Dietary phytoestrogens accelerate the time of vaginal opening in immature CD-1 mice

The purpose of the study reported here was to determine the effects of dietary phytoestrogens on the time of vaginal opening (VO) in immature CD-1 mice, and to correlate it with phytoestrogen and total metabolizable energy (ME) contents of the diet in an effort to determine the most appropriate diets(s) for comparing or evaluating the estrogenic or antiestrogenic activity of endocrine disruptor compounds (EDC). Mice were weaned at postnatal day (PND) 15 and fed the test diets from PND 15 to 30. Vaginal opening was recorded from PND 20 to 30. The phytoestrogen content of the diet was highly predictive (P < 0.0001) of the proportion of mice with VO at PND 24. Total ME content also was significantly (P < 0.01) correlated with time of VO, although this variable was somewhat less predictive than was phytoestrogen content. Time of VO in mice was significantly (P < 0.05) accelerated in mice fed diets high in phytoestrogens, compared with those containing low phytoestrogen content. It was concluded that: dietary daidzein and genistein can significantly (P < 0.01) accelerate the time of VO in CD-1 mice; the advancement in time of VO is more highly correlated with daidzein and genistein contents of the diets than with total ME content; advancement in the time of VO is a sensitive end point for evaluating the estrogenic activity of EDCs, and should be part of the standard protocol for evaluating EDCs. Phytoestrogen-free diet(s) containing the same amount of ME should be used in bioassays that compare the time of VO, or increases in uterine weight as end points for evaluating the estrogenic activity of an EDC.     

Phytoestrogen content of purified, open- and closed-formula laboratory animal diets.

BACKGROUND AND PURPOSE: Phytoestrogens exert estrogenic effects on the central nervous system, induce estrus, and stimulate growth of the genital tract of female animals. Over 300 plants and plant products, including some used in laboratory animal diets, contain phytoestrogens. Therefore, the source and concentration of phytoestrogens in rodent diets were determined. METHODS: Twelve rodent diets and six major dietary ingredients were assayed for phytoestrogens (daidzein, genistein, formononetin, biochanin A, and coumestrol), using high-performance liquid chromatography. Three rodent diets recently formulated to reduce phytoestrogen content also were assayed. RESULTS: Formononetin, biochanin A, and coumestrol were not detected. Soybean meal was the major source of daidzein and genistein; their concentrations were directly correlated to the percentage of soybean meal in each diet. CONCLUSIONS: High, variable concentrations of daidzein and genistein are present in some rodent diets, and dietary phytoestrogens have the potential to alter results of studies of estrogenicity. Careful attention should be given to diet phytoestrogen content, and their concentration should be reported. A standardized, open-formula diet in which estrogenic substances have been reduced to levels that do not alter results of studies that are influenced by exogenous estrogens is recommended.     

Estrogens and phytoestrogens: brain plasticity of sexually dimorphic brain volumes

Sexually dimorphic brain volumes (sexually dimorphic nucleus of the preoptic area (SDN-POA) and anteroventral periventricular (AVPV) nucleus) are influenced by estrogens. Phytoestrogens, derived from plants (especially soy products), are molecules structurally and functionally similar to estradiol. The purpose of this study was to examine: the consumption of phytoestrogen (using a phytoestrogen-rich (Phyto-600) versus a phytoestrogen-free (Phyto-free)) diets from conception to adulthood (or changing the diets during adulthood) and characterizing (a) circulating plasma phytoestrogen levels, (b) testosterone levels in males, (c) sexually dimorphic brain volumes (i.e. the SDN-POA and AVPV) and (d) the presence of apoptotic cells in these brain structures in Long-Evans rats. Phyto-600 fed animals displayed total serum phytoestrogens levels 37-fold higher compared to Phyto-free values. Circulating testosterone levels were not significantly altered by the diets. Female SDN-POA volumes were not altered by the diets. Whereas, males fed a Phyto-free diet displayed decreased SDN-POA volumes compared to male Phyto-600 values. Females fed the Phyto-600 diet displayed larger AVPV volumes compared to males on the same diet or females on the Phyto-free diet. Males fed the Phyto-free diet had the largest AVPV values compared to Phyto-600 fed males. When the SDN-POA region was examined in lifelong Phyto-free fed males, apoptotic cells were present versus males fed the Phyto-600 diet and in the AVPV region the opposite results were obtained. In summary, consumption of dietary phytoestrogens (estrogen mimics) can alter hormone-sensitive hypothalamic brain volumes in rodents during adulthood.     

Estrogenicity of the isoflavone metabolite equol on reproductive and non-reproductive organs in mice

Equol, a metabolite of the phytoestrogen daidzein, is present at significant levels in some humans who consume soy and in rodents fed soy-based diets. Equol is estrogenic in vitro, but there have been limited studies of its activity in vivo. We evaluated equol effects on reproductive and non-reproductive endpoints in mice. Ovariectomized age-matched (30-day-old) female C57BL/6 mice were fed phytoestrogen-free diets and given a racemic mixture of equol by daily injections (0, 4, 8, 12, or 20 mg [kg body weight](-1) day(-1)) or in the diet (0, 500, or 1,000 ppm) for 12 days. Mice were killed, and serum concentrations of total and aglycone equol were measured. Total serum equol concentrations ranged from 1.4 to 7.5 microM with increasing doses of injected equol, but uterine weight increased significantly only at 12 and 20 mg (kg body weight)(-1) day(-1). Dietary equol at 500 or 1,000 ppm produced total serum equol concentrations of 5.9 and 8.1 microM, respectively, comparable with those in rodents consuming certain high-soy chows; the proportion of equol present as the free aglycone was much lower with dietary administration than injections, which may be a factor in the greater biological effects induced by injections. Dietary equol did not significantly increase uterine weight. Increasing dietary and injected equol doses caused a dose-dependent increase in vaginal epithelial thickness. Uterine epithelial proliferation was increased by equol injections at 8-20 mg (kg body weight)(-1) day(-1) and 1,000 ppm dietary equol. Neither dietary nor injected equol decreased thymic or adipose weights. In conclusion, equol is a weak estrogen with modest effects on endpoints regulated by estrogen receptor alpha when present at serum levels seen in rodents fed soy-based diets, but quantities present in humans may not be sufficient to induce estrogenic effects, although additive effects of equol with other phytoestrogens may occur.     

Effect of estrogenic activity, and phytoestrogen and organochlorine pesticide contents in an experimental fish diet on reproduction and hepatic vitellogenin production in medaka (Oryzias latipes)

Endocrine-disrupting chemicals (EDCs) are giving rise to serious concerns for humans and wildlife. Phytoestrogens, such as daidzein and genistein in plants, and organochlorine pesticides are suspected EDCs, because their chemical structure is similar to that of natural or synthetic estrogens and they have estrogenic activity in vitro and in vivo. We assessed estrogenic activity and dietary phytoestrogen and organochlorine pesticide contents of various fish diets made in the United Kingdom, and compared them with those features of diets made in Japan that were tested in a previous study. Genistein and daidzein were detected in all of the diets. Using an in vitro bioassay, many of these diets had higher activation of estrogen beta-receptors than estrogen alpha-receptors. Organochlorine pesticides such as hexachlorobenzene, beta-benzene hexachloride (BHC), and gamma-BHC were detected in all fish diets. On the basis of these data, we investigated the effect of differing dietary phytoestrogen content in Japanese fish diets on hepatic vitellogenin production and reproduction (fecundity and fertility) in medaka (Oryzias latipes). Assessment of the effects of a 28-day feeding period on reproduction of paired medaka did not indicate significant differences in the number of eggs produced and fertility among all feeding groups. However, hepatic vitellogenin values were significantly higher for male medaka fed diet C (genistein, 58.5 +/- 0.6 microg/g; daidzein, 37.3 +/- 0.2 microg/g) for 28 days compared with those fed diet A (genistein, < 0.8 microg/g; daidzein, < 0.8 microg/g) or diet B (genistein, 1.4 +/- 0.1 microg/g; daidzein, 2.0 +/- 0.1 microg/g). Our findings indicate that fish diets containing high amounts of phytoestrogens, such as diet C, have the potential to induce hepatic vitellogenin production in male medaka, even if reproductive parameters are unaffected. Therefore, some diets, by affecting vitellogenin production in males, may alter estrogenic activity of in vivo tests designed to determine activity of test compounds added to the diet.     

Effects of a phytoestrogen diet on estrogen-dependent reproductive processes in immature female rats.

The study reported here examined the effects of a phytoestrogen diet on progestin receptor induction, vaginal opening, and the onset and maintenance of vaginal cycles in developing female rats. A natural dietary concentration (0.01%) of the isoflavonoid coumestrol was incorporated into the AIN semipurified diet and fed from 21 to 24 days (acute treatment) or from 22 to 60 days (chronic treatment). Progestin receptor induction was observed in the uterus, pituitary, and hypothalamus-preoptic area following acute treatment. Responses were more marked in the uterus and pituitary than in the hypothalamus-preoptic area. Vaginal opening was accelerated by 4 days during chronic coumestrol treatment and occurred at a lighter body weight. Vaginal cycles began on vaginal opening and did not differ in regularity from those of control animals. However, irregular cycles were observed in coumestrol-treated animals at 116 to 131 days, suggesting that chronic coumestrol treatment may have induced some permanent changes in reproductive function. These findings demonstrate that plant estrogens, at natural dietary levels, produce significant, agonistic actions in several estrogen-dependent tissues and processes.     

Effect of the energy density of non-purified diets on growth, gestation and lactation in mice

The effects of dietary energy density on the performance of growing, gestating and lactating C57BL/6J mice were determined in order to develop pelleted non-purified practical diets for use in all stages of the mouse life cycle. Experimental diets with 4 levels of energy at 24% crude protein (CP) were pelleted and the nutritional values were determined using adult rats. The nitrogen-corrected metabolizable energy (MEn) values ranged from 2.86 to 3.73 kcal/g and the digestive CP (DCP) contents ranged from 20.5 to 22.6% on a dry matter (DM) basis. Mice responded to decreased dietary energy by increasing their feed intake to maintain MEn intake levels, except for 1 week after weaning and during lactation periods. During these periods, mice fed lower energy diets could not consume as much MEn as those fed higher energy diets. The lowest energy diet, in comparison with the highest energy diet, resulted in approximately a 33% lower weaning weight of pups at 3 weeks of age, a 13.2 to 34.4% slower growth at 3 to 4 weeks of age, and a 9.3 day delay in the onset of vaginal opening in young females. Lower energy diets, however, did not affect the litter size or the birth weight of pups. The DCP intake usually increased with decreases in dietary energy but apparently this did not affect the performance of the mice. It was concluded that an optimal diet should have an MEn value of 3.73 kcal/g DM or more for both the one week post weaning growth period and during lactation, but a diet with an MEn value of 2.86 kcal/g DM was sufficient for growth after 4 weeks of age and during gestation.     

Tools to evaluate estrogenic potency of dietary phytoestrogens:A consensus paper from the EU Thematic Network "Phytohealth" (QLKI-2002-2453)

Phytoestrogens are naturally occurring plantderived polyphenols with estrogenic potency. They are ubiquitous in diet and therefore, generally consumed. Among Europeans, the diet is rich in multiple putative phytoestrogens including flavonoids, tannins, stilbenoids, and lignans. These compounds have been suggested to provide beneficial effects on multiple menopause-related conditions as well as on development of hormone-dependent cancers, which has increased the interest in products and foods with high phytoestrogen content. However, phytoestrogens may as well have adverse estrogenicity related effects similar to any estrogen. Therefore, the assessment of estrogenic potency of dietary compounds is of critical importance. Due to the complex nature of estrogenicity, no single comprehensive test approach is available. Instead, several in vitro and in vivo assays are applied to evaluate estrogenic potency. In vitro estrogen receptor (ER) binding assays provide information on the ability of the compound to I) interact with ERs, II) bind to estrogen responsive element on promoter of the target gene as ligand-ER complex, and III) interact between the co-activator and ERs in ligand-dependent manner. In addition, transactivation assays in cells screen for ligand-induced ERmediated gene activation. Biochemical in vitro analysis can be used to test for possible effects on protein activities and E-screen assays to measure (anti)proliferative response in estrogen responsive cells. However, for assessment of estrogenicity in organs and tissues, in vivo approaches are essential. In females, the uterotrophic assay is applicable for testing ERa agonistic and antagonistic dietary compounds in immature or adult ovariectomized animals. In addition, mammary gland targeted estrogenicity can be detected as stimulated ductal elongation and altered formation of terminal end buds in immature or peripubertal animals. In males, Hershberger assay in peri-pubertal castrated rats can be used to detect (anti)androgenic/ (anti)estrogenic responses in accessory sex glands and other hormone regulated tissues. In addition to these short-term assays, sub-acute and chronic reproductive toxicity assays as well as two-generation studies can be applied for phytoestrogens to confirm their safety in long-term use. For reliable assessment of estrogenicity of dietary phytoestrogens in vivo, special emphasis should be focused on selection of the basal diet, route and doses of administration, and possible metabolic differences between the species used and humans. In conclusion, further development and standardization of the estrogenicity test methods are needed for better interpretation of both the potential benefits and risks of increasing consumption of phytoestrogens from diets and supplements.     

Effect of dietary components, including lignans and phytoestrogens, on enterohepatic circulation and liver metabolism of estrogens and on sex hormone binding globulin (SHBG)

A brief account of our present knowledge on the enterohepatic metabolism of estrogens and on the origin, metabolism and biological effects of mammalian lignans and phytoestrogens is undertaken. Furthermore, recently published results on the effects of dietary fiber, fat and carbohydrates on estrogen metabolism are reviewed. New preliminary results are presented on quantitative assays of lignans and phytoestrogens in urine of women belonging to various dietary and population groups and in a group of chimpanzees. The highest values of lignans and phytoestrogens were found in the non-human primates, and in macrobiotic, lactovegetarian and Japanese women, all groups considered having a low risk for the development of breast and other hormone-dependent cancer. New results on correlations between intake of various fibers, lignan and phytoestrogen excretion and plasma levels of estrogens, free testosterone and SHBG in women are presented. There is a significant positive correlation between the intake of fiber and urinary excretion of lignans and phytoestrogens, and the concentration of plasma SHBG. Fiber intake and urinary excretion of lignans and equol correlated negatively with plasma percentage free estradiol. Enterolactone excretion correlated negatively with plasma free testosterone. It is concluded that dietary macro- and micronutrients seem to play an important role in estrogen metabolism.     

Antihypertensive and cognitive effects of grape polyphenols in estrogen-depleted, female, spontaneously hypertensive rats

Both endogenous and dietary estrogens reduce hypertension and enhance cognitive abilities in estrogen-depleted female spontaneously hypertensive rats (SHR). Many of the beneficial effects of estrogens/phytoestrogens also appear to be provided by other polyphenols (e.g., proanthocyanidins) in grape seed, which lack appreciable estrogenic receptor binding. The present study tested the hypothesis that similar to phytoestrogens, proanthrocyanidins in grape seed polyphenols reduce salt-sensitive hypertension in young, estrogen-depleted SHR. SHR were ovariectomized at 4 wk of age and placed on phytoestrogen-free diets with or without 0.5% grape seed extract added and with high (8.0%) or basal (0.6%) NaCl. After 10 wk on the diets, grape proanthrocyanidin supplementation significantly reduced arterial pressure in the rats fed the basal (10 mmHg) and high (26 mmHg)-NaCl diet, compared with the nonsupplemented controls. In vitro superoxide production was significantly reduced (23%) by the grape seed polyphenols. Spatial learning (8-arm-radial maze) in the SHR on the basal NaCl diets was improved by dietary grape seed polyphenols. These results indicate that grape seed polyphenols decrease arterial pressure in SHR, probably via an antioxidant mechanism.     

Effect of fatty acid composition of dietary fat on energy balance and expenditure in hamsters

The comparative effects of feeding diets containing corn, olive, coconut, or menhaden fish oil on efficiency of energy deposition and on short term energy expenditure were examined in growing hamsters. Diets comprising oils mixed with laboratory diets at 10% oil w/w were fed ad libitum for 3 weeks. Animals fed laboratory diets were used as controls. Body composition was determined before and after the feeding period using 3H2O distribution space. Oxygen consumption was measured in each animal during the final week. Weight gains of groups fed corn and olive oil diets exceeded those of the group fed laboratory diet alone (p less than 0.05), although metabolizable energy intakes were similar across groups. Corn oil fed animals demonstrated higher carcass energy gains as fat compared with laboratory diet fed or menhaden oil fed groups. This was reflected in an increased fractional deposition of metabolizable energy intake in the group fed corn oil diet compared with the latter two groups. Fecal energy losses were lower in the group fed corn oil diet, and higher in the group fed laboratory diet alone, compared with other groups. Oxygen consumption did not differ between groups. These findings indicate that feeding dietary fish oil, compared with corn oil, favours energy substrate oxidation reducing the fraction of metabolizable energy partitioned for storage.     

Feeding value of sunflower and groundnut cakes for broilers

Proximate analysis of kernels of 30, and amino acid composition of two, varieties of sunflower seed are reported. Replacement of groundnut cake in the diet of growing chickens by sunflower cake improved growth rate and efficiency of utilization of energy and protein. Diets based on sunflower cake needed only 4% fish meal supplementation as against 8% needed with groundnut cake for equal weight gains (1700 g) in broilers to 70 days of age. A mixture of sunflower and groundnut cakes gave almost similar weight gains with only 2% fish meal. Sunflower hull did not appear to contain any harmful constituent and its presence in the diet did not cause any harm, but it decreased the energy content of the diets. At the 18% dietary protein level, a mixture of sunflower and groundnut cakes gave decidedly higher weight gain than either of the two cakes alone. At 18–26% dietary protein, groundnut cake proved inferior to sunflower cake for growth and feed efficiency. With both cakes, broilers performed better at higher levels of dietary protein. Level of incorporation in the diets did not affect the metabolizable energy of sunflower cake. Decorticated and undecorticated sunflower cakes gave metabolizable energy values of 2.95 and 2.65 Mcal/kg, respectively.     

How isoflavone levels in common rodent diets can interfere with the value of animal models and with experimental results

Phytoestrogens are a diverse group of plant-derived compounds structurally or functionally mimicking mammalian oestrogens. A variety of effects, in vitro, in animal models and in humans, have been attributed to these compounds. It is generally not appreciated that many commercial rodent diets are formulated with soy as a source of protein and thus large daily doses of phytoestrogens in the form of isoflavones are delivered to the animals. The content of isoflavones in laboratory diets depends on a number of factors and, therefore, varies considerably, both between different formulations, and also from batch to batch of the same formulation. Dietary isoflavones can influence a number of different endpoints, rendering the dietary levels of such compounds an important experimental factor to consider. The levels of isoflavones in common laboratory diets are reviewed in relation to the dietary levels influencing different endpoints.     

Behavioral effects of endocrine-disrupting substances: phytoestrogens

A major source of endocrine-disrupting substances, usually not considered in laboratory animal experiments, is the diet used in research investigations. Soy represents the main protein source in almost all natural-ingredient commercially available formulated diets. Soy-derived isoflavones are the most abundant and in many ways the most studied phytoestrogens, and phytoestrogens (isoflavones) are known endocrine disruptors. Research is reviewed that identifies the physiological and behavioral endocrine-disrupting effects of dietary phytoestrogens (isoflavones) in animal diets, including most of the isoflavones, which are in a glycoside form and biologically inactive, and those in the gastrointestinal tract, which are biologically active. The isoflavones genistein and daidzein have similar molecular weights and structural characteristics to that of 17-beta estradiol, which may enable them to exert estrogenic and antiestrogenic properties are described and characterized. Daidzein can be further metabolized to the potent and abundant molecule equol, which in rodents is produced in very large amounts and represents the major circulating metabolite among all biologically active isoflavones. Equol has the unique and important ability to specifically bind 5 alpha-dihydro-testosterone, and to act in turn to inhibit the action of this potent androgen. The specific influence of dietary soy phytoestrogens on consumptive, learning and memory, and anxiety-related behaviors is identified. Regulatory behaviors such as food and water intake, adipose deposition and leptin, and insulin levels affected by dietary isoflavones are also discussed.     

Digestive responses of two omnivorous rodents (<Emphasis Type="Italic">Peromyscus maniculatus</Emphasis> and <Emphasis Type="Italic">P. alstoni</Emphasis>) feeding on epigeous fungus (<Emphasis Type="Italic">Russula occidentalis</Emphasis>)

The sporocarps of hypogeous and epigeous fungi are important dietary items for forest dwelling rodents in temperate and tropical forests throughout the world. However, results of some pioneering works have demonstrated that fungi cannot be considered as nutritionally high-quality food items for some mycophagous small rodents. According to these studies, when mycophagous rodents feed on fungus, they showed a minimal digestibility, but whether this applies to most rodent species that include fungi in their diets is unknown. In this study, we experimentally evaluated body mass changes and feed preferences in captive deer (Peromyscus maniculatus) and volcano (P. alstoni) mice when fed on epigeous fungus (Russula occidentalis). In experiment 1, the animals were fed with fungus as the only feedstuff in comparison to regular rodent chow and oat. In experiment 2, the animals were fed with fungus in a free-choice arrangement together with equal amounts of rodent chow and oat. Both species lost ∼15% of their body mass within 4 days when fed on fungus alone, but gained 5–10% body mass during the same time period when ingesting oat and rodent chow, respectively, as the only feedstuff. However, in contrast, in the free-choice arrangement with all three feedstuffs, both species gained 20–30% body mass, and showed the highest feed preference for fungus followed by oat and rodent chow. In addition, apparent digestibility of energy and nitrogen were analyzed in both rodent species, which were 50–60% for fungus, whereas approximately 90–94% for rodent chow and oat. According to our results, animals need to supplement their diets with alternative high-quality food items in order to maintain and increase their body mass, suggesting that epigeous fungi are only of moderate nutritional value for small rodents. Futures studies should focus on exploring the importance of a mixture of fungal species in the diet of small mycophagous rodents.     

An investigation of factors controlling food intake in Arctic charr, Salvelinus alpinus L.

Digestibility values were determined for diets of varying protein content fed to Arctic charr. The use of these values, in conjunction with food intake data made possible the study of the effects of various parameters on the control of food consumption. Dietary energy content may be more important than protein content in regulating consumption. The regulation of food intake may be partially mediated through factors influencing gastric emptying coupled with mechanisms monitoring dietary metabolizable energy.     

Effects of a normal, human-concentration, phytoestrogen diet on rat uterine growth.

The estrogenic action of the prototype natural phytoestrogen coumestrol was examined in rats in in vitro and in vivo tests. To establish the binding specificity of coumestrol and its relation to biological activities, saturation analyses and uterine weight assays were performed. These assays indicated that coumestrol competitively inhibited binding to the estrogen receptor and induced increases in uterine weight in keeping with its estrogen receptor affinity constant. Most importantly, coumestrol was uterotrophic when incorporated in a semipurified diet at natural dietary concentrations. Significant increases occurred in both uterine wet weight and dry weight, indicating that coumestrol produces true uterine growth. Effects appeared to be cumulative, raising questions of time-related interactions with other estrogen-sensitive mechanisms and clearance of isoflavonoids. Coumestrol induced uterine growth over a 90-hour period at dietary concentrations of 0.01 to 0.1%. Lower doses not active over this period were active when provided over a longer period of time: a 0.005% concentration was not active over a 90-hour period, but was active when provided over a 180-hour period. Coumestrol-induced uterine growth was accompanied by the induction of cytosolic progestin receptors and increases in nuclear estrogen binding. Scatchard analyses verified that these changes were due to changes in receptor number. These studies show that the naturally occurring phytoestrogens have dramatic estrogenic effects at natural dietary levels. These actions may be expressed via traditional receptor-mediated actions and therefore may have the same implications for development, health, and disease as do the steroidal estrogens produced by the body. Because rats have no sex hormone-binding globulin, further studies must be conducted in humans. However, these findings suggest that the natural dietary phytoestrogen coumestrol is a potent estrogen that must be considered in calculating the total estrogenic load to which humans are exposed during normal life.     

A method for separating estrogens and phytoestrogens on Sephadex LH-20 columns.

A procedure for separating estrogens from phytoestrogens in animal fluids is presented. The use of Sephadex LH-20 in absolute methanol permits a simple separation and allows the measurement of phytoestrogen levels in plasma and urine by the competitive binding assay. This method facilitates a more accurate measure of the hormonal balance in animals with an exogenous input of phytoestrogens.     

Role of Choline Deficiency in the Fatty Liver?Phenotype of Mice Fed a Low Protein,Very Low Carbohydrate Ketogenic Diet

Though widely employed for clinical intervention in obesity, metabolic syndrome, seizure disorders and other neurodegenerative diseases, the mechanisms through which low carbohydrate ketogenic diets exert their ameliorative effects still remain to be elucidated. Rodent models have been used to identify the metabolic and physiologic alterations provoked by ketogenic diets. A commonly used rodent ketogenic diet (Bio-Serv F3666) that is very high in fat (~94% kcal), very low in carbohydrate (~1% kcal), low in protein (~5% kcal), and choline restricted (~300 mg/kg) provokes robust ketosis and weight loss in mice, but through unknown mechanisms, also causes significant hepatic steatosis, inflammation, and cellular injury. To understand the independent and synergistic roles of protein restriction and choline deficiency on the pleiotropic effects of rodent ketogenic diets, we studied four custom diets that differ only in protein (5% kcal vs. 10% kcal) and choline contents (300 mg/kg vs. 5 g/kg). C57BL/6J mice maintained on the two 5% kcal protein diets induced the most significant ketoses, which was only partially diminished by choline replacement. Choline restriction in the setting of 10% kcal protein also caused moderate ketosis and hepatic fat accumulation, which were again attenuated when choline was replete. Key effects of the 5% kcal protein diet – weight loss, hepatic fat accumulation, and mitochondrial ultrastructural disarray and bioenergetic dysfunction – were mitigated by choline repletion. These studies indicate that synergistic effects of protein restriction and choline deficiency influence integrated metabolism and hepatic pathology in mice when nutritional fat content is very high, and support the consideration of dietary choline content in ketogenic diet studies in rodents to limit hepatic mitochondrial dysfunction and fat accumulation.     

Effect of avilamycin and tylosin on the metabolizable energy in growing and finishing pigs

In two metabolism trials with growing and finishing pigs the influence of the antibiotic feed additives Avilamycin and Tylosin on the metabolizable energy was investigated at different levels of dietary protein content. In the first experiment (growing pigs) the antibiotics were supplied at levels of 0 mg/kg, 40 mg/kg Avilamycin and 40 mg/kg Tylosin to diets containing 18.5%, 17.5%, 16.5% and 14.0% of crude protein. In the second experiment (finishing pigs) 0 mg/kg antibiotics, 20 mg/kg Avilamycin and 20 mg/kg Tylosin were used in diets containing 16.5% and 14.0% of crude protein. The body weight of the animals averaged 46 kg (growing pigs) and 68 kg (finishing pigs).

In growing pigs the supplementation of Tylosin increased the digestibility of dry matter and energy at 1 percentage unit each, while in finishing pigs no effects were observed. Since the urinary energy excretion was not affected by antibiotics, there was only in Tylosin treated growing pigs a slight rise in dietary contents of metabolizable energy by 1.6%. The reduction of the dietary protein content resulted in increased digestibility of dry matter and energy, reduced urinary energy excretions and increased dietary contents of metabolizable energy.