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© 2004 The American Society for Nutritional Sciences J. Nutr. 134:2158S-2161S, August 2004

Supplement: WALTHAM International Science Symposium: Nature, Nurture, and the Case for Nutrition

Dietary Protein Source and Manufacturing Processes Affect Macronutrient Digestibility, Fecal Consistency, and Presence of Fecal Clostridium perfringens in Adult Dogs1,2

Jürgen Zentek3,4, Sonja Fricke, Marion Hewicker-Trautwein*, Britt Ehinger*, Gunter Amtsberg{dagger} and Christoph Baums{dagger}

Institutes of Animal Nutrition, * Pathology, and {dagger} Microbiology, School of Veterinary Medicine Hannover, Hannover D-30173, Germany

3 To whom correspondence should be addressed. E-mail: juergen.zentek{at}vu-wien.ac.at.

KEY WORDS: • dogs • gut morphology • gut histology • protein intake • digestibility


EXPANDED ABSTRACT

Dietary protein sources and concentrations in mixed diets for dogs can affect "nonspecific dietary sensitivity" (or food intolerance) (1–3). Negative gastrointestinal effects such as poor fecal consistency, especially in large-breed or highly active dogs, have been described after dogs were fed certain animal proteins (1,4,5). In the presence of mild clinical signs but in the absence of an allergic response, this condition has been designated a nonspecific dietary sensitivity (3,5). The underlying mechanism that causes this effect is not clear but may be related to an impact on the intestinal microflora by favoring the conditions for growth and toxin formation of Clostridium perfringens and other proteolytic bacteria, by effects on the water-holding capacity of the intestinal contents, and by decreased water-absorption capacity of the gut wall.

In the present study, we investigated the effects of two different dietary protein sources, beef and poultry, either as extruded or canned mixed diet using a similar recipe in two groups of healthy dogs. Eight Beagles were used as nonsensitive dogs, and three German Shorthair Pointers (GSPs)5 were included because of their known problems with nonspecific dietary sensitivity. Fecal quality, digestibility, intestinal microbiology, and gut-wall histology were studied. A standard dry diet was used as a reference food.


   MATERIAL AND METHODS
TOP
 MATERIAL AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
The investigations were performed with three GSPs (2 females and 1 male; age, 6 y; body wt, 27.4 ± 2.7 kg) and eight adult Beagles (6 females and 1 spayed male; age range, 1–11 y; body wt, 12.4 ± 2.2 kg). The dogs were kept separately under identical conditions in a colony. Dogs were vaccinated yearly and dewormed before the experiment and again 6 mo later. Animal care and treatment protocols were approved by the governmental committee according to the German Tier-schutzgesetz.

Two protein sources (beef and poultry) were used in the experimental diets that were either processed as dry or as canned food. The recipes were kept practically constant between the diets. Main ingredients were meat and organs, rice, corn, cellulose, and animal fat. Meat and animal fat were either pure beef or pure poultry products. Production lines were completely cleaned between the processes to avoid cross-contamination and to achieve the highest purity of the compounded diets. A commercial dry diet (main ingredients: poultry meat and byproducts, cereals) was used for comparison. Data on nutrient analyses are presented in Table 1.


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  		TABLE 1 Nutrient analysis of the experimental diets12

 
The metabolizable energy (ME) of the diets was calculated from the results of the digestive trials after correction of digestible energy of the digestible crude protein by –4.38 kJ/g (6).

Feed allowances were adjusted individually according to the energy density of the diet and the calculated maintenance requirements. Food intake amounts were adjusted to keep body weight constant.

Fecal quality was assessed during each feeding period on d 8–23. Feces produced during each defecation were graded on a 1–5 scale, and mean total scores were calculated for each feeding period. Grade 1 represented dry, crumbly feces; grade 2 were ideal, well-formed feces; grade 3 were feces of good quality that were slightly moist; grade 4 feces were moist and poorly formed; and grade 5 was watery diarrhea. Fresh fecal samples were collected during regular kennel inspection and were subjected to additional analyses. Fecal dry-matter content was determined by overnight oven drying (at 103°C), and mean data were calculated from 15 samples per individual and feeding period. Fecal unbound water was determined by centrifugation of 6 g of fecal material at 25,000 x g for 30 min (Biofuge stratos, Kendro Laboratory Products, Osterode, Germany). The supernatant was considered as unbound water, and the amount was weighed and expressed as a percentage of the fresh fecal matter. Four samples were investigated during each feeding period from each dog, and results are summarized as a mean per dog.

Digestibility of organic matter was determined after 7 d of adaptation to the respective diet by quantitative feces collection. Additionally, the colony forming units of Cl. perfringens per g feces were determined using dilutions of 10–1–10–7* (culture on nonselective and selective media) in the trial with the Beagles after an adaptation of 16 d to the respective diets. Forty Cl. perfringens isolates were characterized in a multiplex PCR that detects the major toxin genes as well as the enterotoxin gene (cpe) and the ß2 toxin gene (cpb2) (7).

Fecal pH was measured after dilution of 1 g of fresh feces with 4 g of distilled water using a digital pH meter (pH 526 Multical, WTW, Weilheim, Germany). Ammonia and volatile fatty acids were investigated in the feces as indicators of microbial activity. Ammonia was measured using an ion-selective electrode (model IS 570 NH, Phillips, Kassel, Germany) that was adapted to a digital pH meter (Knick, Berlin, Germany). Volatile fatty acids were determined in fresh fecal water by centrifugation of 1 g of feces mixed with 4 g of distilled water at 9000 x g for 15 min (Varifuge F, Heraeus Sepatech, Osterode, Germany). Supernatant (1 mL) was mixed with 0.1 mL of internal standard solution (10 mL of formic acid with 0.1 mL of 4-methylvalerianic acid). Analysis was performed by gas chromatography (capillary chromatograph PU-4550, Pye Unicam, Offenbach, Germany) on a 2-m glass column filled with GP 10% SP-1000, and 1% H3PO4 on 100–120 mesh Chromosorb WAW (Supelco, Deisenhofen, Germany).

Biopsies were taken after dogs received the diets for 24 d. The biopsies were collected while dogs were under general anesthesia and were obtained from the proximal and middle colon using a flexible endoscope. At least five colonic biopsies were fixed in 10% neutral-buffered formalin solution, embedded in paraffin wax, and stained with hematoxylin and eosin. The tissue morphology was graded by two investigators (B. Ehinger and M. Hewicker-Trautwein) who had no knowledge of the sample origin. Lesion severity was scored (3) on a scale of 0 (normal) to 3 (severe tissue damage).

Statistics

Data were analyzed using Excel 97 and SAS 6.04 software (8) and are summarized as means ± SD. Feeding periods were compared by ANOVA and t test as post-hoc tests with correction for repeated measurements. Fecal consistency and tissue biopsy scores were compared by Wilcoxon signed-rank test. P < 0.05 was taken as significant. Because only three GSPs were available, the data for this group are presented descriptively only.


   RESULTS
TOP
 MATERIAL AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
All dogs were in good health, and the diets were readily accepted during the entire experimental period. The dry-matter intake amounts were 13.2 ± 1.1 to 20.4 ± 2.4 g · kg of body wt/d in the GSPs and 17.8 ± 3.1 to 20.6 ± 3.1 g of body wt/d in the Beagles, which correspond to calculated ME intakes of 0.23 ± 0.02 to 0.38 ± 0.05 and 0.33 ± 0.06 to 0.35 ± 0.05 MJ · kg of body wt–1·d–1, respectively (Table 2). Mean body wt increased from the beginning to the end of the experiment in both the GSPs and the Beagles. The mean wt for GSPs was 27.4 ± 2.7 kg at the beginning of the feeding trial and 28.0 ± 3.7 kg at the end, and for Beagles, mean body wt was 12.4 ± 2.2 kg initially and 13.0 ± 2.0 kg at the end of the experiment.


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  		TABLE 2 Dry-matter and ME intake of the dogs during the experimental periods1

 
The fecal consistency was considerably firmer in Beagles compared with GSPs especially when the canned diets were fed (Table 3). The canned poultry, which was based on poultry meat and byproducts, led to softer feces in GSPs. Also in GSPs, the dry-matter contents of the feces were generally lower during the periods when canned diets were fed (22.3 ± 2.3 to 23.2 ± 2.8%) than when dry diets were given (26.8 ± 3.5 to 29.7 ± 1.6%). Unbound-water concentrations tended to be higher in GSPs compared with Beagles. Values were 24.3 ± 3.0 to 27.4 ± 4.8% and 19.9 ± 2.5 to 22.0 ± 5.1% in GSPs fed dry and canned diets, respectively, compared with 11.2 ± 3.9 to 16.4 ± 3.2% and 12.9 ± 7.8 to 15.0 ± 5.0% in Beagles fed dry and canned diets, respectively.


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  		TABLE 3 Fecal consistency and concentrations of dry matter and unbound water for dogs1

 
The apparent digestibility of organic nutrients showed similar diet-related differences in both breeds. The dry-matter digestibility values were 75.4 ± 0.4 to 86.7 ± 1.5%, crude protein digestibilities were 78.9 ± 0.5 and 88.4 ± 0.7%, and the apparent digestibility of nitrogen-free extracts was between 74.8 ± 1.9 and 90.0 ± 1.6%. The crude fat of the canned diets was less digestible (88.6 ± 1.9 to 91.6 ± 1.4%) than that of the dry diets (93.5 ± 0.6 to 97.9 ± 0.4%) (Table 4).


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  		TABLE 4 Apparent digestibility of dry matter, crude protein, crude fat, and nitrogen-free extract in the diets1

 
Cl. perfringens was found in all fecal specimens in the Beagles (Fig. 1). When dogs were fed canned diet or the poultry-containing dry diet, Cl. perfringens concentrations were between log 7.6 ± 0.5 and log 8.7 ± 0.4/g of feces. These counts were reduced after dogs were fed the dry diet with beef (log 6.1 ± 1.4/g of feces) and decreased to log 3.3 ± 0.6/g of feces with feeding of the commercial diet. Enterotoxigenic or ß2-toxigenic Cl. perfringens strains were not identified.



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  		FIGURE 1  Concentrations of Cl. perfringens in the feces of eight Beagles. Values are means ± SD. Means not sharing a common superscript are significantly different at P < 0.05. Diet abbreviations are as follows: CP, canned poultry; CB, canned beef; DP, dry poultry; DB, dry beef; DS, commercial dry standard diet.

 
Fecal ammonia concentrations were not significantly influenced by diet. Higher relative concentrations of isobutyric, n-valeric, and isovaleric acids in the feces when canned diet was fed might be related to increased microbial fermentation of proteins within the intestine (Table 5).


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  		TABLE 5 Fecal pH values and ammonia and volatile fatty acid concentrations in Beagles1

 
There was no recognizable dietary effect on the microstructure of the colonic mucosa in Beagles. After GSPs were fed the poultry-containing canned diet, the scores of the pathological-histological alterations of the colonic mucosa (score, 0.9 ± 0.2) were higher than after the dogs were fed other diets (scores, 0.3 ± 0.1 to 0.4 ± 0.3). These findings indicate that tissue integrity was maintained even when the dogs were fed diets that decreased fecal consistency.


   DISCUSSION
TOP
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Dietary intolerance is a common problem. Compared with other breeds, GSPs are especially susceptible to having less-formed feces; this was shown by comparison with German Shepherd Dogs and Beagles (9). Similar findings are reported for different breeds with a trend for larger and very active dogs to be susceptible to developing signs of dietary intolerance (1,10). The underlying reason is not fully understood, but differences in the anatomical conditions of the gastrointestinal tract (with larger breeds having relatively small digestive tracts) (11), accelerated or delayed orocecal transit times, and breed-dependent effects on colonic function have been described (3,4,12) and might contribute to the problem. Processing conditions during food production could be a relevant factor. The extrusion process seems to result in a lower frequency of digestive problems compared with the canning and autoclaving processes. In the present study, minor differences in the recipes must be taken into account. The ingredients were identical for the dry and canned diets used in the study, but due to technological demands, the carbohydrate amounts were slightly higher in the dry than in the canned diets. The small differences in recipe and nutrient composition may have interfered with the effects of the different processing techniques. The tendency for dry diets to result in better fecal quality was coincident with a higher fecal dry-matter content and a tendency for higher fecal free-water contents. Increasing the amount of free water might facilitate colonic absorption of fluid against the water-holding capacity of the intestinal chyme.

Diet also influenced the numbers of fecal Cl. perfringens that were present, and this was most obvious when the commercial dry diet was used. Interestingly, enterotoxinogenic or ß2-toxinogenic strains were not detected. This does not rule out the possibility that these strains were present in the upper part of the intestinal tract. Cl. perfringens numbers have been found to increase substantially in dogs that are fed large quantities of low-quality protein diet (13–15). From the volatile fatty acid concentrations found in the present study, it can be expected that the increased number of isoforms reflects an increase in proteolytic bacterial activity in the gastrointestinal tract.

Conclusion

Protein source and manufacturing process affect dogs' tolerance of mixed diets. Both factors must be considered, especially in the production of diets for sensitive dogs.


   FOOTNOTES
 
1 Presented as part of the WALTHAM International Science Symposium: Nature, Nurture, and the Case for Nutrition held in Bangkok, Thailand, October 28–31, 2003. This symposium and the publication of the symposium proceedings were sponsored by the WALTHAM Centre for Pet Nutrition, a division of Mars, Inc. Symposium proceedings were published as a supplement to The Journal of Nutrition. Guest editors for this supplement were D'Ann Finley, James G. Morris, and Quinton R. Rogers, University of California, Davis. Back

2 This work was supported by a grant from Masterfoods company, Verden, Germany. Back

4 Present address is Institute of Nutrition, University of Veterinary Medicine, A 1210 Vienna, Austria. Back

5 Abbreviations used: CB, canned beef diet; CP, canned poultry diet; DB, dry beef diet; DP, dry poultry diet; DS, commercial dry standard diet; GSP, German Shorthair Pointer; ME, metabolizable energy. Back


   LITERATURE CITED
TOP
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 

1. Meyer, H., Zentek, J., Habernoll, H. & Maskell, I. (1999) Digestibility and compatibility of mixed diets and faecal consistency in different breeds of dog. J. Vet. Med. A 46: 155–165.

2. van der Steen, I., Rohde, J., Zentek, J. & Amtsberg, G. (1997) Dietary effects on the occurrence of Clostridium perfringens and its enterotoxin in the intestine of dogs. Kleintierpraxis 42: 871–886.

3. Zentek, J., Hall, E. J., German, A. J., Haverson, K., Bailey, M., Rolfe, V., Butterwick, R. & Day, M. J. (2002) Morphology and immunopathology of the small and large intestine in dogs with nonspecific dietary sensitivity. J. Nutr. 132: 1652S–1654S.[Abstract/Free Full Text]

4. Rolfe, V. (1999) Colonic fluid and electrolyte transport in health and disease. Vet. Clin. North Am. Small Anim. Pract. 29: 577–588.[Medline]

5. Rolfe, V., Adams, C. A., Butterwick, R. & Batt, R. (2002) Relationship between fecal consistency and colonic microstructure and absorptive function in dogs with and without nonspecific dietary sensitivity. Am. J. Vet. Res. 63: 617–622.[Medline]

6. Meyer, H. & Zentek, J. (2001) Ernährung des Hundes, 4. Auflage, Parey Verlag, Berlin.

7. Baums, C. G., Schotte, U., Amtsberg, G., Goethe, R (2004) Diagnostic multiplex PCR for toxin genotyping of Clostridium perfringens isolates. Vet. Microbiol. 100: 11–16.[Medline]

8. SAS Institute Inc. (1991) SAS/STAT User's Guide, Release 6, 4th ed. SAS Institute, Cary, NC.

9. Zentek, J., Kaufmann, D. & Pietrzak, T. (2002) Digestibility and effects on fecal quality of mixed diets with various hydrocolloid and water contents in three breeds of dogs. J. Nutr. 132: 1679S–1681S.[Abstract/Free Full Text]

10. Zentek, J. & Meyer, H. (1995) Normal handling of diets—are all dogs created equal? J. Small Anim. Pract. 36: 354–359.[Medline]

11. Meyer, H., Kienzle, E. & Zentek, J. (1993) Body size and relative weights of gastrointestinal tract and liver in dogs. J. Vet. Nutr. 2: 31–35.

12. Rolfe, V., Adams, C. A., Smith, V. V. & Butterwick, R. (2000) Large intestinal abnormalities in canine non-specific dietary sensitivity. J. Vet. Intern. Med. 14: 349.

13. Zentek, J. (1995) Influence of diet composition on the microbial activity in the gastro-intestinal tract of dogs. II. Effects on the microflora in the ileum chyme. J. Anim. Physiol. Anim. Nutr. (Berl.) 74: 53–61.

14. Zentek, J. (1995) Influence of diet composition on the microbial activity in the gastro-intestinal tract of dogs. I. Effects of varying protein intake on the composition of the ileum chyme and the faeces. J. Anim. Physiol. Anim. Nutr. (Berl.) 74: 43–52.

15. Zentek, J. (1995) Influence of diet composition on the microbial activity in the gastro-intestinal tract of dogs. III. In vitro studies on the metabolic activities of the small-intestinal microflora. J. Anim. Physiol. Anim. Nutr. (Berl.) 74: 62–73.





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