The percentage of average body weight loss per week was within the recommended range for dogs (1 to 2%) . These results are of paramount importance, as they demonstrate that the weight loss program in all animals was carried out in a healthy and appropriate manner.
All animals in the OG had BCS (9/9)  and, when examining body composition, the average percentage of fat was greater than 40%. The key point of a weight loss program is the reduction of energy intake [40, 41], in order to promote the negative energy balance associated with maintaining lean mass , and this was observed in our study, because despite the difference in fat mass composition, lean mass did not change after weight loss, except when expressed as a percentage. This is essential, as the body's muscle tissue is metabolically active and guarantees greater energy expenditure .
In this study, no differences were observed in beta diversity, as well as in the alpha diversity and Faith's phylogenetic diversity and Shannon alpha-diversity indexes as well as the simple OTUs count. However, Pielou's evenness index showed greater uniformity among the microbial species were found in the OG samples, when compared to the WLG and LG groups. This result may suggest that weight loss increased the biodiversity of emaciated dogs.
According to , an important factor related to dysbiosis may be the loss of total microbial diversity. Recently, a study with dogs , concluded that increased diversity may be an important factor or even a marker of a healthy canine microbiota, however, further studies are needed to confirm this hypothesis in dogs. Another study  evaluated beagle dogs both in ideal body condition and obese. They also did not observe differences in the Shannon diversity index (alpha diversity) and the number of operational taxonomic units (OTU) between groups. And another study finally  found a lower number of OTUs for genera and species in dogs after weight loss, however, the estimated total number of species was higher in these same animals, despite the maintenance of uniformity and diversity of species. The work concluded that the biodiversity of the microbiota increased between the beginning of weight loss and the end.
In this study, it is possible to observe that some of the relative frequency of bacterial groups in WLG is intermediate to LG than to OG (Fig. 4). Studies in humans, rats and even dogs have shown that weight loss causes changes in the intestinal microbiota, and some bacterial groups can return to proportions similar to those of lean animals. An example is the relative abundance of Firmicutes phyla, which is found increased in obese individuals, while Bacteroidetes is decreased, but after weight loss individuals tend to have a microbiota more similar to lean individuals, with an increased relative abundance of Bacteroidetes phyla and decreased Firmicutes [27, 46,47,48,49].
In our study, the relative abundance of the phylum Bacteroidetes increased after weight loss, corroborating the above-mentioned studies. On the other hand, Firmicutes phylum was found in greater proportion in WLG and LG than in OG. According to  Firmicutes is the most abundant phylum in dogs despite of BCS and a previous study  indicated that Proteobacteria was the predominant phylum in obese dogs and Firmicutes in lean dogs. Ley et al. were the first to report a 50% reduction in the abundance of the phylum Bacteroidetes and a proportional increase in Firmicutes in obese rats. Shortly afterwards they found similar results in humans . In contrast, other studies have observed the opposite  or no differences in the proportions of Bacteroidetes and Firmicutes between thin and obese individuals [51, 52]. Sanchez et al.  reported that the increase in Bacteroidetes may have occurred due to the increase in the Bacteroides and Paraprevotella genera after weight loss, similar results were observed with dogs in our study, which showed an increase in the genus Bacteroides and an increase in an unconfirmed genus of the Paraprevotellaceae family. As for a study by Park et al. , OG had a greater abundance of Proteobacteria and Fusobacteria phyla when compared to LG, and dogs after weight loss, and decreased the population of Proteobacteria. The Actinobacteria phylum also showed an increase in dogs after weight loss, a similar result was reported in cats , and they associated this increase to the Collinsella genera, which also occurred in this study.
Within the Firmicutes phylum, the families Erysipelotrichaceae, Turicibacteraceae, Veillonellaceae, Peptostreptococcaceae, Clostridiaceae, Lachnospiraceae and Ruminococcaceae were found in all groups. Lachnospiraceae and Ruminococcaceae had greater abundances in the OG when compared to the other two groups, this may have occurred due to the higher concentration of Butyricicoccus and Faecalibacterium genera in obese animals. In a study carried out with rats, a high protein—low carbohydrate diet led to a decrease in the Lachnospiraceae and Ruminococcaceae families, suggesting that they may be important competitors of C. difficile for amino acids in the lumen. They further discuss that the high protein—low carbohydrate diet would lead to an abundance of oligopeptides and free amino acids in the lumen and could provide a selective advantage that leads to C. difficile overgrowth when associated with loss of Lachnospiraceae and Ruminococcaceae . Furthermore, Lachnospiraceae, in particular, is a dominant bacterial family in the intestinal microbial communities of many mammals. . In contrast, the genera Dorea and Ruminococcus, belonging to the families mentioned above, were found in higher concentrations in animals after weight loss. Previous studies have shown a decrease in the Dorea genus after weight loss [37, 45], on the other hand, the increase in Ruminococcus can be explained by the use of the diet, which has high protein, as previously observed by [37, 55] in dogs.
However, in this same phylum, the families Veillonellaceae and Clostridiaceae had greater abundance in the LG, the genera Meganomas belonging to the Veillonellaceae family presented greater abundance in LG and smaller in WLG, which may be related to the weight loss diet, which has already been previously reported in other studies [27, 37]. According to Pilla et al. , the relative abundance of the Clostridiaceae family is positively correlated with dietary protein digestibility and negatively with fecal protein content, the diet used for weight loss in this study contained approximately 10% of dietary fiber, which may lead to decreased protein digestibility.
In the WLG the predominant families were Erysipelotrichaceae, Turicibacteraceae, Peptostreptococcaceae and Lachnospiraceae. According to Bermingham et al.  Erysipelotrichaceae were positively correlated with a number of markers associated with carbohydrate digestion, including diets with high fiber content and short chain fatty acids production. The same authors observed a negative correlation between Erysipelotrichaceae and markers of protein metabolism in the intestinal tract. Among members of the Erysipelotrichaceae family, we found an effect on the genus Allobaculum, Catenibacterium, and Turicibacter which were identified as part of a healthy microbiota in dogs [56, 57]. The increase of Turicibacteraceae in the WLG may be due to the presence of FOS in the diet composition. In other studies [56, 58], inulin-type fructans prebiotics had also increased Firmicutes but from families Erysipelotrichaceae and Turicibacteraceae. Although Peptostreptococcaceae showed greater abundance in the WLG, the Clostridium genera, belonging to this family, was underrepresented. In an experiment with healthy dogs, the presence of high fiber and prebiotics led to a relative increase in this family . Other studies have shown that after weight loss, dogs had a decrease in genus Clostridium  and the greater abundance of the Clostridia class was associated with an obese phenotype in humans, and it is reported to decrease after weight loss . In addition to the increase in Lachnospiraceae, the WLG had an increase in Blautia genera, as well as other families of the phylum Firmicutes, dogs that consume high protein diets show an increase in Lachnospiraceae .
Within the phylum Bacteroidetes, the population of Bacteriodaceae family increased after weight loss, followed by OG and LG, as well as the genus Bacteroides belonging to this family. In a study carried out by Bermingham et al. , they showed a positive correlation between the presence of Bacteroidaceae and the dietary fiber content. Just as in our study Bacteriodes was the only genus found within Bacteriodaceae . Relative count of Prevotellaceae family, was greater in LG followed by WLG and smaller in OG, as well as the genus Prevotella. According to Park et al.  Prevotella is one of the two most abundant genera in healthy dogs, so it is common to be found on LG in higher relative counts.
The phylum Actinobacteria, family Coriobacteriaceae and the genus Collinsella showed lower abundance in OG when compared to WLG. In the study carried out by Pallotto et al. , they compared fecal microbiota of obese cats and after a weight loss program and observed an increase in Actinobacteria with weight loss which was primarily attributable to an increase in Bifidobacterium spp and Collinsella spp.
Fusobacteria phylum showed a decrease in the Fusobacteriaceae family after weight loss, as did the Fusobacterium genus. Diverging results were found by Sanchez et al. who observed an increase in this bacterial group in dogs after weight loss. However, in this study when compared to the lean group, we observed that the WLG presented intermediate values between the OG and LG, thus showing that there was an approximation of the standard microbiota.
Finally, in the Proteobacteria phylum, the Enterobacteriaceae and Helicobacteraceae families increased their proportion after weight loss, as did the genus Helicobacter. While Succinivibrionaceae followed by greater abundance in LG, by WLG and smaller in OG. No previously published studies have shown differences between dogs after weight loss for these families and genera. Meanwhile Succinivibrio and Anaerobiospirillum are both succinate-producing members of the Succinivibrionaceae family of the Gammaproteobacteria, and are recognized as part of the normal fecal microbiota of dogs and cats .
Particularities and limitations
The fecal microbiota can be influenced by several factors, such as sequencing method, sample type, genotype, age, sex, environment and diet [63, 64]. Therefore, in this study, in addition to taking care of animal standardization, collection and sequencing methods, the design was carried out to had special attention in two moments: the period of diet acclimation and the weight loss program. The diet’s acclimation period, in which obese and lean dogs were included at the beginning of the study, aimed to reduce the variation in composition between the different diets consumed by the animals before participating in the study, a factor cited as important in the characterization of the fecal microbiota [27, 64]. Thus, the results of the microorganism profile observed among obese and control animals had less dietary influence, despite of living in different households.
This study is one of the few that evaluated the effect of weight loss on the fecal microbiota of naturally obese dogs . It provided a detailed experimental comparison of phyla, family, genera and bacterial species found in the feces of dogs in the obese condition and after the reduction of 20% of the body weight of the same animals and of dogs in an ideal BCS.
A fact that can be considered as a limitation is the variability between control and low-calorie diets. However, in this study, the aim was to evaluate the influence of WLP (high-protein-high-fiber diet and energy restriction) on the fecal microbiota of obese dogs and after loss of 20% of the initial weight of the study. In addition, another study  had evaluated the effect of a commercial diet for weight loss, with a similar profile to that used in this study, on the fecal microbiota of obese dogs before and after period of consumption of a weight loss diet. They also evaluated animals that did not complete the WLP, and these dogs showed no significant differences in their fecal microbiota before and after starting the WLP. Similar results were shown in the study by Kieler et al. , which evaluated the fecal microbiota of overweight dogs after a WLP. Still on this study, the dogs were followed for 12 weeks, and it is not clear how many dogs reached an ideal body weight. The main finding was the decrease in abundance of the Megamonas genera, which correlated with a higher rate of weight loss during the 12-week weight loss of the WLP . In our study, we also observed a decrease in the Megamonas genera (Fig. 4) after weight loss, which can be attributed to of the use of commercial diets for weight loss. However, the role of Megamonas in obesity is unclear and deserves further investigation.