases of colic in the east Austrian horse population are large colon impaction or displacements. Seasonal distribution and specific causes of gastrointestinal disorders should be considered essential components of the initial clinical assessment of horses presenting with colic.

33 Profiling miRNA transcripts in the equine hindgut by quantitative PCR

Gastrointestinal (GI) homeostasis in a horse results from dynamic interactions between a horse's gut physiology and the microbes in the various compartments. MicroRNAs (miRNA), single-stranded 20–22 basepair RNA molecules involved in post-transcriptional control of gene expression, have been implicated as an essential mechanism for control of gastrointestinal physiology and communication between host and microbe. Researchers have described how host-derived miRNAs influence microbial communities' composition in the GI. We have recently described how the pelvic flexure separates distinct microbial populations in the equine hindgut and subsequently have wondered if equine miRNA transcripts expressed by GI tissues could have a role in maintaining this separation. Investigations to characterize miRNAs' expression profile and other non-coding RNAs in the equine GI tract are limited. To address this, it is critical that we first know something about the expression of non-coding transcripts in the equine GI. This study investigated miRNA expression in tissues of the hindgut surrounding the pelvic flexure. RNA was isolated from the intestinal epithelium of 3 4-year-old American Quarter Horses collected from the right and left ventral colon (VC), pelvic flexure (PF), and right and left dorsal colon (DC). The miRNA transcripts were reverse transcribed using the miScript II RT kit, and relative abundance was quantified using the miScript SYBR Green PCR kit and primer sets for 286 annotated mature equine miRNAs. Biological replicates were pooled, and differential expression was determined following normalization by the delta Ct = Ct(target) – Ct(sample) method. A total of 230 miRNA transcripts were expressed in at least one GI location, with 60 expressed in all 5 locations. Twenty-eight transcripts had expression restricted to a single site, 5 of which were only expressed in the pelvic flexure. Additionally, 42 transcripts have patterns of expression across a subset of anatomical locations. Notably, 10 transcripts were expressed in the VC and PF, 5 transcripts were expressed in the DC and PF, and 15 transcripts were expressed in the VC and DC but not PF. Pathway and target analysis for the miRNAs with compartment restricted expression was performed using DIANA TOOLS. Of note, miRNA transcripts expressed only in the PF were predicted to target genes in the Hippo signaling pathway, which is associated with the regulation of intestinal tissue proliferation and homeostasis. Further investigation of miRNA expression in the equine hindgut will improve our understanding of gastrointestinal homeostasis's regulatory processes.

138 Expression profiling of the ventral colon, pelvic flexure, and dorsal colon epithelium by RNA-seq

The equine large intestine is essential for nutrient absorption and as a location for microbial digestion. The pelvic flexure is an important anatomical landmark that divides distinct microbial communities in the equine hindgut. Previous work from our group showed that several taxonomic groups were restricted to the cecum and ventral colon. Since no physical barrier separates these compartments from the more distal areas, we became curious about alternative mechanisms that support the segregation of different microbial groups. Physiological differences in each compartment likely result in different requirements from the microbiota at each location. This study evaluated gene expression in the mucosal and submucosal layers of the right and left ventral colon (VC), pelvic flexure (PF), and right and left dorsal colon (DC). Samples were collected from 3 4-year-old American Quarter Horses, and total RNA was isolated. Single-end libraries were prepared using the NEBNextⓇ Ultra II Directional RNA library kit and sequenced on an IlluminaⓇ NextSeq 550 platform. The sequencing generated an average of 22.5 million reads per sample (range 14.5 to 30.9). Gene expression analysis was performed using the Galaxy platform by aligning quality trimmed sequence reads to EquCab3 using the HISAT2, assembling transcripts with StringTie, and quantifying transcript abundance with Salmon. DESeq2 was used to assess differential expression between the intestinal compartments. Expression (present in 2 of 3 biological replicates) was detected in at least one intestinal compartment for 21,987 transcripts and ranging from 14,766 to 15,134 transcripts per sample. A total of 13,944 transcripts were detected in all 5 areas, and 739 transcripts demonstrated a tissue-restricted expression pattern (detected above threshold in only on anatomical location). Gene ontology was used to identify the functional roles of transcripts expressed in all or only one hindgut compartment. Transcripts expressed in all tissues were associated with internal cell metabolic processes, which are common despite cell type. In contrast, transcripts with tissue-restricted expression patterns were associated with cellular differentiation and secretion processes. Matrix metalloproteinases, transcription factors, and integrin proteins were identified as differentially expressed (P < 0.05) in the PF compared with the surrounding VC and DC compartments. Continued investigation of these transcripts in the equine hindgut will enhance our understanding of digestive physiology and support future studies of interactions between host and microbiota.

The pelvic flexure separates distinct microbial communities in the equine hindgut

As hindgut fermenters, horses are especially dependent on the microbiota residing in their cecum and large intestines. Interactions between these microbial populations and the horse are critical for maintaining gut homeostasis, which supports proper digestion. The current project was motivated to determine if any features of the fecal microbiota are informative of the microbial communities from the cecum, ventral colon, or dorsal colon. Digesta from the cecum, ventral colon, dorsal colon and feces were collected from 6 yearling miniature horses. Microbial DNA was isolated and the microbiota from each sample was characterized by profiling the V4 region of the 16S rRNA. Principal coordinate analysis of the beta diversity results revealed significant (p = 0.0001; F = 5.2393) similarities between the microbial populations from cecal and ventral colon and the dorsal colon and fecal samples, however, there was little overlap between the proximal and distal ends of the hindgut. These distinct population structures observed in our results coincide with the pelvic flexure, which itself separates intestinal compartments with distinct roles in digestive physiology. An indicator species analysis confirmed the population differences, supported by the identification of several microbial families characteristic of the compartments upstream of the pelvic flexure that were not represented following it. Our data suggest that the fecal microbiota is not informative of the proximal hindgut but can provide insight into communities of the distal compartments. Further, our results suggest that the pelvic flexure might be an important anatomical landmark relative to the microbial communities in the equine large intestine.

Expression of Proteinase-Activated Receptor 2 During Colon Volvulus in the Horse.

Large colon volvulus in horses is associated with a poor prognosis, especially when ischemic-reperfusion injury of the affected intestinal tract develops. Proteinase-activated receptor 2 (PAR2) plays an important role in the pathogenesis of inflammation in the gastrointestinal tract. The aim of this study was to evaluate the distribution and expression of PAR2 in colonic pelvic flexure of horses spontaneously affected by large colon volvulus (CVH group). Eight horses admitted for severe abdominal colon volvolus and which underwent surgery were included. Colon samples were collected after enterotomy. Data previously obtained from healthy horses were used as a control group. Histologic evaluation was carried out to grade the severity of the colon lesions. Immunofluorescence, western blot and quantitative polymerase chain reaction (RT-qPCR) were carried out on colon samples to evaluate PAR2 expression. In addition, the transcriptional profile of cytokines and chemokines was evaluated using RT2 Profiler™ PCR Array Horse Cytokines & Chemokines. Three out of the eight patients were euthanised due to clinical deterioration. Immunostaining for PAR2 was observed in the enterocytes, intestinal glands and neurons of the submucosal and myenteric plexi. In the CVH horses, the expression of PAR2 mesenger RNA (mRNA) did not differ significantly from that of the healthy animals; western blots of the mucosa of the colon tracts showed a clear band of the expected molecular weight for PAR2 (~44 kDa) and a band smaller than the expected molecular weight for PAR2 (25kDa), suggesting its activation. The gene expressions for C-X-C motif ligand 1 (CXCL1); interleukin 8 (IL8), macrophage inflammatory protein 2 beta (MIP-2BETA) were upregulated in the colic horses as compared with the colons of the healthy horses. Therefore, in the present study, the expression and activation of PAR2 in the colons of horses in the presence of an inflammatory reaction like that occurring in those with spontaneous colon volvulus was confirmed.

Comparison of histomorphometric characteristics of dorsal colon and pelvic flexure biopsy specimens obtained from horses with large colon volvulus that underwent resection.

To determine the degree of histomorphometric damage in dorsal colon and pelvic flexure biopsy specimens (DCBSs and PFBSs, respectively) obtained from horses with large colon volvulus (LCV) and assess the accuracy of predicting short-term outcome for those horses on the basis of DCBS or PFBS characteristics. 18 horses with ≥ 360° LCV that underwent large colon resection. During surgery, biopsy specimens from the dorsal colon resection site and the pelvic flexure (when available) were collected from each horse. Interstitial-to-crypt (I:C) ratio (ratio of the lamina propria space occupied by the interstitium to that occupied by crypts), hemorrhage within the lamina propria (mucosal hemorrhage score [MHS] from 0 to 4), and percentage losses of glandular and luminal epithelium were determined in paired biopsy specimens and compared to determine optimal cutoff values for calculating the accuracy of DCBS and PFBS characteristics to predict short-term outcome (survival or nonsurvival after recovery from surgery). Paired biopsy specimens were obtained from 17 of the 18 horses. The I:C ratio and percentage glandular epithelial loss differed between DCBSs and PFBSs. For DCBSs, an I:C ratio ≥ 0.9 and MHS ≥ 3 each predicted patient nonsurvival with 77.8% accuracy. For PFBSs, an I:C ratio ≥ I and MHS ≥ 3 predicted patient nonsurvival with 70.6% and 82.4% accuracy, respectively. Although different, histomorphometric measurements for either DCBSs or PFBSs could be used to accurately predict short-term outcome for horses with LCV that underwent large colon resection, and arguably PFBSs are easier to collect.