Charlotte Deschamps

Delphine Humbert

Jürgen Zentek

Sylvain Denis

Nathalie Priymenko

Emmanuelle Apper

Stéphanie Blanquet-Diot
email: stephanie.blanquet@uca.fr

From Chihuahua to Saint-Bernard: how did digestion and microbiota evolve with dog sizes

Keywords: canine, digestive physiology, gut microbiota, petfood, veterinary products

scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Canis lupus familiaris, also known as the domesticated dogs, belong to the Canidae family like the grey wolf (Canis lupus) and the dingo, a domestic dog returned to the wild. Descending from the grey wolf, dogs might have been the first animal domesticated by humans around 20.000 to 40.000 years ago [START_REF] Botigué | Ancient European dog genomes reveal continuity since the Early Neolithic[END_REF]. Dogs were initially strict carnivores, but during the agricultural revolution they probably acquired the ability to digest starch and became facultative carnivores. Genes playing key roles in starch digestion (i.e. encoding for pancreatic amylase, membrane-bound intestinal maltase-glucoamylase and gene involved in glucose uptake) were selected during dog domestication [START_REF] Axelsson | The genomic signature of dog domestication reveals adaptation to a starch-rich diet[END_REF]. Depending on their usefulness for humans, the Canis lupus familiaris subspecies have differentiated slowly, with the development of new canine species designated for specific tasks, such as herd protection (Mastiff), hunting (Pointer), cold hardiness (Siberian husky) or companion (Pekinese). Nowadays, the canine species includes approximatively 400 breeds with high size variability and weight ranging from 1 kg for a Chihuahua to 100 kg for a Saint-Bernard [START_REF] Grandjean | Encyclopédie du chien Royal Canin[END_REF]. Dogs now occupy a full place in many families. Their health and well-being are therefore of paramount importance to their owners, to the extent that 7 % of

Ivyspring International Publisher

French dogs have their own health insurance against 30 % of dogs homed in the United Kingdom and 80 % of dogs homed in Sweden (SantéVet/Ipsos, 2018). Digestion, a complex process involving many physicochemical, mechanical, and microbial mechanisms, is a key parameter in dog health. In particular, gut microbiota and its involvement in canine nutrition and health have increasingly been studied during the last decade. Developing new food or pharma products needs to consider all these multi-faceted aspects of canine digestion, to answer important questions such as food digestibility, micronutrient bioaccessibility, probiotic survival and activity, or drug release and absorption in the gut. Petfood manufacturers and veterinary companies aim to develop personalized ranges adapted to size (e.g. long-term growth of large breeds puppies, poor digestive tolerance and gastric dilatation volvulus for large dogs) or to address certain breed predispositions such as obesity in Labrador Retrievers or enteropathies in Terriers [START_REF] Boillat | Assessment of the relationship between body weight and gastrointestinal transit times measured by use of a wireless motility capsule system in dogs[END_REF][START_REF] German | Cytokine mRNA expression in mucosal biopsies from German shepherd dogs with small intestinal enteropathies[END_REF][START_REF] Osto | Translational value of animal models of obesity-Focus on dogs and cats[END_REF][7]. Nevertheless, the impact of dog size or breed on digestive parameters remains poorly described despite its full interest in canine nutrition and health.

This review paper provides for the first time an exhaustive survey of the literature on the impact of body size on dog's digestive physiology, in the entire gut from mouth to colon and feces, by gathering digestive anatomy, physicochemical parameters and gut microbiota variations. Relevant studies were identified, and information extracted regarding involved dogs (i.e. number of dogs, age, weight, breed, sex, reproduction state, living environment), nutrition (i.e. food type, feeding frequency, food's principal components) and analysis methods. Only in vivo studies on healthy adult dogs, fed with dry food or ingesting water were included. Here, canine body sizes were classified into three groups: "small" under 10 kg, "medium" between 10-30 kg and "large" up to 30 kg according to usual practice of main petfood suppliers. Then, the selected data were analyzed according to dog sizes and clarified through graphical representations to highlight a potential size effect on digestive parameters.

General observations of canine digestion and associated organs

External morphological differences observed between extreme dog sizes such as Chihuahua and Saint-Bernard obviously reveal internal anatomical modifications. The canine mature digestive tract length can represent 2.8 % to 7 % of the total body weight (BW), in a 60 kg and a 5 kg dog, respectively [START_REF] Weber | Influence of size on the dog's digestive function[END_REF]. Since gastrointestinal tract (GIT) absolute length (in centimeters) is a reflect of dog height at the shoulder with a 6:1 ratio [START_REF] Morris | Comparative aspects of nutrition and metabolism of dogs and cats[END_REF], it leads to the question: how does the size of dog impact digestive anatomy? Canine digestive anatomy is adapted to their facultative carnivorous diet (i.e. high-protein and high-fat diet) with a short and simple digestive tract. Digestion starts in the mouth with mastication process, helped by saliva. After swallowing, food boluses are transported through the esophagus into the stomach which is a J-shaped organ of glandular type, characterized by three anatomical compartments (i.e. fundus, corps and antrum) leading to the pylorus sphincter [START_REF] Kararli | Comparison of the gastrointestinal anatomy, physiology, and biochemistry of humans and commonly used laboratory animals[END_REF]. Canine gastric mucosal cells secrete hydrochloric acid (HCl), pepsin and lipase, which makes stomach essential in protein and lipid digestion. Canine stomach has a high dilatation capacity, varying from a maximal volume of 0.5 L for small dogs to 8 L in large dogs, which corresponds to the extreme quantity of food that a dog can ingest [START_REF] Kararli | Comparison of the gastrointestinal anatomy, physiology, and biochemistry of humans and commonly used laboratory animals[END_REF]. Digestion continues along the small intestine which is distributed as 10 % length for duodenum, 85 % for jejunum and 5 % for ileum [START_REF] Kararli | Comparison of the gastrointestinal anatomy, physiology, and biochemistry of humans and commonly used laboratory animals[END_REF][START_REF] Albors | Morphometry of the canine intestine with reference to the use of double balloon endoscopy[END_REF]. Small intestine length measured post mortem is positively correlated (Pearson correlation of 0.672) to canine BW (from 240 cm for a 5 kg to 640 cm for a 33 kg dog), as well as small intestine width (weaker correlation, R 2 = 0.36) [START_REF] Albors | Morphometry of the canine intestine with reference to the use of double balloon endoscopy[END_REF]. Canine small intestine, together with peripheral organs such as pancreas and liver, have a key role in canine digestion process. Pancreas produces pancreatic juice delivered into duodenum and associated with protein, carbohydrate and lipid digestion. Liver, coupled with gallbladder, has a central role in lipid digestion through bile acid (BA) production and induction of increased intestinal peristalsis [START_REF]Les enterpathies excudatives chez le chien: actualités, diagnostiques et analyse retrospective de series de cas cliniques[END_REF]. Small intestine is also a central player in nutrient absorption, allowed by the presence of microvilli at the surface of enterocytes. When measuring intestinal wall thickness at different levels of the GIT (descending duodenum, proximal and distal jejunum, proximal and distal ileum), higher values were observed for male dogs compared to female (except for distal ileum) but no correlation was found with dog sizes whatever the intestinal compartment [START_REF] Sarriá | Morphometric study of the layers of the canine small intestine at five sampling sites[END_REF]. Regarding small intestinal villus length, an old study from 1978 showed no correlation between dog weight and mucosal dimensions [START_REF] Hart | The quantitative assessment of normal canine small intestinal mucosa[END_REF]. In adult dogs from various sizes, duodenal villus length was 722 ± 170 µm [START_REF] Washabau | Endoscopic, biopsy, and histopathologic guidelines for the evaluation of gastrointestinal inflammation in companion animals[END_REF]. Jejunal villi were longer in small dogs like Pomeranian and Fox Terrier (900 μm) than in medium ones such as Newfoundland (500 µm) [START_REF] Baum | Age-related histomorphologic changes in the canine gastrointestinal tract: a histologic and immunohistologic study[END_REF]. Lastly, ileal villus length was measured in medium size Greyhound female and values around 796-823 µm were found [START_REF] Feldman | Effects of oral versus intravenous nutrition on intestinal adaptation after small bowel resection in the dog[END_REF]. Canine large intestine measures around 20-80 cm with 2-3 cm diameter in medium dogs [START_REF] Kararli | Comparison of the gastrointestinal anatomy, physiology, and biochemistry of humans and commonly used laboratory animals[END_REF]. The three parts of the canine colon (i.e. ascending, transverse and descending) are not so well defined when compared to humans, with the particularity to be non-sacculated and devoid of sigmoid colon [START_REF] Kararli | Comparison of the gastrointestinal anatomy, physiology, and biochemistry of humans and commonly used laboratory animals[END_REF]. Ascending colon represents in medium size dog 20% of the colon length, while transverse and descending correspond to 30 % and 50 %, respectively. The two first parts are used for transport, electrolyte and water modification as well as for bacterial fermentation and storage areas, while descending colon mainly functions as conduit ending with rectum. Canine large intestine is involved in water and electrolyte absorption but also degradation of residual nutrients thanks to the fermentation activity of resident microorganisms called gut microbiota. Large intestine total length appears to vary according to dog's BW, from 32 cm for Miniature Poodles to 99 cm for Great Danes [START_REF] Weber | Digestive sensitivity varies according to size of dogs: a review[END_REF]. Volume and surface are also increased from Miniature Poodle to Great Dane (volume of 92 versus 2106 cm 3 , surface of 191 versus 1612 cm 2 ). As the large intestine length increases with BW, the same positive relation is observed for absorption surface with a higher number of villi in large compared to small dogs [START_REF] Weber | Digestive sensitivity varies according to size of dogs: a review[END_REF]. Colonic crypts length was around 500-600 µm but without correlation with dog size [START_REF] Baum | Age-related histomorphologic changes in the canine gastrointestinal tract: a histologic and immunohistologic study[END_REF]. To conclude, scarce anatomy data (only five publications) evidenced morphological differences depending on dog's BW (mainly related to the colonic compartment), even if important parameters have not been evaluated such as gastric wall thickness, intestinal microvilli characteristics (i.e. length or number) or peripheral organs anatomy and functions. Variations in digestive anatomy can obviously affect physicochemical parameters such as pH, digestive secretions and transit time, and consequently gut microbiota.

Methods of literature research

Our literature search was performed using PubMed (https://pubmed.ncbi.nlm.nih.gov) and Google Scholar (https://scholar.google.fr) with the key words "dog" OR "canine" AND "stomach", "small intestine", "intestine", "duodenum", "jejunum", "ileum", "ileal", "colonic", "large intestine", "rectum", "feces", AND "anatomy", "digestion", "pH", "enzyme", "digestive secretion", "digestibility", "permeability", "absorption", "microbiota", "bile acids", "transit time", "fatty acids", "fermentation", "gas", "mucus" in all available years. The online database search was last performed in January 2022 on titles, abstracts and key words including original articles, reviews, thesis, and books. Relevant studies were identified after consultation of the main text, figures, and supplementary materials. Information regarding involved dogs (i.e. number of dogs, age, weight, breed, sex, reproduction state, living environment), diet (i.e. type of food, feeding frequency, composition of food), health (i.e. healthy dogs only) and analysis methods were collected. Only in vivo studies on adult dogs, fed with dry food or ingesting water were included in the literature survey.

We found a total of 163 studies, including 87 providing information on a single dog size, with only small dogs involved in 7 publications, only medium dogs in 71 and only large dogs in 9 (Figure S1). The three dog sizes (i.e. small, medium and large dogs) were compared together in 8 additional studies. In the remaining 68 studies, 45 integrated dogs without specifying their characteristics and other 22 included different sizes of dogs but didn't discriminate them in their analysis (both classed in the "unclassified" group). Concerning publication date, 40 studies were performed over 30 years, 76 studies have been done between 5 and 30 years ago and 47 were performed in the last 5 years. Only 10 studies were directly targeting the influence of dog size on canine digestive physiology.

Impact of body size on digestive physicochemical parameters

Gastrointestinal pH

Gastrointestinal pH changes along the dog digestive tract (Fig. 1, Table S1). Mean salivary pH of medium dogs is around 7.3-7.8 and quickly decreases by 0.5 point with a stimulation using a piece of solid sugar on the tongue [START_REF] Smeets-Peeters | A review of the physiology of the canine digestive tract related to the development of in vitro systems[END_REF][START_REF] Durand | Entéropathies exsudatives chez le chien: proposition d'une stratégie thérapeutique diététique et médiacale[END_REF][START_REF] Larmas | Studies on dog saliva. I. Some physico-chemical characteristics[END_REF]. In the stomach, the arrival of food bolus stimulates HCl production. This compartment shows the lowest pH value along the GIT, allowing dogs to partially digest bones [START_REF] De Godoy | In vitro disappearance characteristics of selected categories of commercially available dog treats[END_REF] and putrescent meat and largely depends on feed status. However, due to the paucity of data, it remains difficult to know how BW affects gastric, small intestinal and colonic pH (Fig. 1, Table S1). To date, gastric pH has not been assessed in small and large dogs [START_REF] Lui | Comparison of gastrointestinal pH in dogs and humans: implications on the use of the beagle dog as a model for oral absorption in humans[END_REF][START_REF] Yamada | Measurement of gastric pH during digestion of a solid meal in dogs[END_REF][START_REF] Akimoto | Gastric pH profiles of beagle dogs and their use as an alternative to human testing[END_REF][START_REF] Sagawa | Fed and fasted gastric pH and gastric residence time in conscious beagle dogs[END_REF]. Regarding medium dogs under fasted conditions, mean gastric pH of Beagles is around 1.5 (range 0.9-2.5), punctuated by occasional pH spikes with high frequency changes due to inter-individual variability [START_REF] Mahar | Gastric pH and Gastric Residence Time in Fasted and Fed Conscious Beagle Dogs using the Bravo® pH System[END_REF]. Those values measured in laboratory animals are in accordance with pH found in mixed-breed owner dogs [START_REF] Garcia-Mazcorro | Abundance and short-term temporal variability of fecal microbiota in healthy dogs[END_REF]. Small intestinal pH increases to value close to the neutrality because of the buffering capacity of pancreatic juice enriched in bicarbonate ions and bile [START_REF] Kararli | Comparison of the gastrointestinal anatomy, physiology, and biochemistry of humans and commonly used laboratory animals[END_REF]. It also increases from the proximal to the distal parts, from 6.5 to 8 in medium size dogs [START_REF] Koziolek | Characterization of the GI transit conditions in Beagle dogs with a telemetric motility capsule[END_REF]. To date, there is no available study that investigates the influence of the dog size on duodenal and ileal pH [START_REF] Martinez | Applying the biopharmaceutics classification system to veterinary pharmaceutical products Part II. Physiological considerations[END_REF]. The few studies investigating the canine jejunal pH measured a mean pH of 6.8 and 6.0 for medium and large dogs, respectively [START_REF] Mentula | Comparison between cultured small-intestinal and fecal microbiotas in beagle dogs[END_REF][START_REF] Kalantzi | Canine Intestinal Contents vs. Simulated Media for the Assessment of Solubility of Two Weak Bases in the Human Small Intestinal Contents[END_REF]. Only few studies investigated colonic pH using colonic cannula or wireless capsules, and once new, most of them do not discriminate dogs in terms of BW. Colonic pH is more acidic than the small intestine one, with mean values of 5-6.5 and 6.2, respectively for medium and large dogs, whereas there is no data concerning small dogs [START_REF] Koziolek | Characterization of the GI transit conditions in Beagle dogs with a telemetric motility capsule[END_REF][START_REF] Smith | Observations on the flora of the alimentary tract of animals and factors affecting its composition[END_REF][START_REF] Lidbury | Assessment of the Variation Associated with Repeated Measurement of Gastrointestinal Transit Times and Assessment of the Effect of Oral Ranitidine on Gastrointestinal Transit Times Using a Wireless Motility Capsule System in Dogs[END_REF][START_REF] Warrit | Minimally invasive wireless motility capsule to study canine gastrointestinal motility and pH[END_REF]. Most of the time, colonic pH is estimated using faeces and there is no information on how pH varies depending on colonic sections. The average canine faecal pH values are in accordance with colonic pH, mainly around 6.4-6.6, as observed in Fig. 1. For small dog group, three studies used faeces of 43 dogs and pH values vary weakly from 6.4 to 6.8 [START_REF] Weber | Effect of size on electrolyte apparent absorption rates and fermentative activity in dogs[END_REF][START_REF] Beloshapka | Apparent total tract macronutrient digestibility, fecal characteristics, and fecal fermentative end-product concentrations of healthy adult dogs fed bioprocessed soy protein[END_REF][START_REF] Igarashi | Analysis of fecal short chain fatty acid concentration in miniature dachshunds with inflammatory colorectal polyps[END_REF]. There are also plenty of data on the faecal pH of medium (more than 121 dogs) and large (18 dogs) size dogs, with a pH range of 6-6.9 and 5.6-6.5, respectively [START_REF] Cutrignelli | In vitro fermentation characteristics of different carbohydrate sources in two dog breeds (German shepherd and Neapolitan mastiff)[END_REF][START_REF] Sandri | Raw meat-based diet influences faecal microbiome and end products of fermentation in healthy dogs[END_REF][START_REF] Eisenhauer | Effects of Brewer's spent grain and carrot pomace on digestibility, fecal microbiota, and fecal and urinary metabolites in dogs fed low-or high-protein diets1[END_REF][START_REF] Nogueira | Dietary supplementation of a fiber-prebiotic and saccharin-eugenol blend in extruded diets fed to dogs[END_REF]. This is an accordance with some studies reporting that colonic and faecal pH of large dogs are more acidic than that of other size dogs fed with the same diet [START_REF] Weber | Digestive sensitivity varies according to size of dogs: a review[END_REF][START_REF] Oswald | Canine gastrointestinal physiology: Breeds variations that can influence drug absorption[END_REF].

Digestive secretion

Enzymes. First digestion step occurs in the oral cavity with salivary enzymes (Table S2). Numerous recent studies measured amylase activity in saliva of healthy dogs [START_REF] Contreras-Aguilar | Detection and measurement of alpha-amylase in canine saliva and changes after an experimentally induced sympathetic activation[END_REF][START_REF] Iacopetti | Salivary pH, calcium, phosphorus and selected enzymes in healthy dogs: a pilot study[END_REF][START_REF] Sanguansermsri | Comparative proteomic study of dog and human saliva[END_REF][START_REF] Tecles | Evaluation of adenosine deaminase in saliva and serum, and salivary α-amylase, in canine pyometra at diagnosis and after ovariohysterectomy[END_REF][START_REF] Ricci | Effect of storage on long-term stability of salivary α-amylase, lysozyme, lactate dehydrogenase, calcium and phosphorus in dogs[END_REF][START_REF] Hong | Salivary alpha-amylase as a stress biomarker in diseased dogs[END_REF]. Mean amylase activity varies from 26.5 to 37.3 UI/L of saliva in medium dogs according to literature (Table S2). One study involves 75 dogs from 8 to 42 kg (52 mixed breeds and 23 pure breeds) and measured 35.9 ± 41 UI/L amylase in saliva but results weren't discussed regarding dog sizes [START_REF] Ricci | Effect of storage on long-term stability of salivary α-amylase, lysozyme, lactate dehydrogenase, calcium and phosphorus in dogs[END_REF]. Lactate dehydrogenase and adenosine deaminase activities were also quantified in saliva, without classification with canine BW [START_REF] Iacopetti | Salivary pH, calcium, phosphorus and selected enzymes in healthy dogs: a pilot study[END_REF][START_REF] Ricci | Effect of storage on long-term stability of salivary α-amylase, lysozyme, lactate dehydrogenase, calcium and phosphorus in dogs[END_REF][START_REF] Lavy | pH Values and Mineral Content of Saliva in Different Breeds of Dogs[END_REF]. Gastric mucosa secretes gastric juice containing proteolytic (pepsin, chymosin) and lipolytic (lipase) enzymes [START_REF] Durand | Entéropathies exsudatives chez le chien: proposition d'une stratégie thérapeutique diététique et médiacale[END_REF][START_REF] Aspinall | Anatomy and Physiology of the Dog and Cat 8. The Digestive System[END_REF]. In laboratory Beagles, gastric juice volume output increases with meal viscosity, from a total of 37.2 mL secreted for a low viscosity to 190 mL for a high viscosity meal [START_REF] Ehrlein | Effect of viscosity of test meals on gastric emptying in dogs[END_REF]. Pancreatic juice, discharged in canine duodenum, has an alkaline pH (7.4-8.3). It contains amylase (2013 U/kg BW), lipase (9.8-33.3 mL 0.05 N NaOH/mL -no longer used unit of measure), phospholipases, cholesterases, proteases (old value of 407.5-2440 mg tyrosin/mL -no longer used unit of measure) and nucleases, without further detailed information [START_REF]Les enterpathies excudatives chez le chien: actualités, diagnostiques et analyse retrospective de series de cas cliniques[END_REF][START_REF] Kienzle | Enzyme activity in pancreatic tissue, intestinal mucosa and chyme of dogs in relation to age and diet[END_REF]. Digestive secretions were mainly studied before 2000s, but values were not discriminated depending on dog sizes, and no study focuses on small and large dogs. However, enzymatic activities may vary according to the different diet composition (i.e. protein, lipid, fiber contents) adapted to each dog size.

Bile salts. Bile is produced by liver, partially stored in gallbladder then discharged to duodenum during postprandial phase, allowing stimulation of intestinal motility, intestinal lipids saponification and vitamins A, D, E and K absorption. In liver, primary BA such as cholic acid (CA) and chenodeoxycholic acid (CDCA) are synthesized from cholesterol and conjugated to taurine or glycine [START_REF] Kakimoto | Effect of a high-fat-high-cholesterol diet on gallbladder bile acid composition and gallbladder motility in dogs[END_REF]. Studies evaluating bile production in healthy dogs never discriminate dog sizes. Bile production was only evaluated in medium dogs and was found to be 29 mL/kg/24 h [START_REF] Madrid | Use of a bidirectional cannula to study biliary secretion in conscious dogs[END_REF]. Once reached gallbladder, bile is up to 10 fold more concentrated than in liver with a total concentration around 50 mmol/L [START_REF] Kararli | Comparison of the gastrointestinal anatomy, physiology, and biochemistry of humans and commonly used laboratory animals[END_REF][START_REF] Kakimoto | Effect of a high-fat-high-cholesterol diet on gallbladder bile acid composition and gallbladder motility in dogs[END_REF][START_REF] Nagahara | Effect of prednisolone administration on gallbladder emptying rate and gallbladder bile composition in dogs[END_REF][START_REF] Larcheveque | Etat des connaissances sur la mucocèle biliaire chez le chien: étude bibliographique[END_REF]. Here, it contains up to 15 different BA but the three majors count for 99% of total pool, with 72.8% taurocholic acid, 20.3% taurodeoxycholic acid and 6.2% taurochenodeoxycholic acid [START_REF] Washizu | Changes in bile acid composition of serum and gallbladder bile in bile duct ligated dogs[END_REF]. In the small intestine, BA are deconjugated by gut microbiota and converted into secondary BA. 95% of BA are reabsorbed in ileum, return into liver and the 5% remaining fraction crosses colon [START_REF] Nagahara | Effect of prednisolone administration on gallbladder emptying rate and gallbladder bile composition in dogs[END_REF]. Faecal BA concentrations were measured in three recent studies involving all dog sizes but curiously without BW distinction (Fig. 2A, Table S2). Authors found coherent results with concentrations ranged from 5.8 to 7.5 µg of total BA per mg of dry faeces [START_REF] Schmidt | The fecal microbiome and metabolome differ between dogs fed Bones and Raw Food (BARF) diets and dogs fed commercial diets[END_REF][START_REF] Blake | Altered microbiota, fecal lactate, and fecal bile acids in dogs with gastrointestinal disease[END_REF][START_REF] Manchester | Long-term impact of tylosin on fecal microbiota and fecal bile acids of healthy dogs[END_REF]. Another recent study evaluated faecal BA concentrations in 24 healthy dogs [START_REF] Guard | Longitudinal assessment of microbial dysbiosis, fecal unconjugated bile acid concentrations, and disease activity in dogs with steroid-responsive chronic inflammatory enteropathy[END_REF]. After data retreatment (classification in size groups), small, medium and large dogs present respectively 5.1, 4.7 and 3.4 µg/mg of total BA per mg of dry faeces. This suggests a decrease in faecal BA concentrations with BW increase. Further analysis from 8 studies (Fig. 2B) indicates that relative percentages of faecal secondary BA (BA II: 84.9%) are higher than primary BA (BA I: 15.5%). Moreover, proportions of primary BA such as CA and CDCA seem to be inversely correlated to canine BW whereas the contrary is observed for secondary BA (only one study) (Fig. 2C). These results suggest that the microbiota activity, and notably the BA recycling, differs from small to large breed sizes.

Mucins. Mucins are produced by goblet cells all along the dog GIT [START_REF] Kararli | Comparison of the gastrointestinal anatomy, physiology, and biochemistry of humans and commonly used laboratory animals[END_REF]. Mucus thickness has been evaluated only in gastric compartment and stomach presents a mucosa covering mucin-layer of 576 μm and 425 μm, respectively in the antrum and fundus [START_REF] Kararli | Comparison of the gastrointestinal anatomy, physiology, and biochemistry of humans and commonly used laboratory animals[END_REF][START_REF] Bickel | Gastric gel mucus thickness: effect of distention, 16,16-dimethyl prostaglandin e2, and carbenoxolone[END_REF][START_REF] Etienne-Mesmin | Experimental models to study intestinal microbes-mucus interactions in health and disease[END_REF]. This mucin-layer allows protection of the epithelium against acidic pH of stomach and withstands bone fragments [START_REF] Moon | Metagenomic insights into the roles of Proteobacteria in the gastrointestinal microbiomes of healthy dogs and cats[END_REF]. Influence of dog size on mucin secretion and mucin-layer thickness whatever the digestive compartment has never been assessed.

Nutrient digestibility

Digestibility defines the degree to which organic matter is digested by an animal. Its measure provides a qualitative and quantitative indicator of food's quality, i.e. the more digestible a food is, the higher the proportion of absorbed nutrients will be. Figure 3A gives an overview of canine dry food composition in dogs according to BW. Digestibility performances can be evaluated in dogs by measuring ileal or total (in faeces) apparent digestibility of a tested diet, and standardized digestibility could be obtained by deducing endogen products such as enzymes or metabolites delivered from intestinal cell desquamation. As previously observed for physicochemical parameters, digestibility studies are mainly focused on medium dogs and there are only two publications on small [START_REF] Weber | Influence of age and body size on the digestibility of a dry expanded diet in dogs[END_REF][START_REF] Nery | Influence of dietary protein content and source on fecal quality, electrolyte concentrations, and osmolarity, and digestibility in dogs differing in body size1[END_REF] and one on large dogs [START_REF] Weber | Influence of age and body size on the digestibility of a dry expanded diet in dogs[END_REF] (Table S3).

Due to their invasive nature, only 4 studies have been performed with ileal cannula (to measure ileal digestibility), including 3 on medium dogs [START_REF] Bednar | Selected animal and plant protein sources affect nutrient digestibility and fecal characteristics of ileally cannulated dogs[END_REF][START_REF] Flickinger | Nutrient digestibilities, microbial populations, and protein catabolites as affected by fructan supplementation of dog diets[END_REF][START_REF] Propst | A dose-response experiment evaluating the effects of oligofructose and inulin on nutrient digestibility, stool quality, and fecal protein catabolites in healthy adult dogs[END_REF][START_REF] Hendriks | Comparison of ileal and total tract nutrient digestibility of dry dog foods[END_REF]. Lipid digestibility seems to be almost complete at the ileum level (i.e. 89.3-96.5%), with only around 3-5% increased digestibility when evaluating total digestibility in faecal samples. Ileal protein digestibility appears to be lower (51.3-76.2%), with higher variations certainly related to protein quality which largely influences this parameter [START_REF] Nery | Influence of dietary protein content and source on fecal quality, electrolyte concentrations, and osmolarity, and digestibility in dogs differing in body size1[END_REF][START_REF] Zentek | Dietary protein source and manufacturing processes affect macronutrient digestibility, fecal consistency, and presence of fecal Clostridium perfringens in adult dogs[END_REF][START_REF] Pinna | Influence of dietary protein and fructooligosaccharides on fecal fermentative end-products, fecal bacterial populations and apparent total tract digestibility in dogs[END_REF][START_REF] Carrière | Gastric and pancreatic lipase levels during a test meal in dogs[END_REF]. Surprisingly, the only study investigating total dietary fibre digestibility found an ileal digestibility of 17.8%, while according to their definition fibres are only degraded in the large intestine [START_REF] Bednar | Selected animal and plant protein sources affect nutrient digestibility and fecal characteristics of ileally cannulated dogs[END_REF]. Given the lack of data, it is impossible to conclude on a possible effect of dog BW on ileal nutrient digestibility. Total apparent protein (82-88%) and lipid (95-95.8%) digestibilities appear to be equal between different dog sizes, whatever the initial proportion of dietary proteins or lipids (Fig. 3B-C). In contrast, total apparent dietary fibre digestibility (Fig. 3D) appears to be higher in large than in small and medium dogs (52.5 ± 4% for Great Dane versus 39 ± 7.4% for Miniature poodle, and 26-38% for medium dogs) [START_REF] Nogueira | Dietary supplementation of a fiber-prebiotic and saccharin-eugenol blend in extruded diets fed to dogs[END_REF][START_REF] Weber | Influence of age and body size on gastrointestinal transit time of radiopaque markers in healthy dogs[END_REF][START_REF] Detweiler | Effects of high inclusion of soybean hulls on apparent total tract macronutrient digestibility, fecal quality, and fecal fermentative end-product concentrations in extruded diets of adult dogs[END_REF]. Indeed, it seems that fibre digestibility would be quite similar between small and medium dogs, while it would be improved in large dogs. In addition, faecal apparent digestibility of dry matter, organic matter and gross energy appears to be significantly higher for large compared to small dogs [START_REF] Weber | Influence of age and body size on the digestibility of a dry expanded diet in dogs[END_REF]. All in all, those results mean that the colonic fermentation seems to be more important in large than in medium and small breed size dogs.

Intestinal absorption

Permeability. During digestion process, food is broken down into small soluble compounds (amino acids, fatty acids, monosaccharides, minerals and vitamins), able to be absorbed mainly through the villi-covered wall of the small intestine. Nutrient passage through the epithelial wall is modulated by intestinal permeability, which is the property of epithelium to allow some molecules to be absorbed passively or actively through mucosa while avoiding the passage of microorganisms and macromolecules. Lactulose to L-rhamnose or lactulose to sucralose urinary ratios could be used to monitor changes in canine small and large intestine permeability, respectively [START_REF] Hernot | Colonic permeability is higher in Great Danes compared with smaller breed-dogs[END_REF]. A higher lactulose to L-rhamnose ratio is associated with a leakier small intestine, while a lower lactulose to L-rhamnose ratio indicates a higher colonic permeability. Using these methods, Weber et al. [START_REF] Weber | Influence of age and body size on intestinal permeability and absorption in healthy dogs[END_REF] observed an increased intestinal permeability in Giant Schnauzer and Great Danes (large dogs; lactulose to L-rhamnose ratio: 0.31) compared to small dogs (0.16), and Hernot et al. [START_REF] Hernot | Colonic permeability is higher in Great Danes compared with smaller breed-dogs[END_REF] found a higher colonic permeability in large dogs (lactulose to sucralose ratio: 0.35) than in small ones (0.51). Those differences could be related to modifications associated with dog size in intestinal area, pore size, frequency of tight junctions, differences in tightness of tight junctions or accessibility of luminal content to intestinal crypts [START_REF] Bjarnason | Intestinal permeability: an overview[END_REF]. Of note, breed differences were particularly noticed with a higher colonic permeability in Great Danes, as previously described [START_REF] Zentek | Normal handling of diets--are all dogs created equal?[END_REF][START_REF] Meyer | Digestibility and compatibility of mixed diets and faecal consistency in different breeds of dog[END_REF]. An increased permeability could affect both nutrient, metabolite and electrolyte absorption but also microorganism's translocation. This may explain the weaker digestive tolerance of resistant-starch and higher digestive sensibility of large dogs compared to small ones, as discussed by Goudez et al. [START_REF] Goudez | Influence of different levels and sources of resistant starch on faecal quality of dogs of various body sizes[END_REF].

Passive absorption. Water, electrolytes and vitamins are absorbed through passive mechanisms in the small and large intestinal lumen. In healthy dogs, around 90% fluids crossing the colon are reabsorbed by mucosa [START_REF] Durand | Entéropathies exsudatives chez le chien: proposition d'une stratégie thérapeutique diététique et médiacale[END_REF][START_REF]Colonic fluid and electrolyte transport in health and disease[END_REF]. Meyer et al. [START_REF] Meyer | Digestibility and compatibility of mixed diets and faecal consistency in different breeds of dog[END_REF] demonstrated that total faecal water increases with dog BW, but the percentage of free faecal water decreases. This is of high interest because an increase in free faecal water content is associated with a higher colonic water content that can in turn influence in vivo drug dissolution, in the case of poorly soluble drugs for which dissolution continues in the large intestine [START_REF] Oswald | Canine gastrointestinal physiology: Breeds variations that can influence drug absorption[END_REF]. Whereas small dogs tend to have drier stools, a tendency of poorer faecal consistency and higher water content is observed in larger dogs. Potassium and bicarbonate ions are secreted into the colonic lumen, whereas sodium and chloride ions are passively absorbed from luminal contents [START_REF]Colonic fluid and electrolyte transport in health and disease[END_REF]. Uptake of sodium ions creates an hypertonic environment next to crypts, generating an electrochemical gradient across colonic mucosa which drives water uptake from luminal contents by osmosis [START_REF]Colonic fluid and electrolyte transport in health and disease[END_REF].

Based on observation that large digestibility variations are observed within the same breed and between different breeds, Zentek and Meyer [START_REF] Zentek | Normal handling of diets--are all dogs created equal?[END_REF] compared mineral digestibility of four food types in Great Danes and Beagles. There was no breed difference for calcium, magnesium and phosphorous absorption (Table S3), while net colonic sodium absorption tended to be 9-23% lower in Great Danes compared to Beagles. These data were further supported by Weber et al. [START_REF] Weber | Effect of size on electrolyte apparent absorption rates and fermentative activity in dogs[END_REF] describing an increase in sodium faecal content with an increase in BW (2.1 ± 0.7 g/kg DM in Miniature Poodle versus 6.1 ± 1.3 g/kg DM in Great Dane), traducing a lower sodium absorption by large dogs. Moreover, a reduction of colonic absorption of sodium has been particularly observed in Beagle, Labrador Retriever, Springer Spaniel and Münsterländer, suggesting a breed sensitivity [START_REF] Weber | Digestive sensitivity varies according to size of dogs: a review[END_REF]. Besides, Neri et al. [START_REF] Neri | Canine ileocolonic sphincter: flow, transit, and motility before and after sphincterotomy[END_REF] reported a significantly greater faecal potassium concentration in large compared to smaller dogs. Independently of dog sizes, 90% vitamin D, 80-90% vitamin A, 40-90% vitamin K and 35-50% vitamin E are absorbed by passive absorption in the proximal small intestine [START_REF] Durand | Entéropathies exsudatives chez le chien: proposition d'une stratégie thérapeutique diététique et médiacale[END_REF].

Active absorption. Active absorption processes in the small intestine implicate co-transporters (e.g. glucose or sodium-dependent transports) and concerns monosaccharides from carbohydrate degradation and peptides from protein degradation. Thus, 95% of monosaccharides are absorbed in the duodenum and proximal jejunum [START_REF] Durand | Entéropathies exsudatives chez le chien: proposition d'une stratégie thérapeutique diététique et médiacale[END_REF], and 30% of amino acids and 70% of tripeptides are absorbed and assimilated in the proximal jejunum [START_REF]Les enterpathies excudatives chez le chien: actualités, diagnostiques et analyse retrospective de series de cas cliniques[END_REF]. Regarding lipids, 80% of fatty acids and monoglycerides are absorbed in form of micelles in the small intestine and resulting in chylomicrons that passed into the intestinal lymphatic capillary of villus by endocytosis. There is no available information on the influence of dog size on nutrient absorption. Moreover, the overall active transport capacity of small intestine has been assessed by examining urinary excretion ratio of D-xylose to 3-O-methyl-D-glucose [START_REF] Weber | Influence of age and body size on intestinal permeability and absorption in healthy dogs[END_REF]. Non-significantly different ratios of 0.57, 0.58 and 0.59 for small, medium and large dogs respectively have been reported, suggesting that small intestinal active transport is relatively consistent between sizes.

Mechanical digestion and gastrointestinal transit time

Motility. Canine gut motility was firstly evaluated using radiopaque markers, plastic beads or breath test. Recently, wireless motility capsule was developed to measure pressure, forces and gut contractions frequency. Using this method, Boscan et al. [START_REF] Boscan | Effect of prolonged general anesthesia with sevoflurane and laparoscopic surgery on gastric and small bowel propulsive motility and pH in dogs[END_REF] observed in fed medium dogs a lower maximal amplitude contraction in the stomach compared to small intestine (52 mmHg versus 75 mmHg, respectively), coupled with higher gastric contraction frequency, with 3.7 contractions/min in the stomach versus 0.5 contraction/min in the small intestine. Another study involving dogs from different sizes observed similar tendency on maximal amplitude contraction (lower in stomach than in small intestine, with 0.2 versus 4.1 mmHg), but opposite results on frequency (0.8 in stomach versus 10.9 contractions/min in small intestine) [START_REF] Warrit | Effect of hospitalization on gastrointestinal motility and pH in dogs[END_REF]. Moreover, in this study, large intestinal contraction frequency seems to be similar to the gastric ones (0.6 contraction/min). Authors also calculated a motility index defined as the area under the pressure curve and the higher motility index was observed in small intestine (306.2 compared to 20 in stomach and 76.1 in colon). Using wireless motility capsule, Farmer et al. [START_REF] Farmer | Regional gastrointestinal contractility parameters using the wireless motility capsule: inter-observer reproducibility and influence of age, gender and study country[END_REF] found that motility indexes were higher in large intestine (199 mmHg*second/min) compared to small intestine (134 mmHg*second/min) and stomach (55 mmHg*second/min) with a similar maximum of 3.7 contractions/min in gastric compartment [START_REF] Farmer | Linaclotide increases cecal pH, accelerates colonic transit, and increases colonic motility in irritable bowel syndrome with constipation[END_REF]. Lastly, no study has investigated how dog BW or size influences gut peristalsis.

Transit time. There is no available data on the duration of oral phase in dogs, but they are well known to quickly swallow their whole food. Data on gastric emptying time (GET), small intestinal transit time (SITT), orocaecal transit time (OCTT), large intestinal transit time (LITT) and total transit time (TTT) can be found in the literature with homogeneous definition between studies (Fig. 4, Table S4). Three different studies evaluate the impact of dog size on GET fed animals. Weber et al. [START_REF] Weber | Influence of age and body size on gastrointestinal transit time of radiopaque markers in healthy dogs[END_REF] showed no significant difference in half-gastric emptying time between four breeds dogs (i.e. Miniature Poodle, Standard Schnauzer, Giant Schnauzer and Great Danes) using radiopaque markers ingested with food (T50 = 6.4-7.8 h). Without specifying any values, Bourreau et al. [START_REF] Bourreau | Gastric emptying rate is inversely related to body weight in dog breeds of different sizes[END_REF] concluded on a longer GET in large compared to small breeds after ingestion of a dry food meal using breath test method. Contrarily, Boillat et al. [START_REF] Boillat | Assessment of the relationship between body weight and gastrointestinal transit times measured by use of a wireless motility capsule system in dogs[END_REF] described a shorter GET in large compared to medium breeds (range 6.8-15 h), using wireless motility capsule immediately administered after a dry food meal. Thus, there is apparently no relationship between BW and GET not only in fed, but also in fasted animals (Fig. 4). Besides, a liquid meal conduced to a shorter GET compared to meat, with 90% emptying in 0.4 h and 50% in 1-3 h, respectively (unknown dog size and method) [START_REF] Dressman | Comparison of canine and human gastrointestinal physiology[END_REF]. This suggests that canine gastric emptying is influenced by food consistency [START_REF] Ménard | Gastric Emptying and Dynamic In Vitro Digestion of Drinkable Yogurts: Effect of Viscosity and Composition[END_REF]. There is also no consensus on the effect of dog size on SITT. Oswald et al. [START_REF] Oswald | Canine gastrointestinal physiology: Breeds variations that can influence drug absorption[END_REF] and Weber et al. [START_REF] Weber | Influence of size on the dog's digestive function[END_REF] found no influence of breed or BW, while Boillat et al. [START_REF] Boillat | Assessment of the relationship between body weight and gastrointestinal transit times measured by use of a wireless motility capsule system in dogs[END_REF] measured a shorter SITT in largest dogs, ranging 1.6-3.7 h without linking transit time and dog size [START_REF] Boillat | Assessment of the relationship between body weight and gastrointestinal transit times measured by use of a wireless motility capsule system in dogs[END_REF][START_REF] Weber | Influence of size on the dog's digestive function[END_REF][START_REF] Oswald | Canine gastrointestinal physiology: Breeds variations that can influence drug absorption[END_REF]. OCTT was evaluated in dogs using very different methods. Some studies used sulfasalazine (converted into sulfapyridine in plasma) but do not employ the same threshold to define OCTT, i.e. either 50% conversion or first appearance in plasma [START_REF] Weber | Influence of age and body size on gastrointestinal transit time of radiopaque markers in healthy dogs[END_REF][START_REF] Hernot | Evaluation of association between body size and large intestinal transit time in healthy dogs[END_REF], whereas more recent studies used wireless motility capsule. As a consequence, extremely variable results of OCTT are provided, from 2.2-2.8 h with sulfapyridine [START_REF] Weber | Influence of age and body size on gastrointestinal transit time of radiopaque markers in healthy dogs[END_REF][START_REF] Hernot | Evaluation of association between body size and large intestinal transit time in healthy dogs[END_REF] to 20.7 h with capsule [START_REF] Balsa | Effect of Laparoscopic-assisted Gastropexy on Gastrointestinal Transit Time in Dogs[END_REF]. Whatever the method used, these authors conclude to an absence of correlation between OCTT and BW. Studies of Boillat et al. [START_REF] Boillat | Assessment of the relationship between body weight and gastrointestinal transit times measured by use of a wireless motility capsule system in dogs[END_REF] and Warrit et al. [START_REF] Warrit | Minimally invasive wireless motility capsule to study canine gastrointestinal motility and pH[END_REF] assessed LITT in dogs from several breeds and various BW using wireless motility capsule. Both works conclude on the absence of correlation between LITT and BW (Fig. 4), with T1/2 ranged 7.1-42.9 h [START_REF] Boillat | Assessment of the relationship between body weight and gastrointestinal transit times measured by use of a wireless motility capsule system in dogs[END_REF] and 25.0 h (1.1-49.1 h) [START_REF] Warrit | Effect of hospitalization on gastrointestinal motility and pH in dogs[END_REF]. However, using plastic beads, researches revealed a longer LITT in large dogs (29.3 h for great Dane) than in small dogs (9.1 h for Miniature Poodle) and a significant positive correlation between LITT and BW, but also between LITT and shoulder height was demonstrated [START_REF] Hernot | Evaluation of association between body size and large intestinal transit time in healthy dogs[END_REF]. In this study, LITT accounts for 39% of mean TTT for small breed dogs and 70% for large ones, which means that longer transit times observed in large dogs could be related to a longer LITT. Lastly, TTT showed a clear positive correlation with BW, as highlighted in Fig. 4 [START_REF] Weber | Digestive sensitivity varies according to size of dogs: a review[END_REF]. When gathering the data obtained in all the available studies, TTT ranged from 22.9-31 h (calculated median 24 h) in small dogs, 19.1-55 h (median 32.9 h) in medium dogs and 18.2-45 h (median 43.2 h) in large dogs. Especially, using plastic beads in small and large breeds, TTT observed was 22.9 h in Miniature Poodle and 43.3 h in Great Dane, whilst giant Schnauzer showed an even higher TTT of 55 h [START_REF] Hernot | Relationship between total transit time and faecal quality in adult dogs differing in body size[END_REF]. This result was explained by the authors through a high stress sensitivity of giant Schnauzer that could influence their transit time in refraining their defecation, emphasizing a breed effect in addition to body size influence.

Impact of body size on microbial parameters

Gut microbiota composition

Longitudinal variations. In dogs like in other mammals, microorganisms colonize the entire GIT from mouth to rectum. All along GIT, there are longitudinal variations in gut microbiota composition due to changes in pH, substrate concentrations (including oxygen and nutrient availability) and transit time [START_REF] Etienne-Mesmin | Experimental models to study intestinal microbes-mucus interactions in health and disease[END_REF][START_REF] Hooda | Current state of knowledge: the canine gastrointestinal microbiome[END_REF][START_REF] Friedman | Microbes vs. chemistry in the origin of the anaerobic gut lumen[END_REF]. Gut microbiota has been weakly described in dogs (compared to humans) and most of available studies have been performed since 2003 (for detailed information see Table S5). Canine oral microbiota present similar number (around 350 bacterial taxa from 148 genera) but significantly different populations compared to the human ones [START_REF] Dewhirst | Correction: The Canine Oral Microbiome[END_REF] and is mainly colonized by Proteobacteria (45%), Bacteroidetes (25%) and Firmicutes (19%). Most abundant species are Porphyromonas cangingivalis and Porphyromonas gulae [START_REF] Bell | Ecological Characterization of the Colonic Microbiota of Normal and Diarrheic Dogs[END_REF]100]. Regarding the other digestive compartments, studies have been mostly performed on faeces to avoid invasive procedures. Stomach is the less colonized compartment with 10 4 to 10 5 colony forming units (CFU) per gram of content in medium dogs, mainly composed by Proteobacteria (Fig. 5A) including Helicobacter spp. that are potential pathogenic strains [START_REF] Mentula | Comparison between cultured small-intestinal and fecal microbiotas in beagle dogs[END_REF][START_REF] Hooda | Current state of knowledge: the canine gastrointestinal microbiome[END_REF]101]. Small intestine contains 10 5 to 10 7 CFU/g of content [START_REF] Mentula | Comparison between cultured small-intestinal and fecal microbiotas in beagle dogs[END_REF]101]. Duodenum (Fig. 5A) is colonized by Firmicutes (calculated median 47%), Proteobacteria (27%), Bacteroidetes (9%), Fusobacteria (3%) and Actinobacteria (1%), whereas jejunum is characterized by a higher abundance in Proteobacteria (37%), Actinobacteria (11%) and Fusobacteria (10%), together with lower percentages of Firmicutes (33%) and Bacteroidetes (7%) [START_REF] Garcia-Mazcorro | Abundance and short-term temporal variability of fecal microbiota in healthy dogs[END_REF]102,103]. Ileum (Fig. 5A) is dominated by 31% Fusobacteria, 24% Firmicutes, 23% Bacteroidetes and 22% Proteobacteria [104]. These abundances should be considered with caution as they have been found in a single study performed in 6 medium dogs. As for other mammals, large intestine is the most colonized part of the GIT, with up to 10 9 to 10 11 CFU/g of content [START_REF] Hooda | Current state of knowledge: the canine gastrointestinal microbiome[END_REF]. According to a unique publication using 16S Illumina sequencing to investigate microbiota composition from 6 healthy Hound dogs [104], colonic digesta is dominated by 37% Firmicutes, 33% Bacteroidetes, 29% Fusobacteria and 1% Proteobacteria including E. coli-like organisms (Fig. 5A). It's interesting to highlight that majority of taxa colonizing the colon are also found in canine faeces [105] which seems to be rather different from the human situation where a significant number of mucus-adherent bacteria from the colon are not found in faeces [105]. No study has investigated dog size effect on gut microbiota composition elsewhere than in stools, and the main variations in faeces are presented in Figure 5B and 5C. Whatever dog sizes, faecal microbiota of healthy dogs is dominated by three main bacterial phyla: Firmicutes, Bacteroidetes and Fusobacteria [105]. Bacteria from Actinobacteria and Proteobacteria phyla are also found in canine faeces but in a lesser proportion. Of interest, a variable relative abundance of Bacteroidetes was reported and was . Fusobacteria and Proteobacteria seem to be more abundant in dogs than in other omnivorous, probably related to diet changes [107]. Unlike in human where Fusobacterium is frequently associated with diseases, in dogs this genus is related to non-stressful conditions and is therefore probably a marker of an healthy state, especially because its abundance increases when dogs have access to the outside [START_REF] Oswald | Canine gastrointestinal physiology: Breeds variations that can influence drug absorption[END_REF]. In small dogs faeces (Fig. 5B), average Firmicutes proportions vary widely from 30 to 80%, followed by 13-28% Bacteroidetes, while a lower abundance of Proteobacteria (1-15%), Fusobacteria (1-16%) and Actinobacteria (1-3%) was detected [108][109][110]. Medium dogs display similar value ranges of Firmicutes (15-98%), Bacteroidetes (0.1-34%), Proteobacteria (0.1-27%) and Actinobacteria (1%), but a larger proportion of Fusobacteria (0.1-40%) compared to small dogs [108,111]. Only one study investigated faecal microbiota composition in 8 large dogs and quantified 71% Firmicutes, 22% Bacteroidetes, 5% Fusobacteria, and 1% Actinobacteria, with interestingly a much lower abundance of Proteobacteria (1%) than in small and medium dogs [START_REF] Sandri | Raw meat-based diet influences faecal microbiome and end products of fermentation in healthy dogs[END_REF]. In few studies, canine faecal diversity was followed with Shannon index and calculated medians seem to be higher in medium dogs (4.8, four studies) compared to small (3.5, five studies) and large dogs (2.9, a single study) (Fig. 5C). In addition to Bacteria (representing 98%), canine faecal microbiota also contains 1.1% Archaea, 0.4% Fungi and 0.4% viruses, mainly bacteriophages [112,113]. Fungal part of the faecal microbiota is composed by 97.9% Ascomycota and 1% Basidiomycota [114]. Even if methanogen Archaea have been detected in healthy dogs faeces, there is no information on their methanogen potential [114]. Radial variations. In addition to longitudinal variations, there are also radial changes in gut microbial composition that starts to be described in human [115] but are still in infancy in dogs. Indeed, the entire gut epithelium is covered by a mucus layer that offers an alternative source of host-derived nutrients. This mucus is colonized by a specific mucus-adherent microbiota (namely mucosal microbiota) and seems to play a key role in host homeostasis [116]. Of note, there is a lack of studies on the canine mucosal microbiota. Only two studies investigated the mucosa-associated bacteria on the outer mucus layer in the colon of healthy dogs, using targeted FISH approach [117,118]. Analysis of colonic biopsy samples from healthy Boxers revealed that bacteria appear to be restricted to the outer mucus layer, as no bacteria was detected within the mucosa [117]. In addition, Cassmann et al. [118] demonstrated that free ileal and colonic mucus of healthy young dogs (< 2 years old) was mainly colonized by Bacteroidetes spp. and Eubacteria, while Eubacteria represented the major bacteria attached to adherent mucus. Authors reported that there were almost no bacteria attached to surface epithelium or contained within mucosa. Of interest, Akkermansia muciniphila, a well-known mucin-degrading bacteria in humans, inversely correlated to obesity, was not yet identified in canine faeces [119].

Gut microbiota metabolic activities and functions

Gut microbiota is known to play a key role in host homeostasis and health maintenance, as it is implicated in many nutritional (e.g. vitamin synthesis, fibre degradation), immunological (immune system maturation) and physiological processes (e.g. vascularization, epithelium integrity, "barrier" effect against pathogens and lipid digestion via the metabolism of primary BA into secondary BA) [START_REF] Durand | Entéropathies exsudatives chez le chien: proposition d'une stratégie thérapeutique diététique et médiacale[END_REF]120]. At a functional level, whatever the type of food, identified gene content of microbiome from medium dogs was not modified and was associated with the metabolism of carbohydrates (12.5-13%), proteins (8.1-9.1%), DNA (7.1-7.4%), cell wall and capsule (7-7.6%), amino acids and derivatives (6.8-6.9%), cofactors, vitamins, prosthetic groups and pigments (5.7-6%) and bacterial virulence (6.2-7.2%) [112]. These results underline that all microbiota functions are far to be already discovered, as proved by the remaining 42.8% non-affiliated genes. Guard and Suchodolski [121] have studied faeces from 8 healthy dogs (2.7 to 31.8 kg) and observed high inter-variability microbiota composition between animals, while bacteria's functions were very consistent. Thus, even if gut microbiota composition highly vary between dogs, the functional potential seems to be unchanged whatever dog sizes [START_REF] Durand | Entéropathies exsudatives chez le chien: proposition d'une stratégie thérapeutique diététique et médiacale[END_REF]. Gut microbiota metabolic activity leads to gas and short-chain fatty acid (SCFA) production from soluble fibres. SCFAs stimulate intestinal motility and can be further used as an energy source for colonocytes, liver and brain. The three main SCFAs are acetate, propionate and butyrate, with faecal relative percentages of 60:25:15 [122]. Non-digested protein from diet and endogen proteins are also metabolized by gut microbiota, leading to the production of branched chain fatty-acids (BCFA), ammonia, indoles and phenols [START_REF] Weber | Digestive sensitivity varies according to size of dogs: a review[END_REF]. Canine faecal protein degradation products are associated with deleterious effects, such as poor faecal quality, inflammation and kidney diseases in dogs and colorectal cancer in humans [123,124]. Canine SCFA production was only evaluated in faecal samples (Fig. 6A-B, Table S6). Values were mainly obtained in medium dogs (especially Beagles) and are widely variable due to many differences in design study (e.g. type of food, food composition in carbohydrates, methods, type of units). However, in a study performed by Weber et al. [START_REF] Weber | Effect of size on electrolyte apparent absorption rates and fermentative activity in dogs[END_REF] , the authors compared SCFA production between small, medium and large dogs and demonstrated that total SCFA concentration in stool significantly increased with BW, with 448 ± 67, 894 ± 80 and 1184 ± 259 mmol/kg of lyophilized faeces for small, medium and large dogs respectively. This is consistent with a longer LITT in large breed dogs that may promote microbial fermentation. Large quantity of organic acids produced could thus exceeds colonic mucosa absorption capacity, thereby leading to an accumulation in lumen, a decrease of colonic pH and an increased faecal excretion [START_REF] Weber | Digestive sensitivity varies according to size of dogs: a review[END_REF]. Similarly, total BCFAs were measured only in faecal samples, and mainly in medium dogs (Fig. 6C, Table S6). BCFA concentrations seem to be lower in small dogs (a unique value of 17.1 µmol/g) compared to medium ones (calculated median of 22.2 µmol/g). Moreover, BCFA composition was only studied in medium dogs, with a calculated median concentration of 6.8 µmol/g isobutyrate, 10.5 µmol/g isovalerate and 0.8 µmol/g valerate (Fig. 6D). Phenols, indoles and ammonia concentrations were also poorly studied in small and medium dogs and to our knowledge never measured in large dogs (Fig. 6E-F). Based on our calculated medians, it appears that these products are found in higher concentrations in medium than in small dogs. Lastly, to our knowledge there is no data on gas production in dogs and the two studies on gas composition focused on malodorous compounds such as hydrogen sulphide [125,126].

Discussion and general conclusion

In an original way, this review gives an overview of available literature concerning the effect of dog sizes (i.e. "small", "medium" and "large" sizes) on digestive anatomy and associated physicochemical and microbial parameters, illustrating data with both synthetic graphs (Fig. 1 to Fig. 6) and exhaustive tabs (Table S1 to S6). Even if our conclusions may be hampered by the paucity and old age of many data, as well as the huge variability between experimental protocols (diet composition, measurement methods and data analysis processes) and animals (live or dead, anesthetized or not, companion or laboratory animals, environment), we evidenced clear effects of dog's BW on gastrointestinal physiology, mainly in relation with the colonic compartment (Fig. 7). Large intestine length, area and volume clearly increase with dog size. This seems to be associated with a higher colonic transit time that can affect nutrient and water absorption, gut microbiota composition and activity, as well as faecal moisture. Thus, sodium and potassium absorption are lower in larger dogs resulting in a higher concentration in faecal samples. Large dogs are also characterized by a higher intestinal permeability that can induce a backflow of absorbed electrolytes into the colonic lumen, translated into a luminal retention of electrolytes and water [START_REF] Weber | Digestive sensitivity varies according to size of dogs: a review[END_REF]. Besides, a longer colonic residence time in large dogs should promote microbial fermentations and especially a higher fibre degradation by resident bacteria. This higher fermentation capacity results in a stronger production of SCFAs leading to a diminution in faecal pH, and to a potential disturbance of water absorption due to the high osmotic power of SCFAs [START_REF] Weber | Effect of size on electrolyte apparent absorption rates and fermentative activity in dogs[END_REF]. Together with an increased colonic permeability, excessive SCFAs production would induce water retention in the colon, associated with higher faecal water content and loose watery stools frequently observed in large dogs [START_REF] Oswald | Canine gastrointestinal physiology: Breeds variations that can influence drug absorption[END_REF]127]. In addition, faecal concentrations of microbial degradation products from proteins (phenol, indole, ammonium and BCFAs) seem to be positively associated with dog BW, which again may be explained by a longer transit time. Moreover, our data analysis suggests an increase in Fusobacteria according to BW (observed between small and medium dogs), which can be related to an increase in protein metabolites [START_REF] Moon | Metagenomic insights into the roles of Proteobacteria in the gastrointestinal microbiomes of healthy dogs and cats[END_REF]124]. As certain bacteria are fully involved in BA deconjugation, changes in microbiota composition depending on dog's BW can also be linked to modifications in BA concentrations, inversely correlated with BW.

Further studies would be necessary to enhance available data on physicochemical parameters, especially in the upper GIT, but also on gut microbiota that remains very poorly described in each digestive compartment and not described at all in the mucus layer. Lastly, our bibliographic review revealed the large predominance of some breeds (i.e. Miniature Poodle, Beagle, Standard and Giant Schnauzer and Great Dane) and breeds showing well-known specific digestive particularities (like German Shepherd) or specific energy needs (like Husky, Great Danes or Terriers) [START_REF] Zentek | Normal handling of diets--are all dogs created equal?[END_REF]. It would be therefore of high interest to further analyze current data by considering not only the effect of body size but also that of breeds. Taken together, all the specificities raised in large dog digestive physiology may be correlated to their high sensitivity to diet and digestive diseases [START_REF] Weber | Digestive sensitivity varies according to size of dogs: a review[END_REF]. Finally, all these data concerning the effect of dog size on their digestive physiology can be helpful for the development of new food or veterinary products at the individual level, in accordance with a personalization step intended by petfood and pharma companies. In full accordance with the 3R rules (aiming to reduce animal experiments), such in vivo data also provide key information necessary to develop and validate in vitro gut models adapted to each dog sizes for in-depth mechanistic studies on dog digestive physiology [128].

Figure 1 .Figure 2 .Figure 3 .

 123 Figure 1. Impact of dog sizes on pH values in all digestive compartments. Results from studies measuring in dog's pH values in the stomach (under fasted or fed conditions), small intestine, large intestine and faeces are presented. Small dogs are plotted in green, medium dogs in yellow, large dogs in orange and unclassified dogs in grey. Raw data were pooled in "all" group (in black). Calculated median values are in italic bold, values for a single point in italic. Black bars represent 95% confidence intervals. The number of dogs involved in studies is indicated as "N="

Figure 4 .

 4 Figure 4. Impact of dog sizes on gastrointestinal transit time. Results from studies in dogs evaluating gastric emptying time (GET) under fasted or fed status, small intestinal transit time (SITT), large intestinal transit time (LITT) and total transit time (TTT) are represented. The same caption as used in Fig. 1 was applied

Figure 5 .

 5 Figure 5. Variations in gut microbiota composition along the canine digestive tract and impact of dog sizes. Main bacteria populations found in the different compartments of the dog gastrointestinal tract are represented in (a). Bacteria counts are expressed in colony forming units (CFU) per gram of digestive content. Results from studies exploring by 16S rRNA Illumina sequencing canine microbiota composition (regardless of dog size) in the different digestive compartments are presented in (b). Influence of dog sizes on faecal microbiota composition at the phylum level is shown in (c) and corresponding Shannon index in (d). Canine main phyla are Firmicutes (Firm), Bacteroidetes (Bact), Fusobacteria (Fuso), Proteobacteria (Proteo) and Actinobacteria (Actino). The same caption as used in Fig. 1 was applied

Figure 6 .

 6 Figure 6. Impact of dog sizes on faecal microbial products production. Results from studies in dogs measuring total faecal major short-chain fatty acids (SCFA, i.e. acetate, propionate and butyrate) are presented in (a) and detailed in (b). Similarly, influence of dog size on major branched-chain fatty acids production (BCFA, i.e. isobutyrate, isovalerate and valerate) is shown in (c) and detailed in (d). Effect of dog size on other microbial metabolites are presented in (e) for phenols and indoles and (f) for ammonia. The same caption as used in Fig. 1 was applied

Figure 7 .

 7 Figure 7. Overview of the impact of dog sizes on digestive physiology and faecal microbiota composition and activity. Key parameters of the oral, gastric, intestinal and colonic compartments from the canine digestive tract are summarized. Specified values were obtained from reports comparing in a same study the results obtained for small and large dogs. Lack of data are represented by "?", BA: bile acid, SCFA: short chain fatty acids. *: Lactulose/L-rhamnose ratio, **: Lactulose/sucralose ratio

https://www.ijbs.com

Abbreviations

BA: bile acids; BCFA: branched-chain fatty acids; BW: body weight; CA: cholic acid; CDCA: chenodeoxycholic acid; CFU: colony forming units; DCA: deoxycholic acid; GET: gastric emptying time; HCl: hydrochloric acid; LCA: lithocholic acid; LITT: large intestine transit time; OCTT: orocaecal transit time; SCFA: short-chain fatty acids; SITT: small intestine transit time; TTT: total transit time.

Supplementary Material

Supplementary tables. https://www.ijbs.com/v18p5086s1.pdf

Author contributions

SBD, EA and DH had the idea to make a literature review on this topic and designed the review. CD performed the literature survey, data analysis and figures design. SBD and CD wrote the first draft of the manuscript. All authors critically revised and approved the manuscript.

Competing Interests

The authors have declared that no competing interest exists.


Clément Measson

Faugeras

I Breslavetz

A Delhomme

T Pelini

A Pawbake

D Vaclavkova

M Orlita

M Potemski

M.-A Measson

C Faugeras

Spatially resolved optical spectroscopy in extreme environment of low temperature, high magnetic elds and high pressure I. Breslavetz,

Spatially resolved optical spectroscopy in extreme environment of low temperature, high magnetic elds and high pressure INTRODUCTION Extreme conditions of low temperatures, high magnetic eld, high pressure or high doping, in condensed matter physics are sought-after as they allow us to drive electronic systems into exotic electronic ground states that cannot exist/emerge in other conditions. Relevant examples of such ground states include the appearance of superconductivity in metals at cryogenic temperatures or in ultra-doped two dimensional semiconductors [START_REF] Costanzo | Gate-induced superconductivity in atomically thin mos 2 crystals[END_REF] , magnetic orders that build up in magnetic materials below a critical temperature and once established, require high external magnetic elds to be altered [START_REF] Hagiwara | A complete frequency-eld chart for the antiferromagnetic resonance in MnF2[END_REF] , pressure induced superconductivity such as, for instance, in Fe-based compounds like FePSe 3 [START_REF] Wang | Emergent superconductivity in an iron-based honeycomb lattice initiated by pressure-driven spin-crossover[END_REF] , or the coupling between dierent electronic ground states such as superconductivity and charge density wave in transition metal dichalcogenides like TaS 2 [START_REF] Grasset | Pressure-induced collapse of the charge density wave and higgs mode visibility in 2h -tas 2[END_REF] . Particular condensed matter systems also exhibit a rich phase diagram with a triple quantum critical endpoint that can only be established in well-dened pressure-temperature-magnetic eld conditions [START_REF] Mackenzie | PHYSICS: Enhanced: A quantum critical route to eld-induced superconductivity[END_REF] . Reaching such extreme experimental conditions to perform spatially resolved optical spectroscopy can be a real challenge, and combining them within the same experiment is of course even more challenging.

In the last fteen years, microscopic techniques have been implemented in low-temperature and highmagnetic-eld environments produced by resistive solenoids, mainly thanks to the development of commercial piezo inertial motors [START_REF] Meyer | Slipstick step-scanner for scanning probe microscopy[END_REF] compatible with cryogenic temperature and high magnetic elds, and allowing for sub-micrometer displacements 79 . This opened the possibility to perform spatially resolved experiments at low temperatures such as magneto-photoluminescence, a) Authors to whom correspondence should be addressed: marie- aude.measson@neel.cnrs.fr and clement.faugeras@lncmi.cnrs.fr -reectance, -Raman scattering, also with time resolution [START_REF] Zhang | Magnetic brightening and control of dark excitons in monolayer wse2[END_REF] . High pressures can be obtained using a Diamond Anvil Cell (DAC) [START_REF] Jayaraman | Diamond anvil cell and high-pressure physical investigations[END_REF] , which is also compatible with optical measurements as diamond is transparent over a broad interval of energy including the visible and infrared ranges. A recent example is the optical investigation at room temperature of a van der Waals heterobilayer in a DAC for pressures up to ∼ 4 GPa, see Ref. [12]. Low temperatures for optical measurements with a DAC can be achieved in He4 environments with [START_REF] Hudl | Ppms-based set-up for raman and luminescence spectroscopy at high magnetic eld, high pressure and low temperature[END_REF] or without [START_REF] Li | Optical raman measurements of low frequency magnons under high pressure[END_REF] moderate magnetic elds.

In this manuscript, we present an experimental setup for optical measurements with a micrometer spatial resolution in an extreme environment of low temperature (He4 cryostat), high magnetic elds (compatible with 50mm bore resistive magnets at LNCMI-Grenoble producing elds up to B = 31 T) and high pressure with the use of a DAC. This system allows for spatial mapping of the optical response in such conditions, or to investigate the optical response at a specic location (nanostructures, patterned surface, etc ...) while tuning the extreme conditions.

II. EXPERIMENTAL SETUP

Our experimental setup is presented in Fig. 1a and was conceived to be used in a 50 mm bore resistive magnet at LNCMI-G. This environment imposes a strong spatial constraint as the eld center is located ∼ 450 mm from the top of the magnet, within a bore of 50 mm. Our setup includes a miniaturized optical table xed at the top of the experimental probe, and a 1.6 m tube with an inner diameter of 16 mm, and hosting the sample holder. We use a monomode optical ber, at room temperature, to connect the excitation laser source to the top of the probe in order to minimize the eect of vibrations caused by the large ow of deionized water [START_REF] Debray | State of the art and developments of high eld magnets at the laboratoire national des champs magnétiques intenses[END_REF] (up to 1000 m 3 .h -1 ) used to cool down resistive magnets. The excitation beam at the output of the ber is collimated by an optical lens, cleaned by a reection on a volume Bragg lter for photoluminescence or Raman scattering experiments, and then, it is sent to the sample holder via a carbon-ber tube, after a reection on a 30/70 beamsplitter. For reectivity measurement, the volume Bragg lter is replaced by a mirror and the laser source by a white lamp. Polarization optics and optical lters can be used in the excitation or in the collection path.

A circular or linear polarization can be imposed before the last reection the beam splitter. The collimated excitation beam is then focused on the pressure cell with a 12 mm working distance objective of numerical aperture NA=0.35. The spot size oers a micrometer spatial resolution. A schematic of this part of the probe is presented in Fig. 1c. The DAC is of the Tozer design [START_REF] Graf | Nonmetallic gasket and miniature plastic turnbuckle diamond anvil cell for pulsed magnetic eld studies at cryogenic temperatures[END_REF] . It is made of Cu-Be and it weights around 7 g in total (see Fig. 1b). This small weight allows us to mount it on piezo stages and to move the DAC below the laser spot with a sub-micrometer spatial resolution. Spatially resolved and hyperspectral images of the optical response of the sample within the DAC can then be reconstructed by spatially scanning the DAC below the laser spot. The optical response is collected with the same objective, goes through the beam splitter and is sent to the detection system using free beam optics. For low-energy (below 100 cm -1 ) Raman scattering measurements, we use three volume Bragg lters in series before the detection system to reject the stray light. For low temperature experiments, the tube holding the objective-DAC-piezo stages is placed in a closed metallic tube (32 mm diameter) lled with 100 to 200 mbar of helium exchange gas for an ecient thermal coupling. The tube is then immersed in a bath of liquid helium in a 48 mm cryostat routinely used in the 50 mm bore of resistive or superconducting magnets. A local heater/thermometer is inserted below the DAC to allow for tuning temperature up to T ∼ 80 K in the previously described conditions.

The gasket has been indented and compressed down to a thickness of 85 µm and a hole of 250 µm was drilled in its center. The test sample, a triangular ake of bulk FePS 3 , a layered antiferromagnet, was placed in the hole together with two ruby balls used to measure the pres-sure [START_REF] Syassen | Ruby under pressure[END_REF] . DAC require the use of a pressure transmitting medium (PTM) and we have used a liquid PTM, a mixture of ethanol and methanol (1:4 in volume) expected to remain hydrostatic up to ∼ 10 GPa, see Ref [18]. At T = 4.5 K, the Raman scattering response of this PTM in the pressure cell is displayed in Fig. 2a. The phonon of diamond is clearly visible at ∼ 1340 cm -1 , together with the response of the PTM [START_REF] Emin | Raman study of mixed solutions of methanol and ethanol[END_REF] . Two energy bands are aected by the response of the PTM, a weak intensity band between 870 and 1550 cm -1 and a more intense band between 2770 and 3050 cm -1 . In the inset, we highlight the fact that the optical response of this PTM close to the laser line (below 800 cm -1 where most phonon and magnon energies in condensed matter system are found) is nearly featureless, being only composed of a smooth background and allowing for low-energy Raman scattering experiments, see the inset of Fig. 2a. These signals are negligible when looking at luminescence signals from direct-band-gap semiconducting materials. The pressure value can be determined by placing the laser spot on the ruby crystal and measuring its photoluminescence signal [START_REF] Mao | Solids, liquids, and gases under high pressure[END_REF] . Fig. 2b shows three low temperature spectra of the ruby crystal in the pressure chamber, at selected pressure values.

Finally, despite the fact that our experimental setup is not confocal, still, the optical response has a depth selectivity which allows us to identify, from the response, if we are focusing on the pressure chamber or not. This can be seen in Fig. 3 which displays two characteristic Raman scattering spectra of the top diamond of the DAC. At T = 4.5 K, depending on the relative distance between the objective and the pressure cell, we can observe changes in the energy position of the doubly degenerate LO-TO phonon at the Γ point of diamond, representative of the strain. The strain evolves continuously when focusing deeper into the top diamond, as it is shown in the inset of Fig. 3, and reaches a maximum at the pressure chamber location ∼ 2 mm below the top surface of the top diamond of the anvil. This eect allows us to identify the correct objective-DAC distance in our experimental setup in which no optical imaging is implemented. After xing the focus distance, one can spatially map the optical response to locate the ruby crystals in the pressure chamber.

III. RESULTS

The low-temperature Raman scattering response of FePS 3 includes a doubly degenerate antiferromagnetic magnon excitation close to 122 cm -1 identied by both its peculiar temperature dependence [START_REF] Sekine | Lightscattering study of dynamical behavior of antiferromagnetic spins in the layered magnetic semiconductor feps 3[END_REF] , and recently, by its evolution with an applied magnetic eld [START_REF] Liu | Direct observation of magnon-phonon strong coupling in two-dimensional antiferromagnet at high magnetic elds[END_REF][START_REF] Mccreary | Quasi-two-dimensional magnon identication in antiferromagnetic FePs 3 via magneto-raman spectroscopy[END_REF][START_REF] Vaclavkova | Magnon polarons in the van der waals antiferromagnet Feps 3[END_REF] . The properties of FePS 3 under pressure have been described recently from the viewpoints of X-ray diraction [START_REF] Haines | Pressure-induced electronic and structural phase evolution in the van der waals compound feps 3[END_REF] and of neutron scattering [START_REF] Coak | Emergent magnetic phases in pressure-tuned van der waals antiferromagnet feps 3[END_REF] . We present in Fig. 4a two lowtemperature Raman scattering spectra of FePS 3 measured at ambient pressure (black line), and in the DAC with an applied pressure of 0.9 GPa (red line). The effect of pressure can be seen through the hardening of the phonon modes. Our experimental setup allows for a spatial mapping of the Raman scattering response and this possibility is displayed in Fig. 4b with a falsed color spatial map of the energy position of the phonon near 260 cm -1 at P = 0.9 GPa. One can compare this mapping of the Raman scattering response with the optical photograph of the pressure chamber in Fig. 4c. Our Raman scattering data at dierent pressures up to 8.7 GPa presented in the inset Fig. 4a, indicate that this phonon energy changes with a rate of 5.1 cm -1 /GPa. One can clearly see a gradual change of the phonon energy along the ake, and hence of the eective pressure in the chamber. These data indicate that the pressure is larger in the middle of the chamber than at its borders, close to the metallic gasket, and this pressure dierence is evaluated to be ±0.1 GPa from the center of the chamber to its edge.

When applying a magnetic eld transverse to the FePS 3 layers, the two-fold degeneracy of the magnon excitation is lifted, the energy of one branch increases with the magnetic eld while the energy of the other branch decreases [START_REF] Mccreary | Quasi-two-dimensional magnon identication in antiferromagnetic FePs 3 via magneto-raman spectroscopy[END_REF] . When a pressure of 8.7 GPa is set in the DAC, bulk FePS 3 is expected to be in the HP1 phase [START_REF] Haines | Pressure-induced electronic and structural phase evolution in the van der waals compound feps 3[END_REF] . As can be seen in Fig. 5, the magnon excitation is also observed in the Raman scattering response, at an energy very close to that of the magnon at ambient pressure, but within a phonon spectrum modied by the high pressure. When applying a transverse magnetic eld the magnon splits into two components and we can observe this ex-Figure 4. a) Raman scattering spectra of bulk FePS3 measured at T = 4.5 K and at ambient pressure (black line) and at P = 0.9 GPa (red line). Vertical dashed lines indicate selected phonon energies without applied pressure. Inset: pressure dependence of the ∼ 260 cm -1 phonon in FePS3, redline is a linear t of this evolution with a slope of 5.1cm -1 /GPa. b) False-color map of the energy of the ∼ 260 cm -1 phonon in FePS3 at P = 0.9 GPa. A gradual change of the phonon energy from the center of the pressure chamber to its border clearly indicates a gradient of pressure within the chamber. The color scale is presented as the energy position of this phonon in cm -1 or as associated relative pressure change within the chamber in MPa. c) Optical image of the pressure chamber containing the sample (FePS3, of a triangular shape in the image), ruby balls (of a circular shape) to measure the pressure, and loaded with a mixture of ethanol:methanol of 1:4 in volume.

citation up to highest value of the magnetic eld. This result illustrates the possibility to probe magnetic and phonon excitations in the very specic environment of low temperature, high pressure and high magnetic elds.

IV. CONCLUSIONS

To conclude, we have presented an experimental set-up for spatially resolved optical investigations (Raman scattering, photoluminescence and reectance) of condensed matter systems in an extreme environment of low temperature, high magnetic elds provided by the resistive magnets of LNCMI-Grenoble (France), and of high pressure produced in a diamond anvil cell. This set up allows for the spatial mapping of the optical response of the sample with a sub-micrometer resolution at B = 0 or with an applied magnetic eld, or to investigate a well-dened location or systems with reduced dimensions such as van der Waals heterostructures. This work unlocks for condensed matter physics the novel and exciting possibility of inducing exotic electronic ground states at low temperature and high pressure, and to trace the evolution of their elementary excitations as a function of the magnetic eld produced by a superconducting or a resisitive magnet, by optical means. Additional functionalities such as measurements of reectance or photoluminescence with a time resolution are in the process of being implemented.

V. ACKNOWLEDGEMENT

This work has been partially supported by the EC Graphene Flagship project and by the ANR projects ANR-17-CE24-0030 and ANR-19-CE09-0026. M-A. M. acknowledges the support from the ERC (H2020) (Grant agreement No. 865826). We acknowledge the support of the LNCMI-EMFL, CNRS, Univ. Grenoble Alpes, INSA-T, UPS, Grenoble, France.

VI. DATA AVAILABILITY

The data that support the ndings of this study are available from the corresponding author upon reasonable request.

Figure 1 .

 1 Figure 1. a) Schematics of the experimental setup where L is a laser system, BF a Bragg lter to spectrally clean the excitation laser, P are polarizers or optical lters, OW an optical window, BS a 30/70 (R/T) beam splitter and M a metallic mirror. For reectance measurements, BF is changed for a metallic mirror and the laser is replaced by a white light source. The magnet is presented in purple. b) Optical photograph of the pressure cell with a 1 Euro cent coin. c) Schematic of the sample holder showing the long working distance objective (blue), the pressure cell (orange) attached to its holder (grey) and the piezo stages (red).

Figure 2 .

 2 Figure 2. a) Raman scattering spectrum of the pressure transmitting medium (Eth:Meth (1:4)) at T = 4.5 K and P = 0.9 GPa. Inset: zoom on the 0 -800 cm -1 part of the spectrum. b) Photoluminescence spectra of ruby crystals at three dierent pressures and at T = 4.5 K.

Figure 3 .

 3 Figure 3. Raman scattering spectra of the top diamond obtained by focusing on the surface (blue spectrum) or close to the pressure chamber (red spectrum) showing the eect of strain on the high energy phonon of diamond. Inset: False color map of the Raman scattering from the high energy phonon in diamond when focusing from the top diamond surface (z ∼ 2000µm) towards the pressure chamber at z = 0. A schematics of the top diamond is overlaid on the gure.

Figure 5 .

 5 Figure 5. Low temperature (T = 4.5 K) Raman scattering response of bulk FePS3 at P = 8.7 GPa for selected values of the magnetic eld up to B = 27 T. Black stars indicate the energy position of the magnetic eld dependent magnon excitation.


Germain Arribarat

Muriel Mescam

Franck Caroline Fonta

Franck Desmoulin

Atlas statistique de la vascularisation cérébrale chez le singe marmouset

Atlas statistique de la vascularisation cérébrale chez le singe marmouset

Germain Arribarat 1 , Muriel Mescam 1 , Caroline Fonta 1 , Franck Desmoulin

Objectifs:Figure 1 -

 1 Figure 1 -Pré-traitement et Construction Atlas

  2,3 1 CerCo, UMR 5549 CNRS. Université Paul Sabatier, Toulouse 2 ToNIC, Toulouse NeuroImaging Center, Université de Toulouse, Inserm, UPS, 3 CREFRE, Toulouse.


CHSP), S. GOROSTIZA (CHSP T Souquet

G Parrinello

A WAR ON SEDIMENT ? UNDERSTANDING AND GOVERNING SOLID TRANSPORT IN THE RHÔNE WATERSHED (1840-1920)

INTRODUCTION

In 1978, a sediment flushing of the Génissiat dam was blamed for a massive fish die-off in the Rhône. The resulting mobilisation encouraged the CNR, the manager of the Rhône dams, to revise its flushing methods and take into account the impact of sediment on fauna. In many respects, the 1978 "great flush" marked the emergence of new concerns for the ecological "health" of the river and of political movements targeting sediment management. Nevertheless, the links between sociopolitical configurations, sediment knowledge and technical management are not original to the late 20th century. Sediments in the Rhône have been at the heart of technical, cognitive and political processes involved in river development for at least two centuries.

SEDIMENTS IN HISTORY

In the 19th century, Ponts et Chaussées engineers and foresters were the principal mediators of the relationship between river and society. To study how sediments were understood and managed we have thus examined records of engineers and foresters in charge of the Rhône basin management at the National Archives (serie F) and at the Archives départementales du Rhône (serie S), the main treatises of river management in the 19th century, and the entire collection of journals of these corps: Annales des Ponts et Chaussées (1833Chaussées ( -1940)), Annales forestières , Revue des Eaux et Forêts , Génie Civil , and Houille Blanche .

These sources reveal two opposite understandings of sediments. For agriculture, sediments were a valuable agent of plant nutrition. For river development projects they were an obstacle to water flows. We have found that this negative understanding of sediments became dominant in river management and justified ambitious forms of control of sediment production and circulation.

CLEARING A WATERWAY

At the turn of the 19th century, French authorities hoped to transform the Rhône into a major waterway. The river, however, was unsuitable for steam navigation and companies demanded a clear and deeper channel. In 1836, the state established the Rhône Special Service (Service spécial du Rhône) to improve navigability by controlling solid transport. Over the decades, the Special Service perfected methods to deepen the river by narrowing its channel and flushing out the sediments. The chart below, produced in 1892, visualizes the results, graphically representing the opposition between clear water (blue), a vector of transport, and sediments (dark), a nuisance to eliminate.

Graphic representation of the

SEDIMENTS AND HYDROELECTRICITY

The congestion of the delta also gave rise to programs of mountain soil management. In 1797, engineer Jean-Antoine Fabre proposed to intensively reforest the Alps in order to limit the deposits in the Rhône delta, which "can only come from the debris of the mountains" (1). Trees, by limiting erosion, would make it possible to moderate sediment production and thus, to reduce the deleterious deposition that clogs the delta. This is one of the first expressions of an explicit desire to control solid transport via reforestation at the scale of the watershed.

By the mid 19th century, flood control rose to preminence in river management alongside navigation. In 1840 and 1856, the main rivers of France flooded. The Rhône valley was particularly damaged. In this context, both the public opinion and the government demanded a management scheme that could prevent floods altogether, fulfilling Napoleon III promise that "rivers, like the revolution, return to their beds, and can no longer leave them" (2).

Maps of the Rhône delta in 1760 (left) and in 1842 (right)

showing the significative growth undergone by the mouths, Archives nationales, F/14/6541.

The program of sediment reduction drafted by Fabre found an audience with Alexandre Surell's 1841 book Etude sur les torrents des Hautes-Alpes. Surell argued that soil erosion upstream and deposition downstream generated hydrological and geomorphological instability in the Rhône River. Reforestation in the Alps was thus a necessary condition to the domestication of the watershed.

It was not an entirely new idea but found an echo in a context of disastrous floods. By the midcentury, repeated flooding was blamed on sedimentary congestion and riverbed aggradation. Curbing sediment production with forests promised to solve flooding at the roots.

Surell's book had a long-lasting influence. In its wake, curbing sediment production through massive upland reforestation became a core principle of river management. This principle was at the hearth of the French forest law of 1860, which launched a broad reforestation programme aimed at sediment reduction.

Representation of a torrent watershed in the

Etude sur les torrents des Hautes-Alpes (2nd Ed. 1870).

The translation of Surell's theories into practice was contested. Local communities often opposed reforestation programs and the state had to revise its reforestation ambitions in 1864 and 1882. Faced with resistance, foresters were eager to reassert the virtues of sediment scarcity, to compensate their lack of authority on mountain territories. At the turn of the twentieth century, the emergence of hydroelectricity in the Alps, quickly nicknamed "white coal" (houille blanche) gave to the forest administration a new tool to increase the legitimity of its craft. The above remarks prove that Alpine hydroelectric development was coupled with a struggle, if not declared, at least implicit, to clarify the waters of the Rhône. Moreover, mountain restoration continued to be pursued throughout the 20th century and curbing sediment production remained a declared goal. In 1951, Windmann, an Eaux et Forêts officer, was still arguing that trees protected dams by preventing the erosion of mountainous soils and therefore, in the end, reservoirs sedimentation (5). The war on sediment was now an axiom.

To calculate the lifespan of projected dams new research emerged that mathematized the growth of sedimentary deposits, particularly on the tributaries of the Durance or at Génissiat, on the Upper Rhône (Haut-Rhône) (4).

The foresters program of curbing sediment production had a new appeal in this context, as apparent in publications such as La Houille Blanche, the main venue of the hydroelectric industry. From 1900 to 1920, it served as a forum for foresters who ardently defended reforestation to prevent reservoirs sedimentation (see image on the left).

Graphic representation of the filling of the Pont-du-Loup reservoir, adapted by the authors from R. Walther (3).

Headlines linking reforestation, sediment control and hydroelectric development in the 1900s.

  Rhône shoals before the 1878 works, attached to the general report on the situation of the Special Service in 1892, Archives départementales du Rhône, S/3243.Table summarizing the estimation of the future growth of the Rhône delta, from A. Surell, Mémoire sur l'amélioration des embouchures du Rhône, 1847. 1) Jean-Antoine Fabre, Essai sur la théorie des torrens et des rivières, Paris, Bidault, 1797. 2) « Discours de S. M. l'Empereur », Le Moniteur universel, 16/02/1857. 3) Robert Walther, "Note sur l'engravement et le dégravement d'un bassin d'accumulation. Histoire du lac du Motty", Revue de Géographie Alpine, vol. 24, n° 2, 1936, p. 315-328. 4) Félix Drouhet, "Le charriage des alluvions par les cours d'eau", Le Génie civil, vol. 68, n° 24, 1916, p. 378-383. 5) M. Widmann, "Barrages hydro-électriques et érosion des terrains en montagne", Revue forestière française, 1951, p. 640-646. For more informations about sediment management and government, see Titouan Souquet, "Au-delà de l'eau. Le 'souci sédimentaire' dans l'aménagement du bassin du Rhône (1835-1940)", master thesis, Sciences Po Paris, 2022.CURBING SEDIMENT PRODUCTIONSedimentary congestion was particularly problematic at the river mouths. The challenge of sedimentary deposits that extended the delta and clogged the waterway led to the first experiments to estimate solid transport and the rate of deposition and sea filling by the sediments of the Rhône.In 1749, engineer Bernard Forrest de Bélidor estimated the rate of progradation of the Rhône delta at over 100 meters per year. A century later, Alexandre Surell estimated anew the rate of progradation and envisioned to improve the mouths by narrowing the main channel and concentrating the flow.

"

  White coal" industrialists feared sediment. Sand and gravel could quickly fill up reservoirs, thus rendering them useless. The graph on the right depicts the rapid filling of the 1920s Pont-du-Loup reservoir, almost entirely clogged after only three years. From the 1920s onwards, technological changes and the decline of the scholarly authority of Surell's theories reduced the appeal of the foresters program for hydroelectricity. However, sediment control did not disappear from view. Armand, a Ponts et Chaussées engineer in charge of energy development in the Rhône, pointed out in 1918 what he saw as a happy corollary of Alpine dams. Once all tributaries of the Rhône were dammed, sediment discharge would be considerably reduced in the river channel itself, which would facilitate river development.