Effects of Lactobacillus Reuteri NBF 1 DSM 32203 Supplementation on Healthy Dog Performance

In this study was evaluated the effectiveness of a specific probiotic strain, NBF 1 DSM 32203, on the intestinal health of healthy Golden Retriever adult dogs by analyzing their Bodyweight (BW), Body Condition Score (BCS), fecal quality and fecal moisture. In addition, microbiological analyses were carried out at specific time points during the experiment in order to quantify bacterial species such as E. coli (for the total coliform count) and Lactobacilli. The dogs included in the study were divided into two groups: control group, fed the standard commercial diet with the addition of a placebo, and treated group to which the probiotic was administered. The study lasted 35 days in line with the time needed to assess any effects. BW data showed no differences between the two groups of dogs. The fecal moisture was significantly lower at the end of the trial in the treated group compared with the control group; the beneficial effect of Lactobacillus reuteri NBF 1 DSM 32203 was also confirmed by the values of fecal score recorded among the two groups of dogs. Additionally, at the end of the study period, there was a significant increase of Lactobacilli in the treated group respect to the control group (P= <0.001). The data collected in this study report the ability of the probiotic strain L. reuteri NBF 1 DSM 32203 to improve fecal quality parameters such as fecal moisture and fecal quality in healthy adult dogs showing an increase in the Lactobacilli count and a little reduction of total coliforms.


Ethical Statement
The research was conducted according to the directive 2010/63/EU, (article 1 (paragraph 5f) (EUR-Lex -02010L0063-20190626 -EN -EUR-Lex (europa.eu)); the study did not imply any form of animal suffering or health risk, since it focused on the administration of a natural substance. However, compliance with the decree ensured a further safeguard for the patient's health

ARTICLE INFO SUMMARY
In this study was evaluated the effectiveness of a specific probiotic strain, L. reuteri NBF 1 DSM 32203, on the intestinal health of healthy Golden Retriever adult dogs by analyzing their Bodyweight (BW), Body Condition Score (BCS), fecal quality and fecal moisture. In addition, microbiological analyses were carried out at specific time points during the experiment in order to quantify bacterial species such as E. coli (for the total coliform count) and Lactobacilli. The dogs included in the study were divided into two groups: control group, fed the standard commercial diet with the addition of a placebo, and treated group to which the probiotic was administered. The study lasted 35 days in line with the time needed to assess any effects. BW data showed no differences between the two groups of dogs. The fecal moisture was significantly lower at the end of the trial in the treated group compared with the control group; the beneficial effect of Lactobacillus reuteri NBF 1 DSM 32203 was also confirmed by the values of fecal score recorded among the two groups of dogs. Additionally, at the end of the study period, there was a significant increase of Lactobacilli in the treated group respect to the control group (P= <0.001). The data collected in this study report the ability of the probiotic strain L. reuteri NBF 1 DSM 32203 to improve fecal quality parameters such as fecal moisture and fecal quality in healthy adult dogs showing an increase in the Lactobacilli count and a little reduction of total coliforms.

Animals and Study Design
The trial took place on 4 th October 2020 in the Dietinger Laura breeding, region: Benne Bicocca,3; 10060 -Scalenghe (TO). Healthy adult male and female non pregnant (age > 1year) dogs (n = 40, Golden Retriever; 19 females, 21 males; 19 + 21 = 40) were selected for the study and were randomly assigned to the control group (CTR; n=20; male: female = 9:11) and to the treated group (LACTO; n=20; male: female = 3:2); (Tables 1A & 1B) reports age and weight of each dog included in the study. The design of the practice is a blind trial: the operator on the farm is aware of the animals belonging to the two experimental groups while the operator of the analysis laboratory is not aware of the origin of the sample; in particular, the analyzed dogs were assigned randomly (SurveyMonkey Excel) between the control group and the treated group (as indicated below), thus minimizing selection bias errors. In this way two similar groups were obtained, allowing to better identify the effect of the treatment. Group assignment was organized according to kennel management standard procedure. The sample number was calculated using the formula for the comparison between means, fixing an alpha equal to 0.10 and a beta equal to 0.80; in this way it results that the minimum number of subjects to analyze is roughly equal to 20 for each group (see statistical analysis paragraph). In order to minimize the bias, the subjects involved in the experiment have been divided in a completely random way between the two groups; in order to carry out the randomisation, each subject was labeled with a unique numeric code and then twenty numeric codes were randomly extracted from those labeled. The subjects with those numeric codes were assigned in the treatment group, the rest in the control group.

Feed Supplement and Diet
A dry extruded commercial petfood for adult dogs (
Then, a total of 20 g of this pre-mixture was daily added to each 980 g of commercial feed in the bowl. The CTR group received the commercial diet, with the addition of 20 g of maltodextrin in 980g of dog feed (placebo).   Crude Fiber (%) 1.84 Crude ash (%) 7.49 NFE(%)* 37 ME** (kcal/Kg) 3944.7 Note: *NFE= 100 -(humidity + crude protein + crude fat + crude fiber + crude ash).
**Predictive equations (NRC 2006a) for ME in prepared pet foods for dogs and cats (FEDIAF -Nutritional guidelines for complete and complementary pet food for cats and dogs -September 2020).

Data Collection
Bodyweight (BW) and body condition score (BCS) were and their species count was investigated. Fecal score was evaluated using a 7-point scoring chart according to Bybee and colleagues, as described in Table 5, at all six sampling times (T0-T5). Log-shaped; Leaves residue on ground and loses form when picked up.

5
Very moist but has a distinct shape; Present in piles rather than logs; Leaves residue on ground and loses form when picked up. 6 Has texture, but no defined shape; Present as piles or spots; Leaves residue on ground when picked up.   10.26717/BJSTR.2021.37.005956 In the laboratory, collected fecal samples were analyzed to determine FM . Fecal sampling was carried out at T0, T1, T2, T3, T4 and T5 and the collected samples were stored at +4°C until they are brought to the laboratory, where they are stored at −20 °C.

Statistical Analysis
Unlike cats that can be considered 'narrow carnivores', dogs can be called 'optional carnivores' because they have all the characteristics that define carnivores, both anatomically and physiologically, but they are also able to digest and assimilate nutrients such as carbohydrates present in foods such as cereals, legumes and fruits. It's known that carnivorous animals have a different intestinal microflora from herbivores and omnivores; this intestinal microflora serves, among other things, to ferment certain foods, such as carbohydrates. In dogs, the ability to ferment carbohydrates is poor, although it varies depending on the breeds. In fact, there are breeds that assimilate certain nutrients well while others fail to assimilate them at all [3]. However, the source of carbohydrates would not seem to be indispensable for dog as it has alternative metabolic processes through which it produces glucose from proteins, so, according to the nutritional epigenetics, during the process of domestication of the dog, environmental pressures may have caused a change in the synthesis of enzymes in charge of digestion of nutrients, so that dogs adapted to live on a diet based on the waste of the human diet; this would explain why nowadays dogs can assimilate numerous substances of plant origin [4].
where σ represents the standard deviation, derived from pilot studies or literature, while δ (µ1-µ2) is the minimum clinically relevant difference. As regards the identification of σ and δ, we also used the data of a previous study [9], considering the variable of the fecal score. Considering that the dogs included in this study are healthy (without any gastrointestinal pathology) and living in controlled breeding conditions (without any variation in the daily food ration), the expected variability will be low and therefore a variation of 0.4 points in the fecal score can be considered biologically relevant with a standard deviation of 0.512.
Applying the above values to the above formula we will have: 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 were been statistically tested using Student's t test (with Tukey p-value adjustment). In order to be able to describe the goodness of the fit of the mixed model, we used the R squared described by [12], No outliers and missing data were found.

Discussion
In this study were analyzed the effects of a specific probiotic:

Conclusion
The data collected in this study report the ability of the probiotic