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Comparison of n-alkanes or chromium oxide methods for estimation of herbage intake by sheep
Small Rwninant Research Small Ruminant
Research18 ( 1995) 27-32
Comparison of n-alkanes or chromium oxide methods for estimation of herbage intake by sheep E. Piasentier *, S. Bovolenta, F. Malossini, P. Susmel Dipartimento di Scienze della Produzione Animale, Universitcidi Udine, Via S. Mauro. 2. 33010 Pagnacco (UD), Italy
Accepted15 January 1995
Abstract Herbage intake (HI) was estimated with two procedures: (a) with the n-alkanes method (HI,,), which combines the use of odd-chain n-alkanes, naturally occurring in plants, and dosed even-chain n-alkanes, as internal and external markers, respectively; (b) with the ratio between faecal excretion, estimated with chromium sesquioxide (Cr*Oa), and the herbage indigestibility, estimated in vitro (HI,,). The two techniques were compared in an indoor experiment and under grazing conditions. In the indoor trial, 12 wethers were fed 40 g dry matter (DM) kg- ’ BW”.75day-’ of one of the following diets: herbage only (diet H) ; herbage plus maize (diet M; 26% of DM intake); herbage plus maize plus maize gluten meal (diet MG; 13% of DM intake each). Each wether was dosed once a day for 12 days with the external indicators impregnated on paper and inserted in gelatine capsules: n-dotriacontane, C3*.600 mg day-‘; Cr203, 500 mg day-‘. In the second experiment, six adult, lactating ewes grazing the pasture used for the indoor trial were divided into groups of three receiving 700 g day - ’ of two different supplements: maize (diet M) or a 5050 mix of maize and maize gluten meal (diet MG) .The external indicators ( Cr203 and t&) were administered for 8 consecutive days. The trial was repeated four times with the same procedure between 10th and the 40th day after lambing. Recoveries of Cr,Ol, C3*and herbage n-alkanes (n-hentriacontane, Csir and n-tritriacontane, C3s) were incomplete, with lower values for the natural n-alkanes than the dosed n-alkane (Cr203, 94.9* 5.5%; Ca,, 86.7+ 8.7%; &, 89.6+ 8.5%; C33, 85.1 + 9.9%). Therefore, HI,, gave an overestimate of the true herbage intake of 5.5% and HI,, an underestimate of 3.0%, with a significant discrepancy between methods (803 vs. 733 g organic matter (OM) day-‘), not affected by diet. In the grazing trial, HI,-, was higher than HI,, but the difference between methods (1503 vs. 1273 g OM day-‘) was higher than that obtained in the indoor experiment and was influenced by diet (380 g OM day- ’ vs. 81 g OM day- ’ with diet M and diet MG, respectively). A possible explanation of the different results obtained indoor or at pasture is discussed. Keywords:
n-alkanes; Chromiumoxide; Herbage intake; Grazing ewes
1. Introduction
Of the methods available for estimating herbage intake by grazing animals, the most commonly used is based on the ratio between faecal output, estimated with an external marker, usually Cr,03, and herbage indi* Corresponding
author
0921~4488/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSD10921-4488(95)00712-l
gestibility, which is either known or estimated in vitro (Le Du and Penning, 1982; Minson, 1990). As an alternative, Mayes et al. ( 1986) proposed the combined use of odd-chain n-alkanes, naturally occurring in the cuticular waxes of plants, and even-chain nalkanes present in much smaller amounts in herbage and commercially available, as internal and external
E. Piasentier/Small Ruminant Research 18 (1995) 27-32
28 Table 1 Chemical composition of feeds
Herbage
DM CP Ether extract Ash NDF ADF ADL
(%) (% DM) (%DM) (% DM) (% DM) (% DM) (% DM)
Supplement
At pasture
Indoor a
Maize
Maize gluten meal
22.2 16.3 4.8 7.6 56.6 33.1 3.2
23.0 13.3 3.4 8.2 61.9 35.0 3.6
87.6 7.7 1.1 0.5
90. I 62.9 3.3 6.5
-
a The herbage used for the indoor trial was collected towards the end of the grazing period. CP, crude protein; NDF, neutral detergent fibre; ADF, acid detergent fibre; ADL, acid detergent lignin.
markers to estimate digestibility and faecal production respectively. Carbon-chain lengths of the main n-alkanes detected in herbage species are usually in the range from npentacosane ( CZs) to n-pentatriacontane ( Cs5). Of these hydrocarbons, n-hentriacontane ( C3i ) and n-tritriacontane (C,,) are of particular interest as internal markers because they are dominant alkanes in all species and have high faecal recoveries, which are similar to those of the dosed adjacent homologue, n-dotriacontane (C& (Dove et al., 1989; Dove and Mayes, 1991). This latter condition, as discussed by Mayes et al. ( 1986), provides an estimate of herbage intake which is not biased by the incomplete recovery of markers and is independent of the diet digestibility. The aim of the present research, conducted as part of a study about the effect of dietary protein level for grazing ewes, was to evaluate the two intake estimation techniques. The first trial, conducted with animals held in digestibility crates, aimed to evaluate the faecal recovery of the markers and the accuracy of the techniques while a second experiment, with grazing ewes, compared the two methods under field conditions.
2. Materials and methods 2.1. Indoor trial Twelve Bergamasca wethers (body weight (BW) 73.2f 10.3 kg), held in digestibility crates, were fed
40 g dry matter (DM) kg-’ Bw.” day- ’ of one of the following diets (four animals per diet) : herbage only (H) ; herbage plus maize (26% of DM intake) (M) ; herbage plus maize plus maize gluten meal ( 13% of DM intake each) (MG) . The herbage, represented by a second regrowth of a monoculture fescue meadow (Festuca arundinacea, cv. “Manade”), was cut daily to a height of 5 cm from protected areas of the pasture grazed by the ewes. Each wether was dosed once a day for 12 days with the external indicators impregnated on paper and inserted in gelatine capsules: Cs2, 600 mg day-‘; Cr203, 500 mg day- ‘. In the last 7 days, which corresponded to the final phase of the grazing period, a digestibility trial was performed. Samples of herbage, concentrates and faeces were collected daily and dried for analysis. The feeds were analysed for their proximate composition, Van Soest fibre fractions and n-alkane concentrations (Mayes et al., 1986)) while the faeces samples were used for the determination of their concentrations of n-alkanes and Cr203 (Williams et al., 1962). Table 1 reports the average chemical composition of feeds. The dietary OM digestibility (OMD), the percentage recovery of each marker in the faeces and the correlation coefficients between the faecal recovery of the dosed and natural n-alkanes were calculated. OMD of the supplements (OMD,) was determined indirectly from digestibility values of diet H (OMD,,) and the mixed diets. The quantity of herbage ingested (HZ, kg OM day- ’ ) was calculated with the chromium method HI,,=[Dij/Fjj-Is(l-OMD,)]/(l-OMD,)
E. Piasentier/Small
Ruminant Research 18 (1995) 27-32
and with the alkanes method HI,,= [F,/F,(Dj+IsXSj)
-SXSi]/
(Hi-Fi/FjxHj) where D (mg day- ‘) is the external marker ( Dj represents C3* and Djj Cr,03), F (mg kg- ’ OM) is the faecal concentration (Fj represents C3?, Fi Cfl and Fii Cr,O,), H (mg kg- ’ OM) is the herbage concentration (Hi represents C,, and Hi C3,), Is (kg OM day-‘) is the quantity of any supplement fed and S (mg kg- ’ OM) is the supplement concentration (Sj represents C32 and Si C3i ) . The accuracy of the intake estimate with the two methods was expressed as a percentage difference from true intake. Calculated variables (Yij) were analysed with a monofactorial model Y, =
/_l +
29
Intake estimate and the calculation of the discrepancy between the methods were performed, as in the indoor trial, by using the same OMD, values and adopting in vitro measurements, calibrated from the in vivo digestibility, for OMD,. Intake estimated with each method and the difference between them were analysed with a split-plot design for repeated measures (Gill, 1978) using the following model yijk=
p”+
aiis
P(i)j
+
yk+
( ay)ik+
E(i)jk
where Qr is the fixed effect of diet (i = 1..2), P~i,j is the nested effect of animal (j = 1..3) and the experimental error to test Cui,yk is the fixed effect of period (k= 1..4) and lcijjk the residual error.
3. Results and discussion
ai + Eij
where (Y is diet, with three levels repeated on four wethers (j = 1..4).
(i = 1..3), each
2.2. Field trial Six adult lactating Bergamasca ewes (73.9 + 8.0 kg SW) grazed the same pasture utilised for the indoor trial from 29 June to 6 August. Sheep, reunited into a single flock, were moved daily to a fresh pasture area ( 10 m2 per head) enclosed with a mobile electric fence. Three ewes received a supplement of 700 g day-’ of maize (diet M), the other three a 50:50 mix of maize and maize gluten meal (diet MG) offered in two meals during milking. From the 4th day after lambing, the ewes were dosed daily with the same quantity of Ca2 and Cr,Oa used in the indoor trial. Herbage intake was estimated four times, in different areas on the same pasture, with the same procedure between the 10th and the 40th day after lambing. During the last 2 days of each period, three grab samples (at 07:00, 12:OO and 18:OO h) were taken from each animal; these were later bulked to form a single period sample for the analysis of Cr203 and nalkanes. Herbage samples were hand plucked from two 4 m2 isolated areas, simulating removal by the animals. Chemical composition, in vitro digestibility (Tilley and Terry, 1963) and n-alkane content were determined. Table 1 reports the average chemical composition of feeds.
3.1. Marker concentrations in herbage The most common n-alkanes in the herbage (Table 2) were CZ9, Ca, and Ca3, with a net predominance of C3, (about 62% of the total). The quantitative importance of CZ9 and C3i alkanes, in temperate grass, has been reported previously (Mayes et al., 1986; Dove et al., 1990; Malossini et al., 1990; Laredo et al., 1991). Concentrates had a very low n-alkane content, with concentrations never exceeding 3.1 mg kg- ’ OM. 3.2. In vivo digestibility, faecal recovery of markers and accuracy of methods Table 3 reports the in vivo digestibility of the herbage and the M and MG diets; these values were used to estimate the digestibility of the supplements (90.7% for the maize and 8 1.7% for the maize and gluten mix). In the same table the recoveries of Cr,Oa, the dosed alkane Ca2 and the two natural adjacent homologues C,, and Cs3 are reported. In all cases, the recoveries were incomplete and not influenced by the diet, with higher values for Cr203 and C32 than the two natural alkanes. The C3i-Ca2 alkane pair had, on average, the most similar recoveries. The observed values fell within the range of data reported in the literature (Le Du and Penning, 1982; Dove and Mayes, 1991; Bovolenta et al., 1994). The correlation between Ca2 and C3i (r=0.73**) was higher than that between Cs2 and C33 (r = 0.54).
E. Piasentier/SmaN Ruminant Research 18 (1995) 27-32
30 Table 2 n-alkanes content of feeds
Herbage
n-alkane (mg kg-’ OM) C?, C28 C2’) C30 C3, C3? C33 C35
Supplement
At pasture
Indoor a
Maize
Maize gluten meal
14.4 6.8 79.7 13.2 307.3 5.6 63.0 2.1
12.7 5.0 73.6 12.0 304.1 5.2 68.5 2.0
0.6 1.4 1.1 0.4 2.2 0.5 2.0 0.2
1.5 1.5 3.3 0.9 2.7 0.3 0.9 0.0
’ The herbage used for the indoor trial was collected towards the end of the grazing period.
Table 3 Indoor trial: in vivo organic matter digestibility of diets and faecal recovery of markers Diet ’
No. of wethers OMD (%) Faecal recovery (%) Cr2G3 C31 C32 C33
Mean
SEM
H
M
MG
4 62.7 b
4 70.6 a
4 67.9 a
67.1
3.28
93.0 86.0 90.3 83.5
93.7 84.8 85.3 85.3
98.0 89.3 93.3 86.5
94.9 86.7 89.6 85.1
5.53 8.70 8.51 9.91
a H, herbage only; M, herbage plus maize; MG, herbage plus maize plus maize gluten meal. Means followed by different letters are significantly different (P< 0.05). Table 4 Indoor trial: differences between methods determining intake of herbage organic matter and differences between true intake and values obtained by indirect methods Diet ’
Herbage intake (g OM day- ’ ) Measured Chromium based n-alkanes based Differences between methods Differences from true intake (%) Chromium based n-alkanes based
Mean
SEM
H
M
MG
956 a 1033 a 913 a 120
663 b 711b 660 b 51
656 b 665 b 625 b 40
758 803 733 70*
92.0 127.9 98.0 66.3
7.5 -4.7
6.9 - 0.5
2.2 - 3.8
5.5 -3.0
9.85 6.94
a See footnote to Table 3. Means followed by different letters are significantly different (P < 0.05) ; * difference from 0 (P Q 0.05).
E. Piasentier/Small Ruminant Research 18 (1995) 27-32
31
Table 5 Field trial: effect of diets on intake of herbage organic matter in grazing ewes Diet M No. of observations ” Herbage intake ( g OM day _ ’ ) Chromium based b n-alkanes based Differences between methods
Mean
SEM
1503 1273 230 *
210.9 73.8 178.4
MG 12
12
1693 a 1313 a 380a
1313 a 1232 b 81b
a Three ewes for four experimental periods. b Calculated using one IVOMD value per period; the average of the four periods was 63.1+ 1.9%. Means followed by different letters are significantly different (P < 0.05); * difference from 0 (P < 0.05).
Recovery, correlation and content in herbage were assessed and C,, was chosen for estimating HI,,. HI,, exceeded HI,, in all diets (Table 4). With respect to the true value, the chromium method overestimated herbage intake by an average of 5.5%. This was exclusively due to the incomplete faecal recovery of the marker, as the calculation of HE, involved the use of mean digestibilities effectively measured. In contrast, herbage intake was underestimated (3% on average) with the double marker method, owing to the higher recovery of the dosed than the natural alkane. 3.3. Herbage intake of grazing ewes
Estimates of intake at pasture and the difference between methods are reported in Table 5. Both HI, and HI,, were influenced by diet and were higher with M than MG. As for the wethers, the intake by grazing ewes was lower when estimated by n-alkanes, but with a high difference between the methods (230 g equal to 16.6% of the mean estimated value). Feeding treatment significantly influenced this difference which was higher with diet M than diet MG. The different discrepancies between methods observed in the indoor and field trials could have been due to a lack of correspondence between the samples taken from the pasture and the forage effectively consumed. However, Vulich et al. ( 1993) did not observe significant differences in n-alkane concentrations between samples collected with hand plucking or from oesophageal fistulae, a reference method which, for ethical reasons, is now used much less frequently.
Another possible explanation of the fact that the difference observed between the two methods was greater in the field trial than the indoor trial could be the presence of associative effects between concentrates and forages, which would be much more evident in lactating ewes with their higher feeding level than in wethers at maintenance. In contrast, the differences between the diets could have depended upon the different rumen degradabilities of maize or maize gluten meal (Susmel et al., 1991). These effects would have altered the intake estimate calculated from Cr,03, in which a single digestibility value per period was adopted, but not that based on alkanes, which assign an estimate to each animal, as they take into account individual digestibility values. Dove et al. (1989) have also shown that the relationship between the two methods could be affected by the effect of level of intake on the digestibility of the grazed grass.
4. Conclusions
The trial showed the difficulties associated with obtaining accurate estimates of herbage intake with indirect methods, which, however, are the only techniques applicable with grazing animals. Of the two methods, the n-alkanes technique has some advantages: it involves less analytical work and appears not to be influenced by factors which could affect digestibility, such as supplementation with concentrates.
32
E. Piasentier/Small
Ruminant Research 18 (1995) 27-32
Acknowledgements This research was funded by a grant from Commission of the European Communities, Project CAMAR, Contract 90-002 1.
References Bovolenta, S., Piasentier, E. and Malossini, F., 1994. n-alkanes as markers in feeding trials. In: I.J. Gordon and R. Rubino (Editots), Proc. EEC Workshop on Grazing Behaviour of Goats and Sheep. Cahiers Options Mediterraneennes, Vol. 5. CIHEAM, Zaragoza, pp. 29-38. Dove, H. and Mayes, R.W., 1991. The use of plant wax alkanes as marker substances in studies of the nutrition of herbivores: a review. Aust. J. Agric. Res., 42: 913-952. Dove, H., Mayer., R.W., Freer, M., Coombe, J.B. and Foot, J.Z., 1989. Faecal recoveries of the alkanes of plant cuticular waxes in penned and in grazing sheep. Proc. XVI Int. Grassland Congress, Nice, France, pp. 1093-1094. Dove, H., Milne, J.A. and Mayes, R.W., 1990. Comparison of herbage intakes estimated from in vitro or alkane-based digestibilities. Proc. N.Z. Sot. Anim. Prod., 50: 457459. Gill, J.L., 1978. Design and Analysis of Experiments in the Animal and Medical Sciences. Iowa State University Press, Ames, 410 PP.
Laredo, M.A., Simpson, G.D., Minson, D.J. and Orpin, C.G., 1991. The potential for using n-alkanes in tropical forages as a marker for the determination of dry matter intake by grazing ruminants. J. Agric. Sci., 117: 355-361. Le Du, Y.L.P. and Penning, P.D., 1982. Animal based techniques for estimating herbage intake. In: J.D. Leaver (Editor), Herbage Intake Handbook. British Grassland Society, Hurley, UK, pp. 37-76. Malossini, F., Piasentier, E. and Bovolenta, S., 1990. n-alkane content of some forages. J. Sci. Food Agric., 53: 405409. Mayes, R.W., Lamb, C.S. and Colgrove, P.M., 1986. The use of dosed and herbage n-alkanes as markers for the determination of herbage intake. J. Agric. Sci., 107: 161-170. Minson, D.J., 1990. Intake of grazed forage. In: T.J. Cunha (Editor), Forage in Ruminant Nutrition. Academic Press, San Diego, CA, pp. 60-84. Susmel, P., Spanghero, M., Stefanon, B. and Mills, C.R., 1991. Performance of lactating Simmental cows fed two diets differing in the content of digestible intestinal protein (PDI) . Livest. Prod. Sci., 27: 157-175. Tilley, M.A. and Terry, R.A., 1963. A two stage technique for the in vitro digestion of forage crops. J. Br. Grassl. Sot., 18: 104-l 11. Vulich, S.A., Han&an, J.P. and O’Riordan, E.G., 1993. Pasture sampling for the estimation of herbage intake using n-alkanes: evaluation of alternative sampling procedures. Ir. J. Agric. Food Res., 32: l-l 1. Williams, C.H., David, D.J. and Iismaa, O., 1962. The determination of chromic oxide in faeces samples by atomic absorption spectrophotometry. J. Agric. Sci., 59: 381-385.
Research18 ( 1995) 27-32
Comparison of n-alkanes or chromium oxide methods for estimation of herbage intake by sheep E. Piasentier *, S. Bovolenta, F. Malossini, P. Susmel Dipartimento di Scienze della Produzione Animale, Universitcidi Udine, Via S. Mauro. 2. 33010 Pagnacco (UD), Italy
Accepted15 January 1995
Abstract Herbage intake (HI) was estimated with two procedures: (a) with the n-alkanes method (HI,,), which combines the use of odd-chain n-alkanes, naturally occurring in plants, and dosed even-chain n-alkanes, as internal and external markers, respectively; (b) with the ratio between faecal excretion, estimated with chromium sesquioxide (Cr*Oa), and the herbage indigestibility, estimated in vitro (HI,,). The two techniques were compared in an indoor experiment and under grazing conditions. In the indoor trial, 12 wethers were fed 40 g dry matter (DM) kg- ’ BW”.75day-’ of one of the following diets: herbage only (diet H) ; herbage plus maize (diet M; 26% of DM intake); herbage plus maize plus maize gluten meal (diet MG; 13% of DM intake each). Each wether was dosed once a day for 12 days with the external indicators impregnated on paper and inserted in gelatine capsules: n-dotriacontane, C3*.600 mg day-‘; Cr203, 500 mg day-‘. In the second experiment, six adult, lactating ewes grazing the pasture used for the indoor trial were divided into groups of three receiving 700 g day - ’ of two different supplements: maize (diet M) or a 5050 mix of maize and maize gluten meal (diet MG) .The external indicators ( Cr203 and t&) were administered for 8 consecutive days. The trial was repeated four times with the same procedure between 10th and the 40th day after lambing. Recoveries of Cr,Ol, C3*and herbage n-alkanes (n-hentriacontane, Csir and n-tritriacontane, C3s) were incomplete, with lower values for the natural n-alkanes than the dosed n-alkane (Cr203, 94.9* 5.5%; Ca,, 86.7+ 8.7%; &, 89.6+ 8.5%; C33, 85.1 + 9.9%). Therefore, HI,, gave an overestimate of the true herbage intake of 5.5% and HI,, an underestimate of 3.0%, with a significant discrepancy between methods (803 vs. 733 g organic matter (OM) day-‘), not affected by diet. In the grazing trial, HI,-, was higher than HI,, but the difference between methods (1503 vs. 1273 g OM day-‘) was higher than that obtained in the indoor experiment and was influenced by diet (380 g OM day- ’ vs. 81 g OM day- ’ with diet M and diet MG, respectively). A possible explanation of the different results obtained indoor or at pasture is discussed. Keywords:
n-alkanes; Chromiumoxide; Herbage intake; Grazing ewes
1. Introduction
Of the methods available for estimating herbage intake by grazing animals, the most commonly used is based on the ratio between faecal output, estimated with an external marker, usually Cr,03, and herbage indi* Corresponding
author
0921~4488/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSD10921-4488(95)00712-l
gestibility, which is either known or estimated in vitro (Le Du and Penning, 1982; Minson, 1990). As an alternative, Mayes et al. ( 1986) proposed the combined use of odd-chain n-alkanes, naturally occurring in the cuticular waxes of plants, and even-chain nalkanes present in much smaller amounts in herbage and commercially available, as internal and external
E. Piasentier/Small Ruminant Research 18 (1995) 27-32
28 Table 1 Chemical composition of feeds
Herbage
DM CP Ether extract Ash NDF ADF ADL
(%) (% DM) (%DM) (% DM) (% DM) (% DM) (% DM)
Supplement
At pasture
Indoor a
Maize
Maize gluten meal
22.2 16.3 4.8 7.6 56.6 33.1 3.2
23.0 13.3 3.4 8.2 61.9 35.0 3.6
87.6 7.7 1.1 0.5
90. I 62.9 3.3 6.5
-
a The herbage used for the indoor trial was collected towards the end of the grazing period. CP, crude protein; NDF, neutral detergent fibre; ADF, acid detergent fibre; ADL, acid detergent lignin.
markers to estimate digestibility and faecal production respectively. Carbon-chain lengths of the main n-alkanes detected in herbage species are usually in the range from npentacosane ( CZs) to n-pentatriacontane ( Cs5). Of these hydrocarbons, n-hentriacontane ( C3i ) and n-tritriacontane (C,,) are of particular interest as internal markers because they are dominant alkanes in all species and have high faecal recoveries, which are similar to those of the dosed adjacent homologue, n-dotriacontane (C& (Dove et al., 1989; Dove and Mayes, 1991). This latter condition, as discussed by Mayes et al. ( 1986), provides an estimate of herbage intake which is not biased by the incomplete recovery of markers and is independent of the diet digestibility. The aim of the present research, conducted as part of a study about the effect of dietary protein level for grazing ewes, was to evaluate the two intake estimation techniques. The first trial, conducted with animals held in digestibility crates, aimed to evaluate the faecal recovery of the markers and the accuracy of the techniques while a second experiment, with grazing ewes, compared the two methods under field conditions.
2. Materials and methods 2.1. Indoor trial Twelve Bergamasca wethers (body weight (BW) 73.2f 10.3 kg), held in digestibility crates, were fed
40 g dry matter (DM) kg-’ Bw.” day- ’ of one of the following diets (four animals per diet) : herbage only (H) ; herbage plus maize (26% of DM intake) (M) ; herbage plus maize plus maize gluten meal ( 13% of DM intake each) (MG) . The herbage, represented by a second regrowth of a monoculture fescue meadow (Festuca arundinacea, cv. “Manade”), was cut daily to a height of 5 cm from protected areas of the pasture grazed by the ewes. Each wether was dosed once a day for 12 days with the external indicators impregnated on paper and inserted in gelatine capsules: Cs2, 600 mg day-‘; Cr203, 500 mg day- ‘. In the last 7 days, which corresponded to the final phase of the grazing period, a digestibility trial was performed. Samples of herbage, concentrates and faeces were collected daily and dried for analysis. The feeds were analysed for their proximate composition, Van Soest fibre fractions and n-alkane concentrations (Mayes et al., 1986)) while the faeces samples were used for the determination of their concentrations of n-alkanes and Cr203 (Williams et al., 1962). Table 1 reports the average chemical composition of feeds. The dietary OM digestibility (OMD), the percentage recovery of each marker in the faeces and the correlation coefficients between the faecal recovery of the dosed and natural n-alkanes were calculated. OMD of the supplements (OMD,) was determined indirectly from digestibility values of diet H (OMD,,) and the mixed diets. The quantity of herbage ingested (HZ, kg OM day- ’ ) was calculated with the chromium method HI,,=[Dij/Fjj-Is(l-OMD,)]/(l-OMD,)
E. Piasentier/Small
Ruminant Research 18 (1995) 27-32
and with the alkanes method HI,,= [F,/F,(Dj+IsXSj)
-SXSi]/
(Hi-Fi/FjxHj) where D (mg day- ‘) is the external marker ( Dj represents C3* and Djj Cr,03), F (mg kg- ’ OM) is the faecal concentration (Fj represents C3?, Fi Cfl and Fii Cr,O,), H (mg kg- ’ OM) is the herbage concentration (Hi represents C,, and Hi C3,), Is (kg OM day-‘) is the quantity of any supplement fed and S (mg kg- ’ OM) is the supplement concentration (Sj represents C32 and Si C3i ) . The accuracy of the intake estimate with the two methods was expressed as a percentage difference from true intake. Calculated variables (Yij) were analysed with a monofactorial model Y, =
/_l +
29
Intake estimate and the calculation of the discrepancy between the methods were performed, as in the indoor trial, by using the same OMD, values and adopting in vitro measurements, calibrated from the in vivo digestibility, for OMD,. Intake estimated with each method and the difference between them were analysed with a split-plot design for repeated measures (Gill, 1978) using the following model yijk=
p”+
aiis
P(i)j
+
yk+
( ay)ik+
E(i)jk
where Qr is the fixed effect of diet (i = 1..2), P~i,j is the nested effect of animal (j = 1..3) and the experimental error to test Cui,yk is the fixed effect of period (k= 1..4) and lcijjk the residual error.
3. Results and discussion
ai + Eij
where (Y is diet, with three levels repeated on four wethers (j = 1..4).
(i = 1..3), each
2.2. Field trial Six adult lactating Bergamasca ewes (73.9 + 8.0 kg SW) grazed the same pasture utilised for the indoor trial from 29 June to 6 August. Sheep, reunited into a single flock, were moved daily to a fresh pasture area ( 10 m2 per head) enclosed with a mobile electric fence. Three ewes received a supplement of 700 g day-’ of maize (diet M), the other three a 50:50 mix of maize and maize gluten meal (diet MG) offered in two meals during milking. From the 4th day after lambing, the ewes were dosed daily with the same quantity of Ca2 and Cr,Oa used in the indoor trial. Herbage intake was estimated four times, in different areas on the same pasture, with the same procedure between the 10th and the 40th day after lambing. During the last 2 days of each period, three grab samples (at 07:00, 12:OO and 18:OO h) were taken from each animal; these were later bulked to form a single period sample for the analysis of Cr203 and nalkanes. Herbage samples were hand plucked from two 4 m2 isolated areas, simulating removal by the animals. Chemical composition, in vitro digestibility (Tilley and Terry, 1963) and n-alkane content were determined. Table 1 reports the average chemical composition of feeds.
3.1. Marker concentrations in herbage The most common n-alkanes in the herbage (Table 2) were CZ9, Ca, and Ca3, with a net predominance of C3, (about 62% of the total). The quantitative importance of CZ9 and C3i alkanes, in temperate grass, has been reported previously (Mayes et al., 1986; Dove et al., 1990; Malossini et al., 1990; Laredo et al., 1991). Concentrates had a very low n-alkane content, with concentrations never exceeding 3.1 mg kg- ’ OM. 3.2. In vivo digestibility, faecal recovery of markers and accuracy of methods Table 3 reports the in vivo digestibility of the herbage and the M and MG diets; these values were used to estimate the digestibility of the supplements (90.7% for the maize and 8 1.7% for the maize and gluten mix). In the same table the recoveries of Cr,Oa, the dosed alkane Ca2 and the two natural adjacent homologues C,, and Cs3 are reported. In all cases, the recoveries were incomplete and not influenced by the diet, with higher values for Cr203 and C32 than the two natural alkanes. The C3i-Ca2 alkane pair had, on average, the most similar recoveries. The observed values fell within the range of data reported in the literature (Le Du and Penning, 1982; Dove and Mayes, 1991; Bovolenta et al., 1994). The correlation between Ca2 and C3i (r=0.73**) was higher than that between Cs2 and C33 (r = 0.54).
E. Piasentier/SmaN Ruminant Research 18 (1995) 27-32
30 Table 2 n-alkanes content of feeds
Herbage
n-alkane (mg kg-’ OM) C?, C28 C2’) C30 C3, C3? C33 C35
Supplement
At pasture
Indoor a
Maize
Maize gluten meal
14.4 6.8 79.7 13.2 307.3 5.6 63.0 2.1
12.7 5.0 73.6 12.0 304.1 5.2 68.5 2.0
0.6 1.4 1.1 0.4 2.2 0.5 2.0 0.2
1.5 1.5 3.3 0.9 2.7 0.3 0.9 0.0
’ The herbage used for the indoor trial was collected towards the end of the grazing period.
Table 3 Indoor trial: in vivo organic matter digestibility of diets and faecal recovery of markers Diet ’
No. of wethers OMD (%) Faecal recovery (%) Cr2G3 C31 C32 C33
Mean
SEM
H
M
MG
4 62.7 b
4 70.6 a
4 67.9 a
67.1
3.28
93.0 86.0 90.3 83.5
93.7 84.8 85.3 85.3
98.0 89.3 93.3 86.5
94.9 86.7 89.6 85.1
5.53 8.70 8.51 9.91
a H, herbage only; M, herbage plus maize; MG, herbage plus maize plus maize gluten meal. Means followed by different letters are significantly different (P< 0.05). Table 4 Indoor trial: differences between methods determining intake of herbage organic matter and differences between true intake and values obtained by indirect methods Diet ’
Herbage intake (g OM day- ’ ) Measured Chromium based n-alkanes based Differences between methods Differences from true intake (%) Chromium based n-alkanes based
Mean
SEM
H
M
MG
956 a 1033 a 913 a 120
663 b 711b 660 b 51
656 b 665 b 625 b 40
758 803 733 70*
92.0 127.9 98.0 66.3
7.5 -4.7
6.9 - 0.5
2.2 - 3.8
5.5 -3.0
9.85 6.94
a See footnote to Table 3. Means followed by different letters are significantly different (P < 0.05) ; * difference from 0 (P Q 0.05).
E. Piasentier/Small Ruminant Research 18 (1995) 27-32
31
Table 5 Field trial: effect of diets on intake of herbage organic matter in grazing ewes Diet M No. of observations ” Herbage intake ( g OM day _ ’ ) Chromium based b n-alkanes based Differences between methods
Mean
SEM
1503 1273 230 *
210.9 73.8 178.4
MG 12
12
1693 a 1313 a 380a
1313 a 1232 b 81b
a Three ewes for four experimental periods. b Calculated using one IVOMD value per period; the average of the four periods was 63.1+ 1.9%. Means followed by different letters are significantly different (P < 0.05); * difference from 0 (P < 0.05).
Recovery, correlation and content in herbage were assessed and C,, was chosen for estimating HI,,. HI,, exceeded HI,, in all diets (Table 4). With respect to the true value, the chromium method overestimated herbage intake by an average of 5.5%. This was exclusively due to the incomplete faecal recovery of the marker, as the calculation of HE, involved the use of mean digestibilities effectively measured. In contrast, herbage intake was underestimated (3% on average) with the double marker method, owing to the higher recovery of the dosed than the natural alkane. 3.3. Herbage intake of grazing ewes
Estimates of intake at pasture and the difference between methods are reported in Table 5. Both HI, and HI,, were influenced by diet and were higher with M than MG. As for the wethers, the intake by grazing ewes was lower when estimated by n-alkanes, but with a high difference between the methods (230 g equal to 16.6% of the mean estimated value). Feeding treatment significantly influenced this difference which was higher with diet M than diet MG. The different discrepancies between methods observed in the indoor and field trials could have been due to a lack of correspondence between the samples taken from the pasture and the forage effectively consumed. However, Vulich et al. ( 1993) did not observe significant differences in n-alkane concentrations between samples collected with hand plucking or from oesophageal fistulae, a reference method which, for ethical reasons, is now used much less frequently.
Another possible explanation of the fact that the difference observed between the two methods was greater in the field trial than the indoor trial could be the presence of associative effects between concentrates and forages, which would be much more evident in lactating ewes with their higher feeding level than in wethers at maintenance. In contrast, the differences between the diets could have depended upon the different rumen degradabilities of maize or maize gluten meal (Susmel et al., 1991). These effects would have altered the intake estimate calculated from Cr,03, in which a single digestibility value per period was adopted, but not that based on alkanes, which assign an estimate to each animal, as they take into account individual digestibility values. Dove et al. (1989) have also shown that the relationship between the two methods could be affected by the effect of level of intake on the digestibility of the grazed grass.
4. Conclusions
The trial showed the difficulties associated with obtaining accurate estimates of herbage intake with indirect methods, which, however, are the only techniques applicable with grazing animals. Of the two methods, the n-alkanes technique has some advantages: it involves less analytical work and appears not to be influenced by factors which could affect digestibility, such as supplementation with concentrates.
32
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Ruminant Research 18 (1995) 27-32
Acknowledgements This research was funded by a grant from Commission of the European Communities, Project CAMAR, Contract 90-002 1.
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