Microbial seeding in early life is critical for the host’s short- and long-term health, and the mother is the first source of bacteria for the newborn. The objective of this study was to characterize the maternal fecal, vaginal, and colostral microbiotas in the canine species one day after parturition and to evaluate the relationship between the microbial profiles of 36 dams and the neonatal outcomes of 284 newborns. The first part of the study revealed the presence of 2 fecal, 3 vaginal, and 2 colostral microbial clusters on the basis of the core microbiota of the dams. Among these three maternal microbiotas, only the vaginal microbiome was found to be associated with neonatal outcomes. Compared with those in the other clusters, females in Cluster 1, with the lowest stillbirth and neonatal mortality ratios, presented a greater abundance of Moraxellaceae in their vaginal microbiota; Cluster 2, with a greater abundance of Pasteurellaceae, mostly from the Haemophilus genus; and Cluster 3 (with the highest stillbirth and neonatal mortality ratios), a greater abundance of Enterobacteriaceae, mostly E. coli. Moreover, Cluster 3 dams presented significantly lower species richness according to the Shannon index than did dams from the other clusters. This study underscores the strong association between maternal microbiota, particularly the vaginal microbiota, and newborn health. The results of this study call for further research to gain a deeper understanding of the optimal vaginal microbiota composition in canine species and the ways to modulate it to improve neonatal outcomes.

Nutritional programming is a manipulation of fetal and neonatal development through maternal feeding. In humans and pigs, maternal yeast supplementation was demonstrated as a promising approach to positively to modulate newborns' health. This study aimed to investigate the effects of Saccharomyces cerevisiae var. boulardii CNCM I-1079 (SB) supplementation in pregnant and lactating bitches on the newborns' early growth rate (EGR, between birth and 2 days of life), metabolic profiles, and the association between both of them. A total of 17 female dogs and their 81 puppies were included. From day 28 of gestation until the end of the study, bitches were divided into two groups, one of which received orally 1.3 × 109 colony forming units of live yeast per day. Puppies from mothers receiving the live yeast were defined as the SB group (n = 40) and the others were defined as the placebo group (n = 4 1). For each puppy, EGR was calculated, and blood and urine samples were collected at D2 for metabolome analysis using liquid chromatography-mass spectrometry (LCMS). Puppies from the SB group presented higher EGR compared with the placebo group (12% vs. 7%; p = 0.049). According to the Sparse Partial Least Squares Discriminant Analysis (sPLS-DA), both urine and serum metabolome profiles were significantly different between the two groups with a total of 29 discriminating metabolites in urine and serum. Fourteen of them were implicated in the nitrogen metabolism pathway including, gamma-aminobutyrate, 3-methyl-l-histidine and xanthosine (less abundant in SB compared with placebo group, all p < 0.05), adenine, aspartate and proline (more abundant in SB compared with placebo group, all p < 0.05). Metabolic pathways pointed to proline synthesis, a crucial component in collagen synthesis and osteoarticular system development. Urinary proline abundance was positively correlated with EGR (r = 0.45; p < 0.001). These findings highlight the potential benefits of maternal supplementation with SB promoting early neonatal growth, essential for the neonatal survival, through nitrogen metabolism orientation.

Time around parturition is a stressful period for both bitches and their puppies. The use of probiotics has been proposed, e.g., in pigs, to improve health status of sows, their reproductive performances and in turn, the health and performance of their progeny. The objective of the present study was to evaluate the impact, on both dams and puppies, of a supplementation of bitches with the live yeast Saccharomyces cerevisiae var. boulardii CNCM I-1079 (SB-1079) during the second part of the gestation and the lactation period. A total of 36 bitches of medium and large-sized breeds were enrolled. They were divided into two groups, one of which received 1.3 × 109 colony forming units of live yeast per day. At dam’s level, SB-1079 yeast shaped a different microbiota structure between the two groups just after whelping, impacted alpha diversity and some plasma metabolites related to energy metabolism. Regarding reproductive performances, SB-1079 improved gross energy of the colostrum (1.4 vs. 1.2 kcal of ME/g) as well as the concentration of protein in milk at Day 7 after parturition (10.4 vs. 7.6%). SB-1079 also reduced the odds of having low birth weight in the litter. At puppy’s level, a modulation of immunometabolic phenotype is suggested by the observation of increased growth rates during the early pediatric period (i.e., between 21 and 56 days of life, 225 vs. 190%) and a decrease of the IL-8:IL-10 ratio after vaccination against rabies (4.2 vs. 16.9). Our findings suggest that SB-1079 supplementation during gestation and lactation has the potential to enhance health of bitches and in turn health of puppies through maternal programming.

Different dog sizes are associated with variations in large intestinal physiology including gut microbiota, which plays a key role in animal health. This study aims to evaluate, using the CANIM-ARCOL (Canine Mucosal Artificial Colon), the relative importance of gut microbes versus physicochemical and nutritional parameters of the canine colonic environment in shaping microbiota structure and functions. CANIM-ARCOL was set up to reproduce nutrient availability, bile acid profiles, colonic pH, and transit time from small, medium, or large dogs according to in vivo data, while bioreactors were all inoculated with a fecal sample collected from medium size dogs (n = 2). Applying different dog size parameters resulted in a positive association between size and gas or SCFA production, as well as distinct microbiota profiles as revealed by 16S Metabarcoding. Comparisons with in vivo data from canine stools and previous in vitro results obtained when CANIM-ARCOL was inoculated with fecal samples from three dog sizes revealed that environmental colonic parameters were sufficient to drive microbiota functions. However, size-related fecal microbes were necessary to accurately reproduce in vitro the colonic ecosystem of small, medium, and large dogs. For the first time, this study provides mechanistic insights on which parameters from colonic ecosystem mainly drive canine microbiota in relation to dog size. The CANIM-ARCOL can be used as a relevant in vitro platform to unravel interactions between food or pharma compounds and canine colonic microbiota, under different dog size conditions. The potential of the model will be extended soon to diseased situations (e.g. chronic enteropathies or obesity).

As in humans, antibiotics are widely used in dogs to treat gastrointestinal infections, contributing to the global burden of antimicrobial resistance on both human and animal health. Close contact between pets and their owners can lead to horizontal transfer of gut microbes, including transmission of antibiotic resistance. Nevertheless, until now, the impact of antibiotics on the canine gut microbiota has been poorly described. The aim of this study was to adapt the canine mucosal artificial colon (CANIM-ARCOL) model, reproducing the main nutritional, physicochemical and microbial parameters found in the large intestine of the dog to simulate an antibiotic-induced perturbation. Following initial investigation of five antibiotic cocktails at in-field doses, a 5-day regimen of metronidazole/enrofloxacin (ME) was selected for further model development. Two CANIM-ARCOL bioreactors were inoculated with a faecal sample ( n = 2 donors) and run in parallel for 26 days under control or antibiotic conditions. ME reduced microbial diversity and induced major shifts in bacterial populations, leading to a state of dysbiosis characterized by an increase in the relative abundance of Streptococcaceae, Lactobacillaceae and Enterobacteriaceae, and a decrease in the relative abundance of Bacteroidaceae, Fusobacteriota and Clostridiaceae. Overall, mucusassociated microbiota were less impacted by antibiotics than luminal microbes. Microbial alterations were associated with drastic decreases in gas production and short-chain fatty acid concentrations. Finally, the model was well validated through in-vitro-in-vivo comparisons in a study in dogs. The CANIM-ARCOL model provides a relevant platform as an alternative to in-vivo assays for an in-depth understanding of antibiotic-microbiota interactions and further testing of restoration strategies at individual level. (c) 2024 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ )

IntroductionElderly dogs often suffer from chronic diseases, in part attributed to immunosenescence, characterized by reduced blood CD4+ T cells (helper T cells) and elevation in the CD8+ T cells subset (cytotoxic T cells). Studies conducted in adult dogs suggested that supplementing short chain fructo-oligosaccharides (scFOS) or postbiotics derived from yeasts may positively influence the immune response. The aim of the current study was to investigate whether dietary supplementation with a combination of scFOS with a new yeast postbiotic (Profeed ADVANCED® called scFOS+) could have a positive influence on the immune status of senior dogs subjected to an immune challenge (Lyme disease vaccination).MethodsTo this end, 22 healthy senior client-owned dogs were divided into two groups: one group received a placebo diet without scFOS+ and the other group the basal diet supplemented with 1.1% scFOS+. In order to assess immune function, complete blood count, serum acute phase proteins, immunoglobulins, cytokines, T-cell subsets and antibody secreting cells were analyzed. Furthermore, faecal score and pH were recorded.Results and discussionDogs fed the scFOS+ supplement had decreased total serum IgA concentrations (p < 0.01), which might suggest a more local IgA response in agreement with what was previously found when adult dogs were supplemented with β-1, 3/1, 6-glucan, a yeast-based product. More importantly, the present study demonstrated that feeding 1.1% scFOS+ to healthy senior dogs increased the CD4+:CD8+ T-cell (Helper:Cytotoxic T cell) ratio (p < 0.001) during and after vaccination against Lyme disease. Combining scFOS and yeast-derived postbiotics in the diet can therefore counter certain characteristics of T-cell immunosenescence in dogs.

Differences in dog breed sizes are an important determinant of variations in digestive physiology, mainly related to the large intestine. In vitro gut models are increasingly used as alternatives to animal experiments for technical, cost, societal, and regulatory reasons. Up to now, only one in vitro model of the canine colon incorporates the dynamics of different canine gut regions, yet no adaptations exist to reproduce size-related digestive parameters. To address this limitation, we developed a new model of the canine colon, the CANIne Mucosal ARtificial COLon (CANIM-ARCOL), simulating main physiochemical (pH, transit time, anaerobiosis), nutritional (ileal effluent composition), and microbial (lumen and mucus-associated microbiota) parameters of this ecosystem and adapted to three dog sizes (i.e., small under 10 kg, medium 10–30 kg, and large over 30 kg). To validate the new model regarding microbiota composition and activities, in vitro fermentations were performed in bioreactors inoculated with stools from 13 dogs (4 small, 5 medium, and 4 large). After a stabilization period, microbiota profiles clearly clustered depending on dog size. Bacteroidota and Firmicutes abundances were positively correlated with dog size both in vitro and in vivo, while opposite trends were observed for Actinobacteria and Proteobacteria . As observed in vivo, microbial activity also increased with dog size in vitro, as evidenced from gas production, short-chain fatty acids, ammonia, and bile acid dehydroxylation. In line with the 3R regulation, CANIM-ARCOL could be a relevant platform to assess bilateral interactions between food and pharma compounds and gut microbiota, capturing inter-individual or breed variabilities. Key points • CANIM-ARCOL integrates main canine physicochemical and microbial colonic parameters • Gut microbiota associated to different dog sizes is accurately maintained in vitro • The model can help to move toward personalized approach considering dog body weight

Numerous studies have demonstrated the importance of digestion and gut microbiota in the nutrition and health of dogs. Artificial digestive systems represent a relevant alternative to in vivo assays, in accordance with the Euro-pean 3Rs rules. Today, a dozen static or dynamic in vitro models reproducing one or more parts of the dogs gas-trointestinal tract have been developed, considering the physicochemical, mechanical and/or microbial parameters of the canine digestion. These models make it possible to study in the laboratory the fate of food and veterinary products in the dogs digestive environment, particularly in terms of macronutrient digestibility, micronutrient bioaccessibility, microorganisms’ survival and activity or drugs’ pharmacokinetics. In order to expand their poten-tial, in the near future these models may be coupled with cellular models or adapted to reproduce pathological conditions, such as obesity.

Inoculants combining Lentilactobacillus buchneri and Lentilactobacillus hilgardii have been shown to improve the aerobic stability of high-moisture corn (HMC) and whole-plant corn silage, but the mode of action of this co-inoculation remains to be elucidated. This study used metatranscriptomics to evaluate the effects of inoculation with L. buchneri alone or combined with L. hilgardii on the bacterial community, gene expression, fermentation profile, and starch digestibility in HMC. High-moisture corn not inoculated (Control) or inoculated with L. buchneri NCIMB 40788 (LB) or L. buchneri NCIMB 40788 combined with L. hilgardii CNCM-I-4785 (Combo) was ensiled in mini silo bags for 30, 60, 120, and 180 days. The fermentation profile was evaluated at all time points. Metatranscriptomics was performed on samples collected on day 120. Combo had a greater alpha diversity richness index of contigs than LB and Control, and inoculation with Combo and LB modified the beta-diversity of contigs compared to Control. Out of 69 genes of interest, 20 were differentially expressed in LB compared to Control and 25 in Combo compared to Control. Of those differently expressed genes, 16 (10 of which were associated with carbohydrate metabolism and six with amino acid metabolism) were differently expressed in both LB and Combo compared to Control, and all those genes were upregulated in the inoculated silages. When we compared Combo and LB, we found seven genes expressed differently, four associated with carbohydrate metabolism and downregulated in Combo, and three associated with amino acid metabolism and upregulated in Combo. At day 120, the inoculated silages had more culturable lactic acid bacteria, higher Lactobacillus relative abundance, and lower Leuconostoc relative abundance than Control. The concentration of acetic acid remained low throughout ensiling in Control, but in LB and Combo, it increased up to day 60 and remained stable from day 60 to 180. The 1,2-propanediol was only detected in LB and Combo. Inoculation did not affect the concentration of starch, but starch digestibility was greater in Combo than in Control. Inoculation of HMC with Combo modified the gene expression and fermentation profile compared to Control and LB, improving starch digestibility compared to uninoculated HMC.

With the rising awareness of antimicrobial resistance, the development and use of functional feed additives (FFAs) as an alternative prophylactic approach to improve animal health and performance is increasing. Although the FFAs from yeasts are widely used in animal and human pharma applications already, the success of future candidates resides in linking their structural functional properties to their efficacy in vivo. Herein, this study aimed to characterise the biochemical and molecular properties of four proprietary yeast cell wall extracts from S. cerevisiae in relation to their potential effect on the intestinal immune responses when given orally. Dietary supplementation of the YCW fractions identified that the α-mannan content was a potent driver of mucus cell and intraepithelial lymphocyte hyperplasia within the intestinal mucosal tissue. Furthermore, the differences in α-mannan and β-1,3-glucans chain lengths of each YCW fraction affected their capacity to be recognised by different PRRs. As a result, this affected the downstream signalling and shaping of the innate cytokine milieu to elicit the preferential mobilisation of effector T-helper cell subsets namely Th17, Th1, Tr1 and FoxP3+-Tregs. Together these findings demonstrate the importance of characterising the molecular and biochemical properties of YCW fractions when assessing and concluding their immune potential. Additionally, this study offers novel perspectives in the development specific YCW fractions derived from S. cerievisae for use in precision animal feeds.

Low birth weight puppies present an increased risk of neonatal mortality, morbidity, and some long-term health issues. Yet it has not been investigated if those alterations could be linked to the gut microbiota composition and evolution. 57 puppies were weighed at birth and rectal swabs were performed at 5 time points from birth to 28 days of age. Puppies were grouped into three groups based on their birth weight: low birth weight (LBW), normal birth weight (NBW) and high birth weight (HBW). 16S rRNA gene sequencing was used to highlight differences in the fecal microbiota. During the first three weeks, the relative abundance of facultative anaerobic bacteria such as E. coli, C. perfringens and Tyzzerella was higher in LBW feces, but they catch back with the other groups afterwards. HBW puppies showed higher abundances of Faecalibacterium and Bacteroides during the neonatal period, suggesting an earlier maturation of their microbiota. The results of this study suggest that birth weight impact the initial establishment of the gut microbiota in puppies. Innovative strategies would be desired to deal with altered gut microbiota in low birth weight puppies aiming to improve their survival and long term health.

It is generally accepted that microbes play a critical role in maintaining gut barrier function, making them ideal to target in order to mitigate the effects of intestinal diseases such as inflammatory bowel disease with specialist supplementations such as probiotic or postbiotic preparations. In this study, specific strains of Lactobacillus helvictus both live and inactivated and Lactobacillus plantarum inactivated were fed to zebrafish at an inclusion level of 6 × 106 cells/g in order to assess the effects on gut barrier function and protection. Taken together, our results indicate that dietary administration of pro- or postbiotics strengthens the gut barrier function and innate immunity of healthy zebrafish in a strain-specific and process-dependent way. With some differences in the response intensity, the three treatments led to increased intestinal villi length and proportion of IELs, reinforcement of the GC population and up-regulated expression of biomarkers of AMP production and tight junction zona-occludin 2a (zo-2a). In addition, LPPost had an impact on the adaptive immune response, and we hypothesized that it conferred the potential to drive Th17/ILC3 immunity, as suggested by its effect on the gene expression of il22, of different AMPs, and the expression of zo2a. Moreover, LPPost showed the potential to drive Th1/ILC1-like immunity, with a higher percentage of CD8+ cells and higher ifnγ gene expression. In summary, the use of inactivated Lactobacilli species in this study represented a promising strategy for improving barrier function and regulating the immune fate of the intestinal mucosa in a strain-specific way.

Triticale, a small-grain forage, was harvested for an ensiling experiment evaluating different silage inoculants. Fresh material (mean dry matter 404.1 g kg−1) was wilted, chopped, and assigned to one of four treatment groups including water only (Control), heterofermentative strains Lentilactobacillus buchneri (LB), Lentilactobacillus hilgardii (LH), or combination (Combo) of both. Inoculants were applied at 4.0 x 105 CFU per g of fresh forage, and the Combo contained both strains at 2.0 x 105 CFU per g. Treated forage was packed into 7.57 L mini-silos for openings after 15, 30, and 130 d. Samples were collected at each opening for microbial enumeration of lactic acid bacteria, enterobacteria, yeasts, and moulds. Additional samples were collected for analysis of fermentation profiles, and nutritional analysis following dry matter determination and grinding. Aerobic stability was also evaluated at each opening through a 10-d period. Fermentation profiles were notably affected, including increases in acetic acid (g/kg DM) in LB and Combo treated silage after 15 d which resulted in reduced pH. Aerobic stability was vastly improved through inoculation by prolonging the time to reach 2˚ C above ambient, which was likely a result of decreased yeast counts. Our research validates the use of combined heterofermentative lactic acid bacteria strains on an ensiled small-grain specie, and further agrees with previous findings of prolonged aerobic stability through increases in lactate and acetate in response to co-inoculation.

Aims This study evaluated changes in epiphytic microbial population of alfalfa (Medicago sativa) during the growing season. First cut forage was harvested to study the effects of an inoculant combining two obligate heterofermentative lactic acid bacteria strains on the bacterial and fungal communities and the fermentation of alfalfa silage. Methods and Results The epiphytic microbiome of alfalfa was evaluated 10-times during the growing season. Alfalfa wilted to 395.0 g/kg was treated with water (Control) or with a combination of L. buchneri NCIMB 40788 and L. hilgardii CNCM-I-4785 (LBLH). Mini-silos were opened after 1, 4, 8, 16, 32, and 64 days of ensiling. The relative abundance (RA) of the epiphytic bacterial and fungal families varied during the growing season. After 1 day, Weissella was the most abundant genus and present at similar RA in the two treatments (average 80.4%). Compared with Control, LBLH had a higher RA of Lactobacillus at day 1, 16, 32, and 64, and a lower RA of Weissella from day 8 to 64. Control contained more bacteria belonging to the Enterobacteriales than LBLH up to day 16. Inoculated silage had more acetate than Control at day 32 and 64. The fungal population were similar between treatments. The enhanced development and dominance of Lactobacillus in inoculated silage led to greater accumulation of acetate and propionate, which reduced the numbers of culturable yeasts but did not markedly affect the fungal community structure. Conclusions The bacterial community composition of alfalfa stands in the filed changed over time and was affected by cutting. For the ensiling trial, inoculation modified the composition of the bacterial community of alfalfa, increasing the RA of Lactobacillus while reducing the RA of Weissella and of Enterobacteriaceae. Significance and Impact of Study Inoculation increased the RA of Lactobacillus, hampering the dominance of Weissella in the early stages of ensiling, improving antifungal compounds production and reducing the numbers of culturable yeasts.

Health and well-being of dogs are of paramount importance to their owners. Digestion plays a key role in dog health, involving physicochemical, mechanical and microbial actors. However, decades of breeding selection led to various dog sizes associated with different digestive physiology and disease sensitivity. Developing new products requires the consideration of all the multi-faceted aspects of canine digestion, the evaluation of food digestibility, drug release and absorption in the gut. This review paper provides an exhaustive literature survey on canine digestive physiology, focusing on size effect on anatomy and digestive parameters, with graphical representation of data classified as “small”, “medium” and “large” dogs. Despite the huge variability between protocols and animals, interesting size effects on gastrointestinal physiology were highlighted, mainly related to the colonic compartment. Colonic measurements, transit time permeability, fibre degradation, faecal short-chain fatty acid concentration and faecal water content increase while faecal bile acid concentration decreases with body size. A negative correlation between body weight and Proteobacteria relative abundance was observed suggesting an effect of dog body size on faecal microbiota. This paper gathers helpful in vivo data for academics and industrials and supports the development of new food and pharma products to move towards canine personalized nutrition and health.

Microorganisms of the gastrointestinal tract play a crucial role in the health, metabolism and development of their host by modulating vital functions such as digestion, production of key metabolites or stimulation of the immune system. This review aims to provide an overview on the current knowledge of factors shaping the gut microbiota of young dogs. The composition of the gut microbiota is modulated by many intrinsic (i.e., age, physiology, pathology) and extrinsic factors (i.e., nutrition, environment, medication) which can cause both beneficial and harmful effects depending on the nature of the changes. The composition of the gut microbiota is quickly evolving during the early development of the dog, and some crucial bacteria, mostly anaerobic, progressively colonize the gut before the puppy reaches adulthood. Those bacterial communities are of paramount importance for the host health, with disturbance in their composition potentially leading to altered metabolic states such as acute diarrhea or inflammatory bowel disease. While many studies focused on the microbiota of young children, there is still a lack of knowledge concerning the development of gut microbiota in puppies. Understanding this early evolution is becoming a key aspect to improve dogs' short and long-term health and wellbeing.

Dogs occupy a full place in the family, and their well-being is of paramount importance to their owners. Digestion, a complex process involving physicochemical, mechanical, and microbial parameters, plays a central role in maintaining canine health. As in vivo studies in dogs are increasingly restricted by ethical, regulatory, societal, and cost pressures, an alternative option is the use of in vitro models simulating the different compartments of the canine gastrointestinal tract. This review introduces digestion and gut microbiota as key factors in dog nutrition and health under both healthy and diseased conditions (obesity and inflammatory bowel disease) and highlights similarities and differences between the human and canine digestive tract and processes. We provide the first in-depth description of currently available models of the canine digestive tract, discuss technical and scientific challenges that need to be addressed, and introduce potential applications of in vitro gut models in the food and veterinary fields. Even if the development of some in vitro models is still limited by a lack of in vivo data in dogs that is necessary for relevant configuration and validation, translation of long-term expertise on human in vitro gut models to dogs opens avenues for model optimization and adaptation to specific canine digestive conditions associated with various dog ages, sizes, breeds and/or diets, in both physiological and diseased states.