Establishment of Bacteroides succinogenes and measurement of the main digestive parameters in the rumen of gnotoxenic lambs

We attempted to determine the degree of diversification of the microflora that allow the establishment of Bacteroides succinogenes S85 in the rumen of gnotoxenic lambs. Four lambs (group I) received an inoculum orally, composed of 182 noncellulolytic bacterial strains (inoculum 1) previously isolated from the rumen of conventional young lambs. Two lambs (group II) were inoculated with 32 strains (inoculum 2) selected among the 182 strains of inoculum 1. Two lambs (group III) received an inoculum (inoculum 3) composed of 106 noncellulolytic bacterial strains previously isolated from the rumen of meroxenic lambs. Two lambs (group IV) were inoculated with 16 strains (inoculum 4) chosen among the 106 strains of inoculum 3. All lambs were inoculated from birth except two lambs of group I, which were inoculated from 1 month of age. Each lamb then received orally a pure culture of B. succinogenes. This strain became established more easily in the rumen of lambs that had received complex inocula (group I). Its population reached a level close to that generally observed in conventional lambs (10(7)-10(8) bacteria.mL-1). In contrast, B. succinogenes became established in only one lamb of group II, but bacterial numbers varied considerably. In group III, repeated inoculations were necessary to obtain its definitive establishment (10(7)-10(8) bacteria.mL-1 after weaning). In spite of several inoculations, this cellulolytic species failed to establish in the rumen of lambs of group IV, which had received the less complex inoculum. The volatile fatty acid levels were very different from one lamb group to another. The more complex the inoculum administered to the animals, the higher the concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fibrolytic Bacteria Isolated from the Rumen of North American Moose (Alces alces) and Their Use as a Probiotic in Neonatal Lambs

Fibrolytic bacteria were isolated from the rumen of North American moose (Alces alces), which eat a high-fiber diet of woody browse. It was hypothesized that fibrolytic bacteria isolated from the moose rumen could be used as probiotics to improve fiber degradation and animal production. Thirty-one isolates (Bacillus, n = 26; Paenibacillus, n = 1; and Staphylococcus, n = 4) were cultured from moose rumen digesta samples collected in Vermont. Using Sanger sequencing of the 16S rRNA gene, culturing techniques, and optical densities, isolates were identified and screened for biochemical properties important to plant carbohydrate degradation. Five isolates were selected as candidates for use as a probiotic, which was administered daily to neonate lambs for 9 weeks. It was hypothesized that regular administration of a probiotic to improve fibrolysis to neonate animals through weaning would increase the developing rumen bacterial diversity, increase animal production, and allow for long-term colonization of the probiotic species. Neither weight gain nor wool quality was improved in lambs given a probiotic, however, dietary efficiency was increased as evidenced by the reduced feed intake (and rearing costs) without a loss to weight gain. Experimental lambs had a lower acetate to propionate ratio than control lambs, which was previously shown to indicate increased dietary efficiency. Fibrolytic bacteria made up the majority of sequences, mainly Prevotella, Butyrivibrio, and Ruminococcus. While protozoal densities increased over time and were stable, methanogen densities varied greatly in the first six months of life for lambs. This is likely due to the changing diet and bacterial populations in the developing rumen.     

Effect of Absence of Ciliate Protozoa From the Rumen on Microbial Activity and Growth of Lambs

A survey of the components of the rumen ciliate population in a series of adult sheep, raised in the Faculty of Agriculture, University of Alexandria, has shown that a mixture of Entodinium, Isotricha, Ophryoscolex, Diplodinium, and Polyplastron species was found in the rumen contents of Egyptian sheep; no Epidinium and a negligible number of Dasytricha ruminantium were also observed. The microbial population, reducing sugars, ammonia, volatile fatty acids (VFA) production, and growth rate of 14 lambs inoculated with whole rumen contents from a mature sheep were compared over a 6-month period with those of 13 lambs maintained under the same conditions, except that they were strictly isolated from other ruminants. Certain large oval organisms and large numbers of flagellates and Oscillospira were frequently observed in the rumen contents of the isolated lambs. The reducing sugars, ammonia, and VFA levels, measured before and at intervals after feeding, in the inoculated lambs showed a pronounced rise above the values found in the ciliate-free animals. The propionic acid-acetic acid ratio in the rumen contents of the faunated lambs was considerably higher than in the nonfaunated controls. The inoculated lambs grew faster than the isolated lambs. Differences in weight gain which ranged from 15 to 17% were statistically significant. The inoculated animals impressed the observers by their good appearance which was superior to that of the ciliate-free lambs. It was, therefore, concluded that the rumen ciliate protozoa are essential for the metabolism and growth of young lambs.     

Establishment of cellulolytic bacteria and development of fermentative activities in the rumen of gnotobiotically-reared lambs receiving the microbial additive Saccharomyces cerevisiae CNCM I-1077

We studied the effects of a yeast additive used in ruminant nutrition on the establishment of cellulolytic bacteria, on plant cell wall degradation and on digestive functions in the rumen of gnotobiotically-reared lambs. Cellulolytic bacteria inoculated to the lambs tended to become established earlier in the presence of Saccharomyces cerevisiae CNCM I-1077 (SC). In addition, their population was maintained at a higher level, when the physico-chemical conditions of the biotope were altered. In these lambs, specific activities of fibrolytic enzymes were greater, and in sacco degradation of wheat straw tended to increase. In the presence of SC there was a decrease in ruminal ammonia concentration and a higher volatile fatty acid (VFA) concentration when lambs were 20 to 50 days old. These data suggest that this yeast strain may stimulate the development of cellulolytic microflora and enhance microbial activity in the rumen of young ruminants. Such activity could be beneficial in preventing microbial imbalance and a reduction of rumen function efficiency in the case of nutritional transitions. Further studies with conventional animals will soon be performed in order to verify these dings.     

PCR-DGGE analysis reveals a distinct diversity in the bacterial population attached to the rumen epithelium

Bacteria attached to the rumen epithelium (or epimural community) are not well characterised and their role in rumen functioning is not totally understood. There is just one published report of a clone library from one cow that suggests that this epimural community differs from the bacteria associated with the rumen digestive contents. However, this time-consuming approach is not adapted for examining microbial population changes in groups of animals. In in vivo studies, when samples from several animals have to be analysed simultaneously, a simpler technique has to be used. In this study, a genetic fingerprinting technique, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), was used to characterise the structure of the bacterial population attached to the rumen epithelium. This community was compared with that present in the solid and liquid phases of rumen content under two contrasting diets. Rumen samples were obtained from four forage-fed and four high-concentrate-fed (80 : 20, wheat grain : hay) 5-month-old lambs. After slaughter, samples from five epithelial sites and the solid and liquid digesta phases were taken for DNA extraction and analysis. Bacterial communities were profiled by PCR-DGGE using bacterial-specific 16S rDNA primers. Analysis of the fingerprint revealed that the epithelial community differed from those of rumen content in both diets. As expected, the nature of the feed influenced the bacterial communities from the solid and liquid rumen phases but no diet effect was observed in the rumen epithelial profiles suggesting a strong host effect on this bacterial population. Additionally, no differences were observed among the five epithelial sampling sites taken from each animal. The profile of the bacterial population attached to the rumen epithelium presented a high inter-animal variation, whether this difference has an influence in the function of this community remains to be determined.     

Influence of the composition of the cellulolytic flora on the development of hydrogenotrophic microorganisms, hydrogen utilization, and methane production in the rumens of gnotobiotically reared lambs

We investigated the influence of the composition of the fibrolytic microbial community on the development and activities of hydrogen-utilizing microorganisms in the rumens of gnotobiotically reared lambs. Two groups of lambs were reared. The first group was inoculated with Fibrobacter succinogenes, a non-H(2)-producing species, as the main cellulolytic organism, and the second group was inoculated with Ruminococcus albus, Ruminococcus flavefaciens, and anaerobic fungi that produce hydrogen. The development of hydrogenotrophic bacterial communities, i.e., acetogens, fumarate and sulfate reducers, was monitored in the absence of methanogens and after inoculation of methanogens. Hydrogen production and utilization and methane production were measured in rumen content samples incubated in vitro in the presence of exogenous hydrogen (supplemented with fumarate or not supplemented with fumarate) or in the presence of ground alfalfa hay as a degradable substrate. Our results show that methane production was clearly reduced when the dominant fibrolytic species was a non-H(2)-producing species, such as Fibrobacter succinogenes, without significantly impairing fiber degradation and fermentations in the rumen. The addition of fumarate to the rumen contents stimulated H(2) utilization only by the ruminal microbiota inoculated with F. succinogenes, suggesting that these communities could play an important role in fumarate reduction in vivo.     

Establishment of ciliate protozoa in the rumen of conventional and conventionalized lambs: influence of diet and management conditions

The establishment of ciliate protozoa in the rumen was studied in conventional lambs reared under different conditions of management. The role of the microflora in the kinetics of this establishment was also investigated in conventionalized lambs. In lambs reared under farm conditions ciliate protozoa appeared in the following order: Entodinium (15-20 days), Polyplastron, Eudiplodinium, and Epidinium (20-25 days), and Isotricha (50 days). Entodinium was the most abundant (10(5)-10(6) ciliates mL-1). During the 3rd month, ciliates disappeared spontaneously in about 60% of the lambs during a period that varied from 1 to 4 weeks. In lambs fed only cow's milk Entodinium spp. and Polyplastron multivesiculatum became established at low levels. The results obtained with the conventionalized lambs demonstrate that the establishment of the ciliates in the rumen requires that the bacterial flora be well established beforehand.     

Methanogens and Methanogenesis in the Rumens and Ceca of Lambs Fed Two Different High-Grain-Content Diets

The amount and nature of dietary starch are known to influence the extent and site of feed digestion in ruminants. However, how starch degradability may affect methanogenesis and methanogens along the ruminant''s digestive tract is poorly understood. This study examined the diversity and metabolic activity of methanogens in the rumen and cecum of lambs receiving wheat or corn high-grain-content diets. Methane production in vivo and ex situ was also monitored. In vivo daily methane emissions (CH₄ g/day) were 36% (P < 0.05) lower in corn-fed lambs than in wheat-fed lambs. Ex situ methane production (μmol/h) was 4-fold higher for ruminal contents than for cecal contents (P < 0.01), while methanogens were 10-fold higher in the rumen than in the cecum (mcrA copy numbers; P < 0.01). Clone library analysis indicated that Methanobrevibacter was the dominant genus in both sites. Diet induced changes at the species level, as the Methanobrevibacter millerae-M. gottschalkii-M. smithii clade represented 78% of the sequences from the rumen of wheat-fed lambs and just about 52% of the sequences from the rumen of the corn-fed lambs. Diet did not affect mcrA expression in the rumen. In the cecum, however, expression was 4-fold and 2-fold lower than in the rumen for wheat- and corn-fed lambs, respectively. Though we had no direct evidence for compensation of reduced rumen methane production with higher cecum methanogenesis, the ecology of methanogens in the cecum should be better considered.     

Establishment and development of ruminal hydrogenotrophs in methanogen-free lambs

The aim of this work was to determine whether reductive acetogenesis can provide an alternative to methanogenesis in the rumen. Gnotobiotic lambs were inoculated with a functional rumen microbiota lacking methanogens and reared to maturity on a fibrous diet. Lambs with a methanogen-free rumen grew well, and the feed intake and ruminal volatile fatty acid concentrations for lambs lacking ruminal methanogens were lower but not markedly dissimilar from those for conventional lambs reared on the same diet. A high population density (10(7) to 10(8) cells g(-1)) of ruminal acetogens slowly developed in methanogen-free lambs. Sulfate- and fumarate-reducing bacteria were present, but their population densities were highly variable. In methanogen-free lambs, the hydrogen capture from fermentation was low (28 to 46%) in comparison with that in lambs containing ruminal methanogens (>90%). Reductive acetogenesis was not a significant part of ruminal fermentation in conventional lambs but contributed 21 to 25% to the fermentation in methanogen-free meroxenic animals. Ruminal H(2) utilization was lower in lambs lacking ruminal methanogens, but when a methanogen-free lamb was inoculated with a methanogen, the ruminal H(2) utilization was similar to that in conventional lambs. H(2) utilization in lambs containing a normal ruminal microflora was age dependent and increased with the animal age. The animal age effect was less marked in lambs lacking ruminal methanogens. Addition of fumarate to rumen contents from methanogen-free lambs increased H(2) utilization. These findings provide the first evidence from animal studies that reductive acetogens can sustain a functional rumen and replace methanogens as a sink for H(2) in the rumen.     

Natural genetic transformation in the rumen bacterium Streptococcus bovis JB1

Natural transformation of Streptococcus bovis JB1 was demonstrated after development of competence in normal culture medium. Transformation efficiencies were not significantly increased when heat-inactivated horse serum was added to the medium before growth. This is the first time that a resident rumen bacterial species has been shown to be naturally transformable. Transformation allowed the acquisition of plasmids or integration of sequences into the chromosome. No transformation was observed in the presence of undiluted autoclaved or filter-sterilised ovine rumen fluid or filter-sterilised ovine saliva, suggesting that transformation in the ruminant digestive tract is a rare event, although transformation was observed in the presence of 1% and 0.5% filter-sterilised rumen fluid. The use of natural transformation of S. bovis should facilitate further molecular biological studies on this species.     

Establishment and Development of Ruminal Hydrogenotrophs in Methanogen-Free Lambs

The aim of this work was to determine whether reductive acetogenesis can provide an alternative to methanogenesis in the rumen. Gnotobiotic lambs were inoculated with a functional rumen microbiota lacking methanogens and reared to maturity on a fibrous diet. Lambs with a methanogen-free rumen grew well, and the feed intake and ruminal volatile fatty acid concentrations for lambs lacking ruminal methanogens were lower but not markedly dissimilar from those for conventional lambs reared on the same diet. A high population density (10⁷ to 10⁸ cells g⁻¹) of ruminal acetogens slowly developed in methanogen-free lambs. Sulfate- and fumarate-reducing bacteria were present, but their population densities were highly variable. In methanogen-free lambs, the hydrogen capture from fermentation was low (28 to 46%) in comparison with that in lambs containing ruminal methanogens (>90%). Reductive acetogenesis was not a significant part of ruminal fermentation in conventional lambs but contributed 21 to 25% to the fermentation in methanogen-free meroxenic animals. Ruminal H₂ utilization was lower in lambs lacking ruminal methanogens, but when a methanogen-free lamb was inoculated with a methanogen, the ruminal H₂ utilization was similar to that in conventional lambs. H₂ utilization in lambs containing a normal ruminal microflora was age dependent and increased with the animal age. The animal age effect was less marked in lambs lacking ruminal methanogens. Addition of fumarate to rumen contents from methanogen-free lambs increased H₂ utilization. These findings provide the first evidence from animal studies that reductive acetogens can sustain a functional rumen and replace methanogens as a sink for H₂ in the rumen.     

Spatial and temporal variations of the bacterial community in the bovine digestive tract

Aims:  Improved knowledge of the bacterial community of the digestive tract is required to enhance the efficiency of digestion in herbivores. This work aimed to study spatial and temporal variations of the bacterial communities in the bovine digestive tract and their correlation with gut environmental parameters.
Methods and Results:  Rumen content and faeces of five cows were sampled for 3 weeks. In addition, reticulum content was sampled during the third week. Bacterial communities were assessed by studying capillary electrophoresis single-stranded conformation polymorphism (CE-SSCP) profiles of 16S rRNA genes. The bacterial community structure differed between the forestomach and faecal contents. The abundance of several operational taxonomic units changed from week to week. Bacterial community structure of the rumen was correlated to propionic acid and NH₃–N concentrations.
Conclusions:  The bacterial community of the bovine digestive tract varied in space and time.
Significance and Impact of the Study:  The study of the bacterial communities of the digestive tract in herbivores should be widened from the rumen to the large intestine. The amplitude and origin of the temporal variation of the ruminal bacterial community need to be better understood to improve the control of the fermentative activity in herbivores.     

Total mixed ration pellets for light fattening lambs: effects on animal health

Fifty male Merino lambs (6 to 8 weeks, 14.1 kg; n=10 per group) were used to study the effect of feeding system: barley straw in long form and concentrate pellets in separate troughs (Control), ad libitum alfalfa supplemented with concentrate in separate troughs (Alfalfa) or including various levels of ground barley straw in concentrate pellets (B05, B15 and B25 for 50, 150 and 250 g barley straw/kg), on rumen characteristics, acid-base status, blood cell counts and lymphocyte stimulation. Alfalfa lambs had the heaviest digestive tract contents, highest rumen pH values, lowest volatile fatty acid concentration, highest papillae counts and best mucosa colour and the greatest blood pCO₂ values, lowest sodium and chloride and highest potassium concentrations (P<0.05). Including ground barley straw in the concentrate pellet or providing straw in long form separately from the concentrate reduces rumen pH and darkens ruminal mucosa as compared with alfalfa-fed lambs, thus affecting acid-base status.     

Electron microscopy study of the bacteria adherent to the rumen wall in young conventional lambs

The lamb rumen walls were rapidly colonized by an abundant bacterial population after birth. This colonization was examined by electron microscopy in neonatal conventional lambs. The sequence of establishment of the epimural species during the 3 weeks following birth, and the distribution of bacteria on the different sacs of the rumen, were examined by scanning electron microscopy. The population was very dense and consisted of a limited number of morphological types by 2 days after birth. Three types of rods were dominant at that time. The microflora was more complex 2 weeks later. Observations by transmission electron microscopy of desquamated epithelial cells revealed the presence of adherent bacteria that are surrounded by fibrous carbohydrate coats and sometimes partially enclosed by invaginations of the epithelial cell.     

Rumen chemical and bacterial changes during stepwise adaptation to a high-concentrate diet in goats

The correlation between rumen chemical and bacterial changes was investigated during a four periodical stepwise adaptation to a high-concentrate diet (concentrate level at 0%, 30%, 50% and 70% for diet I to IV, respectively) in goats. The results showed that ruminal pH decreased from 6.7 to 5.5 after switching from diet I to II, and was maintained at about 5.5 on diet III. Denaturing gradient gel electrophoresis results showed that the rumen bacterial community was relatively stable during the initial three feeding periods, except for the appearance of three bands when diet changed from I to II, suggesting that an appropriate concentrate level can promote the proliferation of some bacteria. After 12 days of feeding diet III, total volatile fatty acid (VFA) concentration and butyrate proportion decreased. At days 2 and 3 of feeding diet IV, ruminal pH declined sharply to 5.3 and 4.7, respectively, and total VFA concentration decreased further while lactic acid concentration increased markedly, suggesting a relation between lactic acid accumulation and ruminal pH decline. At the same time, many bacteria disappeared, including most fibrolytic-related bacteria while Streptococcus bovis and Prevotella-like species dominated. Interestingly, Succinivibrio dextrinosolvens-like species maintained throughout the experiment, suggesting its tolerance to low pH. In conclusion, rumen bacterial community was relatively stable feeding 0% to 50% concentrate diets, and it was observed that appropriate concentrate levels in the diet could increase the diversity of rumen bacteria. However, concentrate-rich diets caused lactic acid accumulation and low ruminal pH that caused the disappearance of most fibrolytic-related bacteria sensitive to low pH while S. bovis and genus Prevotella persisted.     

Methanogen Colonisation Does Not Significantly Alter Acetogen Diversity in Lambs Isolated 17?h After Birth and Raised Aseptically

Reductive acetogenesis is not competitive with methanogenesis in adult ruminants, whereas acetogenic bacteria are the dominant hydrogenotrophs in the early rumen microbiota. The ecology of hydrogenotrophs in the developing rumen was investigated using young lambs, raised in sterile isolators, and conventional adult sheep. Two lambs were born naturally, left with their dams for 17?h and then placed into a sterile isolator and reared aseptically. They were inoculated with cellulolytic bacteria and later with Methanobrevibacter sp. 87.7 to investigate the effect of methanogen establishment on the rumen acetogen population since they lacked cultivable representatives of methanogens. Putative acetogens were investigated by acetyl-CoA synthase and formyltetrahydrofolate synthetase gene analysis and methanogens by methyl coenzyme reductase A gene analysis. Unexpectedly, a low abundant but diverse population of methanogens (predominantly Methanobrevibacter spp.) was identified in isolated lambs pre-inoculation with Mbb. sp 87.7, which was similar to the community structure in conventional sheep. In contrast, potential acetogen diversity in isolated lambs and conventional sheep was different. Potential acetogens affiliated between the Lachnospiraceae and Clostridiaceae in conventional sheep and with the Blautia genus and the Lachnospiraceae in isolated lambs. The establishment of Mbb. sp. 87.7 (1,000-fold increase in methanogens) did not substantially affect acetogen diversity.     

Similarity of the ruminal bacteria across individual lactating cows

Dairy cattle hold enormous significance for man as a source of milk and meat. Their remarkable ability to convert indigestible plant mass into these digestible food products resides in the rumen - an anaerobic chambered compartment - in the bovine digestive system. The rumen houses a complex microbiota which is responsible for the degradation of plant material, consequently enabling the conversion of plant fibers into milk and meat and determining their quality and quantity. Hence, an understanding of this complex ecosystem has major economic implications. One important question that is yet to be addressed is the degree of conservation of rumen microbial composition across individual animals. Here we quantified the degree of similarity between rumen bacterial populations of 16 individual cows. We used real-time PCR to determine the variance of specific ruminal bacterial species with different metabolic functions, revealing that while some bacterial strains vary greatly across animals, others show only very low variability. This variance could not be linked to the metabolic traits of these bacteria. We examined the degree of similarity in the dominant bacterial populations across all animals using automated ribosomal intergenic spacer analysis (ARISA), and identified a bacterial community consisting of 32% operational taxonomic units (OTUs) shared by at least 90% of the animals and 19% OTUs shared by 100% of the animals. Looking only at the presence or absence of each OTU gave an average similarity of 75% between each cow pair. When abundance of each OTU was added to the analysis, this similarity decreased to an average of less than 60%. Thus, as suggested in similar recent studies of the human gut, a bovine rumen core microbiome does exist, but taxa abundance may vary greatly across animals.     

Rumen fluid transplantation affects growth performance of weaned lambs by altering gastrointestinal microbiota,immune function and feed digestibility

Although rumen fluid transplantation (RT) has been developed to confer benefits for adult ruminants by altering gastrointestinal tract microbiota, the question remains whether RT can also benefit weaned lambs. Hence, in this study, thirty-eight pre-weaning lambs were randomly assigned to one of three treatment groups: control lambs (CON) received 25 ml of normal saline solution, and lambs in two RT groups received 25 ml of rumen fluid either from 3-month-old lambs (LT) or from one-year-old adult ewes (AT). The effects on their growth performance, nutrient digestibility, some blood parameters and gastrointestinal tract microbiota were monitored. There were differences (P < 0.05) in rumen bacterial composition between the groups at weaning, at 3 months and at 1 year. Rumen fluid transplantation decreased (P < 0.05) average daily feed intake, average daily gain in live weight and apparent digestibility of ether extract in the LT group, and it decreased (P < 0.05) apparent digestibility of NDF and ADF in the AT group. Rumen fluid transplantation also increased (P < 0.05) concentrations of serum immunoglobulin A in the AT group and increased (P < 0.05) serum concentrations of interleukin-6, interferon alpha and D-lactate in both LT and AT groups. Bacterial α-diversity in the rumen and rectum was not affected by RT (P > 0.05), but a bacterial community change was observed after RT, and the abundance of some dominant bacteria in both rumen and rectum changed after RT (P < 0.05). Analysis of correlations between the parameters indicated that the altered gastrointestinal microbiota and accelerated maturity of rumen microorganisms induced by RT caused some impairment of gastrointestinal integrity and immunity, which led to decreased feed intake, reduced feed digestibility and lower growth performance of the weaned lambs. In conclusion, rumen fluid transplantation altered the gastrointestinal microbiota causing adverse effects on feed intake, feed digestibility and growth performance of the weaned lambs.     

Faculative Anaerobic Bacteria in the Digestive Tract of Chum Salmon (Oncorhynchus keta) Maintained in Fresh Water Under Defined Culture Conditions

The bacterial flora in the digestive tract of chum salmon growing in fresh water under defined and controlled culture conditions was examined both qualitatively and quantitatively. The predominant species present in the digestive tract were identified as Aeromonas, with Aeromonas hydrophila being the most common isolate. These aeromonads were not isolated from the diet. Other bacterial species commonly isolated included Bacillus, Enterobacter, nonpigmented pseudomonads, Micrococcus, and Acinetobacter. These species were also isolated from the diet or tank water. As many as 10-8 viable bacteria per g (wet weight) of digestive tract plus contents were counted. After 75 days of starvation, 10-6 viable bacteria were counted, whereas fish fed a sterile feed contained 10-5 viable bacteria per g (wet weight) of digestive tract plus contents.