Does Body Size Matter? Probiotic Response to Limosilactobacillus reuteri DSM 32203 in Small-Breed Dogs

1. Introduction

The domestic dog (Canis lupus familiaris) represents a unique model in which marked differences in body size have developed within a single species because of selective breeding. Adult body weight may range from less than 2 kg in some toy breeds to more than 50 kg in giant breeds, and this variation is accompanied by differences in gastrointestinal anatomy and physiology. Body size has been associated with changes in gastrointestinal dimensions, colonic transit, intestinal permeability, fermentative activity, and fecal water content, suggesting that the digestive environment is not uniform across canine breeds [1,2,3,4,5]. These differences are of relevance when nutritional interventions are intended to modify intestinal function, because the physiological conditions encountered by a dietary component or microorganism may vary substantially among dogs of different sizes [2,3,6]. The intestinal microbiota is an integral component of the canine gastrointestinal ecosystem, contributing to nutrient metabolism and fermentation, modulation of intestinal barrier function, and bidirectional interactions with the host [7,8,9,10]. Although the general structure and functional relevance of the canine gut microbiota are increasingly well characterized, substantial inter-individual variation has been reported, with microbial composition influenced by multiple intrinsic and extrinsic factors, including breed, age, body condition, body size, diet, and living environment [11,12,13]. Importantly, evidence also suggests that body size may contribute to differences in the canine fecal microbial ecosystem. Middleton et al. [6] identified metabolic, clinical, and microbiota differences between small and larger dogs maintained on the same diet, suggesting that some metabolic and microbial characteristics may be associated with host body size rather than being solely attributable to dietary differences. More recently, a comprehensive review of canine digestive physiology highlighted associations between body size and several gastrointestinal and fecal parameters, particularly within the colonic compartment, including intestinal transit time, permeability, fiber degradation, fecal short-chain fatty acids, and fecal water content [3]. Subsequent experimental work further demonstrated that nutritional and physicochemical conditions representative of different dog sizes can reshape canine microbiota structure and function, including microbial fermentation activity [5]. These observations are particularly relevant to the use of probiotics. Probiotics are defined as live microorganisms that, when administered in adequate amounts, confer a health benefit on the host, and their effects are increasingly investigated as a means of modulating intestinal microbial communities and gastrointestinal function [14,15,16,17]. In dogs, probiotic supplementation has been associated with changes in fecal characteristics, intestinal microbial communities, microbial metabolites, and selected host responses. However, the available evidence remains heterogeneous, and comparisons among studies are complicated by differences in probiotic strains, doses, administration protocols, diets, host characteristics, and outcome measures [18,19]. Consequently, probiotic effects should not be attributed solely to the bacterial species, as strains belonging to the same species may exhibit distinct biological properties and probiotic potential [18,20]. The strain-dependent nature of probiotic effects raises an important question that has received comparatively little attention in canine nutrition: to what extent can characteristics of the host influence the response to a defined probiotic strain? Recent evidence in healthy dogs indicates that the magnitude of the microbiota response to probiotic supplementation can vary among individuals and is associated with the microbial profile at baseline, supporting the relevance of host-related factors in probiotic responsiveness [19]. If dogs of different sizes differ in gastrointestinal anatomy, intestinal transit, water handling, fermentation, and microbial composition, the intestinal environment in which an exogenous microorganism acts may also differ according to host size [3]. It is therefore plausible that the magnitude or temporal pattern of the response to a given probiotic strain may vary across canine phenotypes. Among lactic acid bacteria investigated for canine applications, Limosilactobacillus reuteri has attracted interest because of its potential to interact with the intestinal microbiota and influence microbial composition and activity [18,21]. Nevertheless, the biological effects of L. reuteri remain strain-dependent, and effects observed with one strain should not be assumed to be transferable to other strains of the same species [18,20]. The strain L. reuteri DSM 32203 has previously been investigated in dogs, including French Bulldog and Golden Retriever, and has shown potential to improve fecal consistency through a reduction in fecal moisture when administered as a feed additive [22,23,24]. The preparation containing L. reuteri DSM 32203 was subsequently authorized in the European Union as a zootechnical feed additive for dogs [25]. However, the response of small-breed dogs to this defined strain remains comparatively underexplored. Small-breed dogs are particularly relevant in this context because they cannot simply be considered scaled-down versions of larger dogs: body size is associated with differences in gastrointestinal anatomy and physiology, intestinal transit, fecal water content, fermentation-related parameters, and aspects of the fecal microbiota [3]. Studies comparing healthy dogs have also demonstrated that individual characteristics can be associated with measurable differences in fecal microbial communities, emphasizing the importance of considering host-related variables when interpreting microbiota-directed interventions [26]. Nevertheless, experimental studies investigating whether a defined probiotic strain produces consistent effects among dogs differing in body size remain limited. The present study was designed to investigate the effects of L. reuteri DSM 32203 supplementation in two small-breed populations, Chihuahua and Dachshund dogs. These breeds were selected because, despite both being classified as small dogs, they differ substantially in body weight and therefore provide an opportunity to investigate the response to the same probiotic strain in two distinct small-breed phenotypes. The effects of supplementation were evaluated longitudinally using body weight, body condition score, fecal score, fecal moisture, and selected fecal bacterial populations as outcome measures. The primary objective was to determine whether daily supplementation with L. reuteri DSM 32203 could improve fecal quality and modulate selected bacterial populations in healthy small-breed dogs. More broadly, the study aimed to extend previous observations obtained with the same probiotic strain in other canine breeds and to investigate whether the response to a defined probiotic strain is comparable between small-breed dogs differing in body weight. We hypothesized that L. reuteri DSM 32203 would improve fecal consistency and modify selected bacterial populations in both Chihuahua and Dachshund dogs, while the comparison of these two small-breed phenotypes would provide preliminary insight into whether the magnitude and pattern of the response to the same probiotic strain are broadly consistent or differ according to body weight.

2. Materials and Methods

2.1. Animals and Study Design

Sixty healthy Chihuahua (n=30) and Dachshund dogs (n=30), 26 males (12 Chihuahuas + 14 Dachshunds = 26) and 34 non-pregnant females (18 Chihuahuas + 16 Dachshunds = 34), age> 1 year were involved in the study and randomly assigned to the control group (CTR; n= 30: 15 Chihuahuas + 15 Dachshunds) and experimental group (LACTO; n= 30: 15 Chihuahuas + 15 Dachshunds). Animals were divided randomly (SurveyMonkey Excel) into the two groups and the group assignment was organized according to kennel management standard procedure. The experimental group was supplemented with Limosilactobacillus reuteri DSM 32203 while the control group did not receive the probiotic and was supplemented by maltodextrin used as placebo. The two dog breeds were analyzed individually to evaluate any differences inherent to the breed itself or to the diet administered. Table 1 reports age, sex and body weight of each dog of the two breeds belonging to the control and experimental groups. Cleaning and disinfecting procedures of the single fences were carried out and the animals have been individually stabulated. Moreover, the animals were evaluated daily by a veterinarian for any health and welfare concerns throughout the experimental period. The study lasted a total of 49 days: 35 days of study and 14 days of acclimatization.

2.2. Feed Supplement and Diet

All dogs had free access to potable water and were fed with two different commercial petfood: Chihuahua dogs consumed Royal Canin mini adult extruded feed while the dachshund dogs ate Eukanuba adult small breed food. For the entire duration of the study, feed was administered to each animal once a day based on the energy requirements of each dog (adult dogs: 110 kcal x BW^0.75 kg) [27]; table 2 reports the analytical components of each petfood administered. Dogs belonging to the experimental group received commercial feed with the addition of 10 g/100 kg of Limosilactobacillus reuteri DSM 32203, corresponding to 5 x 10⁹ colony-forming units (CFU)/kg food; the process to obtain the right probiotic amount consisted in using 50 g of the feed additive (standard concentration ≥ 1.0 x 10¹¹ CFU/g), pre-mixed in laboratory with 9950 g of maltodextrins. Then, a total of 20 g of this pre-mixture was daily added to each 980 g of commercial feed in the bowl. The control group received the commercial diet, with the addition of 20 g of maltodextrin in 980 g of dog feed (placebo).

Table 2. Analytical components of Royal Canin mini adult extruded feed (A) and Eukanuba adult small breed food (B).

A. Royal Canin mini adult extruded feed
Parameter	Amount (%)
Crude protein	27.00
Crude fats	16.00
Raw fiber	1.30
Raw ash	6.80
Metabolizable energy	3920 Kcal/Kg

Table 2. Analytical components of Royal Canin mini adult extruded feed (A) and Eukanuba adult small breed food (B).

A. Royal Canin mini adult extruded feed
Parameter	Amount (%)
Crude protein	27.00
Crude fats	16.00
Raw fiber	1.30
Raw ash	6.80
Metabolizable energy	3920 Kcal/Kg

Composition: dehydrated poultry protein, wheat flour, maize flour, animal fats, maize, wheat gluten, barley, maize gluten, wheat, hydrolysed animal proteins, minerals, beet pulp, soya oil, yeast products, fructooligosaccharides, fish oil, algae oil.

Nutritional additives: Vitamin A: 15700 IU, Vitamin D3: 1000 IU, Iron (3b103): 36 mg, Iodine (3b201, 3b202): 3.6 mg, Copper (3b405, 3b406): 11 mg, Manganese (3b502, 3b504): 47 mg, Zinc (3b603, 3b605, 3b606): 133 mg, Selenium (3b801, 3b811, 3b812): 0.09 mg - Preservatives - Antioxidants.

B. Eukanuba adult small breed food
Parameter	Amount (%)
Crude protein	30.00
Crude fats	18.00
Raw fiber	2.00
Raw ash	6.70
Metabolizable energy	3923 Kcal/Kg

Composition: dehydrated chicken and turkey (28% of which chicken 17%, natural source of glucosamine, chondroitin sulphate and taurine), fresh chicken (15%), corn, wheat, poultry fat, oats, barley, millet, beet pulp (3.5%), chicken gravy, fish meal (natural source of Omega 3 fatty acids), minerals (including sodium hexametaphosphate 0.35%), dehydrated whole eggs, fructo-oligosaccharides (0.38%, natural prebiotics).

Nutritional additives: Vitamin A (47,749 IU/kg), Vitamin C (60 mg/kg), Vitamin D3 (1,584 IU/kg), Vitamin E (265 mg/kg), Beta-Carotene (5.2 mg/kg), L-Carnitine (50 mg/kg), Copper (9 mg/kg), Iodine (1.1 mg/kg), Manganese (4 mg/kg), Zinc (100 mg/kg), Selenium (0.01 mg/kg). Antioxidants: tocopherol-rich vegetable oil extracts (86 mg/kg); flavourings: organic rosemary extract (46 mg/kg), tea extract (23 mg/kg).

2.3. Data Collection

Body Weight (BW), Body condition Score (BCS), Fecal Score (FS) and Fecal Moisture (FM) were recorded at the beginning of the experiment (day 0= T0), after one week (T1), after 2 weeks (T2), after 3 weeks (T3), after 4 weeks (T4) and at the end of the study (T5) according to the American Animal Hospital Association (AAHA) Nutritional Assessment Guidelines for Dogs and Cats [28]. The Body Weight of each dog was measured by the same operator at the same time (morning, before feed administration), with the same instrument while the body composition assessment was carried out by visual examination and palpation of the animal on a scale between 1 and 9 where a score of 4 or 5 is reflecting the ideal body condition [29]. A 7-point scoring chart was used for the analysis of the fecal score according to the Nestle Purina Fecal Scoring System while for the analysis of fecal moisture (FM) the fecal samples were collected from each dog at T0, T1, T2, T3, T4 and T5 and then stored at +4°C until they are brought to the laboratory, where they are stored at −20°C. 5–10 g of stool was weighed and dried in an oven at a temperature of 105°C–110°C for 20–24 hours, cooled down in a desiccator for another 20–24 hours, after which the Fecal Moisture content was calculated as lost weight after desiccation. In addition, a microbiological analysis was conducted to identify and quantify specific intestinal bacterial species, one gram of fresh stool was used to carry out this analysis at four specific time points: T0, T1, T3 and T5. Briefly, fresh stool was diluted in sterile saline solution with a ratio of 1:10 and then vortexed for two minutes to have a homogeneous suspension which was plated on different culture media for total bacterial counts and identification; for Escherichia coli and total coliforms was used the eosin methylene blue agar (Oxoid, Italy) with an incubation of 24 hours at 37°C. E. coli colonies show growth with a green metallic reflex, while coliforms show growth with blue, red, or uncolored colonies. For lactobacilli identification was used the Man Rogosa and Sharpe agar (Oxoid) and the plates were incubated for 48 hours at 37°C under anaerobic condition. All the analysis was performed in duplicate.

2.4. Statistical Analysis

For the statistical analysis a Mixed Model with repeated measurements has been used, which allows to estimate the parameters considering both random effect and fixed effect [30]. The model has been estimated as the following:

yi,j,k =μ+Si +Gj +Tk +Gj * Tk +ei,j,k

where y = dependent variable (FM, FS, BW, BCS, LB, COLI); μ = overall mean; Si = fixed effect of the ith sex (I = 1, 2); Gj = fixed effect of the jth group (j = 1, 2); Tk = fixed effect of the kth time (k = 0, 5) and ei,j,k = error. The software used was R Core Team (2020), R: A language and environment for statistical computing [31] and for the different analysis was used the Mixed Model [32] and the Least Squares [33]. Time was used as repeated measurement and therefore each subject has been analyzed in every different temporal instant. The autoregressive covariance structure was used. Least Square Means were estimated, and they have been statistically tested using Student’s t test (with Tukey p-value adjustment). To be able to describe the goodness of the fit of the mixed model, we used the R squared described by Nakagawa et al. [34]. No outliers and missing data were found.

3. Results

The results of the study show how L. reuteri 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 moisture (Table 5) decreased in the group of dogs treated with the probiotic L. reuteri 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 DSM 32203 showed a decrease in fecal moisture of 0.20 (g/g) compared to T0 (beginning of the study), while the dachshund dogs showed a decrease of 0.24 (g/g) (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 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 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 DSM 32203, a significant increase in lactobacilli was recorded, going from an initial concentration of 4.72 ± 0.06 log CFU/g to 5.63 ± 0.06 log CFU/g after 35 days of study. As for the Dachshunds, the LACTO group went from an initial concentration of 4.80 ± 0.06 log CFU/g to reach a concentration of 5.62 ± 0.06 log CFU/g at the end of the experiment (Table 7).

In addition, total coliforms decreased in the probiotic-treated groups compared to the control groups. Specifically, the LACTO group in Chihuahua dogs reported an initial concentration of 4.65±0.08 log CFU/g and a final concentration of 3.94±0.08 log CFU/g, while the control group maintained an initial concentration of 4.73±0.08 log CFU/g and a final concentration of 4.71±0.08 log CFU/g (Table 8A, Figure 2A). A similar decrease was observed in Dachshunds: the LACTO group showed an initial coliform concentration of 4.55±0.08 log CFU/g, which dropped to 3.96±0.08 log CFU/g at the end of the trial, whereas the control group showed an initial count of 4.60±0.08 log CFU/g and a final count of 4.68±0.08 log CFU/g (Table 8B, Figure 2B).

5. Conclusions

As previously mentioned, this study is the last in a series of 3 experiments performed to evaluate the efficacy of the probiotic Limosilactobacillus reuteri 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 DSM 32203 can improve also 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 coliforms.