Julian R. Pleasants

REARING GERMFREE CESAREAN‐BORN RATS, MICE, AND RABBITS THROUGH WEANING*

At the present time the method of choice for obtaining germfree rats, mice, and rabbits is the hand feeding of sterilized diets to cesarean-born young. Alternative possibilities have not yet proved satisfactory. Complete decontamination of conventional adult animals has not been reported. Crosssuckling cesarean-born young on germfree foster mothers of other species might create problems for the virologist, oncologist, immunologist, or biological purist. A third type of alternative to hand rearing might be found in the work of Rowntree,l who injected thymus extract into rats for 9 generations. By the tenth generation his rats were born with their ears open and their incisors erupted. At 48 hours, they had their eyes open and were able to eat solid food. Rowntree himself found the method difficult to duplicate, and no one has yet tried it for obtaining germfree animals. The hand rearing of germfree mammals was first reported for such species as the guinea pigz and the goat: whose stage of development a t birth is relatively close to that of the weaned animal. The hand rearing of rats and mice, which are much more helpless a t birth, remained an unexplored possibility until much later. Obtaining germfree colonies of these species has therefore required not only pioneering in the germfree phase of the work, but also an original investigation into the entire technique of hand rearing. The hand rearing of rats was first reported by Gustafsson in 1946.4 The first detailed report on the technique involved was published by Reyniers et ul. in Lobund Report No. l5 later in the same year. Gustafsson gave the details of his method in 1948 in “Germfree Rearing of Rats.”6 At Lobund Institute the rats were offered, a t hourly intervals, an opportunity to suck autoclaved milk formula from a rubber nipple. In the method of Gustafsson, a thin rubber tube was inserted halfway down the esophagus, and sterilized milk formula, including some predigested casein, was forced inlo the stomach gradually over a period of several minutes. The stomach could thus be well filled and the feeding interval prolonged to 4 to 5 hours. Since the publication of Lobund Report No. 1 a different technique of germfree rat rearing has been tested a t Lobund Institute with more consistently reproducible results than the method originally reported. The new technique involved forced feeding with a short, tapered nipple and the use of a relatively simple milk formula. This new technique then proved successful in weaning, for the first time, Swiss albino and C3H mice. After earlier preliminary experiments, germfree rabbits were successfully hand-reared a t Lobund Institute * The work reported in this paper was supported in part by Contract NONR-1623(04) between the Office of Naval Research, Department of the Navy, Washington, D. C., and the University of Notre Dame.

Efficiency of digestion in germ-free and conventional rabbits.

1. Germ-free (GF) and conventional (CV) rabbits wearing collars to prevent coprophagy were fed an autoclaved diet with added cellulose. Their faecal excretion was analysed to determine nutrient digestibility. 2. Clearly distinguishable hard faeces were excreted by the GF rabbit only if the diet contained at least 15% cellulose. Unlike CV rabbits, the GF rabbits did not consume their soft faeces even when permitted to do so. Soft faeces made up a larger proportion of the total output of GF than of CV rabbits. Food intake and total dry-matter excretion per kg body-weight were similar in both groups. 3. Although digestibility of dry matter was similar in the two groups, in the GF rabbits there was a higher digestibility of crude fat and true protein and a lower digestibility of crude fibre and nitrogen-free extract. GF rabbits excreted a higher percentage of ingested calcium and phosphorus in the urine than did CV rabbits. 4. The results suggest that intestinal microbes, even without the enhancing effect of coprophagy, aid in the digestion of carbohydrate by rabbits. The greater faecal excretion of crude fat and true protein by CV rabbits could result from poorer digestion and absorption, but could also represent nutrients synthesized by microbes from simpler materials. The reingestion of faecal crude fat and true protein might therefore improve the quality of the total nutrient intake. The results suggest ways of assuring an adequate dietary intake by GF rabbits in the absence of contributions from an intestinal microflora.

The Effect of Microbial Flora, Diet, and Age on the Tumoricidal Activity of Natural Killer Cells

This study examined the effects of diet (chemically defined vs natural‐ingredient), age, and microbial flora on the tumoricidal activity of natural killer (NK) cells from the spleens of mice. Results from a 4‐h 51Cr‐release assay indicate the following: 1. Germfree C3H/HeCr mice raised on a chemically defined diet had significantly greater NK cell activity than their germfree or “clean‐conventional” (i.e., barrier‐maintained) counterparts who were raised on a sterilized natural‐ ingredient diet. 2. The NK activity of germfree mice was dramatically increased after their alimentary tract was colonized with a complex intestinal flora. 3. Conventional mice raised under clean (barrier) conditions had significantly less NK cell activity than nonbarrier‐maintained mice. 4. Switching germfree mice from a chemically defined diet to a sterile natural‐ingredient diet did not enhance NK cell activity. 5. No significant differences in NK activity were evident with C3H/HeCr mice of different (6–10 wk vs 29–36 wk) ages. These results indicate that diet and microbial flora can modulate the NK cell activity of mice.

Effect of Protein-Calorie Restriction on Brain Amino Acid Pool in Neonatal Rats

Summary Newborn rats fed restricted calories during the first 2 days of life showed a depression of brain weight, and the levels of glutamic acid, glutamine, alanine, serine, and GAB A compared to those allowed to suckle their mothers normally. Also it was found that the levels of valine, leucine, isoleucine, histidine, arginine, cystine, ornithine, urea, and ammonia were increased in newborn rats maintained on restricted calories. It is concluded that calorie malnutrition, even for a short period, during the neonatal development is associated with alteration in the brain amino acid pool.

The Germ-Free Animal Fed Chemically Defined Diet: A Unique Tool

Conclusion With the development of CD diet L-489E 14SE, it is possible to maintain prodction colonies of BALB/c mice under GF conditions with totally defined nutritional intake after weaning. These mice are exposed to a minimum of antigenic stimulation and thus provide a unique model in which all exogenous factors may be controlled.

Metabolic enzymes in liver and kidney of the germfree rat

Abstract The effect of intestinal microflora on the activities of NADP-dependent dehydrogenases in liver and kidneys, and on fatty acid synthetase, ATP-citrate lyse, cytochrome oxidase and succinate dehydrogenase activities in liver of rats were studied. Germfree rats showed a decrease in liver succinate dehydrogenase activity, and no effect on liver cytochrome oxidase activity. The absence of an intestinal microflora resulted in a significant decrease of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities, and a substantial increase of ATP-citrate lyase and fatty acid synthetase activities in the liver. Liver malate dehydrogenase activity was comparable in both germfree and conventional animals. Germfree status had no effect on the activities of NADP-dependent dehydrogenases in the kidney. It is concluded that the absence of an actively metabolizing microflora was associated with quantitative shifts in the activity of hepatic enzymes involved in the metabolism of carbohydrates and fats.

Immunoglobulin isotypes and antibody specificity repertoire of "spontaneously" occurring ("background") immunoglobulin-secreting cells in germfree mice fed chemically defined ultrafiltered "antigen-free" diet.

“Spontaneously” occurring (“background”) immunoglobulin (Ig)-secreting cells have been observed in all lymphoid organs of non-immunized mice. Their frequency was found to depend on the genetic background, age and antigenic load of the mice studied (Benner et al., 1982). Even in germfree (GF) mice “background” antibody-secreting cells occur, as has been assessed in antigen-specific plaque assays with sheep red blood cells (SRBC) as targets (Nordin, 1968; Hof et al., 1975; Benner et al., 1981b). The total number of IgM-secreting cells as detected with the protein A plaque assay is virtually normal in GF mice, but the numbers of IgG- and IgA-secreting cells are severely decreased as compared to conventionally raised mice (Benner et al., 1981b). The latter indicates that exogenous antigenic and mitogenic stimulation is a major driving force for the “background” IgG- and IgA-synthesis. For IgM this is unclear, because of the crucial question whether the GF animals used in those experiments can really be regarded as devoid of exogenous stimuli. It has been shown that a substantial mitogenic and/or antigenic activity can be exerted by food substances or dead bacteria in the diet (Wostman et al., 1971). With the successful breeding of GF mice fed an ultrafiltered solution of chemically defined (CD) low molecular weight nutrients, these exogenous stimuli are reduced to the presently achievable minimum (Pleasants et al., 1981; Wostmann et al., 1982; Pleasants, 1984).

Protein Metabolism in Germfree Rats Fed Chemically Defined, Water-Soluble Diet and Semisynthetic Diet

The importance of germfree animals and chemically defined, water-soluble diets developed at Lobund Laboratory in nutritional and immunological studies was discussed in detail by Pleasants (1). Nutritional adequacy of this diet has been demonstrated by the fact that it supported reproduction of germfree mice into fifth generation and the growth of germfree rats from birth through maturity (2). Although these results indicate that the diet is at least qualitatively adequate for germfree rats and mice, it is of considerable importance to determine if the animal is physiologically “normal” in a chemical environment so limited and so defined. So, as a further test of nutritional adequacy and physiological normality, germfree and conventional rats fed this diet were compared to similar rats fed a semisynthetic diet based on casein-starch using morphology, serum amino nitrogen, pancreatic enzyme levels, liver protein, nitrogen balance and protein efficiency ratio (PER) as criteria. In addition, these studies would indicate to what extent the intestinal microflora could influence the protein metabolism of the host.

Sudden Death in Germfree Mice Reared Through Successive Generations on Chemically Defined Liquid Diet

A diet for germfree animals which consists of chemically defined, low-molecular-weight nutrients, and which can be prepared in liquid form for sterilization by filtration, brings the experimental animal one step farther toward complete control of nutritional and antigenic variables. Such a diet, consisting of purified amino acids, sugars, ethyl linoleate, vitamins and minerals, all dissolved in a single water solution, has been fed to germfree rats (1) and to germfree mice (2,3). The fat-soluble portion of the diet had been solubilized in the water solution by use of Polysor-bate 80, These preliminary experiments had implicated the nonionic detergent as a cause of persistent diarrhea in some mice. Changes in the lymph nodes of germfree mice fed the detergent diet also resembled those observed by Mori and Kato (4) in conventional rats fed polysorbate. For these reasons the detergent was eliminated and the fat-soluble portion of the diet was prepared and fed separately from the water-soluble portion, according to a procedure reported at the 1966 meeting of this Association. Diet prepared in this way has now been subjected to long-term testing of its nutritional adequacy.

FEEDING AND NUTRITION

This chapter discusses germfree animal nutrition and feeding. Successful germfree rearing of many animal species indicates that they have no requirements for unknown nutrients supplied, specifically by bacteria. The rearing of germfree rats and mice on chemically defined diets indicates no requirement for nutrients other than those already known to be required by these species. It is possible that the cecal distention seen in germfree rodents is caused by a deficiency of some nutrient(s), but recent hypotheses tend to link this condition with an accumulation of pharmacologically active substances within the germfree intestinal tract. Although germfree guinea pigs show a somewhat retarded growth rate, germfree rats and mice show growth equal to that of conventional animals, and germfree chickens, turkeys, pigs, and monkeys show superior growth. Milk substitutes for caesarian-derived hand-fed mammals have not provided pre-weaning growth equal to that of mother-fed young, but such substitutes are no longer necessary after reproducing colonies. Germfree guinea pigs do not require milk substitutes even when caesarian derived.