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Pattern of some internal and external markers along the gastrointestinal tract of cattle
Animal
Feed Science
and Technology,
37 (1992)
143-I
143
59
Elsevier Science Publishers B.V., Amsterdam
Pattern of some internal and external markers along the gastrointestinal tract of cattle B. Stefanon, C.R. Mills and E. Piasentier Islifufo Produzione
Animale,
Universitb
degli Sudi Udine,
di Udine,
via S. Mauro,
Z-33010
Pagnacco,
&a/y
(Received g March 199 I; accepted 22 August I99 1)
ABSTRACT Stefanon, B., Mills, CR. and Piasentier, E., 1992. Pattern of some internal and extcmal markers along the gastrointestinal tract ofcattle. Anim. Feed Sri. Technol., 37: 143-l 59. The pattern of distribution ofsome internal and external markers aloat: the gastrointestinal tract of cattle was measured and used to estimate diet digestibility and transit time. On the day before slaughter, part of the cows’ hay diet was replaced with chromium and ytterbium mordanted hay. The cows were subsequently slaughtered and their digestive tracts dissected and the contents analysed. Chromium concentrations along the gastrointestinal tract had a lower variability ,..a” ytterbium, ashadthecutinsincomparison withthelignin.Theexternalma,kersalwayshadlower~ncentrations than in the feed whilst the internal markers generally had higher concentrations. There was a general decrease of external and internal marker concentrations up to the small intestine; thereafter, internal markers increased and approximated the values observed in the rumen while the concentrations of the external markers remained lower than in the rutnen. The estimated t’umen rate of passage did not differ between lignin and cutins, with an average value of 0.01 I h-‘, hut these were both significantly lower (P~0.01) than those estimated with external markers (0.022 h-’ and 0.024 h-’ for chromium and ytterbium, respectively). Calculated mmen digestibility showed significant variations between external and internal marken forcrudeprotein and neutraldetergent tibre (NDF), but not for drymatter (DM) andorganicmatter (OM) degradability; generally, data based on chromium or ytterbium gave higher degradation values in comparison with the internal markers. The rate ofdigestion (16) digestibility values calculated at the abomasum wera more variable and lower than values measured in the nunen. Total tract digestibility (meawed at the second section of the colon ) varied widely between markers for DM, OM, crude protein and NDF.
INTRODUCTION
Prediction of gastrointestinal flow of nutriests in ruminants can be studied by feeding a pulse dose of an indigestible marker. It is generally assumed that the observed change in marker concentration in the rumen or in the faeces simulates the flow of the food, the hypothesis being wpported by the research of Grovum and Williams (1973). For this purpose, the most widely used @ 1992 Elsevier Science Publishers B.V. All rights reserved 0377-8401/92/$05.00
144
B.STEFANON ET AL.
markers for the particulate phase are the rare earth elements and chromium (Grovum and Williams, 1973; Uden et al., 1980, Allen, 1982, Colucci et al., 1982; Eliman and Orskov, 1984; Stefanon and Ovan, 1988; Beauchemin and Buchanan-Smith, 1989). The chromium binds strongly the cell walls and consequently causes almost total feed indigestibility (Uden et al., 1980), while the rare earth elements, ytterbium in particular, are less tenaciously attached to the feed which results in a less evident decrease in the digestibility of the mordanted feed, but the marker can migrate from one particle to another (Allen, 1982). As a consequence of this, the measured gastrointestinal flow differs according to the type of marker, site of sampling and mathematical model used to fit the excretion curves (Dhanoa et al., 1985; Pond et al., 1988; Beauchemin and Buchanan-Smith, 1989; Susmel et al., 1990a). The apparent digestibility of dry matter and that of the other feed components (i.e. protein, tibre fractions) in ruminants can be measured by means of indigestible compounds, naturally present in the foodstuffs, or by feeding indigestible markers for a few days, until a steady state between intake and faecal output of the marker is reached (Siddons et al., 1985; Sein and Todd, 1988). Data obtained from the former markers (internal) are not necessarily comparable with those obtained from the latter (external) and these are frequently claimed to underestimate digestibility owing to their incomplete recovery. The aim of the present trial was to examine the pattern of distribution of some internal and external markers along the gastrointestinal tract of cattle and to estimate digestibility and transit time of the diet. To avoid complementary effects between forage and concentrate, animals received a diet containing only hay. MATERIAL AND METHODS
Chromium mordanted hay was prepared according to the procedure described by Uden et al. (1980), using 40 g sodium dichromate kg-’ hay dry matter (DM) to avoid a marked decrease in feed digestibility (Stefanon and Ovan, 1988). Ytterbium labelled hay was prepared by immersing 3 kg hay in 30 1of water containing 77 g of YbCls for 24 h (25.67 g YbCb kg-’ hay); the liquid was discharged and the wet hay was soaked twice in 30 I of water for 4 h, with a 3 h interval and then dried in a 60°C forced air oven for 24 h. Animals and experimental procedure Four mature Simmental cows (average liveweight 679 ? 19 kg) were housed in a barn at a slaughterhouse and were fed for a 3 week adaptation period
USE OF MARKERS TO ESTlMATE DIET DlGESTIBlLlTY
145
with hay. An amount of hay equal to 9 g DM kg-’ liveweight (recorded value 9. I 2 0.04) was offered at 08:OOh, and the cows were trained to consume it within 2 h. The day before slaughter, 300 g of chromium mordanted hay and 600 g of ytterbium labelled hay were given to the cows instead of 900 g normal hay. Two hours after feeding, dietary hay, chromium and ytterbium bound hay refusals were collected, if present, for analysis. The entire hay residue samples were dried to measure the amount of marker uneaten. The day after marker administration (about 09:OOh), cows were led from the stalls and weighed, cows were slaughtered and the reticula-ntmen and intestines quickly removed and delivered to a working area in the slaughterhouse. The gastrointestinal tract was divided into I2 parts: rumen, reticulum, omasum, abomasum, duodenum (about 500 mm); first and second jejunum; ileum (about 10 m each); caecum; first and second colon (half length each) and rectum. Four cuts were made in the rumen corresponding to four sampling sites (dorsal sac, ventral sac, dorsal blind sac and ventral blind sac) and, for each site, pH was measured with a portable electronic pH meter. The rumen was weighed and rumen contents were emptied, mixed and sampled. Each part of the gastrointestinal tract was weighed full, emptied and washed and left to drain whilst dissection proceeded and then reweighed; the small intestine was closed at varying lengths along the tract with ligatures to prevent fluids moving from one section to another. The pH of samples was taken immediately after collection. Chemical analysis Samples were placed in tared aluminium trays, weighed and dried in a forced dry oven at 60°C for at least 4 days; 1 or 2 days after the samples went into the oven, the drier samples were mixed and the wet samples had the dry surface “skin” broken to help the drying process. Hay and samples were analysed for proximate analysis according to the Association of Official Analytical Chemists ( AOAC, 1980) and fibre was also fractionated as described by Goering and Van Soest ( 1970); with the following sequential treatments: neutral detergent tibre (NDF), acid detergent tibre (ADF), 72% HzS04, KMnO, and tinally ashing, to give NDF (NDF-ash), hemicellulose (NDF-ADF-ash), cellulose (ADF-H2S04 residue-ash), lignin (H2S04 residue-KMn04 residue-ash) and cutins (KMnO, residue-ash). Extraction of chromium from hay and faeces was performed using the method proposed by Williams et al. ( 1962 ) and of ytterbium according to the procedure described by Hart and Polan ( 1984). Concentrations in feed, residues and intestinal samples were measured after ashing using an atomic absorption spectrophotometer. Table 1 shows the chemical analysis for the dietary and marked hays.
8. STEFANON ET AL.
146 TABLE
1
Dry matter content and chemical composition of hay, chromium mordanted and ytterbium labelled hay (% DM
)
Dry maxer Crude protein Crude fibre Ether extract Ash NDF Hemicellulose C&dOX KMnO&gin Cutins Chromium Ytterbium
Hay
Cr-hay
Yh-hay
88.3 8.2 33.3
92.1 6.6 41.9
93.6 6.4 41.3
2.2 a.1 62.3 25.7 30.0 1.9 2.1
1.6 4.8 83.6 39.0 34.9 4.5 5.1 1.2
2.2 4.5 77.1 34.0 37.0 2.5 3.4 I.1
Data management
As concentrations and contents along the small and large intestine varied widely, only three parts were considered: “intake” to rumen+reticulum; rumen+reticulum to abomasum; abomasum to second colon. Dry matter degradability (DgDM) and digestibility (DMD) were estimated according to both the internal and external markers, using the following assumptions. Internal markers
Dry matter degradability and digestibility were calculated in three different compartments based on the increase in concentration of lignin or cutins observed in the rumen, abomasum and in the faeces (second colon) in comparison with the hay kdlti)= (IM-XM,)/IM,
where kdlcIj is the digestibility in the jth compartment, IM is the concentration of internal marker in the diet and IM, is that in thejth compartment. Rate of passage (h-’ ) was calculated considering that an internal marker is accumulated in the digestive tract as a function of its indigestibility ~~o,={[IMM-IMMjx(1-kdij)]/ZMM}/24
where k,i is the passage rate (h-’ ) along the jth compartment, and IMM is the amount (g) of internal marker in the diet and IMM, is that in the jth compartment. External markers
To calculate the rate of passage (k,,),
it was assumed that the change of
chromium and ytterbium amounts from one compartment only owing to the passage
to another was
bCo, = [ (EMM-EMM,)/EMM]/24 along the jth compartment, EMM is the amount (g) of external marker in the diet and EMM, is that in the jth compartment. The decrease in the concentrations of external markers was considered to be a result of the dilution in the undigested feed, further divided by the daily passage (kpevj x24)
where kpe is the passage rate (h-l)
LoI = (EMjlEM)l(hqx24) where kde is the digestibility in thejth compartment, EM is the concentration of internal marker in the diet and EM, ; that in thejth compartment.
Data were analysed using the SAS package (Statistical Analysis Systems Institute SAS, 1988) and analysis of variance was performed using the general linear model procedure. The different concentrations of nutrients and of external (i.e. chromium and ytterbium) and internal (KMn04 lignin and cutin) markers along the gastrointestinal tract were standardised by dividing by their concentrations in the feed. Data were analysed by a repeated measures analysis (SAS, 1988) and comparisons between means were carried out with the Ryan-Einot-Gabriel-Welsch multiple Ftest. The statistical model for the analysis is as follows where y is the dependent variable, a is the effect owiug to the marker (i = 1,4), b is the effect of cow nested within marker (k= 1,4), c is the effect of compartment (j= I, 12) and, e is the random error. RESULTS
The rumen contained 4564 g of dry matter (0.734 of the daily ration) and this was significantly (PcO.01) higher than that in the reticulum, omasum or abomcsum (Table 2). The smallest DM weight was observed in the first part of the duodenum (PiO.01); from here, DM weight increased significantly (PcO.01) up to the terminal ileum. In the large intestine, the amount of DM was lower (PcO.01) than in the ileum but no differences were observed between the parts of the large intestine. It must be pointed out that the total forestomach DM weight (6225 g) represented almost the whole gas-
148 TABLE
B. STEFANON ET AL. 2
pH values, dry Tract
matter content and DM to empty weight ratio in the gastrointestinal DM content
PH
DM to empty weight (g kg-’ )
(g) Forestomachs Rumen Reticulum Omasum Abamasum Total Small intestine Duodenum Jejunum Jeiunum 2
I
II&l
Total Caecum Large intestine Colon 2 Rectum Total SEM’
I Colon
6.2gcdc 6.545.85’ 4.44’
6.30 6.27 6.82 7.21 6.29”b
0.12
‘Pooled standard error ofthe means. Means in the same column with different
4564” I68d 12590 234’ 6225
519’ 143c
9’ 866 64d 174’ 333 1036
16* 64d 48’ 106r 57 270b
304b 119” 383
1306 IOld 346 265 42.95
6.70bcd 6.74” 6.71b’*
superscripts
tract
differ significantly
84’ 64’1 I8* 55 9.3
(PcO.01).
trointestinal content, whilst the amounts weighed in the small and large intestines plus caecum were very similar and small (333 g vs. 368 g, respectively). The repeated measured analysis of the DM weight expressed as a fraction of the weight of each tract differed between sections: the DM content of the caecum was higher (P~0.01) than in the small and large intestine and similar to the value observed in the omasum. The pH values (Table 2) were lower in the omasum and abomasum than in the rumen (Pt0.01) and thereafter increased significantly; only in the final part of the small intestine was a slightly basic pH observed. The hay labelling procedure with both chromium and ytterbium increased the Rbre fractions content. This was particularly evident for the lignin and cutins in the chromium mordanted forage (Table 1); only crude protein content decreased. The repeated measure analysis (Table 3) showed a significant difference (P~O.001) in the variation of nutrients content along the gastrointestinal tract, as shown by the significance of the nutrient effect; the interaction of nutrientxcompartment was significant (P~O.001). The differences in feed nutrient concentrations through the forestomachs and intestine also appear in Fig. 1.In particular, dry matter content decreased from 136 g kg- ’ in the
149
“SEOFMARKERSTOESTlMATEDlETDlGESTlRILlTY
TABLE
3
Repeated measures analysis of DM (g kg-‘) and of the standardised concentrations (percentage of each nutrient with respec? to its concentration in the ingested feed) of ash, crude protein and tibre fractions along the gastrointestinal tract (see Fig. the actual concentrations in the digesta)
I for
DM
Ash
CP
NDF
Cellulose
130’ 2980b 52CdC 363’ 152dC 204kdE
ll9b l34b
Caecum Colon I Colon 2 Rectum SEM’
IOY 108’ 116” ll7k 3.67
249”* 196kdc 220&d’ I88kdC 6.95
107” 103”b IOl”b 98”” 9’ Is”= 25d 46’ 94”b 92b 90b 98” 0.93
104’ 96ab
Ducdewm Jejunum I
I 36w 172’O 199’ 138& I22k 109’
Rumen Reticulum Omasum AiwnV.jiilr.
Nutrient Error Compartment Nutrientxcompartment EWX
I
181b I SO” 588” 481” 448” 24L” l04b l16b 161” l25b 9.57
Hemicellulose
104” 60d 9’ ID 25’ 4oc 81’ 81” 86% 86W 0.95
d.f.
Mean square
Fsig.
5 I8 II 55 I98
326782.3 1826.6 5686.9 28725.7 1210.5
0.0001
‘PC&~’ standard error of the means. Means in the same column with different superscripts differ significantly
O.ooLO 0.0001
(PC
0.01).
rumen to 95 g kg-’ at the end of the small intestine, and increased again in the faeces ( 117 g kg- ’ ) . The standardised concentration of the ash accumulated (PcO.01) in the reticulum (298%) and abomasum (363%), decreased (P
Fig. I. Change in nutrient concentrations along the gastrointestinal tein and ash; (b) NDF, cellttloseattd hemicellulose.
tract: (a) DM, crude pm-
and internal (lignin and cutin) markers along the gastrointestinal tract. From these figures the lower variability (i.e. standard deviation) of chromium in comparison with the ytterbium is evident, as is that of the cutins in comparison with the lignin. The repeated measure analysis for markers (Table 4) revealed a statistically significant effect of ‘marker’ and ‘markerx compartment’. From the values of standardised concentrations, expressed as percentage of ingested markers, reported in Table 4, it can be seen that the external
IS1
Fig. 2. External marker concentrations ytterbium.
along the gastrointestinal
tract: (a) chromium:
(b)
markers always had lower concentrations than in the feed (i.e. less than 100%) whilst the internal markers generally had higher concentrations than in the feed (higher than 100%). The general decrease of external and internal marker percentages up to the small intestine is evident (PC 0.01); thereafter, internal marker percentage increased and approximated the values observed in the rumen while the percentage of external markers remained lower than in the rumen.
Fig. 3. internal marker concentrationsalong the gastrointestinal tract: (a) cutins; (b) KIdnO,lignin.
The estimated rumen rate of passage did not differ between lignin and cutins (Table S), with an average value of 0.011 hb’, but these were both significantly lower (PcO.01) than those estimated with external markers (0.022 h-’ and 0.024 h-r for chromium and ytterbium, respectively). The rates of passage measured in ihc abomasum and colon were also statistically lower (PcO.05) when calculated from internal (kPi) rather than external (k,,) markers, even though the biological significance of the differences is negligible.
“SEOF MARKERSTO ESTIMATE DIET DIGESTIBILITY
153
TABLE 4 Repeated measures analysis of standardised concentrations (percentage of each marker with respect to its concentration in the ingested feed) of cxtcrnal and internal markers along gastrointestinal tract (see Fias, 2 and 3 for the actual concentrations in the digena) External markers’
Internal markers
Cr
Yb
RU”X” Reticulum Omasum Abomasum Duodenum Jejunum Jejunum 2 Ileum Caecum Colon 2 Rectum SEM’
I
I Colon
Repealed nwwsur~” analysrs o/variance Effect Nutrient Error Compartment Nulrientxco.npaRment EITOT
d.f. 3 I2 :: 132
Llgnin
Cutins
32Wb 2870b 226182s~~ 3od 76dc 1 I8&* 339"b 414' 383-b 3OT 299=' 9.11
27P 257=b 249"b 163'w 69“' 66* 22Y=h 29P 244=b 239=b 352' 7.42
Mean square
Fsig.
580746.2 3764.9 61162.8 14749.4 3424.2
0.0001 0.0001 0.000 I
‘External markers administered in a pulse dose. *Pooled standard error of the means. Means in the same column with different sllperscripts differ significantly (P
using
k,i ,, ) k QG(II SEM’
Markers
Rumen
Abomasum
COIO”
Lignin Cutins Chromium Ytterbiunl
0.01 lb 0.01 lb 0.022” 0.024” 0.0035
0.040’ 0.040’ 0.041” 0.0416 0.0001
0.040’ 0.040’ 0.0416 0.0416 O.OcQl
‘External markers administered in a pulse dose. ‘Pooled standard error of the means. =,“Means in the same column with different superscripts differ significantly (k WI ). “Means in the same column with different superscripts differ significantly (PcO.05).
B. STEFANON ET Al.
154 TABLF Digestion calculated
6 coefficients usingeither
kdC,,)
frr DM, OM, crude protein and NDF in various internal (lignin, cutins) or external’ (chromium,
k.3, cj, SEM
tract
Markers
Rumen
Ab0illZISUIt?
C&Xl
Lignin CIltins Chromium Ytterbium
O.G33
0.433””
0.669
0.636 0.659 0.720 0.0225
0.370b 0.54P 0.651’ 0.0136
0.602 O.S9B 0.535 0.025
I
Lignin Cutins Chromium
0.646 0.649 0.610 0.687 0.0235
0.597” 0.560’ 0.351b 0.507=~ 0.0116
0.698 0.623 0.546 0.474 0.032
I
Lignin Cutins Chromium Ytterbium
0.567b 0.567” 0.866” 0.893= 0.0253
0.148’ 0.067’ 0.705d 0.775d 0.0167
0.514 0.494 0.694 0.642 0.0505
Lignin Cutins Chromium Ytterbium
0.611 0.613 0.787 0.735 0.0208
0.455 0.377 0.498 0.615 0.0221
0.706 0.625 0.546 0.487 0.0255
SEM NDF k.+,,,
sections of the intestinal ytterbium) markers
‘External markers administered in a pulse dose. ‘Pooled standard error of the means. “~“Means in thesame column with different superscriptsdiffcrsigniAcanlly ‘.“Means in the same column with different superscripts differ significantly
(P
Calculated rumen degradability values (i.e. digestibility occurring in the rumen) (Table 6) showed significant differences between internal and external markers for crude protein and NDF, but not for DM and OM degradability; generally, data based on chromium and ytterbium gave higher degradation values in comparison with the internal markers. The digestibility coefftcients (/cd,and kde) calculated at the abomasum were more variable and lower than values measured in the rumen. Total tract digestibility (measured at the second section of the colon) varied widely between markers for DM, organic matter (OM), crude protein and NDF. DlSCUSSlON
The pH values (Table 2) were significantly lower in the abomasum and
higher in the duodenum and along the small intestine than other segments of the gastrointestinal tract and had a trend comparable with that reported by Bittante et al. ( 1985, 1987). In these experiments the pH value in the abomasum ranged from 3.20 to 4.43 for all the trials and increased in the small intestine, even though it always remained below 7.00 along the whole tract. The dry matter to gastrointestinal weight ratios, reported in Table 2, indicate that not only the rumen but also the omasum and caecum act as dry matter reservoirs. This could support the idea of a multicompartmental process in feed digestion and transit, as reported by Dhanoa et al. ( 1985) and Susmel et al. (1990a). The trends of dry matter concentration and of nutrients and of internal and external marker percentages (Tables 3 and 4) were comparable to those observed by Bittante et al. ( 1987) for dry matter, crude protein, ash and chromium oxide, by Rogerson ( 1958, cited by Warner, 1981) for dry matter, lignin and soluble ash and by Vidal et al. ( 1969) for chromium oxide. It is well known that in ruminants the degradation process starts in the Nmen and finishes in the abomasum, as well as that the rumen can absorb metabolic products from fermentation processes. Digestion of bacterial matter and undegradable feeds occurs mainly in the proximal intestine with absorption in the distal section. According to Van Soest ( 1982) this can also be deduced from the variation of the concentration of an indigestible substance (internal or external markers) along the gastrointestinal tract: an increase in concentration indicates digestion and absorption, whilst a decrease means di-
lution owing to secretion. From Fig. 1 and Table 3, it appears that mainly crude protein contributes to the dilution of the other nutrients considered (i.e. NDF, hemicellulose and cellulose) in the duodenum; the dilution can also be seen from the decrease of the concentration of internal and external markers from the rumen to the duodenum (Table 4 ) . Digestion and absorption of feed, mainly occurring from the duodenum to caecum, is confirmed from the increase of indigestible markers observed in the small intestine (Table 4, Figs. 2 and 3). It must be noted that external markers were administered in a puise dose while iulernal markers were effectively given continuously in the forage and this caused a different pattern of chromium and ytterbium concentrations in comparison with those of lignin and cutins (Table 4, Figs. 2 and 3). In this paper we have assumed that the change in the amount of external marker, administered in a pulse dose, from one compartment to another can be used to estimate the passage rate, as stated by other authors (Warner, 198 1; Bittante et al., 1987). The outflow rates based on chromium and ytterbium were similar (Table S), as can be also derived +,=c+in~t +=-t from ihe respective trends inthe gastroin.,,...., .___ (Tab!e 4, Fig. 2). It has been reported that outflow rate measured using ytterbium is generally higher in the rumen, but not in the post-ruminal tracts, than that obtained with chro-
156
El.srEF.mONETAt.
mium mordanted fibre (Beauchemin and Buchanan-Smith, 1989), and this is attributed to the lower digestibility and higher density of hay bound with chromium. In the present experiment the amount of sodium dichromate used to mordant the fibre was very low (62.3 g kg-’ NDF) in comparison with other workers (Ganev et al., 1979; Uden et al., 1980; Beauchemin and Buchanan-smith, 1989), to avoid the marked decrease in the digestibility of the mordanted feeds (Stefanon and Ovan, 1988). The mean values for the outflow rates observed agree with the suggested values of the Agricultural Research Council (ARC, 1984) for rations fed at maintenance and with data reported by Susmel et al. ( 199Ob) for forage diets fed at maintenance level. This could be a methodological artefact caused by the use of chromium mordanted forages administered in a pulse dose, so does not necessarily mean that our values are correct. However, given that they are widely accepted, we took the values of external markers as a reference in the comparisons with estimates obtained using lignin and cutins. The change in amount of internal markers from one compartment to the following one has been considered to be the result of different processes: the decrease is only a result of the passage, but it is counteracted by the differential digestion of the indigestible markers in comparison with digestible nutrients (carbohydrates, protein, lipids). To calculate the transit time based on internal markers, the amounts of lignin and cutins in the rumen, abomasum and colon were separated from the calculated DM digestibility, based on the same markers. The latter estimate was based on the consideration that the variation in concentration of internal markers can effectively be used to estimate the digestibility value of the diet (Krysl et al., 1988; Susmel et al., 199Ob). The results reported in Table 5 show that internal markers produced lower passage rates in all the compartments in cosnparison with chromium and ytterbium, especially in the rumen. This could be owing to variable delays in the commencement of digestion in different plant tissues. The difference in rumen outflow rates between external and internal markers, highlighted in Table 5, could be an artefact of the marker administration techniques, in that it is not unreasonable to assume that a pulse dose of marked hay will behave differently to the regular input of indigestible material (internal marker) for the dietary hay. Data obtained from chromium and ytterbium concentrations were based on the assumption that the change in concentration of external markers administered in pulse doses is owing to dilution of undigested feed by gastric secretion (decrease) and diet absorption (increase). The digestibility values calculated using lignin as the internal marker were more variable (see Fig. 3) than those obtained using the other markers. The complex chemistry of lignin digestion, with variable interconversion of phenolic monomers owing to microbial activity or rumen conditions (Bourquin et al., 1990) makes the simple analysis of lignin prone to considerable variation, which would need to be taken into account when estimating rate of pas-
sage and digestibility using iotemal markers. The formation of a soluble lignin-carbohydrate complex in the rumen, as a result of microbial activity was also reported by Hartley (1973) and by Gaillard and Richards (1975). The complex is not recovered as lignin in the faeces and this can cause the disappearance of a variable proportion of the lignin present. The results of dry matter degradability and digestibility (Table 6), obtained using either internal or external markers are consistent with physiological knowledge and laws of digestion. The higher proportion of DM, OM and NDF degradation occurs in the rumen, whilst the small intestine and caecum play more marginal roles: gastric secretion contributes to a dilution of indigestible material and a temporary decrease of digestibili ty coefficient in comparison with the rumen. CONCLUSIONS
From the results presented, it seems that the use of the slaughter technique with a pulse dose of external markers to estimate the outflow rates produce values similar to those obtained from faecal grab samples. However, these estimates differ considerably from those estimated from internal markers (Table 5) and these differences need to be resolved; this is probably a result of the different distribution of concentrations of interral and external markers which are described with different mathematical models. Of the digestibility values obtained, those calculated from the lignin demonstrate more variability than with the external markers. More experiments are needed to evaluate the optimum marker and to validate the equations proposed but, once the technique has been established, it would allow the calculation of degradability and digestibility values of diets at the end of experimental trials with growing ruminants. ACKNOWLEDGEMENTS Research supported by National Research Council of Italy, Special Project RAISA, Sub-project No. 3 Paper No. I65
REFERENCES Agricultural Research Council, 1984. The Nutrient Requirementsof Ruminant Livestock. Suppl. No. I. Commonwealth Agricultural Bureaux, Slough. Association of Offtcial Analytical Chemists, 1980. Official Methods of Analysis. AOAC, Washington, DC. Allen, X.S., 1982. Investigation into the use of rare earth elements as gastrointestinal markers. M.Sc. Txsis, Cornell University, NY. Beauchemin, K.A. and Buchar.an-Smith, J.G., 1989. Evaluation ofmarkers, samplingsitesand
158
a. STEFANON ETAL.
models for estimating mtes of passage of silage or hay in dairy cows. Anim. Feed Sci. Technol., 27: 59-75. Ronrquin, L.D., Garleb, K.A., Merchen, I&R. and Fahey, G.C., 1990. Effects of intake and forage level on site and extent of digestion of plant cell wall monomeric components by sheep. J. Anim. Sci., 68: 2479-2495. Bittante, G., Andrigheto, I. and Galeazzo, M., 1985. Effetto della macinazione della granella di mais e dell’impiego del carbonato di calcio nell’alimentazione di vacche Brune all’ingrasso: presiazioni produttive degli animali e valore nutritive del cereale. Zoot. Nutr. Anim., I I: 93-109. Bittante, G., Andrighetto, 1. and Ramanzin, M., 1987. lmpiego della granella e della farina di mais nell’alimentazione del vitellone: rilievi sull’apparato gastro-intestinale e sul sue contenuto. Zoot. Nutr. Anim., 13: 591-604. Colucci, P.E.. Chase, L.E. and van Soest, P.J., 1982. Feed intake, apparent diet digestibility, and rate of particulate passage in dairy cirttle. J. Dairy Sci., 65: 144% 1456. Dhanoa, M.S., Siddons, R.C., France, J. and Gale, D.L., 1985. A multicompanmental model to describe marker excretion patterns in ruminant faeces. Br. J. Nutr., 53: 663-671. Eliman, M.E. and srskov, E.R., 1984. Estimation of rates ofoutflow ofprotein supplement from the rumen by determining the rate of excretion ofchromium-treated protein supplements in faeces. Anim. Prod., 39: 77-80. Gaillard, B.D.E. and Richards, G.N., 1975. Presence of soluble lignin-carbohydrate complexes in the bovine rumen. Carbohydr. Res., 42: 135-145. Gancv, G., Orskov, E.R. and Smart, R., 1979. The effect of roughage or concentrate intake feeding and lumen retention time on total degradation of protein in the rumen. J. Agric. Sci., 93: 651-656. Goering, H.K. and van Soest, P.J., 1970. Forage Fiber Analyses (Apparatus, Reagents, Procedures and wme Applications). USDA Agricultural Handbook No. 379. Gmvum. W.L. and Williams. V.J.. 1973. Rate of .oassaae - ofdieesta _ in sheen 4. Passage of marker through the alimentary tract and the biological relevance of rate constants derived from the changes in concentratiom of marker in faeces. Br. J. Nutr., 30: 3 13-329. Hart, S.P. and Polan, C.E., 1924. Simultaneous extraction and determination ofytterbium and cobalt ethylene diamine tetraacetate complex in faeces. J. Dairy Sci., 67: 888-892. Hartley, R.D., 1973. Carbohydrate esters of ferulic acid as components of cell walls of Lolium rnu/riJlorun~ Phytochemistry, 12: 66 l-665. Klysl, L.J.. Galyean, M.L., Estell, R.E. and Sowell, B.F., 1988. Estimating digestibility and faecal output in lambs using internal and external markers. J. Agric. Sci., 1 I I: 19-25. Pond, K.R., Ellis, WC., Matis, J.H., Fet’reir0,H.M. andSutton, I.D., 1988.Compartment models for estimating attrihutes of digesta flow in cattle. Br. J. Nutr., 60: 57 l-595. Sei~, T. and Todd, J.R., 1988. Investigations into the use of indicator methods of estimating the digestibilities of feeds by ruminant animals. .I. Agric. Sci., I1 0: 3 15-320. Siddocs, R.C., Paradine, J., Beever, D.E. and Cornell, P.R., 1985. Ytterbium acetate as a particulate-phasedigesta flow marker. Br. J. Nutr., 54: 509-519. Statistical Analysis System Institute, 1988. SAS/STAT User’s Guide. Release 6.03 edn. SAS Institute Inc., Gary, NC. Stefanon, B. and Ovan, M., 1988. Impiego di bicromato sodico per la valutazione della velocit& di transit0 ruminale degli alimenti. Zoot. Nutr. Anim., 15: 431-436. Susmel, P., Stefanon, B., Mills, C.R. and Spanghero, M., 1990a. Evaluation of mathematical models for the study of rumen outflow rate offorages. Zoot. Nutr. Anim., 16: 207-218. Susmel, P., Stefanon, B., Mills, C.R. and Spanghero, M., I990b. Rumen degradability of organic matter, nitrogen and tibre fraction in forages. Anim. Prod., 51: 515-526. Uden. P.. Colucci. P.E. and van Soest. P.J.. 1980. Investiaation ofchromium. cerium and cobalt as markers in digesta rate of passage s&dies. 3. Sci. Fiod Agric., 31: 625632.
“SEOFMARKERSTOESrlMATE
D,ETD,GEsT,EulxrY
159
Van Soesl, P.J., 1982. Nutritional Ecology of the Ruminant. j&B Books, Corvallis, OR, 374 PP. Vidal, H.M., Hague, DE., Elliot, J.H. and Walker, E.F., 196 Digesta of sheep fed different hay-grain ratios. J. Anim. Sci., 29: 62-70. Warner, A.C.I., 1981. Rate of passage of digesra through the 15: of mammals and birds. Nutr. Abstr. Rev. Ser. B, Commonwealth Agricultural Bureau, Lc Anton,51: 783-820. Williams.C.H., David, D.J. and lismaa,O., 1962. Thedetermi \ationofchromicoxideinfa+ces samples by atomic adsorption spectrophotometry. J. Agric. Sci., 59: 381-385.
Feed Science
and Technology,
37 (1992)
143-I
143
59
Elsevier Science Publishers B.V., Amsterdam
Pattern of some internal and external markers along the gastrointestinal tract of cattle B. Stefanon, C.R. Mills and E. Piasentier Islifufo Produzione
Animale,
Universitb
degli Sudi Udine,
di Udine,
via S. Mauro,
Z-33010
Pagnacco,
&a/y
(Received g March 199 I; accepted 22 August I99 1)
ABSTRACT Stefanon, B., Mills, CR. and Piasentier, E., 1992. Pattern of some internal and extcmal markers along the gastrointestinal tract ofcattle. Anim. Feed Sri. Technol., 37: 143-l 59. The pattern of distribution ofsome internal and external markers aloat: the gastrointestinal tract of cattle was measured and used to estimate diet digestibility and transit time. On the day before slaughter, part of the cows’ hay diet was replaced with chromium and ytterbium mordanted hay. The cows were subsequently slaughtered and their digestive tracts dissected and the contents analysed. Chromium concentrations along the gastrointestinal tract had a lower variability ,..a” ytterbium, ashadthecutinsincomparison withthelignin.Theexternalma,kersalwayshadlower~ncentrations than in the feed whilst the internal markers generally had higher concentrations. There was a general decrease of external and internal marker concentrations up to the small intestine; thereafter, internal markers increased and approximated the values observed in the rumen while the concentrations of the external markers remained lower than in the rutnen. The estimated t’umen rate of passage did not differ between lignin and cutins, with an average value of 0.01 I h-‘, hut these were both significantly lower (P~0.01) than those estimated with external markers (0.022 h-’ and 0.024 h-’ for chromium and ytterbium, respectively). Calculated mmen digestibility showed significant variations between external and internal marken forcrudeprotein and neutraldetergent tibre (NDF), but not for drymatter (DM) andorganicmatter (OM) degradability; generally, data based on chromium or ytterbium gave higher degradation values in comparison with the internal markers. The rate ofdigestion (16) digestibility values calculated at the abomasum wera more variable and lower than values measured in the nunen. Total tract digestibility (meawed at the second section of the colon ) varied widely between markers for DM, OM, crude protein and NDF.
INTRODUCTION
Prediction of gastrointestinal flow of nutriests in ruminants can be studied by feeding a pulse dose of an indigestible marker. It is generally assumed that the observed change in marker concentration in the rumen or in the faeces simulates the flow of the food, the hypothesis being wpported by the research of Grovum and Williams (1973). For this purpose, the most widely used @ 1992 Elsevier Science Publishers B.V. All rights reserved 0377-8401/92/$05.00
144
B.STEFANON ET AL.
markers for the particulate phase are the rare earth elements and chromium (Grovum and Williams, 1973; Uden et al., 1980, Allen, 1982, Colucci et al., 1982; Eliman and Orskov, 1984; Stefanon and Ovan, 1988; Beauchemin and Buchanan-Smith, 1989). The chromium binds strongly the cell walls and consequently causes almost total feed indigestibility (Uden et al., 1980), while the rare earth elements, ytterbium in particular, are less tenaciously attached to the feed which results in a less evident decrease in the digestibility of the mordanted feed, but the marker can migrate from one particle to another (Allen, 1982). As a consequence of this, the measured gastrointestinal flow differs according to the type of marker, site of sampling and mathematical model used to fit the excretion curves (Dhanoa et al., 1985; Pond et al., 1988; Beauchemin and Buchanan-Smith, 1989; Susmel et al., 1990a). The apparent digestibility of dry matter and that of the other feed components (i.e. protein, tibre fractions) in ruminants can be measured by means of indigestible compounds, naturally present in the foodstuffs, or by feeding indigestible markers for a few days, until a steady state between intake and faecal output of the marker is reached (Siddons et al., 1985; Sein and Todd, 1988). Data obtained from the former markers (internal) are not necessarily comparable with those obtained from the latter (external) and these are frequently claimed to underestimate digestibility owing to their incomplete recovery. The aim of the present trial was to examine the pattern of distribution of some internal and external markers along the gastrointestinal tract of cattle and to estimate digestibility and transit time of the diet. To avoid complementary effects between forage and concentrate, animals received a diet containing only hay. MATERIAL AND METHODS
Chromium mordanted hay was prepared according to the procedure described by Uden et al. (1980), using 40 g sodium dichromate kg-’ hay dry matter (DM) to avoid a marked decrease in feed digestibility (Stefanon and Ovan, 1988). Ytterbium labelled hay was prepared by immersing 3 kg hay in 30 1of water containing 77 g of YbCls for 24 h (25.67 g YbCb kg-’ hay); the liquid was discharged and the wet hay was soaked twice in 30 I of water for 4 h, with a 3 h interval and then dried in a 60°C forced air oven for 24 h. Animals and experimental procedure Four mature Simmental cows (average liveweight 679 ? 19 kg) were housed in a barn at a slaughterhouse and were fed for a 3 week adaptation period
USE OF MARKERS TO ESTlMATE DIET DlGESTIBlLlTY
145
with hay. An amount of hay equal to 9 g DM kg-’ liveweight (recorded value 9. I 2 0.04) was offered at 08:OOh, and the cows were trained to consume it within 2 h. The day before slaughter, 300 g of chromium mordanted hay and 600 g of ytterbium labelled hay were given to the cows instead of 900 g normal hay. Two hours after feeding, dietary hay, chromium and ytterbium bound hay refusals were collected, if present, for analysis. The entire hay residue samples were dried to measure the amount of marker uneaten. The day after marker administration (about 09:OOh), cows were led from the stalls and weighed, cows were slaughtered and the reticula-ntmen and intestines quickly removed and delivered to a working area in the slaughterhouse. The gastrointestinal tract was divided into I2 parts: rumen, reticulum, omasum, abomasum, duodenum (about 500 mm); first and second jejunum; ileum (about 10 m each); caecum; first and second colon (half length each) and rectum. Four cuts were made in the rumen corresponding to four sampling sites (dorsal sac, ventral sac, dorsal blind sac and ventral blind sac) and, for each site, pH was measured with a portable electronic pH meter. The rumen was weighed and rumen contents were emptied, mixed and sampled. Each part of the gastrointestinal tract was weighed full, emptied and washed and left to drain whilst dissection proceeded and then reweighed; the small intestine was closed at varying lengths along the tract with ligatures to prevent fluids moving from one section to another. The pH of samples was taken immediately after collection. Chemical analysis Samples were placed in tared aluminium trays, weighed and dried in a forced dry oven at 60°C for at least 4 days; 1 or 2 days after the samples went into the oven, the drier samples were mixed and the wet samples had the dry surface “skin” broken to help the drying process. Hay and samples were analysed for proximate analysis according to the Association of Official Analytical Chemists ( AOAC, 1980) and fibre was also fractionated as described by Goering and Van Soest ( 1970); with the following sequential treatments: neutral detergent tibre (NDF), acid detergent tibre (ADF), 72% HzS04, KMnO, and tinally ashing, to give NDF (NDF-ash), hemicellulose (NDF-ADF-ash), cellulose (ADF-H2S04 residue-ash), lignin (H2S04 residue-KMn04 residue-ash) and cutins (KMnO, residue-ash). Extraction of chromium from hay and faeces was performed using the method proposed by Williams et al. ( 1962 ) and of ytterbium according to the procedure described by Hart and Polan ( 1984). Concentrations in feed, residues and intestinal samples were measured after ashing using an atomic absorption spectrophotometer. Table 1 shows the chemical analysis for the dietary and marked hays.
8. STEFANON ET AL.
146 TABLE
1
Dry matter content and chemical composition of hay, chromium mordanted and ytterbium labelled hay (% DM
)
Dry maxer Crude protein Crude fibre Ether extract Ash NDF Hemicellulose C&dOX KMnO&gin Cutins Chromium Ytterbium
Hay
Cr-hay
Yh-hay
88.3 8.2 33.3
92.1 6.6 41.9
93.6 6.4 41.3
2.2 a.1 62.3 25.7 30.0 1.9 2.1
1.6 4.8 83.6 39.0 34.9 4.5 5.1 1.2
2.2 4.5 77.1 34.0 37.0 2.5 3.4 I.1
Data management
As concentrations and contents along the small and large intestine varied widely, only three parts were considered: “intake” to rumen+reticulum; rumen+reticulum to abomasum; abomasum to second colon. Dry matter degradability (DgDM) and digestibility (DMD) were estimated according to both the internal and external markers, using the following assumptions. Internal markers
Dry matter degradability and digestibility were calculated in three different compartments based on the increase in concentration of lignin or cutins observed in the rumen, abomasum and in the faeces (second colon) in comparison with the hay kdlti)= (IM-XM,)/IM,
where kdlcIj is the digestibility in the jth compartment, IM is the concentration of internal marker in the diet and IM, is that in thejth compartment. Rate of passage (h-’ ) was calculated considering that an internal marker is accumulated in the digestive tract as a function of its indigestibility ~~o,={[IMM-IMMjx(1-kdij)]/ZMM}/24
where k,i is the passage rate (h-’ ) along the jth compartment, and IMM is the amount (g) of internal marker in the diet and IMM, is that in the jth compartment. External markers
To calculate the rate of passage (k,,),
it was assumed that the change of
chromium and ytterbium amounts from one compartment only owing to the passage
to another was
bCo, = [ (EMM-EMM,)/EMM]/24 along the jth compartment, EMM is the amount (g) of external marker in the diet and EMM, is that in the jth compartment. The decrease in the concentrations of external markers was considered to be a result of the dilution in the undigested feed, further divided by the daily passage (kpevj x24)
where kpe is the passage rate (h-l)
LoI = (EMjlEM)l(hqx24) where kde is the digestibility in thejth compartment, EM is the concentration of internal marker in the diet and EM, ; that in thejth compartment.
Data were analysed using the SAS package (Statistical Analysis Systems Institute SAS, 1988) and analysis of variance was performed using the general linear model procedure. The different concentrations of nutrients and of external (i.e. chromium and ytterbium) and internal (KMn04 lignin and cutin) markers along the gastrointestinal tract were standardised by dividing by their concentrations in the feed. Data were analysed by a repeated measures analysis (SAS, 1988) and comparisons between means were carried out with the Ryan-Einot-Gabriel-Welsch multiple Ftest. The statistical model for the analysis is as follows where y is the dependent variable, a is the effect owiug to the marker (i = 1,4), b is the effect of cow nested within marker (k= 1,4), c is the effect of compartment (j= I, 12) and, e is the random error. RESULTS
The rumen contained 4564 g of dry matter (0.734 of the daily ration) and this was significantly (PcO.01) higher than that in the reticulum, omasum or abomcsum (Table 2). The smallest DM weight was observed in the first part of the duodenum (PiO.01); from here, DM weight increased significantly (PcO.01) up to the terminal ileum. In the large intestine, the amount of DM was lower (PcO.01) than in the ileum but no differences were observed between the parts of the large intestine. It must be pointed out that the total forestomach DM weight (6225 g) represented almost the whole gas-
148 TABLE
B. STEFANON ET AL. 2
pH values, dry Tract
matter content and DM to empty weight ratio in the gastrointestinal DM content
PH
DM to empty weight (g kg-’ )
(g) Forestomachs Rumen Reticulum Omasum Abamasum Total Small intestine Duodenum Jejunum Jeiunum 2
I
II&l
Total Caecum Large intestine Colon 2 Rectum Total SEM’
I Colon
6.2gcdc 6.545.85’ 4.44’
6.30 6.27 6.82 7.21 6.29”b
0.12
‘Pooled standard error ofthe means. Means in the same column with different
4564” I68d 12590 234’ 6225
519’ 143c
9’ 866 64d 174’ 333 1036
16* 64d 48’ 106r 57 270b
304b 119” 383
1306 IOld 346 265 42.95
6.70bcd 6.74” 6.71b’*
superscripts
tract
differ significantly
84’ 64’1 I8* 55 9.3
(PcO.01).
trointestinal content, whilst the amounts weighed in the small and large intestines plus caecum were very similar and small (333 g vs. 368 g, respectively). The repeated measured analysis of the DM weight expressed as a fraction of the weight of each tract differed between sections: the DM content of the caecum was higher (P~0.01) than in the small and large intestine and similar to the value observed in the omasum. The pH values (Table 2) were lower in the omasum and abomasum than in the rumen (Pt0.01) and thereafter increased significantly; only in the final part of the small intestine was a slightly basic pH observed. The hay labelling procedure with both chromium and ytterbium increased the Rbre fractions content. This was particularly evident for the lignin and cutins in the chromium mordanted forage (Table 1); only crude protein content decreased. The repeated measure analysis (Table 3) showed a significant difference (P~O.001) in the variation of nutrients content along the gastrointestinal tract, as shown by the significance of the nutrient effect; the interaction of nutrientxcompartment was significant (P~O.001). The differences in feed nutrient concentrations through the forestomachs and intestine also appear in Fig. 1.In particular, dry matter content decreased from 136 g kg- ’ in the
149
“SEOFMARKERSTOESTlMATEDlETDlGESTlRILlTY
TABLE
3
Repeated measures analysis of DM (g kg-‘) and of the standardised concentrations (percentage of each nutrient with respec? to its concentration in the ingested feed) of ash, crude protein and tibre fractions along the gastrointestinal tract (see Fig. the actual concentrations in the digesta)
I for
DM
Ash
CP
NDF
Cellulose
130’ 2980b 52CdC 363’ 152dC 204kdE
ll9b l34b
Caecum Colon I Colon 2 Rectum SEM’
IOY 108’ 116” ll7k 3.67
249”* 196kdc 220&d’ I88kdC 6.95
107” 103”b IOl”b 98”” 9’ Is”= 25d 46’ 94”b 92b 90b 98” 0.93
104’ 96ab
Ducdewm Jejunum I
I 36w 172’O 199’ 138& I22k 109’
Rumen Reticulum Omasum AiwnV.jiilr.
Nutrient Error Compartment Nutrientxcompartment EWX
I
181b I SO” 588” 481” 448” 24L” l04b l16b 161” l25b 9.57
Hemicellulose
104” 60d 9’ ID 25’ 4oc 81’ 81” 86% 86W 0.95
d.f.
Mean square
Fsig.
5 I8 II 55 I98
326782.3 1826.6 5686.9 28725.7 1210.5
0.0001
‘PC&~’ standard error of the means. Means in the same column with different superscripts differ significantly
O.ooLO 0.0001
(PC
0.01).
rumen to 95 g kg-’ at the end of the small intestine, and increased again in the faeces ( 117 g kg- ’ ) . The standardised concentration of the ash accumulated (PcO.01) in the reticulum (298%) and abomasum (363%), decreased (P
Fig. I. Change in nutrient concentrations along the gastrointestinal tein and ash; (b) NDF, cellttloseattd hemicellulose.
tract: (a) DM, crude pm-
and internal (lignin and cutin) markers along the gastrointestinal tract. From these figures the lower variability (i.e. standard deviation) of chromium in comparison with the ytterbium is evident, as is that of the cutins in comparison with the lignin. The repeated measure analysis for markers (Table 4) revealed a statistically significant effect of ‘marker’ and ‘markerx compartment’. From the values of standardised concentrations, expressed as percentage of ingested markers, reported in Table 4, it can be seen that the external
IS1
Fig. 2. External marker concentrations ytterbium.
along the gastrointestinal
tract: (a) chromium:
(b)
markers always had lower concentrations than in the feed (i.e. less than 100%) whilst the internal markers generally had higher concentrations than in the feed (higher than 100%). The general decrease of external and internal marker percentages up to the small intestine is evident (PC 0.01); thereafter, internal marker percentage increased and approximated the values observed in the rumen while the percentage of external markers remained lower than in the rumen.
Fig. 3. internal marker concentrationsalong the gastrointestinal tract: (a) cutins; (b) KIdnO,lignin.
The estimated rumen rate of passage did not differ between lignin and cutins (Table S), with an average value of 0.011 hb’, but these were both significantly lower (PcO.01) than those estimated with external markers (0.022 h-’ and 0.024 h-r for chromium and ytterbium, respectively). The rates of passage measured in ihc abomasum and colon were also statistically lower (PcO.05) when calculated from internal (kPi) rather than external (k,,) markers, even though the biological significance of the differences is negligible.
“SEOF MARKERSTO ESTIMATE DIET DIGESTIBILITY
153
TABLE 4 Repeated measures analysis of standardised concentrations (percentage of each marker with respect to its concentration in the ingested feed) of cxtcrnal and internal markers along gastrointestinal tract (see Fias, 2 and 3 for the actual concentrations in the digena) External markers’
Internal markers
Cr
Yb
RU”X” Reticulum Omasum Abomasum Duodenum Jejunum Jejunum 2 Ileum Caecum Colon 2 Rectum SEM’
I
I Colon
Repealed nwwsur~” analysrs o/variance Effect Nutrient Error Compartment Nulrientxco.npaRment EITOT
d.f. 3 I2 :: 132
Llgnin
Cutins
32Wb 2870b 226182s~~ 3od 76dc 1 I8&* 339"b 414' 383-b 3OT 299=' 9.11
27P 257=b 249"b 163'w 69“' 66* 22Y=h 29P 244=b 239=b 352' 7.42
Mean square
Fsig.
580746.2 3764.9 61162.8 14749.4 3424.2
0.0001 0.0001 0.000 I
‘External markers administered in a pulse dose. *Pooled standard error of the means. Means in the same column with different sllperscripts differ significantly (P
using
k,i ,, ) k QG(II SEM’
Markers
Rumen
Abomasum
COIO”
Lignin Cutins Chromium Ytterbiunl
0.01 lb 0.01 lb 0.022” 0.024” 0.0035
0.040’ 0.040’ 0.041” 0.0416 0.0001
0.040’ 0.040’ 0.0416 0.0416 O.OcQl
‘External markers administered in a pulse dose. ‘Pooled standard error of the means. =,“Means in the same column with different superscripts differ significantly (k WI ). “Means in the same column with different superscripts differ significantly (PcO.05).
B. STEFANON ET Al.
154 TABLF Digestion calculated
6 coefficients usingeither
kdC,,)
frr DM, OM, crude protein and NDF in various internal (lignin, cutins) or external’ (chromium,
k.3, cj, SEM
tract
Markers
Rumen
Ab0illZISUIt?
C&Xl
Lignin CIltins Chromium Ytterbium
O.G33
0.433””
0.669
0.636 0.659 0.720 0.0225
0.370b 0.54P 0.651’ 0.0136
0.602 O.S9B 0.535 0.025
I
Lignin Cutins Chromium
0.646 0.649 0.610 0.687 0.0235
0.597” 0.560’ 0.351b 0.507=~ 0.0116
0.698 0.623 0.546 0.474 0.032
I
Lignin Cutins Chromium Ytterbium
0.567b 0.567” 0.866” 0.893= 0.0253
0.148’ 0.067’ 0.705d 0.775d 0.0167
0.514 0.494 0.694 0.642 0.0505
Lignin Cutins Chromium Ytterbium
0.611 0.613 0.787 0.735 0.0208
0.455 0.377 0.498 0.615 0.0221
0.706 0.625 0.546 0.487 0.0255
SEM NDF k.+,,,
sections of the intestinal ytterbium) markers
‘External markers administered in a pulse dose. ‘Pooled standard error of the means. “~“Means in thesame column with different superscriptsdiffcrsigniAcanlly ‘.“Means in the same column with different superscripts differ significantly
(P
Calculated rumen degradability values (i.e. digestibility occurring in the rumen) (Table 6) showed significant differences between internal and external markers for crude protein and NDF, but not for DM and OM degradability; generally, data based on chromium and ytterbium gave higher degradation values in comparison with the internal markers. The digestibility coefftcients (/cd,and kde) calculated at the abomasum were more variable and lower than values measured in the rumen. Total tract digestibility (measured at the second section of the colon) varied widely between markers for DM, organic matter (OM), crude protein and NDF. DlSCUSSlON
The pH values (Table 2) were significantly lower in the abomasum and
higher in the duodenum and along the small intestine than other segments of the gastrointestinal tract and had a trend comparable with that reported by Bittante et al. ( 1985, 1987). In these experiments the pH value in the abomasum ranged from 3.20 to 4.43 for all the trials and increased in the small intestine, even though it always remained below 7.00 along the whole tract. The dry matter to gastrointestinal weight ratios, reported in Table 2, indicate that not only the rumen but also the omasum and caecum act as dry matter reservoirs. This could support the idea of a multicompartmental process in feed digestion and transit, as reported by Dhanoa et al. ( 1985) and Susmel et al. (1990a). The trends of dry matter concentration and of nutrients and of internal and external marker percentages (Tables 3 and 4) were comparable to those observed by Bittante et al. ( 1987) for dry matter, crude protein, ash and chromium oxide, by Rogerson ( 1958, cited by Warner, 1981) for dry matter, lignin and soluble ash and by Vidal et al. ( 1969) for chromium oxide. It is well known that in ruminants the degradation process starts in the Nmen and finishes in the abomasum, as well as that the rumen can absorb metabolic products from fermentation processes. Digestion of bacterial matter and undegradable feeds occurs mainly in the proximal intestine with absorption in the distal section. According to Van Soest ( 1982) this can also be deduced from the variation of the concentration of an indigestible substance (internal or external markers) along the gastrointestinal tract: an increase in concentration indicates digestion and absorption, whilst a decrease means di-
lution owing to secretion. From Fig. 1 and Table 3, it appears that mainly crude protein contributes to the dilution of the other nutrients considered (i.e. NDF, hemicellulose and cellulose) in the duodenum; the dilution can also be seen from the decrease of the concentration of internal and external markers from the rumen to the duodenum (Table 4 ) . Digestion and absorption of feed, mainly occurring from the duodenum to caecum, is confirmed from the increase of indigestible markers observed in the small intestine (Table 4, Figs. 2 and 3). It must be noted that external markers were administered in a puise dose while iulernal markers were effectively given continuously in the forage and this caused a different pattern of chromium and ytterbium concentrations in comparison with those of lignin and cutins (Table 4, Figs. 2 and 3). In this paper we have assumed that the change in the amount of external marker, administered in a pulse dose, from one compartment to another can be used to estimate the passage rate, as stated by other authors (Warner, 198 1; Bittante et al., 1987). The outflow rates based on chromium and ytterbium were similar (Table S), as can be also derived +,=c+in~t +=-t from ihe respective trends inthe gastroin.,,...., .___ (Tab!e 4, Fig. 2). It has been reported that outflow rate measured using ytterbium is generally higher in the rumen, but not in the post-ruminal tracts, than that obtained with chro-
156
El.srEF.mONETAt.
mium mordanted fibre (Beauchemin and Buchanan-Smith, 1989), and this is attributed to the lower digestibility and higher density of hay bound with chromium. In the present experiment the amount of sodium dichromate used to mordant the fibre was very low (62.3 g kg-’ NDF) in comparison with other workers (Ganev et al., 1979; Uden et al., 1980; Beauchemin and Buchanan-smith, 1989), to avoid the marked decrease in the digestibility of the mordanted feeds (Stefanon and Ovan, 1988). The mean values for the outflow rates observed agree with the suggested values of the Agricultural Research Council (ARC, 1984) for rations fed at maintenance and with data reported by Susmel et al. ( 199Ob) for forage diets fed at maintenance level. This could be a methodological artefact caused by the use of chromium mordanted forages administered in a pulse dose, so does not necessarily mean that our values are correct. However, given that they are widely accepted, we took the values of external markers as a reference in the comparisons with estimates obtained using lignin and cutins. The change in amount of internal markers from one compartment to the following one has been considered to be the result of different processes: the decrease is only a result of the passage, but it is counteracted by the differential digestion of the indigestible markers in comparison with digestible nutrients (carbohydrates, protein, lipids). To calculate the transit time based on internal markers, the amounts of lignin and cutins in the rumen, abomasum and colon were separated from the calculated DM digestibility, based on the same markers. The latter estimate was based on the consideration that the variation in concentration of internal markers can effectively be used to estimate the digestibility value of the diet (Krysl et al., 1988; Susmel et al., 199Ob). The results reported in Table 5 show that internal markers produced lower passage rates in all the compartments in cosnparison with chromium and ytterbium, especially in the rumen. This could be owing to variable delays in the commencement of digestion in different plant tissues. The difference in rumen outflow rates between external and internal markers, highlighted in Table 5, could be an artefact of the marker administration techniques, in that it is not unreasonable to assume that a pulse dose of marked hay will behave differently to the regular input of indigestible material (internal marker) for the dietary hay. Data obtained from chromium and ytterbium concentrations were based on the assumption that the change in concentration of external markers administered in pulse doses is owing to dilution of undigested feed by gastric secretion (decrease) and diet absorption (increase). The digestibility values calculated using lignin as the internal marker were more variable (see Fig. 3) than those obtained using the other markers. The complex chemistry of lignin digestion, with variable interconversion of phenolic monomers owing to microbial activity or rumen conditions (Bourquin et al., 1990) makes the simple analysis of lignin prone to considerable variation, which would need to be taken into account when estimating rate of pas-
sage and digestibility using iotemal markers. The formation of a soluble lignin-carbohydrate complex in the rumen, as a result of microbial activity was also reported by Hartley (1973) and by Gaillard and Richards (1975). The complex is not recovered as lignin in the faeces and this can cause the disappearance of a variable proportion of the lignin present. The results of dry matter degradability and digestibility (Table 6), obtained using either internal or external markers are consistent with physiological knowledge and laws of digestion. The higher proportion of DM, OM and NDF degradation occurs in the rumen, whilst the small intestine and caecum play more marginal roles: gastric secretion contributes to a dilution of indigestible material and a temporary decrease of digestibili ty coefficient in comparison with the rumen. CONCLUSIONS
From the results presented, it seems that the use of the slaughter technique with a pulse dose of external markers to estimate the outflow rates produce values similar to those obtained from faecal grab samples. However, these estimates differ considerably from those estimated from internal markers (Table 5) and these differences need to be resolved; this is probably a result of the different distribution of concentrations of interral and external markers which are described with different mathematical models. Of the digestibility values obtained, those calculated from the lignin demonstrate more variability than with the external markers. More experiments are needed to evaluate the optimum marker and to validate the equations proposed but, once the technique has been established, it would allow the calculation of degradability and digestibility values of diets at the end of experimental trials with growing ruminants. ACKNOWLEDGEMENTS Research supported by National Research Council of Italy, Special Project RAISA, Sub-project No. 3 Paper No. I65
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