The effect of dietary consistency on gross and histologic morphology in the craniofacial region of young rats

Three groups of weanling rats and three groups of juvenile rats were fed diets which differed in physical consistency for periods of 5 and 8 weeks, respectively. In both the weanling and juvenile rats, one group was fed a soft diet, a second group was fed a hard diet, and a third group was initially fed the soft diet and then was switched to the hard diet for the remainder of the experimental period. The effects of these differences in dietary consistency on gross and histologic morphology of the craniofacial region were examined. Significant differences were found in the dimensions and morphology of the condyle and condylar cartilage as a result of the differences in dietary consistency in both the weanling and juvenile groups. Soft-diet rats generally had smaller condyles and a thinner layer of condylar cartilage than either hard-diet or soft/hard-diet rats. Little change, however, was found in the overall dimensions of the mandible and maxilla in any of the groups of rats.     

Hard-Diet Feeding Recovers Neurogenesis in the Subventricular Zone and Olfactory Functions of Mice Impaired by Soft-Diet Feeding

The subventricular zone (SVZ) generates an immense number of neurons even during adulthood. These neurons migrate to the olfactory bulb (OB) and differentiate into granule cells and periglomerular cells. The information broadcast by general odorants is received by the olfactory sensory neurons and transmitted to the OB. Recent studies have shown that a reduction of mastication impairs both neurogenesis in the hippocampus and brain functions. To examine these effects, we first measured the difference in Fos-immunoreactivity (Fos-ir) at the principal sensory trigeminal nucleus (Pr5), which receives intraoral touch information via the trigeminal nerve, when female adult mice ingested a hard or soft diet to explore whether soft-diet feeding could mimic impaired mastication. Ingestion of a hard diet induced greater expression of Fos-ir cells at the Pr5 than did a soft diet or no diet. Bromodeoxyuridine-immunoreactive (BrdU-ir) structures in sagittal sections of the SVZ and in the OB of mice fed a soft or hard diet were studied to explore the effects of changes in mastication on newly generated neurons. After 1 month, the density of BrdU-ir cells in the SVZ and OB was lower in the soft-diet-fed mice than in the hard-diet-fed mice. The odor preferences of individual female mice to butyric acid were tested in a Y-maze apparatus. Avoidance of butyric acid was reduced by the soft-diet feeding. We then explored the effects of the hard-diet feeding on olfactory functions and neurogenesis in the SVZ of mice impaired by soft-diet feeding. At 3 months of hard-diet feeding, avoidance of butyric acid was reversed and responses to odors and neurogenesis were recovered in the SVZ. The present results suggest that feeding with a hard diet improves neurogenesis in the SVZ, which in turn enhances olfactory function at the OB.     

Effect of bone strain on cortical bone structure in macaques (Macaca mulatta)

It has recently been shown that the consistency of food significantly affects levels of bone strain in the mandible during mastication (Hylander, '79a). Mandibular bone histology was examined to test the effects of a diet of hard food compared to a diet of soft food in two group of monkeys. One group of rhesus macaques (Macaca mulatta) was fed a diet of commercially prepared hard biscuits. The second group was fed a soft diet the consistency of fudge. Both diets were nutritionally adequate for normal growth and development. As a control for other factors influencing cortical bone structure, fibular morphology was also examined. At the end of the test period, mandibular and fibular tissue samples from the two groups were prepared to determine the amount of secondary Haversian bone present. Mandibular depth at M₂ and fibular anteroposterior diameter were also measured and compared between the two dietary groups. The soft-diet monkeys showed low levels of remodeling in their mandibles. There were large patches of unremodeled bone and resorption spaces were common. The hard-diet monkeys exhibited more extensive evidence of secondary Haversian remodeling in their mandibles. The hard-diet monkeys also had deeper mandibles. In contrast, the fibulae from the two groups had similar mean diameters and showed comparable levels of secondary remodeling. We infer that the higher mandibular bone remodeling levels in the hard-diet monkeys represent an adaptive response to remove and replace fatigued mandibular bone due to higher stress levels associated with the ingestion and mastication of hard foods. We also infer that greater depth of the mandible at M₂ found in the harddiet group represents an adaptive response to higher stress levels associated with the ingestion and mastication of hard foods.     

Effect of altered masticatory function on [3H]-thymidine and [35S]-sulfate incorporation in the condylar cartilage of the rat

The response of the condylar cartilage to alterations in compressive joint reaction forces in vivo has been little studied. In an attempt to reduce or eliminate the occlusal forces resulting from mastication or incision, male weanling rats were fed a soft diet requiring little chewing and/or had their incisors clipped every other day. Incorporation (dpm/micrograms DNA) of [3H]-thymidine and [35S]-sulfate was significantly decreased relative to controls in the incisor-clipped group, but not in the soft-diet group. Animals having both treatments also exhibited significantly lower incorporation values than controls, suggesting the importance of incision for loading at the mandibular joint. These data corroborate in vitro studies which suggest that compressive forces can affect mitotic activity and synthesis of proteoglycans in the condylar cartilage. However, additional factors, both hormonal and biomechanical in nature, may be important in the in vivo environment.     

Effects of Decreased Occlusal Loading during Growth on the Mandibular Bone Characteristics

Background

Bone mass and mineralization are largely influenced by loading. The purpose of this study was to evaluate the reaction of the entire mandibular bone in response to decreased load during growth. It is hypothesized that decreased muscular loading will lead to bone changes as seen during disuse, i.e. loss of bone mass.

Methods and Findings

Ten 21-day-old Wistar strain male rats were divided into two groups (each n=5) and fed on either a hard- or soft-diet for 11 weeks. Micro-computed tomography was used for the investigation of bone mineralization, bone volume, bone volume fraction (BV/TV) and morphological analysis. Mandibular mineralization patterns were very consistent, showing a lower degree of mineralization in the ramus than in the corpus. In the soft-diet group, mineralization below the molars was significantly increased (p<0.05) compared to the hard diet group. Also, bone volume and BV/TV of the condyle and the masseter attachment were decreased in the soft-diet group (p<0.05). Morphological analysis showed inhibited growth of the ramus in the soft-diet group (p<0.05).

Conclusion

Decreased loading by a soft diet causes significant changes in the mandible. However, these changes are very region-specific, probably depending on the alterations in the local loading regime. The results suggest that muscle activity during growth is very important for bone quality and morphology.     

Size and shape of the mandibular condyle in primates

The relationships between the size of the articular surface of the mandibular condyle and masticatory muscle size, tooth size, diet, and biomechanical variables associated with mastication were studied by taking 12 measurements on skulls of 253 adult female anthropoid primates, including three to ten specimens from each of 32 species. In regressions of condylar length, width, or area against body weight, logarithmic transformations substantially improve the fit of the equations compared with untransformed data. There is a strong relationship between condylar measurements and body weight, with all correlations being .94 or higher. The slopes of the allometric regressions of length, width, and area of the condylar head indicate slight positive allometry with body size. Folivorous primates have smaller condyles than frugivorous primates, and colobines have smaller condyles than cebids, cercopithecines, or hominoids. When colobines are eliminated, the differences between frugivores and folivores are not significant. However, the two species with the relatively largest condyles are Pongo pygmaeus and Cercocebus torquatus, suggesting that there may be a relationship between unusually large condylar dimensions and the ability to crack hard nuts between the teeth. Cranial features having strong positive correlations with condylar dimensions include facial prognathism, maxillary incisor size, maxillary postcanine area, mandibular ramus breadth, and temporal fossa area. These data are interpreted as indicating that relatively large condyles are associated with relatively large masticatory muscles, relatively inefficient mandibular biomechanics, and a large dentition. These relationships support the growing evidence that the temporomandibular joint is a stress-bearing joint in normal function.     

Structure and growth activities of the mandibular condyle in monkeys (Macaca fascicularis): II. Synergistic behavior of cell dynamics and metabolism

The mandibular condyles of eight growing male monkeys (Macaca fascicularis) were analyzed by using a combination of radioautographic and morphometric techniques. This was done with the aim of examining the dynamics in the structure and growth activities of the articular tissue covering as well as of the subchondral zone of erosion. The animals received 1 mCi/kg body weight 3H-proline 24 hr and 0.5 mCi/kg body weight 3H-thymidine 3 hr prior to death. Their age was estimated on the basis of skeletal maturation as recorded from radiographs of the hand and wrist. Consistently, the proliferative activity in the intermediate layer, the rates of cell turnover and growth of chondroblasts and chondrocytes, the rates of extracellular matrix production in the intermediate and chondral layers, as well as the resorptive and appositional activities in the zone of erosion were characterized by an in-concert behavior. This behavior suggests a general synergistic control of the various cell dynamic and metabolic processes affecting the rate of normal condylar growth.     

Response of the mandibular joint to loss of incisal function in the rat

In a study of the rat mandibular joint (MJ), Simon [Acta anat. 97: 351-360 (1977)] suggested that reduction in condylar cartilage thickness noted in animals subjected to removal or trimming of incisors resulted from the lessening of joint reaction forces produced during incision. In order to explore this question further, the microanatomy of the MJ in 47-day-old rats whose incisors had been trimmed every other day was compared to that in control animals and in a third group fed a soft diet as a control for reduced joint reaction forces. Both the incisor-clipped and soft-diet groups exhibited reduced size and density of bony trabeculae underlying the condylar cartilage and diminished staining for alcian blue. The thickness of the prechondroblastic layer of the condylar cartilage was significantly (p less than or equal to 0.01) reduced relative to controls in both experimental groups on the superior aspect of the cartilage, but was reduced in the more posterior parts of the cartilage only in the incisor-clipped group. While not denying that joint reaction forces may affect MJ response, the reduced proliferative response noted in the posterior region of the condylar cartilage in incisor-clipped animals is perhaps best explained by a decrease in the frequency and extent of protrusion of the lower jaw due to a lack of incisal preparation of food items.     

Study of histomorphometric changes of the mandibular condyles in neonatal and juvenile rats after administration of cyclophosphamide

Forty 1-week-old Sprague-Dawley rats were divided into 4 groups of 10 animals each. Five rats of each group served as the controls, and another 5 were given weekly intraperitoneal injections of 20 mg cyclophosphamide (CTX, Endoxan, Cytoxan) per kg of body weight. The animals of each group were sacrificed either at 1, 3, 5, or 7 weeks after initial intraperitoneal injection of CTX or water. The mandibular condyles of all rats were assayed histomorphometrically for changes on cartilage and bone cells, and other elements. The results showed a decreasing tendency in the thickness of the cartilage layer, the number of prechondroblasts, the number of chondrocytes, the percentage of cartilage matrix, the percentage of hard tissue, the bone surface perimeter, the number of osteoblasts, the number of osteoclasts, and the average number of nuclei per osteoclast in the condyles at 1 or 3 weeks after initial CTX treatment. After 7 weeks of treatment with CTX there was a significant decrease in the thickness of the cartilage layer, the number of prechondroblasts, and the numbers of both osteoblasts and osteoclasts per square millimeter of bone surface in the condyles, when compared with those of the control groups. In addition, after 5 or 7 weeks of CTX treatment there was a significant decrease in the percentage of hard tissue, the number of osteoblasts and the number of osteoclasts. Furthermore, changes in the morphology and size of the bone cells were also observed in all the rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Light- and electron-microscopic observations on the mandibular condylar cartilages in growing rats on a low-calcium diet.

In order to obtain more insight into the physiologic mechanism of endochondral ossification, histological changes occurring in the mandibular condylar cartilage of growing rats fed on a low-calcium diet were investigated by light and electron microscopy. Twenty-three-day-old rats were fed on a normal diet or a low-calcium diet for 8 weeks. For the histological observations the mandibular condyles were dissected from each animal at 1, 2, 4, 5 and 8 weeks after the initiation of the experiment. Histological changes occurring in the mandibular condylar cartilages of the rats fed on a low-calcium diet were as follows: (1) narrow proliferative and mature cell zones and a wide hypertrophic cell zone, (2) inhibition of development of cell organelles in the mature chondrocytes, (3) decrease in dead cells in the proliferative zone, (4) decrease in glycogen accumulation in the chondrocytes and (5) inhibition of calcification in the extracellular matrix of the hypertrophic cell zone. Additionally at the end of the experimental period, the following findings were observed: (1) appearance of small light cells in the mature cell zone and the hypertrophic cell zone and (2) decrease in proteoglycan granules and appearance of large collagen fibrils in the pericellular region of the hypertrophic cell zone.     

Mechanism of adaptation in the mandibular condyle of the mouse. An organ culture study

The cranial base and whole mandible of 7-day-old mice were cultured for 1 or 2 weeks in a novel organ culture system that provided an articulating movement for the temporomandibular joints. The mandibles were articulating either in a closed or in an open position to simulate the in vivo mouth-breathing pattern. The reactions of the condylar processes were followed macroscopically and microscopically after osteoid or von Kossa staining. After 1 week of culture, small differences were found in the shape of the condylar processes between the two groups. After 2 weeks, significantly increased growth was observed superiorly in those condyles which had been working in a closed position and posteriorly in the condyles which had been working in an open position. Histologic analysis revealed that after 1 week calcification had proceeded faster in the posterior aspect of the condyles in the mandibles which had been working in a closed position as compared to the mandibles which had been working in an open position, and the difference was even more marked after 2 weeks. In the latter condyles calcification had proceeded markedly in the anterosuperior aspect. Osteoid staining showed that perichondrial mesenchymal cells had differentiated into osteoblasts in the posterior aspect of the condyles in those mandibles which had been working in a closed position. This led to a ceasing of expansive growth in the posterior aspect and thus to a more upwardly directed condylar growth. In mandibles which had been working in the open position to simulate the mouth-breathing pattern in vivo, chondrogenesis and thus expansive growth continued in the posterior aspect, leading to a more posteriorly directed condylar growth.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of grape seed proanthocyanidins extract on rat mandibular condyle

We investigated the effects of grape seed proanthocyanidins extract (GSPE) on bone formation by examining total and cortical bone mass, density, architecture, and strength non-invasively using mandibular condyles of Ca-restricted rats. Forty Wistar male rats, each 5 weeks old, were divided into control (C), low-Ca diet (LCaD), low-Ca diet-standard diet (LcaD x SD), and low-Ca diet x Estandard diet with supplementary GSPE (LcaD x SD+GSPE) groups. In LCaD x SD group, after the bone debility was induced by low-Ca diet, a standard diet therapy was given. In LCaD x SD+GSPE group, after the bone debility was induced by low-Ca diet, a standard diet therapy with supplementary GSPE was given. Each mandibular condyle was examined using peripheral quantitative computed tomography (pQCT). There were no significant inter-group differences in body weight seen throughout the experimental period. In LcaD x SD+GSPE, cortical bone cross-sectional area and mineral content were not significantly different from C, while bone mineral content was significantly higher in LcaD x SD+GSPE than in LcaD x SD. Cortical bone density of LcaD x SD+GSPE was not significantly different from that of C, however, that value in LCaD and LcaD x SD was significantly lower than that. The cross-sectional (bending) moment of inertia values in LcaD x SD+GSPE were the highest among all groups, though they did not differ significantly from those in C. Further, the cross-sectional (bending) Stress/Strain Index (SSI) values in LcaD x SD+GSPE were statistically similar to those in C, however, not significant higher than in LcaD x SD. These results suggest that GSPE treatment would increase both bone mass and bone strength on the rat mandibular condyles.     

Replica immunoelectron microscopic study of the upper surface layer in rat mandibular condylar cartilage by a quick-freezing method

A quick-freezing and deep-etching method in combination with replica immunoelectron microscopy was applied for examining localization of hyaluronic acid and fibronectin on the upper surface layer of rat mandibular condylar cartilage. Rat temporomandibular joints were dissected with articular disks in order to leave the articular cartilage surface intact. The disks were slightly cut with razor blades for exposing the condylar articular cartilage surface. They were quickly frozen with the isopentane-propane cryogen (–193°C) and prepared for freeze-fracturing and deep-etching replica membranes. They were additionally treated with 5% SDS and 0.5% collagenase to keep some antigens attached on the replica membranes. After such a treatment, a routine immunogold method was applied for clarifying the localization of hyaluronic acid and fibronectin in the upper surface layer. Small immunogold particles for hyaluronic acid were mainly localized around upper filamentous networks covered with amorphous materials, but large immunogold ones for fibronectin were localized on deep thicker fibrils. We have revealed the native architecture of the upper surface layer of mandibular condylar cartilage on the replica membranes and also three-dimensional localization of hyaluronic acid and fibronectin by the immunogold method.     

Changes in mechanical properties of bone within the mandibular condyle with age

The purpose of the study was to compare indentation modulus (IM) and hardness of condylar bone in young and adult dogs. In addition we desired to examine histologic sections for bone formation activity in the two groups. Mandibular condyles were obtained from adult (1- to 2-year-old) and young (approximately 5-m old) dogs. Two sections/condyle were obtained and one was processed for histomorphometry and the other for mechanical analyses. Indents were made on moist condylar trabecular bone to a depth of 500 nm at a loading rate of 10 nm/s using a custom-made hydration system to obtain IM and hardness. Histomorphometric analyses measured the bone volume/total volume (BV/TV%) and ratio of labeled to unlabeled bone within the condyle. Data were analyzed using a repeated-measures factorial analysis of variance and Tukey-Kramer method. Overall, the IM of the adult condyles (10.0+/-3.4 GPa, Mean+/-SD) were significantly (P<0.0001) higher than in young dogs (5.6+/-2.6 GPa). There was a greater bone mass in the young (60.2%) versus the adult condyles (42%). Also, significantly more labeled bone in the young (66.1%) condylar bone suggested higher bone forming activity than in adult condyles (27.5%). With age there is a change in mass and material properties in the trabecular bone of the mandibular condyle in dogs.     

Aluminum concentrations in tissues of rats: effect of soft drink packaging

Aluminum is a commonly occurring trace element for which no nutritional requirements have been set. Some non-conclusive evidence exists suggesting a need of aluminum for growth, reproduction or health of man and animals. There is concern that exposure or consumption of aluminum may be toxic to humans and animals. The objective of the current study was to compare tissue levels of aluminum of rats fed soft drinks packaged in aluminum cans, glass bottles or distilled water. Thirty male weanling rats (Sprague-Dawley) were divided into three treatment groups of 10 rats each. All rats were fed rodent chow ad libitum throughout the study. Three different fluids, i.e. distilled water, diet soft drinks from aluminum cans and diet soft drinks from glass bottles, were fed for a period of 3 weeks. Aluminum contents of tissues were measured by atomic absorption spectrophotometry. Canned soft drink fed rats had significantly higher blood, liver and bone aluminum concentration than rats that were given glass bottled soft drink. There was a 69% higher bone aluminum concentration and 16% lower femur weight in rats fed aluminum canned soft drinks when compared with rats fed with distilled water.     

Change from a hard to soft diet alters the expression of insulin-like growth factors,their receptors,and binding proteins in association with atrophy in adult mouse masseter muscle

To study the role of insulin-like growth factors (IGFs) in the atrophy of mouse masseter muscle in response to a change from a hard to a soft diet, we analyzed the amounts of mRNA and the immunolocalization for IGF-I, IGF-II, their receptors (IGFRs), and binding proteins (IGFBPs). Sixteen male ICR mice were fed a hard diet after weaning; they were divided into two groups at 6 months of age and fed a hard or a soft diet for 1 week. The soft diet treatment decreased masseter weight by 19% (P<0.01) and the minimal diameter of masseter myofibers by 19% (P<0.01), verifying that a soft diet led to atrophy of mouse masseter muscle. The soft diet treatment induced a 30% reduction in the amount of IGF-I mRNA (P<0.05) in preparations of whole masseter tissues. Immunohistochemical findings suggested that a reduction in the expression of IGF-I protein took place in the neural tissues, not in the masseter myofibers. The soft diet treatment induced a 56% decrease in IGF-II mRNA (P<0.05), a 21% increase in IGFR2 mRNA (P<0.01), and a 38% decrease in IGFBP5 mRNA (P<0.01). Immunohistochemical results suggested that these changes at the protein level occurred in the masseter myofibers. No significant or marked difference in the mRNA amount or immunostaining pattern for IGFR1, IGFBP3, IGFBP4, or IGFBP6 was found between the soft and hard diet groups. No IGFBP1 or IGFBP2 mRNA was detected. Thus, IGF-I, IGF-II, IGFR2, and IGFBP5 seem to play a role in the atrophy of mouse masseter muscle in response to the change from a hard to a soft diet in an autocrine and/or paracrine manner.Part of the present study was supported by a grant-in-aid for funding scientific research (no. 13671955), Bio-ventures and High-Technology Research Center, from the Ministry of Education, Culture, Sports, Science, and Technology of Japan     

Age-related changes in the expression of gelatinase and tissue inhibitor of metalloproteinase genes in mandibular condylar,growth plate,and articular cartilage in rats

Summary Mandibular condylar cartilage acts as both articular and growth plate cartilage during growth, and then becomes articular cartilage after growth is complete. Cartilaginous extracellular matrix is remodeled continuously via a combination of production, degradation by matrix metalloproteinases (MMPs), and inhibition of MMP activity by tissue inhibitors of metalloproteinases (TIMPs). This study attempted to clarify the age-related changes in the mRNA expression patterns of MMP-2, MMP-9, TIMP-1, TIMP-2, and TIMP-3 in mandibular condylar cartilage in comparison to tibial growth plate and articular cartilage using an in situ hybridization method in growing and adult rats. MMP-2 and MMP-9 were expressed in a wide range of condylar cartilage cells during growth, and their expression domains became limited to mature chondrocytes in adults. The patterns of TIMP-1 and TIMP-2 expression were similar to those of MMP-2 and MMP-9 during growth, and were maintained until adulthood. TIMP-3 was localized to hypertrophic chondrocytes throughout the growth stage. Therefore, we concluded that TIMP-1 and TIMP-2 were general inhibitors of MMP-2 and MMP-9 in condylar cartilage, while TIMP-3 regulates the collagenolytic degradation of the hypertrophic cartilage matrix.     

Genotype–environment interaction and the ontogeny of diet-induced phenotypic plasticity in size and shape of Melanoplus femurrubrum (Orthoptera: Acrididae)

The developmental origin of phenotypic plasticity in morphological shape can be attributed to environment-specific changes in growth of overall body size, localized growth of a morphological structure or a combination of both. I monitored morphological development in the first four nymphal instars of grasshoppers (Melanoplus femurrubrum) raised on two different plant diets to determine the ontogenetic origins of diet-induced phenotypic plasticity and to quantify genetic variation for phenotypic plasticity. I measured diet-induced phenotypic plasticity in body size (tibia length), head size (articular width and mandible depth) and head shape (residual articular width and residual mandible depth) for grasshoppers from 37 full-sib families raised on either a hard plant diet (Lolium perenne) or a soft plant diet (Trifolium repens). By the second to third nymphal instar, grasshoppers raised on a hard plant diet had significantly smaller mean tibia length and greater mean residual articular width (distance between mandibles adjusted for body size) compared with full-sibs raised on a soft plant diet. However, there was no significant phenotypic plasticity in mean unadjusted articular width and mandible depth, and in mean residual mandible depth. At the population level, development of diet-induced phenotypic plasticity in grasshopper head shape is mediated by plastic changes in allocation to tissue growth that maintain growth of head size on hard, low-nutrient diets while reducing growth of body size. Within the population, there was substantial variation in the plasticity of growth trajectories since different full-sib families developed phenotypic plasticity of residual articular width through different combinations of head and body size growth. Genetic variation for diet-induced phenotypic plasticity of residual articular width, residual mandible depth and tibia length, as estimated by genotype–environment interaction, exhibited significant fluctuation through ontogeny (repeated measures MANOVA , family × plant × instar, P < 0.01). For example, there was significant genetic variation for phenotypic plasticity of residual articular width in the third nymphal instar, but not earlier or later in ontogeny. The observed patterns of genetic variation are discussed with reference to short-term constraints and the evolution of phenotypic plasticity.     

Effects of ingestion of difructose anhydride III (DFA III) and the DFA III-assimilating bacterium Ruminococcus productus on rat intestine

We have isolated a difructose anhydride III (DFA III)-assimilating bacterium, Ruminococcus productus AHU1760, from human. After an acclimation period of 1 week, male Sprague-Dawley rats (5 weeks old) were divided into four groups (control diet, R. productus diet, DFA III diet, and R. productus + DFA III diet; n = 8) and fed the assigned test diets for 2 weeks. The viable count of administered R. productus was 4.9 x 10(7) CFU/d in R. productus-fed rats and 4.7 x 10(7) CFU/d in R. productus + DFA III-fed rats. Survival in cecal content of this strain was confirmed by randomly amplified polymorphic DNA. The ratio of secondary bile acids in feces in R. productus + DFA III-fed rats decreased the same as that in rats fed only DFA III. The viable count of lactobacilli and bifidobacteria, known as beneficial bacteria, increased more in R. productus + DFA III-fed rats than in control or R. productus-fed rats. A combination of R. productus and DFA III might improve the balance of intestinal microbiota to a healthier condition.