The cuddles, loyalty, companionship and unconditional affection they can give make dogs and cats increasingly present in homes all over the world; furthermore, several studies have shown how the presence of a pet also improves the mental and physical health of humans [1,2,3]. Thanks also to a better quality of life, owners treat these animals as real members of the family, paying close attention to their well-being. Following the discovery of the importance and great influence that the intestinal microbiota can have on animal health, nutritional strategies aimed at maintaining a more balanced intestinal microflora are increasingly. It is well known that probiotics can modulate the composition of the canine and feline intestinal microbiota, promoting the intestinal colonization of beneficial bacterial species able to release metabolites that strengthen the integrity of the intestinal barrier, making the environment more hostile to potentially harmful bacteria. However, it is important to remember that when we talk about probiotics, we are referring to live microorganisms that belong not only to different bacterial phyla but also to different bacterial classes, orders, families, genera and species; it is essential to know this because recent studies have highlighted how bacteria belonging to the same species can perform different functions within the organism based on the specific strain to which they belong, the well-known concept of ‘strain-specificity’. Considering what has been illustrated so far, it is better to evaluate the effects of a specific probiotic strain and not of a specific species, in fact, even if bacteria belonging to the same species will have many common characteristics, it is better to analyze the relative strains to have a more accurate and detailed information about their properties. However, among the species of bacteria that, during evolution, have managed to colonize the intestinal tract, adapting to coexist in a mutualistic way with the organism, we find Lactobacillus reuteri currently known as Limosilactobacillus reuteri. “Limose” from the latin “limosus” means slimy and indicates the ability of this bacterial species to produce exopolysaccharides (EPS). The new classification provides a better environmental and functional view and a better understanding of the differences within lactobacilli. Host-adapted bacteria, for example, are more competitive than those that do not have a common evolutionary history with the host, which is interesting for the effectiveness of a product in fighting pathogens. This reclassification of lactobacilli is a unique opportunity to better explain the world of bacteria, to better understand the specificities of new groups and to develop more targeted products and, ultimately, for greater efficacy. EPS are important food and pharmaceutical additives with beneficial antioxidant, antitumor, and immunological effects for human health. However, EPS are difficult to produce due to complex conditions in fermentation. EPS produced by L. reuteri are important for biofilm formation and adhesion of L. reuteri to epithelial surfaces; EPS synthesized by L. reuteri can inhibit the adhesion of E. coli [4]. In addition, they can reduce the gene expression of proinflammatory cytokines induced by Escherichia coli infection (including IL-1b and IL-6). Other in vivo experiments [5] showed similar results: EPS produced by L. reuteri prevent diarrhea in bacterial infection by reducing the adhesion of E. coli. Furthermore, EPS can act also as a prebiotic. The present study is the last of three research conducted to evaluate the efficacy of a specific strain of L. reuteri, L. reuteri NBF 1 DSM 32203, on the intestinal microbiota and fecal parameters of healthy adult dogs. In the two previously published research were evaluated the effects of L. reuteri NBF 1 DSM 32203 on the intestinal microbiota of medium-large sized dogs (Golden Retriever) and subjects with a predisposition to intestinal diseases like French Bulldogs. We thought it was important to evaluate the effectiveness of this probiotic on small dogs such as Chihuahuas and Dachshunds; in fact, analyzing the growth curves of large and small dogs, those of large dogs show a greater slope underlying how these animals undergo a notable increase in their body weight during the first weeks of life while small breeds show a growth curve with a lesser slope and a moderate increase. These observations suggest that the effort that large breed dogs make during the early stages of growth, when their intestinal microbiota is still unstable, may compromise its integrity in later adulthood by influencing its composition and making it more susceptible to potentially harmful agents; the fact that large dogs live less than small dogs could also be connected to this process. For this reason, it is important to analyze the effect of the same probiotic strain on dogs of different sizes to see if the probiotic can have different effects depending on the intestinal ecosystem it will colonize. L. reuteri NBF 1 DSM 32203 can improve fecal quality parameters in healthy Golden Retriever dogs promoting an increase in the amount of Lactobacillus spp., a beneficial bacterial group able to favor a balanced intestinal ecosystem [7]. Similar results were reported by another study that analyzed the effects of the same probiotic strain on the intestinal microbiota of French bulldogs [8], also in this case, L. reuteri NBF 1 DSM 32203 was able to improve the Fecal Score decreasing the fecal humidity with a production of more consistent and well-formed feces indicator of an adequate intestinal function, in addition, the probiotic enhanced the amount of lactobacilli causing a small reduction in the total coliforms. Except for these two studies, in literature, there are no publications on the effects of L. reuteri NBF 1 DSM 32203 on the intestinal microbiota and fecal parameters of adult dogs. Zhao et al. (2023) [6] investigated the effects of another strain of L. reuteri, specifically Limosilactobacillus reuteri ZJF036, on growth performance and gut microbiota of young Beagle dogs showing that this specific probiotic strain is not able to influence the daily weight gain, but it is able to decrease Chao1 index and ACE index increasing the relative abundance of Firmicutes and Fusobacteria. Obviously, this study is not comparable with those conducted on adult animals because, in young dogs the intestinal microbiota has yet to fully stabilize.

The results of the study show how L. reuteri NBF 1 DSM 32203 supplementation has no influence on the weight (Table 3) and body condition (Table 4) of Chihuahua and Dachshund adult healthy dogs; in fact, the body weight of each dog remained almost constant throughout the entire duration of the study.

However, it is very interesting to see how fecal hmoisture (Table 5) decreased in the group of dogs treated with the probiotic L. reuteri NBF 1 DSM 32203 compared to the control one; similar results were reported in both Chihuahua and Dachshund dogs. At the end of the 35 days of study, Chihuahuas supplemented with L. reuteri NBF 1 DSM 32203 showed a decrease in fecal moisture of 0.2 points compared to T0 (beginning of the study), while the dachshund dogs showed a decrease of 0.24 points (Table 5).

Another interesting effect to consider is the improved fecal score reported in dogs that received the probiotic under study. Starting from the second week of study (T2), the Chihuahua group treated with L. reuteri NBF 1 DSM 32203 reported a Fecal score value of 2.09 (Table 6), very close to the value considered optimal, i.e. 2. The dachshund dogs reported the same excellent score starting from the fourth week of study (T4), while in the respective control group no similar improvement was observed (Table 6).

As regards the microbiological analysis, the trend of lactobacilli is very similar between Chihuahua and Dachshund dogs supplemented with L. reuteri NBF 1 DSM 32203; in both groups a clear increase of this bacterial genus is observed starting from the third week of the study and continuing to increase until the end of the experiment (Table 7, Figure 1). Specifically, in the group of Chihuahuas treated with L. reuteri NBF 1 DSM 32203, a significant increase in lactobacilli was recorded, going from an initial concentration of 4.72 ± 0.06 CFU/ml to 5.63 ± 0.06 CFU/ml after 35 days of study. As for the Dachshunds, the LACTO group went from an initial concentration of 4.80 ± 0.06 CFU/ml to reach a concentration of 5.62 ± 0.06 CFU/ml at the end of the experiment (Table 7).

In addition, total coliforms decreased in the probiotic-treated groups compared to the control ones: Chihuahua dogs reported an initial concentration of 4.65±0.06 CFU/ml and a final concentration of 3.94±0.06 CFU/ml , while the Dachshunds showed an initial concentration of 4.55±0.06 CFU/ml and a concentration of 3.96±0.06 CFU/ml at the end of the study (Table 8, Figure 2).

The beneficial effects deriving from a probiotic administration are now well known, however to date very few studies have focused on the effects of a specific probiotic strain. In fact, a specific probiotic strain can exert a different action compared to other strains belonging to the same bacterial group, it is not a coincidence that today we speak of 'strain -specificity'. In addition, in literature very few studies analyzed the effects of the bacterial species L. reuteri on dog and no study has yet evaluated how the effect of these probiotics may vary according to the size of the animal. Today there are more than 400 dog breeds with high variability in size and weight starting from small dogs like Chihuahuas that can weigh 1 kg up to the 100 kg of a St. Bernard [17]. Digestion is a very important factor for canine health; this process involves physicochemical, mechanical, and microbial mechanisms. In recent years, the intestinal microbiota has gained relevance in dog nutrition and health. However, all the substances used to modulate the intestinal microbiota such as prebiotics, probiotics, postbiotics and other nutraceutical products should consider all the aspects concerning canine digestion and absorption. Recently, different petfood manufacturers produce specific feeds for certain dog breeds based on their size and predispositions [18,19,20,21]. To date, the effect that a dog's size may have on the digestion and absorption of substances remains little investigated. An interesting review published by Deschamps et al. [22] found only 163 studies that focused on the digestive differences that may exist in dogs of different sizes; among these studies small dogs were involved in only 7 publications, medium dogs in 71 and large dogs in 9, the other research has focused on the study of a single breed. It is easy to understand that to date the studies performed on the different digestive peculiarities between dogs of different sizes are very few and we still have little information on the matter. About the gastrointestinal motility and the transit time the studies performed so far do not show consistent results [23,24] suggesting that gastric emptying time is more related to food consistency than animal size [25]. Even regarding the small intestinal transit time, to date it does not appear to be related to the weight of the animal [26]. However, Hernot et al. [27] demonstrated a longer large intestinal transit time in large dogs than in small dogs and a correlation between the large intestinal transit time and the canine size. The length of the large intestine but also its area and volume increases with the size of the animal, this seems to be closely associated with a higher colonic transit time influencing not only the absorption of water and nutrients but also the intestinal microbiota composition and fecal humidity. However, Guard and Suchodolski [28] highlighted that although the intestinal microbiota composition varies greatly between dogs, the functions performed by these microorganisms remain always the same and are unchanged in dogs of different sizes. However, among the most important metabolites released by intestinal microbes we find short-chain fatty acids and it appears that their fecal concentration increases as the animal's weight increases [29]; in fact, a longer large intestinal transit time can be related to a higher microbial fermentation. Excessive bacterial fermentation can create problems in water absorption due to the excessive production of SCFAs with high osmotic power. To what has been described so far, we should add the increased intestinal permeability observed in large breed dogs with a luminal retention of water and electrolytes [30]. Large dogs appear to be more delicate from an intestinal point of view than small dogs and considering this evidence it is appropriate to consider that the same probiotic strain may have different effects on dogs of different sizes as it finds a different intestinal ecosystem. This is why our research group wanted to evaluate the effects of the same probiotic strain on dogs of different breeds and sizes. In the present study we focused on the effects of Limosilactobacillus reuteri NBF 1 DSM 32203 on small dogs, observing how it manages to make the feces more consistent and formed, reducing fecal humidity, reaching a fecal score equal or very close to 2, considered optimal. The improvement in the fecal parameters is an indication of a correct digestive process and adequate intestinal absorption. Furthermore, as expected, the administration of the probiotic caused an increase in total lactobacilli, beneficial bacteria for intestinal health, and a significant reduction in coliforms, which are potentially harmful bacterial species. In previous studies we conducted on Golden Retrievers and French Bulldogs we found the same power of L. reuteri NBF 1 DSM 32203 to increase lactobacilli, however the reduction of coliforms seemed to be less evident than that observed in Chihuahuas and Dachshunds [7,8]. Studies conducted so far would suggest a greater ability of L. reuteri NBF 1 DSM 32203 to reduce the concentrations of potentially pathogenic bacterial species such as coliforms in small animals compared to large ones, perhaps due to the different intestinal ecosystem they colonize.

As previously mentioned, this study is the last in a series of 3 experiments performed to evaluate the efficacy of the probiotic Limosilactobacillus reuteri NBF 1 DSM 32203 on body condition, fecal parameters and intestinal microbiota of healthy adult dogs of different sizes. The result obtained from the present study suggest how L. reuteri NBF 1 DSM 32203 can improve the fecal parameters of small dogs like Chihuahua and Dachshund managing to modulate the composition of the intestinal microbiota with an increase in beneficial bacterial species such as lactobacilli and a decrease in potentially harmful ones such as E. coli. Furthermore, this study showed the greater ability of L. reuteri to reduce total coliforms in small dogs compared to large dogs.