. Technical Note: Mineral Deposits on Dacron Bags During Ruminal Incubation' Jaap van Milgen, Marsha L. Roach, Larry L. Berger2, Michael R. Murphy, and Duane M. Moore3
Department of Animal Sciences, University of Illinois, Urbana 61801
dislodged crystals from plant material. Contamination seemed to occur exponentially within the range of observations (0 to 42 d). Contamination also occurred in steers fed orchardgrass, although to a lesser extent than in steers fed alfalfa hay. was less than .04 per bag The DM (average bag weight was 1.2 gl during the first 10 d of incubation. However, correction for contamina tion might be required for studies involving longterm incubation O r mineral digestion.
Key Words: Minerals, Contamination, Calcium, Phosphates, In Situ Digestibility
J. Anim. Sci. 1992. 70:2551-2555
Introduction In situ ruminal digestion studies routinely are performed to evaluate the nutritive value of feedstuffs. Introduced in the late 1930s (Quin et al., 19381, the in situ technique has been extensively reviewed (Nocek, 1988). Factors influencing the results are known to include bag porosity, sample weight:bag surface area, and substrate particle size. Microbial attachment to substrate is thought to be a requirement for some digestive enzymes, serving as a potential source of contamination. Olubobokun et al. (1990) indicated that the extent of microbial contamination could be as high as 95% for CP, and 20% for DM. Without correcting for this contamination, the substrate that remains
'The authors wish to acknowledge the Center for Electron Microscopy at the Univ. of Illinois a t Urbana-Champaign for support provided to this project. 2To whom correspondence should be addressed: 1207 West Gregory Drive, Urbana, IL 61801. 3X-Ray Diffraction Laboratory, Illinois State Geological Survey. Received November 7, 1991. Accepted March 19, 1992.
easily could be overestimated. Recently, van Milgen et a1. (1992) found extensive mineral contamination of Dacron bags after 42 d of ruminal incubation in steers fed alfalfa hay; however, little mineral contamination was found on bags incubated in the rumen of steers fed a concentrate diet. The purpose of the present study was to further characterize this contamination and to determine the rate at which it occurred.
Materials and Methods A detailed description of the protocol of this experiment was given by van Milgen et al. (1992). Briefly, five feedstuffs (alfalfa, corn stover, orchardgrass, rice straw, and soybean residue) were treated with either water or NaOH (5%; wt/wt) and H202(2%; wt/wtl and dried a t 57°C. Approximately 4 g of substrate was weighed into Dacron bags (7 cm x 13 cm, pore size 50 pn, average bag weight 1.2 g) and incubated for 4 or 42 d in the rumen of two cannulated steers that were fed once per day either mature alfalfa hay (ad libitum) or a concentrate diet (90% of ad libitum). Details of diet composition and nutrient intakes were described by van Milgen et al. (1992).Water was available ad libitum. The steers were housed in a n open-front
2551
Downloaded from https://academic.oup.com/jas/article-abstract/70/8/2551/4705027 by 04860000 user on 23 January 2019
ABSTRACT: Extensive DM contamination was found on Dacron bags that were incubated for Prolonged Periods of time in the rumen of steers fed hay* The ash content Of the contaminant was and most Of it was acidsoluble. X-ray analysis indicated the presence of hydroxylapatite and synthetic calcium magnesium phosphate or whitlockite. The contaminant appeared as a smooth coating on the Dacron fiber, was a gradual suggesting that process rather than the result of entrapment of
2552
VAN MILGEN ET AL.
Table 1. Chemical composition of contamination deposited on Dacron bagsa and isolated contaminant after 42 days of incubation in the rumen of steers fed alfalfa ~
Item
Deposited contaminant %
61.3 6.4 2.1 49.6 17.3 42.5 8.1
of DM 48.1 5.9 2.6 30.4 23.1 19.5 13.0
*Dacron bags contained . 5 % ash, 99.4% NDF, and 99.2% ADF.
barn, bedded with straw, and had approximately 800 m2 of floor space per animal. After incubation, bags were rinsed with cold tap water and frozen. Following completion of the experiment, bags were thawed, rinsed in a two-cycle washing machine, and dried at 57°C. Replicate bags containing a glass marble were incubated as controls. As reported by van Milgen et al. (19921, bags incubated for 42 d in the rumen of steers fed alfalfa were rigid and dark brown in color; control bags increased by approximately 2.2 g of DM in weight. Regular washing procedures only partially removed the contamination. The contamination occurred both in the bag (as a brown powder, further referred to as isolated contaminant) as well as on the bag (referred to as deposited contaminant). To further examine the contamination, bags containing a glass marble were incubated for 42 d in the rumen of steers fed alfalfa. Bags were washed and dried as described above. Isolated contaminant from five bags was pulverized using a mortar and pestle. The DM, OM, Kjeldahl nitrogen, NDF, and ADF contents (Goering and Van Soest, 1970; AOAC, 1984) were determined both on isolated and deposited contaminants. Elemental composition of the minerals (metallic elements atomic number 11 and above) was determined using energy dispersive x-ray analysis (Tracor-Northern 2000) and scanning electron microscopy (ISI-DS 130). Ashed samples were mounted on 13-mm aluminum scanning electron microscopy (SEMI stubs, which were covered with a carbon planchet. The mounted specimens, then, were coated with carbon (Model KDTG-3P, Kenny Vacuum, Boston, MA). Stubs were loaded into the microscope and the stage was tilted 45" to reduce system peaks. The acquire time was 133 s and the accelerating voltage was 15 kV. Data were plotted on a Tracor-Northern Spectral Plot, and elemental composition was calculated. Samples for SEM were prepared as described above. Mounted speci-
Results and Discussion Van Milgen et al. (1992) reported that the indigestible fraction of various substrates (expressed as the residue remaining after 42 d of incubation) was lower in steers fed alfalfa hay than in steers fed a concentrate diet. These workers also found that Dacron bags could be contaminated. The correlation between residue remaining after 4 and 42 d of ruminal incubation is shown in Figure 1. Theoretically, all observations should be below the line y = x, because observations above that line indicate a DM weight increase between 4 and 42 d of incubation. The DM residue remaining for concentrate-fed steers was always lower and for forage-fed steers always higher at 42 d than at 4 d, indicating that diet played a n important role in the extent of contamination. In an effort to minimize the effects of mineral contamination, van Milgen et al. (1992) expressed their data based on ADF. However, acid detergent only partially removed the contaminant. Because little contamination was apparent on bags incubated in concentrate-fed steers, the difference in substrate remaining after 42 d of incubation between bags incubated in concentrate and alfalfa-fed steers will be even greater than in the data reported by van Milgen et al. (19921. Chemical composition of isolated and deposited contaminant is given in Table 1. In both instances, ash content of the contaminant was extremely high. Because most of the ash was acid-soluble, it is unlikely that sand became entrapped in the Dacron mesh. Isolated contaminant was higher in OM, NDF-OM, and ADF-OM contents, suggesting that small feed particles may have entered the bags and served as sites for mineral deposition. Mineral deposits seemed to fill in gaps between Dacron fibers (Figures 2 and 31 and, in some cases, build up dense deposits. Minerals not only were deposited as individual crystals (Figure 3) but also covered the Dacron fibers, because imprints of the
Downloaded from https://academic.oup.com/jas/article-abstract/70/8/2551/4705027 by 04860000 user on 23 January 2019
Ash Acid insoluble ash Nitrogen NDF NDF-OM ADF ADF-OM
Isolated contaminant
mens of contaminated Dacron bags or ashed bags were coated with 120 nm of gold palladium. Micrographs were taken on a n Amray 1000 or ISIDS 130 SEM both at low (100~)and high ( 6 , 0 0 0 ~ ) magnification. Mineralogy of samples was determined using a n automated Scintag theta-theta xray diffractometer (Sunnyvale, CAI, using CuKa radiation. To study whether mineral contamination occurred specifically in steers fed alfalfa and to determine the rate at which this occurred, two steers were given a d libitum access to either alfalfa hay or orchardgrass hay. Four replicate bags containing a glass marble were ruminally incubated for 0, I, 2, 3, 4, 5, 6, 7, 14, 21, 28, 35, and 42 d, after which bags were washed as described before.
MINERAL DEPOSITS ON DACRON BAGS 1.00
.60
c e,
.40
* c
2553
I A
1 1
rrJ
.20
~
.oo
.20
.40
.GO
.80
1.oo
Residue after 4 d (g/g incubated)
Figure 1. Effect of ruminal incubation time on dry matter residue remaining. 0,Jersey fed concentrate; 0 , Simmental fed concentrate; A, Jersey fed forage; A, Simmental fed forage.
fiber mesh remained visible after ashing (Figures 4 and 5). Energy-dispersive x-ray analysis of the crystals indicated that Ca was the most abundant element (79.6%), followed by P (19.2%), Si (1.5%), and K (.6O/o). Ward et al. (1979) identified Ca, Si, and K as the major elements of crystals appearing in feces of cows fed alfalfa. Most of these crystals were either calcium oxalate, potassium oxalate, or a combination of these. X-ray diffraction of isolated contaminant indicated the presence of quartz. In addition, two very broad peaks were found, centered at Bragg angles of approximately 32'28 and 26"28. Given a single compound, the breadth of a peak at half the peak height above background is inversely related to
Figure 2. Scanning electron micrograph of a Dacron bag before ruminal incubation, 81x.
Figure 3. Scanning electron micrograph of a Dacron bag after 42 d of ruminal incubation in alfalfa-fed steers, 81x.
the size of the diffracting domain (Scherrer equation). The size of diffracting domains often is a function of the density of defects in the crystal structure. However, broad peaks also can occur if more than one compound is diffracting x-rays. In an effort to stimulate the material to grow into larger crystals of the same material, or to change into compounds that could be indicative of the original mineralogy, samples were ashed at 450' C in ambient atmosphere. The two broad peaks resolved into discrete, narrow peaks. Other than quartz, two phosphate compounds could account for all but three small, discrete peaks in this tracing (i.e., hydroxylapatite [Ca5(P04)3(0H)Iand synthetic calcium magnesium phosphate [Ca7Mg2P60241 or whitlockite [(Ca,Mg13(P04)211.
Figure 4. Scanning electron micrograph of an ashed Dacron bag after 42 d of ruminal incubation in alfalfsfed steers using magnifications of 8 1 ~ .
Downloaded from https://academic.oup.com/jas/article-abstract/70/8/2551/4705027 by 04860000 user on 23 January 2019
J
.00
2554
VAN MILGEN ET AL. lor-----
i 0
10
----
I
20
30
Tiiiie
40
50
(tl)
Figure 5. Scanning electron micrograph of an ashed Dacron bag after 42 d of ruminal incubation in alfalfafed steers using magnifications of 2,100~.
Figure 6 . Effect of diet on dry matter (DM)contamination of Dacron bags during ruminal incubation. 0 , alfalfa; 0, orchardgrass.
Some small peaks and shoulders of larger peaks remained unaccounted for, indicating the presence of small quantities of other compounds. Crystals appearing on Dacron bags either originate from intact crystals that dislodged from plant material or from solubilized mineral that gradually deposited on the bag. Ward et al. (1979)found that fecal calcium oxalate crystals could be 30 p x 30 p in size. Although some size reduction might occur postruminally, the size of the crystals deposited on the bags seemed to be much larger ( > 100 p; Figure 3). Also, the smooth appearance of crystals on the Dacron (Figure 51 supports the hypothesis that minerals deposited gradually on the bags, rather than representing dislodged crystals of plant origin. Mineral deposits appearing in the gastrointestinal tract are not uncommon in other animals. Blue (1979) reported the presence of large mineral concretions (enteroliths) in the proximal small colon of horses. The enteroliths (up to 20 cm in diameter) were primarily composed of ammonium calcium phosphate with a central nidus of foreign material (metal fragments or small stones). Blue (1979) also reported a case of obstruction of the small colon in a mare caused by mineral deposition on synthetic cloth. The rate of contaminant accumulation is illustrated in Figure 6. Because the SD of replicate observations seemed to be proportional to the mean, data were logarithmically transformed. A linear model was fitted to the logarithmically transformed data using the GLM procedure of SAS (1988). This implies that contamination occurs exponentially when data are expressed on the original scale: contamination = Aoek time, where A0 is a scaling parameter indicating initial con-
tamination (grams), and k is the fractional rate of Contamination (day-'). Theoretically, this model is incorrect because it does not intersect the origin. Moreover, it is unlikely that mineral contamination would proceed exponentially beyond the range of observations. Fractional rate of contamination was .160 and .127 d-l, and A. ,011 and ,010 g, for bags incubated in steers fed alfalfa and orchardgrass, respectively. Thus, contamination does not occur exclusively in steers fed alfalfa hay, but occurs for other roughages as well. The ash and AIA contents of the contaminant were similar for steers fed orchardgrass and alfalfa. Little contamination occurred during the first 10 d of incubation (Figure 6). However, extensive contamination of Dacron bags can occur during long-term incubation, and failure to correct for this will overestimate the substrate remaining.
Conclusions Mineral contamination can be extensive during long-term ruminal incubation studies. However, most studies are relatively short and no significant mineral contamination is anticipated within the first 7 d of ruminal incubation. Calcium phosphates seemed to be the major source of mineral contamination in steers fed alfalfa.
Implications Extensive mineral contamination can occur on Dacron bags that are incubated in the rumen of steers fed forage. Although little dry matter contamination occurred during the first 10 d of
Downloaded from https://academic.oup.com/jas/article-abstract/70/8/2551/4705027 by 04860000 user on 23 January 2019
,001
__
MINERAL DEPOSITS ON DACRON BAGS
incubation, the high mineral content (especially calcium) of the contaminant could seriously affect the outcome of studies focusing on mineral digestion.
Literature Cited
protein and energy digestibility: A review. J. Dairy Sci. 71: 2051.
Olubobukun, J A., W. M. Craig, and K. R. Pond. 1990. Effects of mastication and microbial contamination on ruminal in situ forage disappearance. J. Anim. Sci. 68:3371. SAS. 1988. SAS/STAT@ User’s Guide (Release 6.031. SAS Inst. Inc., Cary, NC. Quin, J. I., J. G. Van Der Wath, and S. Myburgh. 1938. Studies on the alimentary tract of Merino sheep in South Africa. IV. Description of experimental technique. Onderstepoort J. Vet. Sci. Anim. Ind. 11:341. van Milgen, J., L. L. Berger, and M. R. Murphy. 1992. Fractionation of substrate as a n intrinsic characteristic of feedstuffs fed to ruminants. J. Dairy Sci. 75:124. Ward, G., L. H. Harbers, and J. J. Blaha. 1979. Calcium-containing crystals in alfalfa: Their fate in cattle. J. Dairy Sci. 62: 715.
Downloaded from https://academic.oup.com/jas/article-abstract/70/8/2551/4705027 by 04860000 user on 23 January 2019
AOAC. 1984. Official Methods of Analysis (14th Ed.). Association of Official Analytical Chemists, Arlington, VA. Blue, M. G. 1979. Enteroliths in horses-a retrospective study of 30 cases. Equine Vet. J. 11:76. Goering, H. K., and P. J. Van Soest. 1970. Forage fiber analyses (apparatus, reagents, procedures, and some applications). Agric. Handbook 379. ARS, USDA, Washington, DC. Nocek, J. E. 1988. In situ and other methods to estimate ruminal
2555
Department of Animal Sciences, University of Illinois, Urbana 61801
dislodged crystals from plant material. Contamination seemed to occur exponentially within the range of observations (0 to 42 d). Contamination also occurred in steers fed orchardgrass, although to a lesser extent than in steers fed alfalfa hay. was less than .04 per bag The DM (average bag weight was 1.2 gl during the first 10 d of incubation. However, correction for contamina tion might be required for studies involving longterm incubation O r mineral digestion.
Key Words: Minerals, Contamination, Calcium, Phosphates, In Situ Digestibility
J. Anim. Sci. 1992. 70:2551-2555
Introduction In situ ruminal digestion studies routinely are performed to evaluate the nutritive value of feedstuffs. Introduced in the late 1930s (Quin et al., 19381, the in situ technique has been extensively reviewed (Nocek, 1988). Factors influencing the results are known to include bag porosity, sample weight:bag surface area, and substrate particle size. Microbial attachment to substrate is thought to be a requirement for some digestive enzymes, serving as a potential source of contamination. Olubobokun et al. (1990) indicated that the extent of microbial contamination could be as high as 95% for CP, and 20% for DM. Without correcting for this contamination, the substrate that remains
'The authors wish to acknowledge the Center for Electron Microscopy at the Univ. of Illinois a t Urbana-Champaign for support provided to this project. 2To whom correspondence should be addressed: 1207 West Gregory Drive, Urbana, IL 61801. 3X-Ray Diffraction Laboratory, Illinois State Geological Survey. Received November 7, 1991. Accepted March 19, 1992.
easily could be overestimated. Recently, van Milgen et a1. (1992) found extensive mineral contamination of Dacron bags after 42 d of ruminal incubation in steers fed alfalfa hay; however, little mineral contamination was found on bags incubated in the rumen of steers fed a concentrate diet. The purpose of the present study was to further characterize this contamination and to determine the rate at which it occurred.
Materials and Methods A detailed description of the protocol of this experiment was given by van Milgen et al. (1992). Briefly, five feedstuffs (alfalfa, corn stover, orchardgrass, rice straw, and soybean residue) were treated with either water or NaOH (5%; wt/wt) and H202(2%; wt/wtl and dried a t 57°C. Approximately 4 g of substrate was weighed into Dacron bags (7 cm x 13 cm, pore size 50 pn, average bag weight 1.2 g) and incubated for 4 or 42 d in the rumen of two cannulated steers that were fed once per day either mature alfalfa hay (ad libitum) or a concentrate diet (90% of ad libitum). Details of diet composition and nutrient intakes were described by van Milgen et al. (1992).Water was available ad libitum. The steers were housed in a n open-front
2551
Downloaded from https://academic.oup.com/jas/article-abstract/70/8/2551/4705027 by 04860000 user on 23 January 2019
ABSTRACT: Extensive DM contamination was found on Dacron bags that were incubated for Prolonged Periods of time in the rumen of steers fed hay* The ash content Of the contaminant was and most Of it was acidsoluble. X-ray analysis indicated the presence of hydroxylapatite and synthetic calcium magnesium phosphate or whitlockite. The contaminant appeared as a smooth coating on the Dacron fiber, was a gradual suggesting that process rather than the result of entrapment of
2552
VAN MILGEN ET AL.
Table 1. Chemical composition of contamination deposited on Dacron bagsa and isolated contaminant after 42 days of incubation in the rumen of steers fed alfalfa ~
Item
Deposited contaminant %
61.3 6.4 2.1 49.6 17.3 42.5 8.1
of DM 48.1 5.9 2.6 30.4 23.1 19.5 13.0
*Dacron bags contained . 5 % ash, 99.4% NDF, and 99.2% ADF.
barn, bedded with straw, and had approximately 800 m2 of floor space per animal. After incubation, bags were rinsed with cold tap water and frozen. Following completion of the experiment, bags were thawed, rinsed in a two-cycle washing machine, and dried at 57°C. Replicate bags containing a glass marble were incubated as controls. As reported by van Milgen et al. (19921, bags incubated for 42 d in the rumen of steers fed alfalfa were rigid and dark brown in color; control bags increased by approximately 2.2 g of DM in weight. Regular washing procedures only partially removed the contamination. The contamination occurred both in the bag (as a brown powder, further referred to as isolated contaminant) as well as on the bag (referred to as deposited contaminant). To further examine the contamination, bags containing a glass marble were incubated for 42 d in the rumen of steers fed alfalfa. Bags were washed and dried as described above. Isolated contaminant from five bags was pulverized using a mortar and pestle. The DM, OM, Kjeldahl nitrogen, NDF, and ADF contents (Goering and Van Soest, 1970; AOAC, 1984) were determined both on isolated and deposited contaminants. Elemental composition of the minerals (metallic elements atomic number 11 and above) was determined using energy dispersive x-ray analysis (Tracor-Northern 2000) and scanning electron microscopy (ISI-DS 130). Ashed samples were mounted on 13-mm aluminum scanning electron microscopy (SEMI stubs, which were covered with a carbon planchet. The mounted specimens, then, were coated with carbon (Model KDTG-3P, Kenny Vacuum, Boston, MA). Stubs were loaded into the microscope and the stage was tilted 45" to reduce system peaks. The acquire time was 133 s and the accelerating voltage was 15 kV. Data were plotted on a Tracor-Northern Spectral Plot, and elemental composition was calculated. Samples for SEM were prepared as described above. Mounted speci-
Results and Discussion Van Milgen et al. (1992) reported that the indigestible fraction of various substrates (expressed as the residue remaining after 42 d of incubation) was lower in steers fed alfalfa hay than in steers fed a concentrate diet. These workers also found that Dacron bags could be contaminated. The correlation between residue remaining after 4 and 42 d of ruminal incubation is shown in Figure 1. Theoretically, all observations should be below the line y = x, because observations above that line indicate a DM weight increase between 4 and 42 d of incubation. The DM residue remaining for concentrate-fed steers was always lower and for forage-fed steers always higher at 42 d than at 4 d, indicating that diet played a n important role in the extent of contamination. In an effort to minimize the effects of mineral contamination, van Milgen et al. (1992) expressed their data based on ADF. However, acid detergent only partially removed the contaminant. Because little contamination was apparent on bags incubated in concentrate-fed steers, the difference in substrate remaining after 42 d of incubation between bags incubated in concentrate and alfalfa-fed steers will be even greater than in the data reported by van Milgen et al. (19921. Chemical composition of isolated and deposited contaminant is given in Table 1. In both instances, ash content of the contaminant was extremely high. Because most of the ash was acid-soluble, it is unlikely that sand became entrapped in the Dacron mesh. Isolated contaminant was higher in OM, NDF-OM, and ADF-OM contents, suggesting that small feed particles may have entered the bags and served as sites for mineral deposition. Mineral deposits seemed to fill in gaps between Dacron fibers (Figures 2 and 31 and, in some cases, build up dense deposits. Minerals not only were deposited as individual crystals (Figure 3) but also covered the Dacron fibers, because imprints of the
Downloaded from https://academic.oup.com/jas/article-abstract/70/8/2551/4705027 by 04860000 user on 23 January 2019
Ash Acid insoluble ash Nitrogen NDF NDF-OM ADF ADF-OM
Isolated contaminant
mens of contaminated Dacron bags or ashed bags were coated with 120 nm of gold palladium. Micrographs were taken on a n Amray 1000 or ISIDS 130 SEM both at low (100~)and high ( 6 , 0 0 0 ~ ) magnification. Mineralogy of samples was determined using a n automated Scintag theta-theta xray diffractometer (Sunnyvale, CAI, using CuKa radiation. To study whether mineral contamination occurred specifically in steers fed alfalfa and to determine the rate at which this occurred, two steers were given a d libitum access to either alfalfa hay or orchardgrass hay. Four replicate bags containing a glass marble were ruminally incubated for 0, I, 2, 3, 4, 5, 6, 7, 14, 21, 28, 35, and 42 d, after which bags were washed as described before.
MINERAL DEPOSITS ON DACRON BAGS 1.00
.60
c e,
.40
* c
2553
I A
1 1
rrJ
.20
~
.oo
.20
.40
.GO
.80
1.oo
Residue after 4 d (g/g incubated)
Figure 1. Effect of ruminal incubation time on dry matter residue remaining. 0,Jersey fed concentrate; 0 , Simmental fed concentrate; A, Jersey fed forage; A, Simmental fed forage.
fiber mesh remained visible after ashing (Figures 4 and 5). Energy-dispersive x-ray analysis of the crystals indicated that Ca was the most abundant element (79.6%), followed by P (19.2%), Si (1.5%), and K (.6O/o). Ward et al. (1979) identified Ca, Si, and K as the major elements of crystals appearing in feces of cows fed alfalfa. Most of these crystals were either calcium oxalate, potassium oxalate, or a combination of these. X-ray diffraction of isolated contaminant indicated the presence of quartz. In addition, two very broad peaks were found, centered at Bragg angles of approximately 32'28 and 26"28. Given a single compound, the breadth of a peak at half the peak height above background is inversely related to
Figure 2. Scanning electron micrograph of a Dacron bag before ruminal incubation, 81x.
Figure 3. Scanning electron micrograph of a Dacron bag after 42 d of ruminal incubation in alfalfa-fed steers, 81x.
the size of the diffracting domain (Scherrer equation). The size of diffracting domains often is a function of the density of defects in the crystal structure. However, broad peaks also can occur if more than one compound is diffracting x-rays. In an effort to stimulate the material to grow into larger crystals of the same material, or to change into compounds that could be indicative of the original mineralogy, samples were ashed at 450' C in ambient atmosphere. The two broad peaks resolved into discrete, narrow peaks. Other than quartz, two phosphate compounds could account for all but three small, discrete peaks in this tracing (i.e., hydroxylapatite [Ca5(P04)3(0H)Iand synthetic calcium magnesium phosphate [Ca7Mg2P60241 or whitlockite [(Ca,Mg13(P04)211.
Figure 4. Scanning electron micrograph of an ashed Dacron bag after 42 d of ruminal incubation in alfalfsfed steers using magnifications of 8 1 ~ .
Downloaded from https://academic.oup.com/jas/article-abstract/70/8/2551/4705027 by 04860000 user on 23 January 2019
J
.00
2554
VAN MILGEN ET AL. lor-----
i 0
10
----
I
20
30
Tiiiie
40
50
(tl)
Figure 5. Scanning electron micrograph of an ashed Dacron bag after 42 d of ruminal incubation in alfalfafed steers using magnifications of 2,100~.
Figure 6 . Effect of diet on dry matter (DM)contamination of Dacron bags during ruminal incubation. 0 , alfalfa; 0, orchardgrass.
Some small peaks and shoulders of larger peaks remained unaccounted for, indicating the presence of small quantities of other compounds. Crystals appearing on Dacron bags either originate from intact crystals that dislodged from plant material or from solubilized mineral that gradually deposited on the bag. Ward et al. (1979)found that fecal calcium oxalate crystals could be 30 p x 30 p in size. Although some size reduction might occur postruminally, the size of the crystals deposited on the bags seemed to be much larger ( > 100 p; Figure 3). Also, the smooth appearance of crystals on the Dacron (Figure 51 supports the hypothesis that minerals deposited gradually on the bags, rather than representing dislodged crystals of plant origin. Mineral deposits appearing in the gastrointestinal tract are not uncommon in other animals. Blue (1979) reported the presence of large mineral concretions (enteroliths) in the proximal small colon of horses. The enteroliths (up to 20 cm in diameter) were primarily composed of ammonium calcium phosphate with a central nidus of foreign material (metal fragments or small stones). Blue (1979) also reported a case of obstruction of the small colon in a mare caused by mineral deposition on synthetic cloth. The rate of contaminant accumulation is illustrated in Figure 6. Because the SD of replicate observations seemed to be proportional to the mean, data were logarithmically transformed. A linear model was fitted to the logarithmically transformed data using the GLM procedure of SAS (1988). This implies that contamination occurs exponentially when data are expressed on the original scale: contamination = Aoek time, where A0 is a scaling parameter indicating initial con-
tamination (grams), and k is the fractional rate of Contamination (day-'). Theoretically, this model is incorrect because it does not intersect the origin. Moreover, it is unlikely that mineral contamination would proceed exponentially beyond the range of observations. Fractional rate of contamination was .160 and .127 d-l, and A. ,011 and ,010 g, for bags incubated in steers fed alfalfa and orchardgrass, respectively. Thus, contamination does not occur exclusively in steers fed alfalfa hay, but occurs for other roughages as well. The ash and AIA contents of the contaminant were similar for steers fed orchardgrass and alfalfa. Little contamination occurred during the first 10 d of incubation (Figure 6). However, extensive contamination of Dacron bags can occur during long-term incubation, and failure to correct for this will overestimate the substrate remaining.
Conclusions Mineral contamination can be extensive during long-term ruminal incubation studies. However, most studies are relatively short and no significant mineral contamination is anticipated within the first 7 d of ruminal incubation. Calcium phosphates seemed to be the major source of mineral contamination in steers fed alfalfa.
Implications Extensive mineral contamination can occur on Dacron bags that are incubated in the rumen of steers fed forage. Although little dry matter contamination occurred during the first 10 d of
Downloaded from https://academic.oup.com/jas/article-abstract/70/8/2551/4705027 by 04860000 user on 23 January 2019
,001
__
MINERAL DEPOSITS ON DACRON BAGS
incubation, the high mineral content (especially calcium) of the contaminant could seriously affect the outcome of studies focusing on mineral digestion.
Literature Cited
protein and energy digestibility: A review. J. Dairy Sci. 71: 2051.
Olubobukun, J A., W. M. Craig, and K. R. Pond. 1990. Effects of mastication and microbial contamination on ruminal in situ forage disappearance. J. Anim. Sci. 68:3371. SAS. 1988. SAS/STAT@ User’s Guide (Release 6.031. SAS Inst. Inc., Cary, NC. Quin, J. I., J. G. Van Der Wath, and S. Myburgh. 1938. Studies on the alimentary tract of Merino sheep in South Africa. IV. Description of experimental technique. Onderstepoort J. Vet. Sci. Anim. Ind. 11:341. van Milgen, J., L. L. Berger, and M. R. Murphy. 1992. Fractionation of substrate as a n intrinsic characteristic of feedstuffs fed to ruminants. J. Dairy Sci. 75:124. Ward, G., L. H. Harbers, and J. J. Blaha. 1979. Calcium-containing crystals in alfalfa: Their fate in cattle. J. Dairy Sci. 62: 715.
Downloaded from https://academic.oup.com/jas/article-abstract/70/8/2551/4705027 by 04860000 user on 23 January 2019
AOAC. 1984. Official Methods of Analysis (14th Ed.). Association of Official Analytical Chemists, Arlington, VA. Blue, M. G. 1979. Enteroliths in horses-a retrospective study of 30 cases. Equine Vet. J. 11:76. Goering, H. K., and P. J. Van Soest. 1970. Forage fiber analyses (apparatus, reagents, procedures, and some applications). Agric. Handbook 379. ARS, USDA, Washington, DC. Nocek, J. E. 1988. In situ and other methods to estimate ruminal
2555
