Assay and Use o f M a g n e s i u m Ferrite as a Reference in A b s o r p t i o n Trials w i t h Cattle 1 ,2

H. NEUMARK, A. HALEVI, S. AM|R, and S. YERUSHALMI 3 Agricultural Research Organization The Volcani Center Bet Dagan, Israel

ABSTRACT

INTRODUCTION

Magnesium ferrite, an inert ferrimagnetic material, was introduced as reference substances in absorption trials with ruminants. A method for assaying magnesium ferrite was based upon the difference between the weight of the test material on an analytic balance and that of the same material weighed below a permanent magnet. Four heifers in two pairs were in a feeding experiment lasting for 6 wk divided into two 3-wk periods. Each animal received once daily 12 g magnesium ferrite and, as control, 12 g shredded paper impregnated with 4.2 g chromium oxide. The rations were composed of chopped wheat straw and concentrates, given twice daily, with a different straw-concentrate ratio for each group. The treatments were reversed after the first experimental period. Mean recoveries of magnesium ferrite and chromic oxide in total collection of feces were 103 and 90.2%. Mean absorption coefficients of organic matter calculated by total collection and by the ratios of magnesium ferrite and chromic oxide in feces taken from the rectum were 68.1, 70.1, and 65.7%. The method is simple and accurate and may replace methods involving time consuming chemical determinations.

At the beginning of this century, substances such as chromic oxide, ferric oxide, and, later on, lignin and polyethylene glycol were introduced as reference substances (6) to measure the absorption of nutrients in feeding trials, thereby replacing the classic balance technique. In recent years, the introduction of labeled reference substances, such as yttrium-91 (5, 8), cerium-144 (1), chromium-51, and scandium-47 (9), considerably improved the methods. For reliable work with a reference substance, the latter should meet certain requirements. It should not be absorbed. It should move along the intestine at the same rate as the nutrient under study. Its inclusion should not alter the properties of the diet. And, it should be readily assayed. The chemical assay of unlabeled substances is time consuming, whereas the handling of labeled substances involves serious hazards, especially in experiments with large domestic animals. In our present work, magnesium ferrite (MgOFe203) was introduced as a reference substance in feeding experiments with four Israeli-Friesian heifers which also received chromic oxide (Cr203) as an additional reference substance for control. Magnesium ferrite, virtually insoluble in water and hydrochloric acid, was first used in human medicine as a contrast material for X-ray diagnosis by Frei et al. (4) and as reference substance for measuring the passage of food from the rumen to the abomasum in sheep (13).

Received January 13, 1975. 1Contribution from the Agricultural Research Organization, The Volcani Center, P.O.B. 6, Bet Dagan, Israel. 1974 Series, No. 277-E. 2 Supported by a grant of the U. S. Department of Agriculture (P.L. 480). 3Weizmann Institute of Science, Rehovot, Israel. 4Produced under U. S. Patent 3,592,185 (13/VII/71) and U. K. Patent 1,174,360 (15/IV/70) and obtainable from the Department of Electronics, Weizmann Institute of Science, Rehovot, Israel.

MATERIALS AND METHODS

Magnesium ferrite, 4 an inert ferrimagnetic material (spinal type ferrite), was prepared in the pilot plant of the Department of Electronics of the Weizmann Institute of Science, Rehovot, Israel. The material had a grain size of

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5 to 50 ~tm and a bulk specific weight of 4.25 to 4.45 g/cm a . It is nontoxic to animals even at 5 g MgOFe203/kg body weight, by experiments with r a t s : The ferrimagnetic property of MgOFe2Oa did not change after being heated for 24 h at 105 C or after it had been incubated for 48 h with .1 N HCI at room temperature. Preparation of Test Meals

To prevent the reference substance from separating from the nutrients during their passage through the gastrointestinal tract, magnesium ferrite was incorporated into the feedstuffs as follows. Feed was spread at the b o t t o m of the bowl of a dough mixer (15-liter capacity) in a layer several centimeters deep. This was sprayed as evenly as possible with Dow Corning Medical Adhesive, 6 a polysiloxan polymer dissolved in fluorocarbon as propellant and used in human medicine for repairing tissues. After at least 1 min, MgOFe203 was distributed evenly over the layer through a sieve, and a second spray was administered. A second layer of feed was then spread over the first, pressed down firmly, and the whole process repeated several times. By this procedure, 60 g of MgOFe203 were firmly bound to 5 kg of concentrates which were then thoroughly mixed for 10 min. The magnesium ferrite did n o t separate from the nutrient after beeing immersed in water for 24 h. The partial covering of the nutrients by the spray did not affect their digestibility in the in vitro digestion technique of Tilley and Terry (1.2).

FIG. 1. The Samarium-Cobalt magnet fixed to an aluminum rod on an aluminum base.

magnet (Fig. 1). Two U-shaped rails, fixed to the b o t t o m of the balance housing underneath the weighing pan, served as a guide for sliding the base of the magnet into place (Fig. 2). A Satorius digital analytic balance model 2472 was used in which fractional grams are measured by beam displacement shown on a projection scaIe. Since any displacement of the beam alters the distance between the sample and the fixed magnet and, therefore, also the force exterted on the magnetic material by the magnet, constant conditions for weighing had to be provided. By placing two small weighing vessels containing acid-washed sand on the weighing pan, the beam was kept in a virtually constant position, corresponding to 755 +- 1 rag, at the start of the weighing procedure.

Quantitative Assay of M g O F % 0 3

The assay is based on the difference between the weight of the test material, weighed on an analytic balance in the regular manner, and that of the same material weighed below a permanent magnet. To ensure uniform magnetic conditions, a Samarium-Cobalt magnet (field strength at face: 1000 Oersted) was fixed to an aluminum rod which was screwed onto an aluminum plate serving as the base of the

5Toxicological tests were made at the Pharmacological Institute, National Council for Research and Development, Petah Tiqwa, Israel. 6Dow Corning Corp., Medical Products Division, Midland, MI 48640.

: :2

FIG. 2. The magnet in place in the balance housing. Beneath the magnet is a pellet, resting on a support. Journal of Dairy Science Vol. 58, No. 10

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For the assay of MgOFe203, we pelleted the dried feces with the aid of a potassium bromide die (Research and Industrial Instruments Co., London, England). However, any pelleting device would be suitable. After introducing 700 to 750 mg of the test material (depending upon the resistance of the material to pressure) into the die, the appropriate rod was inserted without first inserting a small disc, as is the general practice. An exactly measured load of 10 tons was applied with the aid of a pellet press and a pellet was formed (5.2 mm height and 13.4 mm diameter), having a slight depression on one side and confined by a small rim. The pellet was dipped briefly into hot liquefied paraffin wax (congealing point 54.4 C), covering the pellet with a thin paraffin film to prevent any changes in size due to atmospheric influences. Pellets were placed for weighing in a fixed position on a support resting on the weighing pan (Fig. 2). The distance from the magnet to the pellet (surface to surface) was 10 mm. To ensure uniform conditions, the pellets always were put on the support with their rims downwards, i.e., the rims served as the base of the pellets. All determinations were in triplicates.

17'

-& o g D_ 0

15 1:5 II

g

5

5

7

9

II

15

15

Weight differences (rag)

FIG. 3. Regression of weight differences with standard error of the slope resulting from weighing graded amounts of magnesium ferrite in the regular manner and beneath the magnet, y = 1.489207 (-+ .0217) × -.083379. y = magnesium ferrite (mg) in sample, x = weight difference (rag).

Calibration of Method

Standards were prepared by mixing measured amounts of MgOFe203 with 700 mg dried ground feces collected from cows not previously treated with MgOF% O3. These samples were made into pellets and weighed as described above. The results were used to establish the calibration curve shown in Fig. 3 and the regression equation. The following example demonstrates the weighing procedure: A. Load of the pan bearing support and pellet contairfing 700 mg feces and 8.2 mg MgOFe2 03. 61,320.2 mg B. Load of the pan after adding the weighing vessel. 61,755.1 mg C. Load of the pan with magnet in place. 61,749.4 mg D. Difference in weight (B -- C).

5.7 mg

Chromic oxide was determined quantitatively according to the method of Stevenson and de Langen (11). Journal of Dairy Science Vol. 58, No. 10

Animals and Treatments

Four Israeli-Friesian heifers (Nos. 358, 360, 363, and 366), about 10 mo old, were put in metabolism cages and fitted with harnesses for separate collections of feces and urine. After the animals became accustomed to the cages and the feed, an experiment was carried out lasting 6 wk and divided into two equal periods. During the first periods, heifers 358 and 366 were fed 2 kg concentrates and .5 kg chopped wheat straw at 0800 and 1600 h (treatment A). At the same hours, heifers 360 and 363 received 1.8 kg concentrates and 1.2 kg chopped wheat straw (treatment B). After 3 wk, treatments were reversed, i.e., heifers 358 and 366 received treatment B and heifers 360 and 363, treatment A. During the entire period, all animals had free access to water and consumed all of their rations, only occasionally leaving small amounts of straw, which were weighed and taken into account. Daily during the 6 wk, at 0800 h all animals received a

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TABLE 1. Means and standard deviations (SD) of total excretion of magnesium ferrite and chromic oxide (g/day) of individual collection of four heifers according to treatment (7-day collection). Heifer

Treatment

MgOF% 03

SD 358

A B A B A B A B

360 363 366

Cr2 03 (g/day)

SD

13.04 + 1.466 12.87 + 1.278 11.89 ± 1.184 12.35-+ .883 12.41 _+1.171 12.88 +_1.328 11.59+- .798 11.91 +_ .901

gelatin capsule containing 12 g shredded paper, impregnated with 4.2 g Cr2 0 3 , as described by C o r b e t t e t a [ . (3). The capsules were administered by a ball-gun. A t the same time, all animals were given 12 g MgOFe2 O3 m i x e d with 1 kg of the c o n c e n t r a t e ration. The administration of M g O F e 2 0 3 was interrupted for 3 successive days starting at the end of the first period.

3.70 ± .358 3.87 ± .326 3.77 ± .448 3.84±.253 3.73 ± .403 4.12 ± ,340 3.46±.354 3.83 ± .449

366 in the first period on the 5th collection day at 1400 h, or f r o m No. 363 in the second period on the 4th day at 0100 h. The grab samples were oven dried (80 C), and their magnesium ferrite and chromic oxide concentrations were determined. Absorption coefficients of organic m a t t e r for individual heifers were calculated by total collection and by the magnesium ferrite and chromic oxide ratios in four pooled grab samples, each of 3 days.

Collection of Samples

F o r c o m p l e t e collection (total collection), feces of each heifer were collected daily at 0700, 1500, and 2100 h during the last 7 days of each e x p e r i m e n t a l period. Representative samples were taken and oven dried at 80 C. C o m b i n e d samples of each day were milled, and their c h r o m i c oxide and magnesium ferrite c o n c e n t r a t i o n s were determined. F r o m the 15th to the 20th day of each period, feces f r o m the r e c t u m (grab samples) were taken from the animals twice a day at 12-h intervals, but at different times each day. No grab sample could be o b t a i n e d f r o m No.

TABLE 2. Mean of magnesium ferrite and chromic oxide in feces of four individual heifers, collected during the last 7 days of the two experimental periods, as percentage of amount given.

Heifer

358

MgOF%O 3 108.0 Cr203 90.1

360

363

101.0

(%) 105.3 93.5

90.6

366

Total average

RESULTS AND DISCUSSION

As the aim of this study was to investigate the suitability of magnesium ferrite as a reference substance in absorption trials, we will n o t discuss the effect of the different compositions of the rations upon the absorption ratio. F r o m Table 1, three heifers e x c r e t e d slightly m o r e and one slightly less magnesium ferrite on the average than the actual daily intake (12 g). Mean excretion of chromic oxide in total collection was inferior to that of magnesium ferrite (Tables 2 and 3).

TABLE 3. Mean of magnesium ferrite and chromic oxide in feces collected from each of four heifers on the last 7 days of the two experimental periods and calculated according to treatment, as percentage of quantity given. Treatment

MgOFe 2 03

A B

101.9 104.2

Cr 2 03 (%)

97.8 103.0 86.8 90.2

87.3 93.2

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TABLE 4. Mean concentration (mg/g organic matter), standard deviations (SD) and coefficients of variation of magnesium ferrite and chromic oxide in feces taken twice daily from the rectums of four heifers on 6 consecutive days of each treatment. Heifer

Treatment

MgOFe 2 03

Cr~ 03 (rag)

SD 358

A B A B A** B A** B

360 363 366

C.V.*

9.88 ± 1.653 7.86 +- 1.007 10.15+- 1.145 6.61 ± .889 12.89 ± 1.516 7.12± 1.306 9.37±2.095 7.47± 1.154

(16.7) (12.8) (11.3) (13.4) (11.8) (18.3) (22.3) (15.4)

SD 3.10+-.553 2.56-+.335 2.86-+.372 2.07 + .207 3.83 +.545 2.34+.316 2.75+.693 2.37 + .451

C.V.* (17.8) (13.1) (13.0) (10.0) (14.2) (13.5) (25.2) (19.0)

*C.V. = Coefficient of variation. **Mean of 11 samples.

Coefficients of variation of c o n c e n t r a t i o n of magnesium ferrite and of chromic oxide in grab samples, arranged according to treatment, were relatively high but of the same order (Table 4). The absorption coefficients for organic m a t t e r (Table 5) calculated by the magnesium ferrite and chromic oxide ratio in grab samples were not significantly different f r o m those calculated by the totaI collection method. To the best of our knowledge, this is the first report on the use of an analytic balance used in c o m b i n a t i o n with a p e r m a n e n t magnet for the quantitative d e t e r m i n a t i o n of a ferrimagnetic substance in biological material. As the attractive force b e t w e e n a given magnet and a given a m o u n t of ferrimagnetic material is dependent upon the distance b e t w e e n them, the optimal distance has to be determined. When the distance is small, the weight differences for a given a m o u n t of magnesium ferrite b e c o m e great, but the different dispersion within the pellets will also increase the variation of the results. T o o great a distance, on the other hand,

may render the m e t h o d too insensitive. In our experiments with various types of magnets, we f o u n d that weight differences (mg) should be kept in the range of 60 to 70% of the a m o u n t of magnesium ferrite in the sample. The assays of magnesium ferrite were in triplicates, b u t the results differed only slightly (

Assay and use of magnesium ferrite as a reference in absorption trials with cattle.

Magnesium ferrite, an inert ferrimagnetic material, was introduced as reference substances in absorption trials with ruminants. A method for assaying ...
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