Obtaining and rearing germ-free miniature piglets for biomedical research

The method of obtaining and rearing germfree miniature piglets for medical-biological research is described. The gnotobiotes were reared in rigid organic glass isolation cells up to 3 months. Milk diets supplemented with minerals and vitamins were used for nutrition. The gnotobiotes gained weight well during the whole observation period.     

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 and Monocontaminated Chicks in Rigid Plastic Isolators

A Plexiglas isolator for rearing germfree vertebrates is described. Plastic components were precut and finished by a Plexiglas supplier. This equipment is relatively inexpensive; moreover, neither special facilities nor skills are required in their construction. Advantages of this type of isolator are: readily dismantled for cleaning, durable, excellent visibility of the interior, and minimal maintenance. In 30 separate groups of animals, 27 of these yielded germfree chicks. Many of these chicks were reared germfree for 4 weeks; and several were maintained for about 2 months, without microbial contamination. Pure cultures of Candida albicans, Streptococcus faecalis, and Clostridium perfringens were established as monocontaminations in the intestinal tracts of chickens. In contrast, Alcaligenes faecalis ATCC 9220 failed to grow in the gut of chicks, following peroral administration of this organism. All chicks described were employed on other experiments, and were killed for various assays at times specified.     

Examination of latent infection in germfree mice for some murine viruses.

Sera from some groups of germfree mice were examined for reactivity against Sendai, reo 3, Theiler's GD VII, ectromelia, and mouse hepatitis virus antigens. Of 51 sera collected in September and October 1976 and in August and September 1977 from one germfree mouse breeding colony, there was no positive reaction against all the antigens tested. Moreover, the attempt of virus isolation from homogenates of various organs including lungs, liver, kidneys, and intestines of 6 germfree mice was unsuccessful.     

A New Germfree Chicken Cage—Characteristics and Use

A new germfree chicken cage for rearing chicks up to 3 or 4 weeks of age has been designed and is in use at the University of Missouri. The cage and the accessory parts, the small magnet and hinged door, the “Bactytector,” the built-in air filter assembly, and the glass top with connecting air outlet filter have been described in detail. The complete operating procedure for sterilizing the cage and diet and the method of adding sterile embryonated eggs have been outlined. Data on the effectiveness of the cage as a physical barrier to microbes have been presented.     

Effect of bile acid deconjugation on the fecal excretion of steroids

The effect of microbiological deconjugation of bile acids on total bile acid and neutral sterol fecal excretion by adult male rats has been studied. A screening method utilizing mice allowed selection of a Clostridium perfringens type A strain, which accelerated cholesterol catabolism in mice. When this species of bacteria was associated with germfree rats, the fecal bile acids were excreted as free bile acids (deconjugated), however the quantities of bile acids excreted were not increased compared with those of germfree rats. Conventional rats excrete twice as much bile acids (all deconjugated) as do the germfree and C. perfringens-associated rats. It is, therefore, unlikely that the microbiological deconjugation of bile acids is responsible for the increased fecal excretion of bile acids seen in conventional rats. The C. perfringens-associated rats excreted identical kinds and quantities of fecal neutral sterols as did the germfree rats.     

The histochemistry of mucosaccharides in some organs of germfree rats.

In order to study the histochemical nature of mucosaccharides in germfree animals, the organs in natural contact with bacteria (stomach, small and large intestine) and those naturally remote from bacteria (tracheal and ear cartilage and aorta) were studied by means of light microscopic methods for mucosaccharides in germfree and conventional rats. In the stomach (surface and foveolar cells) of germfree rats the histochemical reactions for acid and neutral mucosaccharides were apparently less intense than in that of conventional rats, whereas in the small and large intestine (goblet cells) of germfree rats the reactions were significantly more intense than in those of conventional rats. In the cartilage (intercellular matrix, lacunar border and chondrocyte cytoplasm) and aorta (interelastic spaces) of germfree animals the reactions were less intense than in those of conventional animals. In addition, some differences in the histochemical nature of mucosaccharides between the organs of germfree and conventional rats were noted, as revealed by the effects of chemical modifications and digestions with enzymes upon the histochemical reactions studied.     

Effect of lighting regimen,rearing in isolation and olfactory bulbectomy on growth and puberty in Yorkshire gilts

This study was designed to determine the effects of isolation or group rearing, total darkness or constant light, and olfactory bulbectomy on growth and onset of puberty in Yorkshire gilts. Forty-eight gilts, 90 days old, were assigned randomly to the following treatments: rearing in isolation with constant darkness, IS-CD; isolation with constant light, IS-CL; isolation with natural summer daylength, IS-NL; group rearing with natural daylength, GR-NL. They were reared in these environments to 245 days of age, without exposure to a boar. Twelve gilts, ～ 100 days old, were olfactory-bulbectomized (OB) and ten sham-operated (SC) littermates served as controls. They were reared in natural daylength in their respective groups, without exposure to a boar. Average weekly gains were similar among all treatment and control groups, ranging from 4.5 to 4.8 kg. Percentages of gilts in groups IS-CD, IS-CL, IS-NL and GR-NL that exhibited pubertal estrus by 245 days of age were 50, 50, 50 and 75, respectively (P>0.05), and the corresponding average ages of them at puberty were 208, 220, 213 and 205 days (P>0.05). In OB gilts, the percentage of them cycling at 270 days (33) was less (P<0.05) than that of SC (80). Sequential profiles of peripheral blood serum concentrations of progesterone confirmed normal estrous cycles after pubertal estrus of individual gilts among all treatment and control groups. These results indicate that rearing of gilts in isolation with either total darkness or constant light has no significant detrimental effect on their growth, age at onset of pubertal estruc and subsequent estrous cycles. Olfactory bulbectomy during the prepubertal period, however, delays onset of puberty but does not result in permanent loss of ovarian function.     

Effects of isolation-rearing on intracranial self-stimulation reward of the lateral hypothalamus: baseline assessment and drug challenges

There is evidence that isolation rearing produces down-regulation of the dopamine D2 receptor. Therefore, isolation rearing should also modify the effects of D2 antagonists on intracranial self-stimulation (ICSS) reward. This study investigated the effect of isolation rearing on ICSS reward, and modulation of that reward by SCH23390, Raclopride and MK-801. Sprague-Dawley rats were reared alone (isolates) or in pairs from day 21 postnatal to day 75 postnatal. At this time, all rats were implanted with monopolar stimulating electrodes in the lateral hypothalamus. The ICSS rate-frequency curve-shift method was used to assess reward and operant motor function at baseline and after administration of SCH-23390 (D1 antagonist: 0.02, 0.06, 0.2 mg/kg), Raclopride (D2 antagonist: 0.01, 0.025, 0.06 mg/kg), and MK-801 (non-competitive NMDA receptor antagonist: 0.1, 0.2 mk/kg). Isolation-reared rats displayed similar measures of both basal reward and motor function when compared to socially reared controls. Isolation-reared rats were subsensitive to the reward decreasing effects of Raclopride. Socially reared rats were observed to have more variant baseline reward measures, and could be divided into distinctly different groups with different basal reward function. Isolation-rearing down-regulates D2 function but does not affect basal reward function, but some unknown factor in the social rearing environment did have a substantial effect on basal reward function.     

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.     

无菌小鼠的人工哺乳培育

采用无菌子宫摘取术,在隔离器中剥离子宫取仔１６只,生存１５只,在我国首次采用每２４小时插胃管灌胃５～６次的人工哺乳方法,哺乳１５只,离乳６只。粪便、垫料、棉拭子经无菌检查,均无细菌生长。同时对人工乳成分进行了分析比较,比较了人工乳灭菌温度与时间对各营养成分破坏情况,比较了人工乳、豚鼠母乳与小鼠母乳之间的差异     

The histochemistry of mucosaccharides in some organs of germfree rats

Summary In order to study the histochemical nature of mucosaccharides in germfree animals, the organs in natural contact with bacteria (stomach, small and large intestine) and those naturally remote from bacteria (tracheal and ear cartilage and aorta) were studied by means of light microscopic methods for mucosaccharides in germfree and conventional rats. In the stomach (surface and foveolar cells) of germfree rats the histochemical reactions for acid and neutral mucosaccharides were apparently less intense than in that of conventional rats, whereas in the small and large intestine (goblet cells) of germfree rats the reactions were significantly more intense than in those of conventional rats. In the cartilage (intercellular matrix, lacunar border and chondrocyte cytoplasm) and aorta (interelastic spaces) of germfree animals the reactions were less intense than in those of conventional animals. In addition, some differences in the histochemical nature of mucosaccharides between the organs of germfree and conventional rats were noted, as revealed by the effects of chemical modifications and digestions with enzymes upon the histochemical reactions studied.This investigation was supported in part by a Grant-in-Aid from the Japanese Education Ministry (1975). A major part of this investigation has been presented at the 10th International Congress of Anatomists held in Tokyo (1975)     

Serum amyloid A, cytokines, and corticosterone responses in germfree and conventional mice after lipopolysaccharide injection.

To determine why germfree mice are less susceptible to lipopolysaccharide (LPS) than conventional mice, we studied serum levels of serum amyloid A (SAA), tumor necrosis factor (TNF), interleukin 1 (IL-1), IL-6, and corticosterone in mice after treatment with LPS. A single injection of LPS caused an elevation of SAA, an acute-phase protein in the mouse, in both conventional and germfree IQI mice, and the response was significantly less in germfree mice. LPS-induced elevations of serum TNF, IL-1, and IL-6 levels were also significantly less in germfree mice, while serum corticosterone levels were greater in germfree mice than in conventional mice. These results suggest that the lower susceptibility to LPS and a smaller response of SAA elevation by LPS in germfree mice may result from less elevation in serum of these cytokines in these mice, which are known to mediate the acute phase response of SAA. High levels of serum corticosterone in germfree mice may be partly responsible for the lower responsiveness of these inflammatory cytokines to LPS in these mice.     

Phase-dependent locomotor activity in first-stadium nymphs of the desert locust, Schistocerca gregaria: effects of parental and progeny rearing density

This study examined the effects of parental and progeny rearing density on locomotor activity of 1st-stadium nymphs of the desert locust, Schistocerca gregaria, using an actograph. Progeny obtained from solitarious (isolated-reared) or gregarious (crowd-reared) locusts were reared in isolation or in a group of 30 nymphs. Crowding after hatching had a slight influence on mean activity shortly after the start of measurements, but no clear effect was detected until day 2, when maximum activity during the 6-24 h of observation was significantly higher than that of the nymphs kept in isolation. On the other hand, the effects of parental rearing density on locomotor activity manifested at all ages examined (0-2 days old). Progeny of gregarious locusts showed consistently higher activity than those of solitarious locusts. In newly hatched nymphs, the effect of parental rearing density was explained by variation in body size at hatching, one of the phase-dependent characteristics. Hatchling body color was also correlated with locomotor activity and body weight. Similar levels of locomotor activity were exhibited when green, solitarious and black, gregarious nymphs were similar in body weight. These results suggested that parental rearing density indirectly influences locomotor activity in the progeny shortly after hatching by affecting their body size as eggs or hatchlings.     

Plate diffusion assay as a rapid method for dosimetry of mutagens.

In the feces of conventional rats, the amount of omega-muricholic and hyodeoxycholic acids vary according to the diet. To understand this phenomenon, we investigated the bacterial formation of these bile acids. The present paper reports the first isolation, from conventional rat feces, of a strain of Clostridium group III which transforms beta-muricholic acid, the main bile acid in germfree rats, into omega-muricholic acid.     

Increased enterocyte production in gnotobiotic rats mono-associated with Lactobacillus rhamnosus GG

There is increasing scientific and commercial interest in using beneficial microorganisms (i.e., probiotics) to enhance intestinal health. Of the numerous microbial strains examined, Lactobacillus rhamnosus GG has been most extensively studied. Daily intake of L. rhamnosus GG shortens the course of rotavirus infection by mechanisms that have not been fully elucidated. Comparative studies with germfree and conventional rats have shown that the microbial status of an animal influences the intestinal cell kinetics and morphology. The present study was undertaken to study whether establishment of L. rhamnosus GG as a mono-associate in germfree rats influences intestinal cell kinetics and morphology. L. rhamnosus GG was easily established in germfree rats. After 3 days of mono-association, the rate of mitoses in the upper part of the small intestine (jejunum 1) increased as much as 14 and 22% compared to the rates in germfree and conventional counterparts, respectively. The most striking alteration in morphology was an increase in the number of cells in the villi. We hypothesis that the compartmentalized effects of L. rhamnosus GG may represent a reparative event for the mucosa.     

Sperm transfer is affected by mating history in the simultaneously hermaphroditic snail Lymnaea stagnalis

Abstract. Males are predicted to strategically allocate sperm across mating partners in order to maximize their chances of paternity. This requires that males have the ability to detect aspects of their partner's mating history or the number of potential mates. We investigated whether simultaneous hermaphrodites mating in the male role strategically adjust sperm transfer depending on rearing conditions. The pond snail Lymnaea stagnalis (Basommatophora) is known to donate sperm repeatedly to different partners during a breeding season and store received sperm for >3 months. The rearing conditions of the donor as well as the recipient affect the amount of sperm transferred. Sperm donors raised in isolation transfer more sperm than those raised in groups. Furthermore, isolated sperm donors transfer less sperm to partners that were raised in groups than to those raised in isolation, i.e., virgins. These findings suggest that snails raised in isolation shift their sex allocation toward the male function and indicate that they can somehow assess the mating status of their partner.     

Higher resistance of germfree mice to dianhydrodulcitol, a lymphotropic cytostatic agent

The same dose of dianhydrodulcitol (DAD) produced a lower mortality rate among germfree mice than among SPF or conventional C3H mice. On the other hand, it caused graver lymphoid atrophy in germfree mice. Their higher resistance, as evidenced by the mortality rate, can be explained on the basis of a histological study of the ileum. It showed milder alterations of the intestinal wall in germfree than in SPF mice. The lymphotropic cytostatic agent had a less direct toxic effect in germfree mice, due to the lacking damaging effect of endotoxin from the normal intestinal flora.     

Age and changes in the splenic lymphoid tissue of germ-free rats during the postnatal period of ontogenesis]

In germfree rats the splenic lymphopoiesis is not demonstrated by the investigation to be suppressed, and lymphoid follicules undergo age changes. Up to 15 days, periarterial zone of T-lymphocytes, responsible for cell immunity, is determined, and by the 30th day, as in control animals, peripheral zone of B-lymphocytes, responsible for hormonal immunity, is distinctly observed. In 4-month-old germfree animals, a large amount of free iron crystals is detected in the spleen, while in the organ of control animals its amount is still small. In 10-month-old germfree rats, with the appearance of reactive centers, the zone of B-lymphocytes widens in lymphoid follicules and iron crystals integrate in the red pulp. Lymphopoiesis in the spleen of the germfree rats seems to be maintained by certain local conditions which are connected with increased haemolytic function of the organ. This produces a discharge of a large amount of the products of erythrocytosis which, like autoantigens, stimulate lymphopoiesis in the spleen even when microflora is absent in the organism, while lymphopoiesis in lymph nodes in germfree animals is sharply inhibited.     

Influence of a cecal volume-reducing intestinal microflora on the excretion and entero-hepatic circulation of steroids and bile acids

From mouse fecal material we have isolated four strictly anaerobic bacteria which, when associated with germfree mice or rats, reduced the cecal volume by 80 and 60%, respectively. This cecal volume-reducing flora did not metabolize estrone-3-sulfate, taurolithocholate-3-sulfate or taurolithocholate but gnotobiotic rats associated with this particular flora (CRF-rats) excreted these compounds faster in feces plus urine than did germfree rats. The time needed for 50% excretion (t1/2) of orally administered estrone-3-sulfate was 32 h in germfree rats versus 13 h in CRF rats; for intraperitoneally injected taurolithocholate-3-sulfate the t1/2 was 63 h in germfree versus 17 h in CRF rats and for taurolithocholate the t1/2 was 199 h in germfree and 96 h in CRF rats. Association of germfree rats with the cecal volume-reducing flora did not change the cecal absorption rate of estrone-3-sulfate, but shortened the 50% small intestinal transit time of [14C]PEG from 10 to 3 h; a value also found in conventional rats. These results stress the important influence of the intestinal microflora on the absorption and excretion of steroids via its effect on the physiology of the whole intestinal tract and point to the deficiencies inherent to the use of germfree animals in excretion studies.