Growth differences of male and female Göttingen minipigs during ad libitum feeding: a pilot study.

Even though minipigs have been used in biomedical research for nearly half a century now, no specific nutrient requirements are available. For that reason a series of studies into the nutrient requirements of Göttingen minipigs were carried out. Firstly, a pilot study was carried out to determine the ad libitum feed intake (FI) during growth, as a reference for later feed restriction studies. Four male and four female minipigs were fed two types of diet, one standard pig diet (20.6% crude protein; 11.7% crude fat; 13.5 mJ/kg DM metabolizable energy) and one diet specially designed for minipigs (12.0% crude protein; 2.9% crude fat; 11.9 MJ/kg DM metabolizable energy). When fed ad libitum for 13 weeks, female Göttingen minipigs developed a significantly (P<0.05) higher body weight (BW) than males (27.4 vs 16.6 kg) on either diet. The large difference in growth between male and female Göttingen minipigs did not appear to be the result from differences in metabolizable energy intake. Metabolizable energy intake of male and female Göttingen minipigs could be predicted by ME=1877 kJxBW(0.61). Both male and female Göttingen minipigs became obese when fed ad libitum, defined by relative backfat thickness. Relative backfat thickness ranged from 5 to 13 cm/100 kg. Females had thicker relative backfat layers than males. Remarkably, no large changes in haematology and clinical chemistry occurred in ad libitum fed Göttingen minipigs as compared to reference values, and no abnormalities other than enlarged fat reserves were observed at necropsy. Apparently, Göttingen minipigs do not restrain FI voluntarily, and restricted feeding is therefore indicated to prevent obesity.

Lowering dietary phosphorus concentrations reduces kidney calcification, but does not adversely affect growth, mineral metabolism, and bone development in growing rabbits.

New Zealand White rabbits were used to investigate the influence of increasing dietary P concentrations on growth performance, mineral balance, kidney calcification and bone development. The minimum dietary P requirement of 0.22 % (National Research Council) is usually exceeded in commercial natural-ingredient chows, leading to undesirable kidney calcifications. In order to study the optimal dietary P level, rabbits were fed semi-purified diets with four different P levels (0.1, 0.2, 0.4, and 0.8 %; w/w) at a constant dietary Ca concentration (0.5 %) during an 8-week period. Body weight and growth were not influenced by the dietary P level. During two periods (days 20-23 and 48-51), faeces and urine were collected quantitatively for the analysis of Ca, Mg and P and balances were calculated. Increased dietary P intake caused increased urinary and faecal P excretion and P apparent absorption and retention. Faecal Ca excretion increased with higher dietary P levels, whereas urinary Ca excretion reacted inversely. The apparent absorption of Ca became reduced at higher dietary P concentrations, but Ca retention was unchanged. The response of Mg was in a similar direction to that of the Ca balance. Kidney mineral content increased with higher dietary P levels, indicating the presence of calcified deposits. Nephrocalcinosis became more severe in kidney cortex and medulla at increasing dietary P levels, as was confirmed by histological analysis. Femur bone length was not differentially influenced by dietary P. Bone density (g/cm(3)) of the femur diaphysis became significantly lower at the 0.8 % dietary P level as compared with the 0.2 % P group only. The bone Mg content was significantly increased on the 0.8 % P diet, both in the diaphysis and epiphysis. Plasma P concentration increased and plasma Ca decreased with higher dietary P levels, whereas plasma Mg levels were unaffected. The present study shows that the current recommended minimum dietary P level of 0.2 % for rabbits, as advised by the National Research Council in 1977, leads to a normal growth and bone development, but also causes some degree of kidney calcifications at a dietary Ca level of 0.5 %. As the dietary P level of 0.1 % virtually prevented kidney calcification and at the same time did not give evidence for any deleterious effects on growth and bone development, this indicates that the current recommended dietary P level for rabbits should be regarded as a maximum advisable concentration, and that a lower P level may be more optimal.

Influence of excessive fluoride consumption on the severity of dystrophic cardiac calcification in DBA/2 mice.

Soft tissue calcifications in inbred laboratory mice are frequently observed and are often associated with dystrophic cardiac calcinosis (DCC). We tested the hypothesis that an excessive intake of fluoride would inhibit pathological calcifications in DCC-susceptible mice. A diet containing either a high (200 mg F/kg added to the diet) or low fluoride content (no F added) was fed to both weanling and retired breeder DBA/2 mice. The high-fluoride diet reduced feed intake and body weight gain when given after weaning. It was found that a high fluoride intake effectively reduces soft tissue calcifications in young mice, but not in retired breeders. Because DCC in mice is a pathological finding that could interfere with certain experimental procedures, it is suggested that the optimum fluoride concentration in the diet for mice of susceptible strains should be established.

The effects of feeding and housing on the behaviour of the laboratory rabbit.

The effects of housing, feeding time and diet composition on the behaviour of the laboratory rabbit were examined. The animals were caged individually in single or double metal cages with perforated metal floors, metal walls, and bars in the front, or kept as a group in floor pens. The light/dark cycle was 12/12 h with light from 04:00 to 16:00 h and 30 min twilight. One experiment compared feeding equal energy levels of a high energy diet (10.1 MJ/kg) and with a low energy diet (7.0 MJ/kg) at 08:00 h. The second experiment compared feeding the high energy diet at 08:00 h and at 14:00 h. In both studies the behaviour of the rabbits was recorded between 08:00 and 14:00 h and between 16:00 and 22:00 h. Feeding the animals at 14:00 h reduced abnormal behaviour during the dark period compared to feeding at 08:00 h, whereas no difference in behaviour could be detected between feeding a high-energy and a low-energy diet at 08:00 h. Animals in floor pens generally showed less abnormal behaviour than caged animals. The results indicate that the welfare for caged rabbits can be improved by feeding the animals in the afternoon rather than in the morning.

The influence of transportation stress on selected nutritional parameters to establish the necessary minimum period for adaptation in rat feeding studies.

The optimal length of the adaptation period after transportation of rats, to be used in nutritional studies, was investigated in this study. After intracontinental transportation of rats by car and by air to and from the laboratory for a total period of 15 h, measurements were carried out for a period of 3 weeks after transport. Control and transported animals were housed in the same laboratory before and after transportation. During transport the animals had access to food and water. As blood collection could also cause stress, a factorial design was carried out with transport and blood collection as main factors. Transport or blood collection did not cause significant effects on the following parameters: body weight, growth, clinical observation, and blood enzyme activities of LDH and ASAT. Water intake was significantly increased after transport. Food intake did not show consistent effects after transport or blood collection. Unexpectedly, blood corticosterone levels were significantly lower in the transported animals at day 1 after transport. After 3 days these levels were back to normal. Blood glucose, blood free fatty acids and blood urea nitrogen concentrations were incidentally decreased, whereas total cholesterol levels showed an incidental rise in the transported rats. The open-field behaviour test revealed no clear-cut results concerning the effects of transport or blood collection on faeces production, rearing and ambulation. Our results indicate that after intracontinental transport, an adaptation period of 3 days appears to be sufficient for rats to be used in nutritional studies.

The association of increasing dietary concentrations of fish oil with hepatotoxic effects and a higher degree of aorta atherosclerosis in the ad lib.-fed rabbit.

The long-term effects of consumption of marine long-chain n-3 polyunsaturated fatty acids (PUFA) on atherosclerosis in the rabbit were examined. Female Dutch rabbits were fed purified diets, containing 40 energy% total fat, for a period of 2.5 years. To study the dose response relationship between fish oil intake and atherosclerosis, four diets were formulated with fish oil levels being 0, 1, 10 and 20 energy%. A fifth and sixth group were fed an alpha-linolenic acid-(C18:3, n-3) and linoleic acid-(C18:2, n-6) rich diet, respectively. Every 6 weeks, blood samples were taken for determination of clinical chemical parameters, triacylglycerol and total cholesterol levels. Feeding 10 and 20 energy% fish oil containing diets, resulted in an increase of liver enzymes (AST, ALT and ALP). Histological evaluation of the liver also revealed adverse effects of fish oil containing diets. Triacylglycerol blood levels were similar in all groups, and remained constant throughout the study. Total cholesterol levels in blood was significantly lower in the animals fed a linoleic acid-rich diet, as compared with the other five groups. An n-3 long-chain PUFA concentration dependent increase in aorta plaque surface area was observed in the fish oil groups. A significant positive relationship was found between the group mean score for severity of liver pathology and the aorta plaque surface area. These results indicate that the long-chain n-3 polyunsaturated fatty acids in fish oil may be hepatotoxic to the herbivorous rabbit, which may interfere with the outcome of atherosclerosis studies. This finding necessitates the exclusion of liver pathology in experimental studies on atherosclerosis in animal models.

Genetic analysis of dystrophic cardiac calcification in DBA/2 mice.

A DBA/2 x D2B6F1 backcross was produced in order to study the genetic background of pathological soft tissue calcification in the mouse. Calcification was assessed in the myocardium, kidney and tongue. Significant co-segregation was found with the genotype of microsatellite markers on the proximal end of Chromosome 7. This region contains a candidate gene, Hrc, coding for the histidine-rich calcium binding protein in the sarcoplasmatic reticulum. The results support the hypothesis that the gene previously reported to be responsible for DCC (dystrophic cardiac calcification) in C3H mice (1) causes generalized soft tissue calcification in DBA/2 mice.

Nutrition of (Göttingen) minipigs: facts, assumptions and mysteries.

Detailed guidelines are available concerning the nutrient requirements of pigs (National Research Council 1988). These nutrient requirements are based on ad libitum feeding and obtaining maximum growth, as they have originated from feeding schedules for slaughter pigs. Whether these nutrient guidelines for pigs can be applied to minipigs as well remains to be answered. Moreover, ad libitum feeding and maximum growth are not considered the optimum in scientific research. The German Society for Laboratory Animal Science has published guidelines for the composition of minipig diets, mainly based on empirical results (1993). Upon comparison of dietary guidelines for pigs and minipigs, differences can be found. At the moment it is unclear which of the two dietary guidelines guarantees that all minimum nutrient requirements of the minipig are met. Restricted feeding is often applied in studies using minipigs, in order to prevent obesity. As the two guidelines are based on ad libitum feeding, this raises the question whether restriction results in (marginal) nutrient deficiencies, which may interfere with experimental results.

Effects of two dietary fat levels and four dietary linoleic acid levels on mammary tumor development in Balb/c-MMTV mice under ad libitum feeding conditions.

The relationship between dietary fat intake (level and type) and the development of breast cancer in humans is a matter of concern in Western society. A high fat intake is associated with a greater mammary cancer risk in humans and in animal models. Higher intake of polyunsaturated fatty acids in humans shows little or no association with mammary tumor development in epidemiologic surveys. From literature data, it appears that a higher intake of polyunsaturated fatty acids (linoleic acid) is related to an increase in mammary tumorigenesis in animal studies in which chemical carcinogens like dimethylbenz[a]anthracene are used as tumor initiator. Mostly the latency period of these chemically induced models in rather short. In this study, the Bald/c-MMTV (mouse mammary tumor virus) mouse strain was chosen as an animal model: MMTV leads to tumor initiation, and dietary factors influence tumor promotion over a relatively long latency period. The mice were fed diets with two fat concentrations: a high [36% of energy (en%)] or low (16 en%) fat level; fat was isocalorically replaced by carbohydrates (cornstarch). At both dietary fat levels, linoleic acid was given at four levels: 2, 3, 6, and 10 en%. Linoleic acid-rich fat was isocalorically replaced by oleic acid-rich fat. The diets were consumed ad libitum over a lifetime. Animals were euthanized as soon as mammary tumor diameter was > or = 1 cm or when the animals were in a poor clinical condition. The incidence of mammary tumors at 18 months was significantly higher in one group only: 36 en% fat and 2 en% linoleic acid. This group also showed the shortest mean latency period for mammary tumor development. Mean mammary tumor incidence was higher and mean onset time shorter in the four high-fat groups than in the low-fat groups. No (linear) dose-response relationship between dietary linoleic acid concentration and mammary tumor incidence and latency period was observed. This indicates that a higher dietary linoleic acid intake does not increase the incidence or shorten the latency period of breast cancer in the Balb/c-MMTV mouse strain at two different dietary fat levels.

Long-term phosphorus restriction prevents corticomedullary nephrocalcinosis and sustains reproductive performance but delays bone mineralization in rats.

In a long-term experiment with three successive generations of rats, the influence of dietary phosphorus restriction (2 instead of 4 g phosphorus/kg diet) on nephrocalcinosis, reproduction and bone mineralization was studied. Nephrocalcinosis in female rats, as based on kidney calcium concentration and histological examination, was prevented by phosphorus restriction. The low phosphorus diet caused reduced femur concentrations of magnesium, calcium and phosphorus in rats of the first and second generation aged 4 to 12 wk. The low phosphorus diet resulted in lower plasma phosphorus concentrations. In the kidneys of female rats, immediately after lactation, a higher degree of tubular hyperplasia was seen after the low phosphorus diet was fed. Reproductive performance was not affected by phosphorus restriction. We conclude that 0.2% phosphorus in the diet prevents nephrocalcinosis in female rats while it sustains reproduction but delays bone mineralization.

Inbred strains of rats have differential sensitivity to dietary phosphorus-induced nephrocalcinosis.

The degree of nephrocalcinosis after increasing the dietary phosphorus concentration from 0.2 to 0.5 g/100 g was measured in weanling female rats of 10 inbred strains. Based on kidney calcium concentrations and histological kidney calcification scores, there were considerable strain differences in nephrocalcinogenesis; 86% of the strain variability in nephrocalcinosis was attributable to genetic factors. Two strains with the most extreme nephrocalcinogenic responses were retested and the strain difference was found to be reproducible. Mean plasma phosphorus concentrations after phosphorus feeding were lower in the sensitive strain than in the insensitive strain. The high phosphorus diet produced greater urinary phosphorus concentrations, with the increase being greater in the sensitive strain. The strain difference in the response of urinary phosphorus concentrations after raising dietary phosphorus level may determine the strain difference in phosphorus-induced nephrocalcinosis. After consuming the high phosphorus diet, RP rats housed in groups in solid-floored cages had significantly higher degrees of nephrocalcinosis than their counterparts housed individually in metabolism cages with wire-mesh bases.

Inhibitory effect of high protein intake on nephrocalcinogenesis in female rats.

Increased intakes of protein have been shown to reduce kidney calcification (nephrocalcinosis) in female rats. Two questions were addressed in the present study. First, can protein-induced inhibition of nephrocalcinosis be demonstrated when the diets used are balanced for calcium, magnesium and phosphorus in the added protein? Second, can the protein effect be explained by the frequently observed magnesiuria after giving high-protein diets? Nephrocalcinosis was induced in female rats by giving purified diets containing 151 g casein/kg and either an increased concentration of P (6 v. 2 g/kg) or a decreased concentration of Mg (0.1 v. 0.4 g/kg). To these diets 151 g ovalbumin/kg was added at the expense of glucose, and the diets were balanced for Ca, Mg and P in ovalbumin. The diets were given for 29 d. In rats fed on the diet containing 151 g protein/kg, an increased intake of P or a decreased intake of Mg caused nephrocalcinosis as measured chemically by analysis of kidney Ca as well as histologically by scoring kidney sections stained according to Von Kossa's method. The addition of ovalbumin to the diet prevented the induction of nephrocalcinosis. High P intake and low Mg intake with the low-protein diets induced enhanced loss of albumin in urine, suggesting that nephrocalcinosis caused kidney damage. Increased protein intake with a non-calcinogenic diet also caused increased albumin excretion in urine. Irrespective of the composition of the background diet, increased protein intake caused increased urinary excretion of Mg. When all dietary groups were considered, differences in nephrocalcinosis and urinary Mg output were not proportionally related.

Nephrocalcinosis in the rat: a literature review.

Nephrocalcinosis is a common disorder in female rats. Various etiological factors are involved in the pathogenesis, e.g. sex, age, genetical and dietary factors. Dietary phosphorus concentration appears to be of crucial importance in the induction of nephrocalcinosis. The pathological changes in calcinotic kidney tissue are described. Possible mechanisms underlying nephrocalcinogenesis are discussed. Phosphorus concentration within the proximal tubule may be the major determinant of nephrocalcinogenesis.

Phosphorus-induced nephrocalcinosis in female rats: a study on regression and clinical abnormalities.

The question addressed was whether preestablished phosphorus (P)-induced nephrocalcinosis would regress after dietary P restriction. Female rats were fed purified diets containing either 0.2% (w/w) P (low P) or 0.6% P (high P). After 29 days, the high-P diet had caused massive nephrocalcinosis as demonstrated chemically (by the analysis of calcium in kidney) and histologically (by inspection of kidney sections stained for calcium phosphate deposits). Switching rats from the high P to the low P diet did not result in a decrease in the degree of nephrocalcinosis within 91 days. Thus, P-induced nephrocalcinosis may not regress upon subsequent P restriction. Rats that had been fed either the 0.2 or 0.6% P diet for 56 days were examined clinically with respect to 14 selected variables. None of the variables discriminated between rats with or without nephrocalcinosis. This might imply that P-induced nephrocalcinosis in female rats does not cause significant discomfort.

Dietary fluoride prevents phosphorus-induced nephrocalcinosis in female rats.

The effect of dietary fluoride (F) on nephrocalcinosis was studied in young, female rats. Nephrocalcinosis was induced by a diet rich in phosphorus (P). F in the diet effectively counteracted P-induced nephrocalcinosis in a dose-dependent fashion. The feeding of increasing amounts of F caused decreasing calcium (Ca) and F concentrations in kidney. This suggests that the amount of Ca in kidney determines F accumulation in this organ, rather than F intake. Increasing amounts of F in the diet caused increasing rates of urinary and fecal excretion and whole-body retention of F. Dietary F did not influence urinary and fecal excretion and plasma concentrations of Ca, magnesium (Mg), and P. The metabolic basis for the protective effect of F against the development of nephrocalcinosis remains to be established.

Commercial rodent diets and nephrocalcinosis in weanling female rats.

This study addresses the questions to what extent commercial rodent diets would induce nephrocalcinosis, and which dietary components would be responsible for inducing this condition. For this purpose, 10 commercial diets were analysed for selected components and fed to weanling female rats. On the basis of histological inspection of kidney sections, two diets were found to produce significant nephrocalcinosis. The condition could be considered relatively mild because concentrations of Ca in kidney tissue were not increased. There was considerable variation between the commercial diets in the (analysed) concentrations of Ca, P, Mg and protein as well as in the diet-induced urinary pH, urinary volume and caecal weight. Of these parameters, only the dietary Ca:P ratio and group mean urinary pH correlated significantly with the observed variation in group mean calcification scores, the relationships being negative. It is suggested that the Ca:P ratio of commercial rodent diets is an important determinant of nephrocalcinosis.

The influence of dietary lactose on phosphorus-induced nephrocalcinosis in female rats.

The effect of dietary lactose, when compared with glucose, on phosphorus-induced nephrocalcinosis was studied in young, female rats. Nephrocalcinogenic diets containing either 0.4 or 0.6% (w/w) phosphorus were used, and lactose was added up to concentrations of 30%. The diets were fed for 28 days. The 0.4 and 0.6% phosphorus diets, when compared with a diet containing 0.2% phosphorus, caused mild and severe kidney calcification, respectively; kidney calcification was demonstrated chemically by the analysis of kidney calcium, and histologically by staining kidney slides for calcium deposits. Dietary lactose caused calciuria, decreased urinary pH and increased cecum weights. The addition of lactose to the diet partly counteracted nephrocalcinogenesis induced by diets containing 0.4% phosphorus, but it did not influence the severity of nephrocalcinosis seen in rats fed diets containing 0.6% phosphorus. It is suggested that high amounts of lactose in the diet have only weak anti-nephrocalcinogenic activity.