Splanchnic net balance of oxygen and metabolites in response to a discontinuous mesenteric vein infusion of ammonium in sheep

To simulate daily episodes of high absorption associated with the intake of diets with high N content, four wethers (42 ± 3.4 kg body weight), fitted with permanent catheters in the femoral artery and splanchnic vessels, were infused with 340 μmol into the mesenteric vein for 3 h, during the morning meal, over seven consecutive days. On the 7th day, mass transfers of , urea, glucose, lactate, ß‐OH‐butyrate and O₂ were measured across portal‐drained viscera (PDV), liver and splanchnic tissues during the last 90 min of the infusion. Measurements were repeated on the following day, at the same time, without the infusion. Plasma concentration in the portal vein (+332 μm ; p = 0.006), portal absorption (+424 μmol/min; p < 0.001), liver uptake (+375 μmol/min; p = 0.003) and urea N production (+338 μmol/min; p = 0.059) were higher during infusion. Mass transfers of urea, glucose, lactate, ß‐OH‐butyrate and O₂ across the PDV, and glucose, lactate, ß‐OH‐butyrate and O₂ across the liver, were not altered by the infusion. Results suggest that a daily, discontinuous increase in portal flow during a meal stimulates liver removal and urea N production but does not significantly affect liver glucose production and O₂ consumption in sheep.     

Effect of low-temperature drying on the nitrogenous compounds and inositol phosphates in broiler chickens and cecectomized laying hen excreta

We investigated how the chemical composition of broiler chicken and cecectomized laying hen excreta is affected by drying in a forced-air drying chamber at low temperatures. Excreta that was immediately frozen after voiding provided the reference values. The excreta were dried in drying chambers for 4 hr, 6 hr, and 12 hr at 23°C or 33°C in the broiler experiment and 19°C or 29°C in the cecectomized laying hen experiment. The total N and inositol phosphate concentrations in the excreta of broiler chickens and cecectomized laying hens were not influenced (p > .050), except for one inositol tetrakisphosphate isomer (p = .026) in broilers. Compared to fresh excreta, drying did not affect the ammonia concentrations in the cecectomized laying hen experiment (p > .050), but the ammonia concentration was lower when dried for 12 hr at 33°C in the broiler experiment (p = .002). Amino acid concentrations in cecectomized laying hen excreta decreased until 4 hr of drying and then increased at both drying temperatures (p < .001). The results indicate that the applicability of drying poultry excreta at low temperatures in forced-air drying chambers to determine the chemical compound concentrations is trait-dependent. Future studies are necessary to investigate whether these results are also dependent upon the amount of excreta stored in the drying chambers.     

Relationship between level of forage intake,blood flow and oxygen consumption by splanchnic tissues of sheep fed a tropical grass forage

Four Polwarth castrated male sheep (42 ± 4.4 kg live weight (LW) surgically implanted with chronic indwelling catheters into the mesenteric, portal and hepatic veins, housed in metabolism cages and offered Cynodon sp. hay at rates (g of dry matter (DM)/kg LW) of 7, 14, 21 or ad libitum, were used in a 4 × 4 Latin square experiment to evaluate the effect of the level of forage intake on blood flow and oxygen consumption by the portal‐drained viscera (PDV), liver and total splanchnic tissues (ST). The portal blood flow and the oxygen consumption by PDV linearly increased at increased organic matter (OM) intake. No effect of level of OM intake was obtained for the hepatic artery blood flow and oxygen consumption by liver. As a consequence, the level of OM intake only tended to directly affect hepatic blood flow and oxygen consumption by total ST. Oxygen consumption was linearly and positively related to blood flow across PDV, liver and total ST. The heat production by PDV and total ST, as proportion of metabolizable energy (ME) intake, decreased curvilinearly at increased ME intake. In conclusion, the oxygen consumption by PDV, but not by liver, was directly related to the level of forage intake by sheep. Moreover, when ingested at levels below maintenance, most of ME was spent as heat produced by ST.     

Gas and short‐chain fatty acid production from feeds commonly fed to red deer (Cervus elaphus L.) and incubated with rumen inoculum from red deer and sheep

Efficient red deer supplementary feeding depends on estimations of the nutritive value of offered feeds, frequently estimated with the use of equations derived from domestic ruminants. The aim of this study was to compare the 24‐hour in vitro true dry matter degradability (ivTD₂₄), in vitro gas production (GP) kinetic parameters, GP in 24 hr of incubation (GAS₂₄) and short‐chain fatty acid (SCFA) and microbial biomass (MBS) produced after 24‐hour incubation of feeds in inoculum prepared from sheep and red deer rumen fluid. Eleven feeds, frequently consumed by red deer in Slovenia, which occur either naturally (two fresh grasses, chestnut fruits and common and sessile oak acorns) or are fed as winter supplemental feeds (two grass hays, two grass silages, apple pomace, fresh sugar beetroot), were investigated. The in vitro GP kinetic parameters, GAS₂₄ and ivTD₂₄, did not differ between animal species. Amounts of SCFAs were greater (p < 0.05) when feeds were incubated in sheep inoculum, while molar proportions of acetic and propionic acids did not differ. Molar proportions of butyric acid produced during incubation of high fibre feeds did not differ between animal species, but were higher (p < 0.05) when feeds high in starch or sugar were incubated in red deer inoculum. Greater production of SCFA by sheep rumen microbes suggests better coverage of host animal with energy precursors, while greater production of MBS by red deer rumen microbes suggests better coverage of host animal with protein. Results also suggest that rumens of sheep and red deer are inhabited by different microbial communities, which did not affect the extent of in vitro GP and degradation of feeds used in the present experiment. However, the possibility exists that the divergent nutrient use could be a consequence of different priming by different feeds of the donor animal diets.     

Responses to betaine and inorganic sulphur of sheep in growth performance and fibre growth

Sulphur‐containing amino acids (SAA) are essential and usually the first limiting amino acids for growth, milk and wool production. The keratin fibre that grows from epidermal tissue is rich in SAA. The rate of fibre growth and its S content are influenced by the availability of SAA. Betaine is a dietary source for a labile methyl group and actively participates in methionine metabolism by donating methyl groups for the remethylation of homocysteine to methionine. Ruminants are capable of synthesizing SAA from inorganic S sources, and most bacteria in the rumen can use inorganic S to meet their requirements for growth. The objective of this study was to examine whether betaine and an inorganic sulphur supplement could provide methyl groups and sulphur amino acids in a way that growth performance and wool production of ewes and lambs are improved. Treatments performed included betaine supplementation, sulphate supplementation and betaine plus sulphate supplementation with five replications for each treatment. The dry matter intake of the ewes was affected by betaine plus sulphate supplementation (p < 0.05). In the ewes, betaine plus sulphate supplementation increased (p < 0.05) the wool growth rate, wool yield, staple length and wool sulphur concentration, while decreasing wool wax and wool yellowness (p < 0.05). In the lambs, wool growth rate, wool yield, fibre diameter, staple length, staple strength, wool sulphur concentration, wool wax and fibre percentage did not differ (p > 0.05) between treatments. In the ewes, plasma methionine concentration increased (p < 0.05) with betaine plus sulphate treatment. No corresponding difference (p > 0.05) was observed in plasma methionine concentration in the lambs. It can be concluded that betaine plus sulphate supplementation has the potential to change wool characteristics in the ewes, while these compounds were without any effect on growth and wool production of the lambs. Combining the two supplements was advantageous.     

Anaesthetic and cardiorespiratory effects of a constant rate infusion of fentanyl in isoflurane‐anaesthetized sheep

ObjectiveTo determine the anaesthetic and cardiorespiratory effects of a constant rate infusion of fentanyl in sheep anaesthetized with isoflurane and undergoing orthopaedic surgery.Study designProspective, randomised, ‘blinded’ controlled study.AnimalsTwenty healthy sheep (weight mean 41.1 ± SD 4.5 kg).MethodsSheep were sedated with intravenous (IV) dexmedetomidine (4 μg kg⁻¹) and morphine (0.2 mg kg⁻¹). Anaesthesia was induced with propofol (1 mg kg⁻¹ minute⁻¹ to effect IV) and maintained with isoflurane in oxygen and a continuous rate infusion (CRI) of fentanyl 10 μg kg⁻¹ hour⁻¹ (group F) or saline (group P) for 100 minutes. The anaesthetic induction dose of propofol, isoflurane expiratory fraction (Fe’iso) required for maintenance and cardiorespiratory measurements were recorded and blood gases analyzed at predetermined intervals. The quality of recovery was assessed. Results were compared between groups using t-tests or Mann–Whitney as relevant.ResultsThe propofol induction dose was 4.7 ± 2.4 mg kg⁻¹. Fe’iso was significantly lower (by 22.6%) in group F sheep than group P (p = 0). Cardiac index (mean ± SD mL kg⁻¹ minute⁻¹) was significantly (p = 0.012) lower in group F (90 ± 15) than group P (102 ± 35). Other measured cardiorespiratory parameters did not differ statistically significantly between groups. Recovery times and recovery quality were statistically similar in both groups.Conclusions and clinical relevanceFentanyl reduced isoflurane requirements without clinically affecting the cardiorespiratory stability or post-operative recovery in anaesthetized sheep undergoing orthopaedic surgery.     

Effects of pH and fermentative substrate on ruminal metabolism of fatty acids during short‐term in vitro incubation

The ruminal biohydrogenation of c9,c12‐18:2 can be affected by the fibre/starch ratio of the diet and the ruminal pH. The objectives of this study were to examine independently in vitro the effects of fermentation substrate (hay vs. corn starch) and buffer pH (6 vs. 7) on the biohydrogenation of c9,c12‐18:2 carried out by grape seed oil, focusing on its t11 and t10 pathways, using 6‐h ruminal incubations. The experimental design was a 2 × 2 factorial arrangement. Fermentation substrate and pH affected the C18 fatty acid balance in incubated media, but few interactions were observed. Compared with starch, hay as the fermentation substrate favoured the production of 18:0 (×2.3), all trans‐18:1 isomers (×12.6) and CLA (×6.1), except c9,t11‐CLA, and the disappearance of unsaturated C18 fatty acids, but decreased the production of odd and branched chain fatty acids. Compared with pH 6 buffer, pH 7 buffer resulted in higher c9,c12‐18:2 disappearance and CLA production. For c9,t11‐CLA, an interaction was noticed between the two factors, leading to the highest production in cultures incubated on hay with the 7 pH buffer. Compared with starch, hay as fermentation substrate favoured the activity of t11 producers, which are fibrolytic bacteria, and the production of t10 isomers, possibly due to the presence of potential t10 producers in hay. Low pH resulted in a decreased t11 isomers production and in a slightly increased t10 isomers production, probably due to a modulation of enzymatic or bacterial activity.     

Effect of dietary energy on digestibilities,rumen fermentation,urinary purine derivatives and serum metabolites in Tibetan and small‐tailed Han sheep

Tibetan sheep are indigenous to the Qinghai–Tibetan Plateau, graze the grassland all year round without supplementation and are well‐adapted to the harsh conditions. Small‐tailed Han sheep were introduced to the plateau and are raised mainly in feedlots. Based on their different backgrounds, we hypothesized that the ability to cope with poor diets would be better in Tibetan than in Han sheep. To test our prediction, we examined the effect of dietary energy on apparent digestibilities, rumen fermentation, urinary purine derivatives and serum metabolites by using a 4 × 4 Latin square design in each sheep breed. Four diets were formulated to be low in crude protein (~7%) but to differ in metabolizable energy concentration. Average daily gain was greater in Tibetan than in Han sheep (p < 0.01) and increased linearly with an increase in energy intake (p < 0.001). The digestibilities of dry matter, organic matter, gross energy, and neutral and acid detergent fibres were greater in Tibetan than in Han sheep (p < 0.05). Ruminal pH was lower (p < 0.05), while volatile fatty acids (VFAs), urea‐N, ammonia‐N and soluble protein‐N concentrations were higher (p < 0.05) in Tibetan than in Han sheep. As a molar proportion of total VFA, acetate decreased (p < 0.001) with an increase in dietary energy whereas propionate and butyrate increased (p < 0.05). Urinary purine derivative excretion was greater in Tibetan than in Han sheep (p < 0.01), as was microbial nitrogen production; both parameters increased with dietary energy (p < 0.01). Serum concentrations of glucose, insulin and insulin‐like growth factor‐1 increased (p < 0.05) as energy level increased, while non‐esterified fatty acids and growth hormone decreased (p < 0.05). It was concluded that Tibetan sheep were better able to cope with low‐protein, low‐energy diets and, consequently, our prediction was supported.     

Contribution of different rumen microbial groups to gas,short‐chain fatty acid and ammonium production from different diets—an approach in an in vitro fermentation system

In this study, the relative contribution of different microbial groups to ruminal metabolism was investigated for different diets. The rumen microbial cultures included whole rumen fluid, fungi + protozoa, bacteria + protozoa, protozoa and bacteria + fungi and were established by physical and chemical methods. Gas production, short‐chain fatty acid (SCFA) and ammonium production were measured at 24 hr in in vitro incubations using the Hohenheim gas test (HGT) procedure. Seven donor animal diets with different concentrate‐to‐roughage ratios (C:R: 10:90, 30:70, 50:50, 70:30, 70:30BC (BC = NaHCO₃), 90:10 and 90:10BC) and five HGT diets (C:R: 10:90, 30:70, 50:50, 70:30 and 90:10) were formulated. Incubations in the HGT were always based on inoculum from sheep diets with the respective C:R ratio. Gas and ammonium production increased (p < 0.001) as a result of a gradual increase in concentrate proportion of the diets. In general, SCFA production followed the same trend. Whole rumen fluid and bacteria + fungi produced approximately 50% higher gas volume than protozoa and fungi + protozoa fractions, whereas gas production with bacteria + protozoa was at an intermediate level. Coculture of protozoa either with bacteria or with fungi produced more ammonium. Populations without bacteria were characterized by a particularly high acetate/propionate ratio. Although an interaction between microbial group and diet was observed for several variables, no clear direction could be established. Manipulating rumen fluid by selectively suppressing specific rumen microbial groups may be a helpful tool in elucidating their role in nutrient degradation and turnover in vitro.     

The effects of early‐age thermal manipulation and daily short‐term fasting on performance and body temperatures in broiler exposed to heat stress

This study was conducted to investigate the effects of thermal manipulation at 5 days of age and short‐term fasting during the warmest part of the day on responses to prolonged heat stress of broilers. A total of 240‐day‐old Ross 308 female broiler chicks were divided into three groups: control, thermal manipulation (chicks were exposed to 36 °C for 24 h at 5 days of age) and short‐term fasting during the warmest part of the day (10.00–17.00 h). Prolonged heat stress was induced daily from 28 to 42 days by heating until the ambient temperature reached 32–35 °C between 10.00 and 17.00 h. Both thermal manipulation and short‐term fasting resulted in a decrease in rectal temperatures and haematocrit values at 35 and 41 days of age. Thermal manipulation improved body weight, feed consumption and feed conversion. However, short‐term fasting caused a reduction in body weight and a deterioration in feed conversion. Short‐term fasting lowered the percentages of carcass, whereas thermal manipulation highered breast yield. Both thermal manipulation and short‐term fasting decreased heart mass and abdominal fat.     

Amino acids profile within peripheral blood and follicular fluid based on high‐performance liquid chromatography methods may explain differences in folliculogenesis between short‐term under/over‐fed treatments during luteal phase of Hu sheep

The nutritional alteration of amino acids (AAs) profile in physiological fluid was poorly characterized in livestock. After oestrus synchronization, 24 ewes were randomly assigned to two groups based on the nutrient requirement recommended for maintenance (M): the feed‐supplemented group (S, 1.5 × M, N = 12) and feed‐restricted group (R, 0.5 × M, N = 12) on days 6–12 of their oestrous cycle, which occurred shortly before ovulation. The concentration of 30 AAs in peripheral blood (PB) and follicular fluid (FF) was quantified to calculate the PB‐to‐FF concentration gap for each AA and determine its correlation with metabolites and hormones in PB and FF. Results showed that the feed restriction enlarged the oestrous cycle length, decreased the number of follicles 2.5–3.5 mm, increased the number of follicles >3.5 mm and augmented the volume of follicles >2.5 mm. Nineteen AAs from PB were significantly different between the groups. The phosphoethanolamine (PEtN) and ration of essential AAs to nonessential AAs (EAA/NEAA) in FF significantly (p < 0.05) increased and decreased in the R group, respectively. Most AAs, except aspartate (Asp) and carnosine (Car) in the R group and alanine (aAla) in both groups, were significantly lower within FF than those within PB. The correlation of AAs with FSH and progesterone (P₄) was more significant than that of AAs with other endocrine milieu characteristics. In conclusion, our results revealed that the influence of short‐term nutritional manipulation during luteal phase on folliculogenesis might not be due to the variation of intrafollicular AAs profile but rather attribute to the peripheral blood AAs profile alteration.     

Performance,serum amino acid concentrations and expression of selected genes in pair‐fed growing pigs exposed to high ambient temperatures

Heat stress (HS) depresses pig performance mainly because of appetite reduction, although other factors involved in the cellular availability of nutrients may also contribute to that depression. An experiment was conducted with twelve pair‐fed pigs (30.3 ± 2.7 kg BW) to examine the effect of severe HS (up to 45 °C) on the expression of genes coding for two cationic amino acid (AA) transporters (b^0,+AT and CAT‐1), leptin, heat‐shock protein (Hsp‐90) and myosin in several tissues; serum concentrations (SC) of AA; and performance. There were two treatments: Comfort, pigs housed at an average temperature of 22 (±2) °C; and HS, pigs housed in a similar room with no climate control, where temperature was raised up to 45 °C. All pigs received the same wheat–soybean meal diet and had similar daily feed intake. Comfort pigs had a higher daily gain and better gain/feed ratio than HS pigs (p < 0.05). The expression of b^0,+AT in jejunum and liver, that of myosin in the Semitendinosus muscle, and leptin in adipose tissue was lower, but CAT‐1 in jejunum and liver, and Hsp‐90 in liver was higher in HS pigs. The SC of Lys and Met in HS pigs were around 55% and 20%, respectively, of that in Comfort pigs (p < 0.05). In conclusion, HS affects the expression of cationic AA transporters, myosin, Hsp‐90, leptin; the SC of Lys and Met; and the performance of pair‐fed pigs. These results suggest that HS‐related changes in gene expression affect the performance of pigs beyond the effect caused by the reduction in voluntary feed intake.     

The effects of experimentally induced fever on the estimated blood flow to and oxygen utilization by the liver and the viscera drained by the portal vein in sheep

Intrajugular injection of a purified E. coli lipopolysaccharide induced a biphasic fever in sheep after a latent period of 12 to 20 min. The changes in the blood flow from the liver and from the viscera drained by the portal vein were: (a) in the latent period, decreases in total hepatic blood flow (THF) due to decreased portal venous blood flow (PVF); (b) during the first febrile phase, increases in THF due to increased hepatic arterial blood flow and, (c) in the second febrile phase, increases in THF due to decreased PVF. Although there were large variations in the oxygen supply to the viscera drained by the portal vein and to the liver, there were relatively small or no changes in their oxygen consumption.     

Digestibility and nitrogen utilization in sheep fed enset (Ensete ventricosum) pseudostem or corm and graded levels of Desmodium intortum hay to wheat straw‐based diets

The aim of this study was to investigate the effects of different levels of Desmodium intortum (Desmodium) hay supplementation in sheep fed fixed amounts of enset pseudostem or corm and a basal diet of wheat straw on intake, digestibility and nitrogen utilization. Eighteen male sheep with a mean (± SD) live weight of 20.5 ± 1.45 kg were assigned to six treatments in a completely randomized design and fed either 108 g dry matter (DM) enset pseudostem or 165 g DM enset corm each with three levels (100, 200 and 300 g) of hay supplementation. For the pseudostem diets, there was no significant difference in total DM intake. Total crude protein (CP) intake and N retention increased with increasing levels of hay in both pseudostem and corm diets. The apparent digestibility of DM, OM, CP, acid detergent fibre and neutral detergent fibre (NDF) and microbial nitrogen supply (MN) at 100 g was lower that other levels of supplementation. For the corm diets, total DM and OM intake and MN supply increased with increasing levels of hay. The digestibility decreased (p < 0.001) with increasing levels of supplementation. The results suggest that at least 300 g (395 g/kg dietary DM) of Desmodium hay is required in pseudostem diets, whereas 200 g (337 g/kg dietary DM) may be sufficient in corm diets for efficient nutrient utilization.     

A preliminary trial of the sedation induced by intranasal administration of midazolam alone or in combination with dexmedetomidine and reversal by atipamezole for a short‐term immobilization in pigeons

ObjectiveTo assess the sedative and immobilization effect of intranasal administration (INS) of midazolam (MID) without or with INS dexmedetomidine (DXM), and some physiological changes induced by the drugs. The ability of INS atipamezole to reverse the DXM component was also assessed.Study designProspective ‘blinded’ experimental study.AnimalsIn total, 15 pigeons.MethodsPigeons were sedated by INS MID alone at a dose of 5 mg kg⁻¹ (group MID, n = 6) or in combination with INS DXM at a dose 80 μg kg⁻¹ (group MID-DXM, n = 6). Measurements were made of heart rate (HR), respiratory rate (f_R) and cloacal temperature (CT). The degree of sedation was assessed at 15 minutes prior to, immediately after, and at intervals until 100 minutes after drug administrations. Following MID-DXM, INS atipamezole (250 μg kg⁻¹) was administered and the same indices measured 5 and 10 minutes later.ResultsMID had no effect on HR and f_R, and although CT decreased, it remained within physiological range. MID-DXM caused significant falls in HR, f_R and CT that persisted until the end of sedation. Atipamezole antagonized sedation and cardiorespiratory side effects of MID-DXM within 10 minutes of application. In addition, for MID compared to MID-DXM, the lowest sedation scores [10 (7–14) and 10.5 (5–14) versus 2 (1–4) and 2 (1–5)] were achieved in the 10th and 20th minute versus the 20th and 30th minute of the sedation, respectively.Conclusions and clinical relevanceMID alone, given INS had minimal side effects on vital functions but caused inadequate immobilization of pigeons for restraint in dorsal recumbency. MID-DXM caused an effective degree of immobilization from 20 to 30 minutes after administration, at which time birds tolerated postural changes without resistance. Atipamezole antagonized both side effects and sedation, but complete recovery had not occurred within 10 minutes after its application.     

Post‐glucose load changes of plasma key metabolite and insulin concentrations during pregnancy and lactation in ewes with different susceptibility to pregnancy toxaemia

Insulin resistance during late gestation may act as a predisposing factor of ovine pregnancy toxaemia (OPT). To evaluate the insulin action on energy metabolism in ewes with different susceptibilities to OPT, intravenous glucose tolerance tests (1 mmol glucose/kg body weight) were performed in 5.6 ± 0.7 year old, slightly underfed German Blackheaded Mutton ewes [high‐risk (HR) ewes] and 2.5 year old, overnourished Finnish Landrace ewes [low‐risk (LR) ewes] during mid and late pregnancy, during early lactation and during the dry period. Plasma samples were analysed for glucose, insulin, non‐esterified fatty acids (NEFA) and β‐hydroxybutyrate (β‐HB). The glucose elimination rate and the glucose‐stimulated first‐phase insulin secretion were significantly (p < 0.05) lower in the HR, in relation to the LR group combining the data of all gestational stages. The basal rate of lipolysis was significantly increased in the HR ewes during late pregnancy, but the NEFA clearance after the glucose load was similar in both groups during all reproductive stages. Plasma β‐HB concentrations decreased only in the LR ewes after the glucose load during late pregnancy. Results indicate an insulin resistance in the HR ewes regarding the glucose utilization and the ketone body formation during late pregnancy. The insulin resistance in the HR ewes may represent one predisposing factor responsible for the susceptibility to OPT. Further scientific work is necessary to elucidate whether this insulin resistance was due to breed, age or nutritional state.     

Gastrointestinal health and function in weaned pigs: a review of feeding strategies to control post‐weaning diarrhoea without using in‐feed antimicrobial compounds

For the last several decades, antimicrobial compounds have been used to promote piglet growth at weaning through the prevention of subclinical and clinical disease. There are, however, increasing concerns in relation to the development of antibiotic‐resistant bacterial strains and the potential of these and associated resistance genes to impact on human health. As a consequence, European Union (EU) banned the use of antibiotics as growth promoters in swine and livestock production on 1 January 2006. Furthermore, minerals such as zinc (Zn) and copper (Cu) are not feasible alternatives/replacements to antibiotics because their excretion is a possible threat to the environment. Consequently, there is a need to develop feeding programs to serve as a means for controlling problems associated with the weaning transition without using antimicrobial compounds. This review, therefore, is focused on some of nutritional strategies that are known to improve structure and function of gastrointestinal tract and (or) promote post‐weaning growth with special emphasis on probiotics, prebiotics, organic acids, trace minerals and dietary protein source and level.