WO2000013589A1

WO2000013589A1 - A method of analysing consumption and digestibility in animals or humans

Info

Publication number: WO2000013589A1
Application number: PCT/DK1999/000462
Authority: WO
Inventors: Henrik Rosendahl Kristiansen
Original assignee: Mobile Nutrients Aps
Priority date: 1998-09-02
Filing date: 1999-09-02
Publication date: 2000-03-16
Also published as: EP1109493A1; AU5408299A

Abstract

The invention relates to a method of analysing consumption and digestibility in animals or humans. Animals or humans may be given a test feed which may contain at least a first marker for X-ray analysis of consumption in the alimentary canal of animals or humans. The test feed may also contain at least a second marker for chemical analysis of digestibility of the test feed. Measurement of both consumption and digestiblity can take place hereby. The measurements are made almost simultaneously, and subsequently analysis results from X-ray pictures may be compared with the chemical measurement results. The double marker technique thus ensures an optimum result which allows for the fact that the digestibility depends on the consumption.

Description

A method of analysing consumption and digestibility in animals or humans

The invention relates to a method of analysing consumption and digestibility in animals or humans, wherein animals or humans are given a test feed which contains at least one marker, wherein the marker is followed through the alimentary canal in animals or humans by X-raying, and wherein faeces from animals or humans are analysed.

It is known from JP 09217697 to use barium sulphate as a digestion marker in X-ray analyses. In connection with X- ray analysis it is generally known to use a number of different contrast agents. In connection with digestibil- ity measurements, the scientific literature describes a large number of markers which are either naturally found in the feed, or which are added to the feed in the form of chemical substances.

The object of the invention is to provide a method of analysing consumption and digestibility in animals or humans of a quantified meal.

The object is achieved by the method stated in the open- ing paragraph if it is performed such that the test feed contains at least a first marker for X-ray analysis of feed consumption, said test feed likewise containing at least a second marker for chemical analysis of the digestibility of the feed consumed.

Hereby, a measurement of both consumption and digestibility may be obtained. The measurements may be performed almost simultaneously, and subsequently analysis results from X-ray pictures may be compared with the chemical measurement results. The double marker technique thus en- sures an optimum result, which allows for the fact that the digestibility depends on the consumption.

Consumption may be analysed by X-ray, while animals or humans are placed permanently relative to an X-ray source where X-ray pictures are taken in the period after consumption of a test meal . X-ray pictures may hereby be taken automatically with limited intervention from staff. Animals or humans are stressed as little as possible, and the measurement result is improved. A large number of pictures may be taken, which may subsequently be processed on a picture processing device suitable for the purpose such that the digestion process can be seen as a time-shortened film.

Another possibility is that in the period after consumption of a test meal animals or humans are moved to an X- ray apparatus in which X-ray pictures are taken. Hereby a single X-ray device may be used for a large number of test individuals, where many small individuals are photographed simultaneously, or many larger individuals are photographed singly.

Advantageously, selected test animals may be kept indi- vidually in confined spaces where the animals are X-rayed simultaneously. This minimizes stress. If the animals are small, more individuals may be included at the same time on each film.

The X-ray pictures may advantageously be taken by a digital camera which is in direct connection with a computer in which automatic picture analysis takes place. Hereby, the entire X-ray analysis may take place with limited intervention from staff. Picture analysis inter alia allows determination of the volume of the feed content in the stomach and also the density of X-ray markers. Advantageously, a first quantification of the second marker in the test feed may be carried out. This determines the concentration of the marker prior to feeding.

After drying of a faeces sample, a second quantification of the second marker may be carried out, and then the first quantification is compared with the second quantification. The concentration of the marker through the gastrointestinal system may be determined hereby.

A microscopic amount of faeces may be used for an analysis pie, where subamounts of the analysis pie are used for various analyses. Hereby, a plurality of parameters, such as e.g. protein, fat, etc., may be measured in the same dung sample.

The second marker may consist of Cr203, where a small dried amount of feed or faeces is analysed by a combina- tion of dry oxidation in a heating furnace and a bleaching agent oxidation, where the process takes place at a high temperature in silicon oil until all chromium oxide has oxidized, before addition of HCL to remove excess of bleaching agent, and then the contents of the second marker are determined by spectrophotometry .

Hereby, the chemical analysis may be performed on a very small amount of faeces, an amount of 10 mg of dry matter being sufficient to achieve a reliable result. The method is thus very suitable for small and young animals, it being possible to measure on the individual subject. Hereby, the same animal may be used for a large number of tests, and digestion may be related to the descent of the individual . The method may thus be used for the selection of animals for breeding with optimum utilization of feed. Also, the dung amount is reduced, and livestock pollution may be reduced. A better feed conversion also improves the economy of livestock production.

The invention will be explained more fully below, and reference will be made to sketches.

Test feed is produced by comminuting human or animal feed in a kitchen blender, and then adding a consumption marker (KM) and a digestion marker (FM) . Barium sulphate (BaS04) ^is used as KM and constitutes 10-20% by weight of the feed. Chromium oxide (Cr2θ3) is used as FM and constitutes about 1% of the feed. Then water is added, and the dough is kneaded and converted into feed pellets in a mincing machine or in a commercial feed production system. The pellets are dried in a furnace or a freeze drier.

It is advantageous to have several KMs , and in certain cases glass globes may advantageously be used instead of barium sulphate. Raw materials such as fish meal and feed phosphates may be added to the feed in a varying amount and quality.

The livestock is given normal feed, and when welfare and behaviour are deemed normal, the double marker (DM) feed is used until the normal feed is out of the gastrointestinal system. Manual or machine feeding may be used.

Consumption is measured by means of X-ray technique. The animals are X-rayed so that the entire gastrointestinal system is seen on photos . Photos can be taken where the animals stand permanently under the X-ray source, or where the animal or the X-ray source is moved when measurements are being performed. A qualitative evaluation (e.g. the duration from the meal and until the feed leaves the stomach and is seen in the duodenum or the in- testine) and a quantitative determination (feed amount consumed in a given period) may be made.

Fig. 1 shows a permanent X-ray source. The test animals may be kept in separate compartments (rats), cages (poultry, mink), buildings (ostriches, pigs) or aquaria (fish). Typically 4-8 animals are used. The X-ray source is placed in the vicinity of the animals so that the source may be moved manually or by machine from animal to animal. Fig. 1 shows an X-ray source 10 arranged over an aquarium 11 which contains fish 12, and a film cassette 13 is present under the aquarium.

The test animals may be out in the open. After feeding, the animals are caught and anaesthetized. The animal is then moved to the X-ray source or conversely.

In a qualitative test of livestock feed it is of interest to know how long the feed stays in the stomach before it has a suitable consistency (combination of water absorption and beginning enzymatic digestion) , so that the stomach begins working and discharging the meal into the intestinal system. In practice, an X-ray photo is taken, showing where the highly white barium sulphate is present in the gastrointestinal system.

Prior to a quantitative determination, a standard curve is made, giving a mathematical relation between known feed amount and amount of X-ray marker. A linear regres- sion curve is made with glass globes between the feed weight of feed and the number of glass particles on a plurality of X-ray photos. With barium sulphate, the area or the intensity of barium sulphate is used on photos. The area and intensity are determined by picture analy- sis. Feed consumption may now be determined quantitatively by photographing the animals, determining the amount of marker and using the standard curve for calculating feed amount in the stomach or the entire gastrointestinal system.

A transportable X-ray device may be used, e.g. Siemens Polymobil 3 with X-ray tubes SR 100/20 is suitable. Typically, the X-ray source is placed 1 rrt from the object. The apparatus is adjusted at 50 kV and 1 mAs (milliampere seconds are the product of 40 mA and 25 mS) . Photos of the type Agfa Curix Ortho HT-Universal may be used as a standard, but a range of types and qualities will be used for different purposes. Development can be carried out in an Agfa developing machine with developers which are optimum for the individual films .

Fig. 2 shows an X-ray photo 14 which shows markings of particles in an alimentary canal. The qualitative and quantitative analysis may be made directly on a light table without any other aids than a pencil and a ruler. The accuracy and the speed of the quantitative analysis may be increased considerably by photographing photos on the light table with a video camera of great resolution. The pictures may be digitized and analysed further by picture analysis programs with suitable transformations. The optimum procedure in future is digitization at the X- ray source and transfer of picture information directly to the computer.

Fig. 3 shows an X-ray source 14 which illuminates a cage 15 containing a test animal 16. Behind the cage there is a screen 17 in a light-tight room 18, which screen may be photographed by a digital camera 19 that is connected to a picture processing device 20, which may consist of a PC. Dung samples may be collected from the individual subjects and the water content is determined by drying. The dried samples are cooled in an desiccator to avoid water absorption from atmospheric air. After drying, FM is ana- lysed as part of an analysis pie. It is of paramount importance to quantify the marker in the feed as well as in the dung, because, otherwise, the digestibility of the individual ingredients of the feed cannot be determined. It is decisive for the chemical analyses that the dung is homogenized, e.g. in a swing mill with ceramic dishes with stone globes .

Fig. 4 shows an analysis pie. Only a very small dung amount can be collected from small animals like chicken, rabbits and fish. The rat is also an important example, because it may be used as a model when testing livestock feed, in particular for pigs. Analysis pie means that a small dung amount of 100-200 milligrams constitutes the entire pie, and the less dung used for the individual analyses, the more information gained on nutrition and digestion. In principle, the dung from a large number of individuals may be grouped, but in the concept described the dung is kept separate as far as possible to obtain the maximum amount of information from each individual, which is important with a view to distinguishing between the importance of heredity and environment.

A possible method is described below for the determination of the concentration of a digestion marker, where fig. 5 shows a flow chart of a possible method.

Oxidation with bleaching agent may take place in a fume cupboard, where the green chromium oxide is converted into orange dichromate . a: Duran borosilicate test tubes (75 mm height, 12 mm diameter) are heated over night in a furnace at 550 °C to remove particles and to be certain that the tubes do not break when the dry matter is to be burned. A new design has been created for the simultaneous burning of 104 tubes .

b: The tubes are weighed on an analysis weight with four decimals (4.5678 g) .

c: About 10 milligrams of dung from each individual are diluted with silicon gel (colloidal solution of silicic acid which is dried after gel formation) . Dilution at least with a factor 2 (10 mg of gel per 10 mg of sample) is necessary to avoid the situation that KM (BaSθ4) impedes the oxidation of FM (Cr2θ3). FM in dung is frequently so high that dilution up to 32 times is possible without any adverse effect on the analysis accuracy. The dilution is very important, and without this element it would not be possible to determine chromium oxide. One cannot rule out the possibility that other diluents will be tested and used.

c: About 10 mg of diluted dung (0.0100 g) are weighed on an analysis weight directly in the test tubes.

d: The samples are heated like in point a, which causes the organic substance to disappear.

e: After heating, ashes and markers (KM and FM) are left in the test tubes . The weight of tubes with contents is determined on the analysis weight.

f: The tubes are moved from the heating racks to the oxi- dation racks, which are adjusted to a water bath with silicon oil. 1 ml of the oxidation agent, "bleaching agent" sodium hypochlorite (NaOCl in 6% solution) is added to each tube with an automatic pipette. Also three pumice stones are added to each tube to avoid bumping.

g: The rack is lowered into a Grant thermostat-controlled water bath (capacity 38 litres) with Dow Corning 200/100 cS silicon oil that flows around. The heating member was turned on one hour before so that the temperature is 125 °C. Racks of stainless steel have been designed to allow oxidation of about 350 samples at a time in a totally uniform environment (heat) . A lid is applied to avoid accidents with the hot oil and reduce heat losses.

h: When most of the liquid has evaporated, 1 ml is added again without removing the rack. When it has evaporated, the tubes are moved to a rack at the side of the water bath.

i: 1 ml of oxidation agent is added again, and then one tube at a time is shaken for 10-20 seconds in a hand mixer (so-called Vortex) adjusted to 1000 revolutions per second. Then the tubes are moved to the water bath. When most of the oxidation agent has evaporated, the last ml is finally added (a total of 4 ml per tube). When most of the oxidation agent has evaporated, about 4 hours have elapsed. Now the tubes are moved again to a rack at the side of the water bath. This treatment will be optimized so that the number of treatments with the oxidation agent as well as the duration of the entire process is fine- tuned and stopped when all chromium oxide has oxidized.

Removal of bleaching agent

j: Now excess of sodium hypochlorite is to be removed by treatment with hydrochloric acid (HC1), because the bleaching agent absorbs light at the same wavelength as the substance (dichromate) which is to be analysed. This is done by adding 1 ml of 0.5M HC1 to each test tube. There is a clear development of free chlorine. When this is over, the tubes are again placed in the water bath. The rest of the procedure is as described for the oxidation and is stopped when 4 ml have been added. The process of removing HCl will be optimized by manipulating the strength of the acid (the molarity, M) and volume added.

Removal of liquid phase

The test tubes remain in the oil bath until all the liquid has evaporated. The reason is that an accurate liquid volume must be used in the subsequent calculations . When the precipitate appears to be dry, and no liquid can be recognized, the work in the oil bath is completed.

Ready making of liquid phase with dichromate

Before the final measurement, exactly two ml of distilled water are added to each test tube. The tubes are manually mixed for 20 seconds, and any salt residues stuck on the tube wall are loosened and moved down into the tube by a plastics spatula. Then the test tubes are centrifuged for 10 minutes at a rate of 5000 revolutions per minute. Then salt particles precipitate, and there is a clear separation between precipitate and the two ml of liquid phase.

Spectrophotometry

Dichromate is analysed by spectrophotometry since the substance has an absorption maximum at 440 nanometers (nm) . A reference spectrophotometer with distilled water is used (absorption is zero) as a reference. First, a di- lution series of the orange potassium dichromate is measured in the range 0.1 to 1 μmole per ml. Then the same dilution series is measured as has been subjected to the described chemical analysis procedure for chromium oxide. This is necessary, because the saturated salt solution in the test tubes reduces the absorption. The slope of the curve where absorption of potassium dichromate (treated with sodium hypochlorite and hydrochloric acid) is plotted against the known concentration (0.1 to 1 μmole per ml) is decisive for the calculations.

Then the samples with FM are measured. The measured values of light absorption are converted into values of chromium oxide on the basis of the formula shown, where

Cr2θ3 is determined as mg per gram of dried dung - ABS440 i^{s tne} measured absorption of each test tube

Slope is the inclination of the standard curve for potassium dichromate

10 shows that the unit μmole is involved The mole weight of chromium oxide is 151.99 gram per mole

The sample volume is 2 millilitres

1000 mg/g are used for conversion from gram into mg per mole - m shows that division by the exact dung weight of about 0.01 gram is to be performed.

If the sample is diluted with silicon gel, multiplication must moreover be performed by a dilution factor which is at least a factor 2 and max. 32.

Formula for calculation

Cr₂0₃(mg/g)

^A^ βl 0^"6 (rnol/ml) 0151.99(g/mol)

Q2ml Ql000(mg/g) Qm^~l(g) Further use of data

When the amount of marker is known in feed and dung, and the rest of the analysis pie (protein, fat, carbohydrate, etc.) has also been determined, accurate coefficients of digestion may be calculated for the individual ingredients in the feed and for the feed as a whole (the dry matter) .

Although the procedure has been designed for measuring consumption and digestion at the same time, there will be either/or cases where just the one parameter is meas- ured.

Fig. 5

1. Livestock consume a quantified test meal with admixed chromium oxide marker, and then a representative dung sample is taken

2. Dung is dried in a vacuum furnace and homogenized in a swing mill so that it is turned into a greenish powder

3. Dung is diluted 2-32 times with silicon gel by means of analytical weight and is homogenized again

4. The diluted dung amount constitutes an analysis pie which is to give as much information on the feed as possible

5. For marker analysis in triplicate about 3x10 mg of dry matter are taken and weighed on analysis weight in three Duran test tubes

6. The organic content is removed in a heating furnace at 550 degrees, leaving ashes and chromium oxide

7. The ash samples are admixed with 1 ml of bleaching agent (sodium hypochlorite 6-15%) and placed in a water bath with silicon oil at 125 degrees

8. The treatment with bleaching agent continues until all chromium oxide has been converted (oxidized) into dichromate

9. Typically, 4xlml of bleaching agent are added, and the samples are manually mixed (vortex) halfway to loosen salt residues on the tube 10. When the oxidization has been completed, the tubes are removed from the hot oil. Hydrochloric acid is added (chlorine gas is formed) until excess of bleaching agent is removed

11. Typically, 1-4 ml are added, one ml at a time

12. The samples are mixed manually and centrifuged to precipitate particles

13. Absorption of the yellowish liquid (dichromate) is measured by spectrophotometry with microcuvettes at the wavelength 440 nm

14. The content of chromium oxide in the dung sample is determined on the basis of a dichromate standard curve

15. Digestibility of the feed is determined by comparing the amount of chromium oxide in feed and dung. The feed is analysed in the same manner, apart from the dilution

Claims

PATENT CLAIMS

1. A method of analysing consumption and digestibility in animals or humans, wherein animals or humans are given a test feed which contains at least one marker, wherein the marker is followed through the gastrointestinal system in animals or humans by X-raying, and wherein faeces from animals or humans are analysed, characterized in that the test feed contains at least a first marker for X-ray analysis of consumption in the alimentary canal of animals or humans, and that the test feed also contains at least a second marker for chemical analysis of digestibility of the test feed.

2. A method according to claim 1, characterized in that consumption is analysed by X-ray while animals or humans are placed permanently relative to an X-ray source, where X-ray pictures are taken in the period after consumption of a test meal.

3. A method according to claim 1, characterized in that animals or humans are moved at time intervals to an X-ray apparatus where X-ray pictures are taken.

4. A method according to claim 1, characterized in that several animals are kept in confined spaces where several animals are X-rayed simultaneously.

5. A method according to one of claims 1-4, character- ized in that the X-ray pictures are taken by a digital camera which is in direct connection with a computer in which automatic picture analysis is performed.

6. A method according to claim 1, characterized in that a first quantification of the second marker in the test feed is carried out.

7. A method according to claims 1 and 6, characterized in that a second quantification of the second marker is carried out after drying of a faeces sample, following which the first quantification is compared with the second quantification.

8. A method according to claim 7, characterized in that a microscopic faeces amount is used for an analysis pie where subamounts of the analysis pie is used for various analyses .

9. A method according to claims 6-8, characterized in that the second marker consists of Cr203, where a small amount of feed or faeces is dried followed by a dry oxidation in a heating furnace and a bleaching agent oxidation, where the process takes place at a high temperature in silicon oil until all chromium oxide has oxidized, before addition of HCL to remove excess of bleaching agent, following which the contents of the second marker are determined by spectrophotometry.