Rearing of germfree rabbits

The present work describes in detail experimental procedures and conditions required for rearing of germfree rabbits. Newborn germfree rabbits were obtained using the “wet” hysterectomy technique. The animals were successfully maintained under germfree conditions up to four months after delivery in flexible plastic isolators. Infant rabbits were first fed a sterile VA2-b diet and after weaning, a modifled diet L-478 according to Reddyet al. (1965). Both diets were shown to be suitable as the development of megacoecum was significantly reduced. The infant germfree rabbits were characterized by a good general clinical condition and body weights were comparable to those found in their conventional counterparts fed mother’s milk.     

Rearing of germfree rabbits and establishment of an SPF rabbit colony]

Baby rabbits hysterectomy-derived from conventional Japanese white rabbits were reared under aseptic condition by feeding with 4 types of artificial diets. Rabbit milk for the artificial diet was obtained from conventional dams at 7--25 days after delivery. The artificial diets was given by stomach tube twice a day. The total volumes of diet given (ml per day) were Y = 2.3X + a (1 to approximately 14 days of age), Y = 32.2 + a (l5 to approximately 25 days of age), or Y = (32.2 + a) - 37.5 (X - 25) (26 to approximately 34 days of age), (X = age in days, a = volumes fed at 0 day of age). After 14 days of age young animals were also fed freely sterilized commercial pellets and weaned at 35 days of age. Out of 155 rabbits, 130 were aseptically reared till 36 approximately 40 days of age, and no difference on weaning rate was seen between the 4 groups of rabbits. Thereafter, they were exposed to a barriered room of SPF rabbitry outside the isolators. The best growth was seen in animals given by artificial diet containing rabbit milk at 40%. The SPF breeding colony of rabbits was found to be free of Pasteurella pneumotropica, Bordetella bronchiseptica, Salmonella spp., Eimeria spp., Encephalitozoon cuniculi and Acaritic otistis.     

Rearing of germfree guinea pigs and establishment of an SPF guinea pig colony]

New born guinea pigs of Hartley strain derived by hysterectomy were fed commercial pellets, cow's milk, egg yolk and vitamin mixture since 0 days of age, when they were kept at 31 +/- 1 degrees C. Out of 33 animals, 30 were reared for 40 days under aseptic state and they were transfered to a barrierred facility to establish an SPF colony free from Bordetella bronchiseptica, Streptococcus zooepidemicus (Animal C), Salmonella spp., Tyzzer's organisms, Mycoplasma spp., Reo 3 virus, Sendai virus, Eimeria spp., Chirodiscoides caviae and Gliriocola porcelli.     

Lymphatic tissue of the intestinal tract of germfree and conventional rabbits

The lymphatic tissue of ileum, sacculus rotundus and appendix was poorly developed with-out germinal centres in germfree rabbits; on the other hand, in conventional rabbits, the lymphatic tissue was abundant with numerous germinal centers. In germfree rabbits, the stroma of villi of the jejunum and ileum possessed low cellularity, in conventional rabbits, the villi were wide with rich cellularity. The basal position of lymphocytes was predominant in the villi of ileum of germfree rabbits, in conventional rabbits a high percentage of lymphocytes was in the apical position.     

Catabolism and elimination of cholesterol in germfree rats

Three-month old germfree and conventional male rats were maintained on a complete steam-sterilized, semisynthetic diet. After intravenous injection of cholesterol-26-(14)C the animals were housed in a plastic metabolism chamber for 72 hr. Expired CO(2) was collected throughout the period. The conventional rats released 50% more (14)C as (14)CO(2) than the germfree animals. The total amount of the label recovered as (14)CO(2) during the 72 hr period amounted to 30% and 19% respectively, of the original dose. In both conventional and germfree rats the release of (14)CO(2) accounted for approximately 75% of the (14)C recovered in forms other than the original cholesterol-26-(14)C; 15-20% was found incorporated in water-soluble and fat-soluble fractions other than 3Beta-OH sterol of liver and carcass while the remainder was excreted with feces and urine. After the 72 hr period the specific activities of the cholesterol in plasma and liver were lower in conventional than in germfree animals. The data express the accelerating effect of the intestinal microflora on systemic cholesterol catabolism. They demonstrate that the release of (14)CO(2) from cholesterol-26-(14)C in the intact rat is a suitable and convenient indicator of the oxidative catabolism of cholesterol.     

Phytate hydrolysis by germfree and conventional rats.

Phytic acid is naturally occurring compound that reduces intestinal absorption of many metals. Early work suggests that some dietary phytate may be hydrolyzed in the large intestines by bacteria, but more recently nutritionists have suggested that a mucosal enzyme is responsible. This paper reports a study intended to resolve this controversy. The hydrolysis of dietary phytic acid was measured in germfree and conventional rats fed either of two diets that differed in their calcium content. Negligible phytate hydrolysis occurred in the germfree rats, whereas 22 and 56% of the phytic acid was hydrolyzed by conventional rats fed high- and low-calcium diets, respectively. We concluded that bacteria were responsible for the hydrolysis of phytate in these diets and that any activity of endogenous enzyme was negligible.     

Fecal neutral steroids and bile acids from germfree rats

The amount and composition of fecal neutral sterols and bile acids excreted by adult male germfree and conventional rats have been determined. The amounts of neutral sterols excreted were 12.8 (germfree) and 19.5 (conventional) mg/kg of body wt per day. The germfree rats excreted cholesterol and lathosterol (methostenol was not assayed); the conventional rats excreted coprostanol and coprostanone in addition. The amounts of bile acids excreted were 11.3 (germfree) and 21.4 (conventional) mg/kg of body wt per day. The bile acids excreted by the rats were tentatively identified as tauro--muricholate, tauro-alpha-muricholate, and tauro-cholate, besides an unidentified component. The conventional rats excreted the corresponding unconjugated acids as well as many other unconjugated bile acids. No significant correlation was found between the amount of coprosterols and the total amount of neutral sterols excreted by the conventional rats. This suggests that bacterial reduction of cholesterol is not an important mechanism of increasing neutral sterol excretion of conventional rats as compared to germfree rats. Evidence is presented that suggests that this difference in neutral sterol excretion is due to changes in intestinal secretion and sloughing between the two types of animal. The factors reponsible for the differences in bile acid excretion have not been identified.     

Exteriorization of the bile duct in germfree rats

A modified T cannula and a cannula housing receptacle was employed to cannulate the bile duct of germfree rats. Bile was collected without anesthesia or sedation. During collection, the rat was restrained in a plastic holder where it was free to eat.     

Ultrastructure of the enlarged cecum in germfree rats

Summary The cecum of germfree rats, as studied by light microscopy and scanning and transmission electron microscopy, differs in many respects from the cecum of conventional rats. Epithelial cells in germfree rats are taller and have larger nuclei and longer microvilli than similar cells in conventional rats. The cecal mucosa of germfree rats shows a larger variability in the arrangement of the crypts of Lieberkühn than does the mucosa of conventional rats. Some crypts are funnel-shaped and connected close to the mucosal surface with adjacent similar crypts to form long valleys. Less wide crypts open on elevated regions of the mucosal surface. The lamina propria of germfree animals is devoid of plasma cells but rich in mast cells. Germfree animals show hypertrophy of the tunica muscularis externa.In conventional rats the cecal lumen contains a large variety of morphologically different bacteria. However, the lumen of the crypts of Lieberkühn contains only one type of elongated bacteria, which are present in large amounts. This finding suggests that symbiotic relations may be of particular importance in the crypts of Lieberkühn in the cecum.Supported by research grants from the Swedish Medical Research Council (206), Knut och Alice Wallenbergs Stiftelse and Stiftelsen Therese och Johan Anderssons Minne.     

Ammonia in the intestinal contents from germfree rats

The values of pH and the concentrations of nitrogen (N) and ammonia in the middle part of the small intestinal and cecal contents of germfree (GF) and conventionalized (CVZ) seven-week-old rats were compared. The pH of the small intestinal and cecal contents of GF rats was higher than that of CVZ rats. There was no difference in total N per fresh weight in contents from the middle part of the small intestine between GF and CVZ, whereas total N per fresh weight of the cecal contents was higher in CVZ than in GF rats. The ammonia concentrations per fresh weight or per total N in the intestinal and cecal contents of CVZ rats were higher than those of GF rats.