Validation of a deep learning-based image analysis system to diagnose subclinical endometritis in dairy cows.

The assessment of polymorphonuclear leukocyte (PMN) proportions (%) of endometrial samples is the hallmark for subclinical endometritis (SCE) diagnosis. Yet, a non-biased, automated diagnostic method for assessing PMN% in endometrial cytology slides has not been validated so far. We aimed to validate a computer vision software based on deep machine learning to quantify the PMN% in endometrial cytology slides. Uterine cytobrush samples were collected from 116 postpartum Holstein cows. After sampling, each cytobrush was rolled onto three different slides. One slide was stained using Diff-Quick, while a second was stained using Naphthol (golden standard to stain PMN). One single observer evaluated the slides twice at different days under light microscopy. The last slide was stained with a fluorescent dye, and the PMN% were assessed twice by using a fluorescence microscope connected to a smartphone. Fluorescent images were analyzed via the Oculyze Monitoring Uterine Health (MUH) system, which uses a deep learning-based algorithm to identify PMN. Substantial intra-method repeatabilities (via Spearman correlation) were found for Diff-Quick, Naphthol, and Oculyze MUH (r = 0.67 to 0.76). The intra-method agreements (via Kappa value) at ≥1% PMN (κ = 0.44 to 0.47) were lower than at >5 (κ = 0.69 to 0.78) or >10% (κ = 0.67 to 0.85) PMN cut-offs. The inter-method repeatabilities (via Lin's correlation) were also substantial, and values between Diff-Quick and Oculyze MUH, Naphthol and Diff-Quick, and Naphthol and Oculyze MUH were 0.68, 0.69, and 0.77, respectively. The agreements among evaluation methods at ≥1% PMN were weak (κ = 0.06 to 0.28), while it increased at >5 (κ = 0.48 to 0.81) or >10% (κ = 0.50 to 0.65) PMN cut-offs. To conclude, deep learning-based algorithms in endometrial cytology are reliable and useful for simplifying and reducing the diagnosis bias of SCE in dairy cows.

Ethiopian Medicinal Plants Traditionally Used for the Treatment of Cancer; Part 3: Selective Cytotoxic Activity of 22 Plants against Human Cancer Cell Lines.

Medicinal plants have been traditionally used to treat cancer in Ethiopia. However, very few studies have reported the in vitro anticancer activities of medicinal plants that are collected from different agro-ecological zones of Ethiopia. Hence, the main aim of this study was to screen the cytotoxic activities of 80% methanol extracts of 22 plants against human peripheral blood mononuclear cells (PBMCs), as well as human breast (MCF-7), lung (A427), bladder (RT-4), and cervical (SiSo) cancer cell lines. Active extracts were further screened against human large cell lung carcinoma (LCLC-103H), pancreatic cancer (DAN-G), ovarian cancer (A2780), and squamous cell carcinoma of the esophagus (KYSE-70) by using the crystal violet cell proliferation assay, while the vitality of the acute myeloid leukemia (HL-60) and histiocytic lymphoma (U-937) cell lines was monitored in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) microtiter assay. Euphorbia schimperiana, Acokanthera schimperi, Kniphofia foliosa, and Kalanchoe petitiana exhibited potent antiproliferative activity against A427, RT-4, MCF-7, and SiSo cell lines, with IC50 values ranging from 1.85 ± 0.44 to 17.8 ± 2.31 µg/mL. Furthermore, these four extracts also showed potent antiproliferative activities against LCLC-103H, DAN-G, A2780, KYSE-70, HL-60, and U-937 cell lines, with IC50 values ranging from 0.086 to 27.06 ± 10.8 µg/mL. Hence, further studies focusing on bio-assay-guided isolation and structural elucidation of active cytotoxic compounds from these plants are warranted.

The TRPA1 Agonist Cinnamaldehyde Induces the Secretion of HCO3- by the Porcine Colon.

A therapeutic potential of the TRPA1 channel agonist cinnamaldehyde for use in inflammatory bowel disease is emerging, but the mechanisms are unclear. Semi-quantitative qPCR of various parts of the porcine gastrointestinal tract showed that mRNA for TRPA1 was highest in the colonic mucosa. In Ussing chambers, 1 mmol·L-1 cinnamaldehyde induced increases in short circuit current (ΔIsc) and conductance (ΔGt) across the colon that were higher than those across the jejunum or after 1 mmol·L-1 thymol. Lidocaine, amiloride or bumetanide did not change the response. The application of 1 mmol·L-1 quinidine or the bilateral replacement of 120 Na+, 120 Cl- or 25 HCO3- reduced ΔGt, while the removal of Ca2+ enhanced ΔGt with ΔIsc numerically higher. ΔIsc decreased after 0.5 NPPB, 0.01 indometacin and the bilateral replacement of 120 Na+ or 25 HCO3-. The removal of 120 Cl- had no effect. Cinnamaldehyde also activates TRPV3, but comparative measurements involving patch clamp experiments on overexpressing cells demonstrated that much higher concentrations are required. We suggest that cinnamaldehyde stimulates the secretion of HCO3- via apical CFTR and basolateral Na+-HCO3- cotransport, preventing acidosis and damage to the epithelium and the colonic microbiome. Signaling may involve the opening of TRPA1, depolarization of the epithelium and a rise in PGE2 following a lower uptake of prostaglandins via OATP2A1.

TRPV3 and TRPV4 as candidate proteins for intestinal ammonium absorption.

AIM: Absorption of ammonia from the gut has consequences that range from encephalitis in hepatic disease to global climate change induced by nitrogenous excretions from livestock. Since patch clamp data show that certain members of the transient receptor potential (TRP) family are permeable to NH4+ , participation in ammonium efflux was investigated. METHODS: Digesta, mucosa and muscular samples from stomach, duodenum, jejunum, ileum, caecum and colon of pigs were analysed via colourimetry, qPCR, Western blot, immunohistochemistry and Ussing chambers. RESULTS: qPCR data show high duodenal expression of TRPV6. TRPM6 was highest in jejunum and colon, with expression of TRPM7 ubiquitous. TRPM8 and TRPV1 were below detection. TRPV2 was highest in the jejunum but almost non-detectable in the colon. TRPV4 was ubiquitously expressed by mucosal and muscular layers. TRPV3 mRNA was only found in the mucosa of the caecum and colon, organs in which NH4+ was highest (>7 mmol·L-1 ). Immunohistochemically, an apical expression of TRPV3 and TRPV4 could be detected in all tissues, with effects of 2-APB and GSK106790A supporting functional expression. In symmetrical NaCl Ringer, removal of mucosal Ca2+ and Mg2+ increased colonic short circuit current (Isc ) and conductance (Gt ) by 0.18 ± 0.06 µeq·cm-2 ·h-1 and 4.70 ± 0.85 mS·cm-2 (P < .05, N/n = 4/17). Application of mucosal NH4 Cl led to dose-dependent and divalent-sensitive increases in Gt and Isc , with effects highest in the caecum and colon. CONCLUSION: We propose that TRP channels contribute to the intestinal transport of ammonium, with TRPV3 and TRPV4 promising candidate proteins. Pharmacological regulation may be possible.

Erratum to "The GadX regulon affects virulence gene expression and adhesion of porcine enteropathogenic Escherichia coli in vitro" [Vet. Anim. Sci. 3C (2017) 10-17].

[This corrects the article DOI: 10.1016/j.vas.2017.04.001.].

Effects of dietary menthol-rich bioactive lipid compounds on zootechnical traits, blood variables and gastrointestinal function in growing sheep.

BACKGROUND: The present study aimed at investigating the influence of 90% menthol-containing plant bioactive lipid compounds (PBLC, essential oils) on growth performance, blood haematological and biochemical profile, and nutrient absorption in sheep. Twenty-four growing Suffolk sheep were allotted into three dietary treatments: Control (without PBLC), lower dose of PBLC (PBLC-L; 80 mg/d) and higher dose of PBLC (PBLC-H; 160 mg/d). Sheep in all groups were fed meadow hay ad libitum plus 600 g/d of concentrate pellets for 28 d. RESULTS: Average daily gain was not affected by treatment. Feeding of PBLC increased hay and total feed intake per kg body weight (P < 0.05). Counts of total leucocytes, lymphocytes and monocytes were not different among treatments. However, neutrophil count decreased (P < 0.05) in PBLC-H with a similar trend in PBLC-L (P < 0.10). Concentrations of glucose, bilirubin, triglycerides, cholesterol, urea and magnesium in serum were not different among sheep fed different doses of PBLC. However, serum calcium concentration tended to increase in PBLC-H (P < 0.10) and serum concentrations of aspartate & asparagine (P < 0.01) and glutamate & glutamine (P < 0.05) increased linearly with increasing PBLC dose. In ruminal epithelia isolated from the rumen after killing, baseline conductance (G t; P < 0.05) and short-circuit current (I sc; P < 0.01) increased in both PBLC groups. Ruminal uptakes of glucose and methionine in the presence of Na+ were not affected by the dietary PBLC supplementation. In the absence of Na+, however, glucose and methionine uptakes increased (P < 0.05) in PBLC-H. In the jejunum, I sc tended to increase in PBLC-H (P < 0.10), but baseline G t was not affected. Intestinal uptakes of glucose and methionine were not influenced by PBLC in the presence or absence of Na+. CONCLUSION: The results suggest that menthol-rich PBLC increase feed intake, and passive ion and nutrient transport, the latter specifically in the rumen. They also increased serum concentrations of urea precursor amino acids and tended to increase serum calcium concentrations. Future studies will have to show whether some of these findings might be commonly linked to a stimulation of transient receptor potential (TRP) channels in the gastrointestinal tract.

Dietary supplementation of menthol-rich bioactive lipid compounds alters circadian eating behaviour of sheep.

BACKGROUND: Plant bioactive lipid compounds (PBLC), commonly known as essential oils, are increasingly evaluated as feed additives in ruminants due to beneficial effects on animal performance and health; however, there is no study evaluating circadian eating behaviour in ruminants. Altered eating behaviour may be implicated in changes of feed intake in ruminants. Therefore, the present study investigated the influence of menthol-rich PBLC on circadian eating behaviour in 24 growing sheep that were equally divided into three treatments, control (without PBLC), a lower dose (80 mg/d) or a higher dose (160 mg/d) of PBLC. Daily doses of PBLC were supplied with 600 g/d of concentrates fed in three equal portions at 07:00, 11:00 and 15:00 h for 4 weeks, whereas, meadow hay was fed ad libitum. RESULTS: The eating behaviour recorded by an automatic transponder-operated feeding system revealed that daily eating time and feeder visits increased with increasing doses of PBLC. The circadian distribution of eating time and feeder visits (with 1-h resolution) was influenced by the treatment. Eating time during concentrate-offering hours and between concentrate-offering hours increased or tended to increase linearly with greater concentrations of PBLC. Feeder visits did not change significantly during concentrate-offering hours, but were greater in the PBLC groups compared with the control between concentrate-feeding hours. Average length of the longest meals (5th percentile) decreased due to PBLC feeding. Daily feed intake was greater in the PBLC groups than the control. CONCLUSIONS: Menthol-rich PBLC in the applied dose range stimulate circadian eating behaviour, which cannot only be attributed to their presence during concentrate feeding hours, but persist during post-concentrate feeding hours.

Dietary Bioactive Lipid Compounds Rich in Menthol Alter Interactions Among Members of Ruminal Microbiota in Sheep.

This study aimed to investigate the effects of two practically relevant doses of menthol-rich plant bioactive lipid compounds (PBLC) on fermentation, microbial community composition, and their interactions in sheep rumen. Twenty-four growing Suffolk sheep were divided into three treatments and were fed hay ad libitum plus 600 g/d of concentrate containing no PBLC (Control) or PBLC at low dose (80 mg/d; PBLC-L) or high dose (160 mg/d; PBLC-H). After 4 weeks on the diets, samples of ruminal digesta were collected and analyzed for short-chain fatty acid (SCFA), ammonia, and microbiota; microbiota being analyzed in the solid and the liquid digesta fractions separately. Ruminal SCFA and ammonia concentrations were not affected by the PBLC treatments. The microbiota in the solid fraction was more diverse than that in the liquid fraction, and the relative abundance of most taxa differed between these two fractions. In the solid fraction, phylogenetic diversity increased linearly with increased PBLC doses, whereas evenness (lowest in PBLC-L) and Simpson diversity index (greatest in PBLC-H) changed quadratically. In the liquid fraction, however, the PBLC supplementation did not affect any of the microbial diversity measurements. Among phyla, Chloroflexi (highest in PBLC-L) and unclassified_bacteria (lowest in PBLC-L) were altered quadratically by PBLC. Lachnospiraceae, Bacteroidaceae (increased linearly), BS11 (increased in PBLC-L), Christensenellaceae (decreased in PBLC treatments), and Porphyromonadaceae (increased in PBLC treatments) were affected at the family level. Among genera, Butyrivibrio increased linearly in the solid fraction, YRC22 increased linearly in the liquid fraction, whereas Paludibacter increased and BF311 increased linearly with increasing doses of PBLC in both fractions. The PBLC treatments also lowered methanogens within the classes Thermoplasmata and Euryarchaeota. Correlation network analysis revealed positive and negative correlations among many microbial taxa. Differential network analysis showed that PBLC supplementation changed the correlation between some microbial taxa and SCFA. The majority of the predicted functional features were different between the solid and the liquid digesta fractions, whereas the PBLC treatments altered few of the predicted functional gene categories. Overall, dietary PBLC treatments had little influence on the ruminal fermentation and microbiota but affected the associations among some microbial taxa and SCFA.

A comparative study of ammonia transport across ruminal epithelia from Bos indicus crossbreds versus Bos taurus.

Absorption of ammonia from the rumen of cattle decreases nitrogen availability for fermentational protein synthesis, leading to increased competition of cattle with humans for protein and enhancing the release of toxic nitrogenous compounds into the environment. Given that differences in feeding and breeding might induce differences in ruminal ammonia transport, we compared electrophysiological, histological, and molecular biological characteristics of ruminal epithelia of Bos indicus crossbreds (Sahiwal-Mix, SWM) with those of Bos taurus (Holstein-Friesian, HF). As in HF, the stratified cornified epithelium of SWM expressed claudin 1 and 4. Measurements of ammonia flux (HF) and serosal pH (both breeds) suggested that at a mucosal pH of 6.4, net transport primarily occurred as NH4 + . As shown previously for HF, NH4 + induced a concentration-dependent rise in short circuit current (Isc ) in SWM that could be further stimulated by the TRP channel agonist menthol. Relative mRNA expression levels for TRPV3, TRPV4, TRPM6, and TRPM7 were significantly lower in SWM than in HF, with TRPA1 expression near the limit of detection. We conclude that uptake of ammonia from the rumen of both breeds occurs electrogenically as NH4 + with functional and molecular biological evidence pointing towards involvement of TRPV3 and TRPV4.

The GadX regulon affects virulence gene expression and adhesion of porcine enteropathogenic Escherichia coli in vitro.

The ability of enteropathogenic Escherichia coli (EPEC) to express virulence factor genes and develop attaching and effacing (AE) lesions is inhibited in acidic environmental conditions. This inhibition is due to the activation of transcription factor GadX, which upregulates expression of glutamic acid decarboxylase (Gad). Gad, in turn, produces γ-aminobutyric acid (GABA), which was recently shown to have a beneficial effect on the jejunal epithelium in vitro due to increased mucin-1 levels. In the present study, we sought to test whether forced GadX activation/overexpression abolishes virulence associated features of EPEC and provokes increased GABA production. EPEC strains were isolated from diarrheic pigs and submitted to activation of GadX by acidification as well as gadX overexpression via an inducible expression vector plasmid. GABA concentrations in the growth medium, ability for adhesion to porcine intestinal epithelial cells (IPEC-J2) and virulence gene expression were determined. Growth in acidified media led to increased GABA levels, upregulated gadA/B expression and downregulated mRNA synthesis of the bacterial adhesin intimin. EPEC strains transformed with the gadX gene produced 2.1-3.4-fold higher GABA levels than empty-vector controls and completely lost their ability to adhere to IPEC-J2 cells and to induce actin accumulation. We conclude that intensified gadX activation can abolish the ability of EPEC to adhere to the intestinal epithelium by reducing the expression of major virulence genes.

Evidence for the functional involvement of members of the TRP channel family in the uptake of Na(+) and NH4 (+) by the ruminal epithelium.

Large quantities of protein are degraded in the fermentative parts of the gut to ammonia, which is absorbed, detoxified to urea, and excreted, leading to formation of nitrogenous compounds such as N2O that are associated with global warming. In ruminants, channel-mediated uptake of NH4 (+) from the rumen predominates. The molecular identity of these channels remains to be clarified. Ruminal cells and epithelia from cows and sheep were investigated using patch clamp, Ussing chamber, microelectrode techniques, and qPCR. In patch clamp experiments, bovine ruminal epithelial cells expressed a conductance for NH4 (+) that could be blocked in a voltage-dependent manner by divalent cations. In the native epithelium, NH4 (+) depolarized the apical potential, acidified the cytosol and induced a rise in short-circuit current (I sc) that persisted after the removal of Na(+), was blocked by verapamil, enhanced by the removal of divalent cations, and was sensitive to certain transient receptor potential (TRP) channel modulators. Menthol or thymol stimulated the I sc in Na(+) or NH4 (+) containing solutions in a dose-dependent manner and modulated transepithelial Ca(2+) fluxes. On the level of messenger RNA (mRNA), ovine and bovine ruminal epithelium expressed TRPA1, TRPV3, TRPV4, TRPM6, and TRPM7, with any expression of TRPV6 marginal. No bands were detected for TRPV1, TRPV5, or TRPM8. Functional and molecular biological data suggest that the transport of NH4 (+), Na(+), and Ca(2+) across the rumen involves TRP channels, with TRPV3 and TRPA1 emerging as prime candidate genes. TRP channels may also contribute to the transport of NH4 (+) across other epithelia.

Determination of Henry's constant, the dissociation constant, and the buffer capacity of the bicarbonate system in ruminal fluid.

Despite the clinical importance of ruminal acidosis, ruminal buffering continues to be poorly understood. In particular, the constants for the dissociation of H2CO3 and the solubility of CO2 (Henry's constant) have never been stringently determined for ruminal fluid. The pH was measured in parallel directly in the rumen and the reticulum in vivo, and in samples obtained via aspiration from 10 fistulated cows on hay- or concentrate-based diets. The equilibrium constants of the bicarbonate system were measured at 38°C both using the Astrup technique and a newly developed method with titration at 2 levels of partial pressure of CO2 (pCO2; 4.75 and 94.98 kPa), yielding mean values of 0.234 ± 0.005 mmol ∙ L(-1) ∙ kPa(-1) and 6.11 ± 0.02 for Henry's constant and the dissociation constant, respectively (n/n = 31/10). Both reticular pH and the pH of samples measured after removal were more alkalic than those measured in vivo in the rumen (by ΔpH = 0.87 ± 0.04 and 0.26 ± 0.04). The amount of acid or base required to shift the pH of ruminal samples to 6.4 or 5.8 (base excess) differed between the 2 feeding groups. Experimental results are compared with the mathematical predictions of an open 2-buffer Henderson-Hasselbalch equilibrium model. Because pCO2 has pronounced effects on ruminal pH and can decrease rapidly in samples removed from the rumen, introduction of a generally accepted protocol for determining the acid-base status of ruminal fluid with standard levels of pCO2 and measurement of base excess in addition to pH should be considered.

GABA selectively increases mucin-1 expression in isolated pig jejunum.

The inhibitory neurotransmitter GABA (γ-aminobutyric acid) is synthesized by glutamic acid decarboxylase, which is expressed in the central nervous system and in various other tissues including the intestine. Moreover, GABA can be ingested in vegetarian diets or produced by bacterial commensals in the gastrointestinal tract. As previous studies in lung have suggested a link between locally increased GABA availability and mucin 5AC production, the present study sought to test whether the presence or lack of GABA (and its precursor glutamine) has an effect on intestinal mucin expression. Porcine jejunum epithelial preparations were incubated with two different amounts of GABA or glutamine on the mucosal side for 4 h, and changes in the relative gene expression of seven different mucins, enzymes involved in mucin shedding, GABA B receptor, enzymes involved in glutamine/GABA metabolism, glutathione peroxidase 2, and interleukin 10 were examined by quantitative PCR (TaqMan(®) assays). Protein expression of mucin-1 (MUC1) was analyzed by Western blot. On the RNA level, only MUC1 was significantly up-regulated by both GABA concentrations compared with the control. Glutamine-treated groups showed the same trend. On the protein level, all treatment groups showed a significantly higher MUC1 expression than the control group. We conclude that GABA selectively increases the expression of MUC1, a cell surface mucin that prevents the adhesion of microorganisms, because of its size and negative charge, and therefore propose that the well-described positive effects of glutamine on enterocytes and intestinal integrity are partly attributable to effects of its metabolite GABA.

Modulation of sheep ruminal urea transport by ammonia and pH.

Ruminal fermentation products such as short-chain fatty acids (SCFA) and CO2 acutely stimulate urea transport across the ruminal epithelium in vivo, whereas ammonia has inhibitory effects. Uptake and signaling pathways remain obscure. The ruminal expression of SLC14a1 (UT-B) was studied using polymerase chain reaction (PCR). The functional short-term effects of ammonia on cytosolic pH (pHi) and ruminal urea transport across native epithelia were investigated using pH-sensitive microelectrodes and via flux measurements in Ussing chambers. Two variants (UT-B1 and UT-B2) could be fully sequenced from ovine ruminal cDNA. Functionally, transport was passive and modulated by luminal pH in the presence of SCFA and CO2, rising in response to luminal acidification to a peak value at pH 5.8 and dropping with further acidification, resulting in a bell-shaped curve. Presence of ammonia reduced the amplitude, but not the shape of the relationship between urea flux and pH, so that urea flux remained maximal at pH 5.8. Effects of ammonia were concentration dependent, with saturation at 5 mmol/l. Clamping the transepithelial potential altered the inhibitory potential of ammonia on urea flux. Ammonia depolarized the apical membrane and acidified pHi, suggesting that, at physiological pH (< 7), uptake of NH4 (+) into the cytosol may be a key signaling event regulating ruminal urea transport. We conclude that transport of urea across the ruminal epithelium involves proteins subject to rapid modulation by manipulations that alter pHi and the cytosolic concentration of NH4 (+). Implications for epithelial and ruminal homeostasis are discussed.