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Use of the N-alkanes to Estimate Intake, Apparent Digestibility and Diet Composition in Sheep Grazing on Stipa breviflora Desert Steppe
Journal of Integrative Agriculture 2014, 13(5): 1065-1072
May 2014
RESEARCH ARTICLE
Use of the N-alkanes to Estimate Intake, Apparent Digestibility and Diet Composition in Sheep Grazing on Stipa breviflora Desert Steppe HU Hong-lian1*, LIU Yong-zhi2, 3*, LI Ya-kui4, LU De-xun1 and GAO Min1 1
Institute of Animal Nutrition and Feed, Inner Mongolia Academy of Agricultural & Animal Husbandry Sciences, Hohhot 010031, P.R.China Inner Mongolia Prataculture Research Center, Chinese Academy of Sciences, Huhhot 010031, P.R.China 3 Institute of Botany, Chinese Academy of Sciences, Beijing 100093, P.R.China 4 College of Animal and Technology, Hebei North University, Zhangjiakou 075000, P.R.China 2
Abstract The application of n-alkane as markers to estimate herbage intake, apparent digestibility and species composition of diet consumed by grazing sheep was studied. Six local Mongolian sheep were used to determine dry matter (DM) intake, apparent DM digestibility and species composition of diet during summer, autumn and winter. Animals were orally dosed twice daily with n-alkane gelatin capsules containing 60 mg C32-alkane as an external marker. Diet composition was estimated by comparing the odd-chain n-alkanes pattern profile (C27-C31) of the consumed plant species with the n-alkanes fecal concentrations of grazing animals, using a non-negative least squares algorithm called EATWHAT software package. The alkane pair C32:C33 and C33 alkane were used to estimate DM intake and diet apparent DM digestibility, respectively. The results showed that daily dry matter intake of the sheep were 1.77, 1.61 and 1.18 kg d-1 in summer, autumn and winter, respectively. Apparent DM digestibility, crude protein (CP), metabolizable energy (ME) and neutral detergent fiber (NDF) intake of diet consumed by sheep decreased significantly (P<0.01) from summer to winter, with no evident changes in ADF and ADL intake. Diet composition indicated Artemisia frigida Willd was the most dominant diet component, contributed 79.68, 68.12 and 86.26% of sheep’s diets in summer, autumn and winter, respectively. Cleistogenes songorica Ohwi and Convolvulus ammannii Desr were the important components of the diet. Although Stipa breviflora Griseb is one of the main plant species in the study area, the sheep rarely choosed it. The study indicated that CP and ME in diet consumed by sheep were deficient in winter. Therefore, appropriate supplementation strategies should be indispensable during this period. Key words: n-alkanes, diet composition, intake, apparent digestibility, sheep
INTRODUCTION China belongs to one of the largest grassland regions of the world (Glindemann et al. 2009). In northern China, there are vast areas of grasslands in both arid and alpine environments, and used for grazing and supporting more than 55% of the sheep population
in China. Grazing is a nutritional necessity for most of the China’s sheep, but information about what and which plants grazing animals consumed are limited, which has restricted progress towards the development of sustainable grazing systems. The nutrient status of the grazing animals mainly depends on the nutritive value of the plants available, botanical composition of the consumed diet and the intake of animal (Dove
Received 27 December, 2012 Accepted 25 April, 2013 HU Hong-lian, Mobile: 13947198168, E-mail: [email protected]; Correspondence LIU Yong-zhi, Tel: +86-471-5295028, E-mail: [email protected] * These authors contributed equally to this study. © 2014, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S2095-3119(13)60502-X
HU Hong-lian et al.
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et al. 1996). Accurate measurement of feed intake and digestibility are important to meet nutritional requirements of the animal and optimize production (Bezabih et al. 2012). However, the estimation of those parameters is very difficult, complicated and laborious in grazing conditions, with errors often unacceptable due to limitations of available methods of measurement (Keli et al. 2008). Fortunately, n-alkanes as markers has been widespread for the last two decades and successfully applied to estimate intake, digestibility and diet composition in ruminants, such as sheep (Lewis et al. 2003; Valiente et al. 2003; Keli et al. 2008), goat (Sun et al. 2008), yak (Ding and Long 2010), beef steer (Premaratne et al. 2005; Chopa et al. 2012) and cattle (Morais et al. 2011; Bezabih et al. 2012). N-alkanes are saturated hydrocarbons with chain length varying from 21 to 35 carbon atoms, which can be found in the wax cuticle of most plants (Dove and Mayes 1996). The n-alkanes with odd-number carbon chains predominate and the relative levels and the patterns of n-alkane components differ among the plant species (Peiretti et al. 2006). As n-alkanes are mainly indigestible and have high feacal recovery rates (Ferreira et al. 2009), they are suitable markers to estimate diet selection and nutrient intake. The advantages of n-alkanes as marker include low invasiveness, accuracy and the possibility of taking into account diet-animal interactions (Bezabih et al. 2012). In addition, if the animals are dosed with even-chain alkane, estimates of total intake and the proportions of plant species in the diet of grazing animals can be obtained simultaneously using only one analytical procedure (Dove and Mayes 1996, 2005).
Although the n-alkane technique is widely applied in other parts of the world, there is limited information on its applications in China desert steppe ecosystems. Therefore, the objective of this study was to use the n-alkane technique to estimate simultaneously dry matter intake, apparent dry matter digestibility and diet composition selected by sheep grazing on desert steppe, and evaluate the quality of the diet during the year. This information is required in order to know aspects of selectivity and optimal timing of grazing, as well as to assure adequate livestock production.
RESULTS Herbage and fecal n-alkanes concentration Herbage n-alkane concentrations (mg kg -1 dry matter, DM) in different grazing seasons were showed in Table 1. As expected, concentration of C32 alkane in the most species was very low and almost zero, which was the reason for using C32 alkane as external marker as its presence in plants was almost negligible. Concentrations of C31 and C33 alkanes were the highest in herbage species, while C27 and C29 alkanes had very low concentrations, which could limit their use for intake calculation. The different herbage species differed in their alkanes patterns. In summer pasture, Stipa breviflora Griseb and Cleistogenes songorica Ohwi had the highest concentration of C 31 alkane (P<0.05),whist Cleistogenes songorica Ohwi and Convolvulus ammannii Desr had higher concentration
Table 1 N-alkanes concentration of diet components consumed by sheep in summer, autumn and winter pasture (mg kg-1 dry matter, DM) Species In summer pasture Artemisia frigida Willd Stipa breviflora Griseb Cleistogenes songorica Ohwi Convolvulus ammannii Desr In autumn pasture A. frigida Willd S. breviflora Griseb C. songorica Ohwi C. ammannii Desr In winter pasture A. frigida Willd S. breviflora Griseb C. songorica Ohwi C. ammannii Desr
C27
C29
C31
C32
C33
148.5±18.0 c 342.3±48.3 a 39.1±4.2 d 221.3±23.2 b
326.4±25.1 a 108.9±16.2 c 137.9±12.5 b 121.9±6.1 b
254.9±19.2 c 1 664.1±26.6 a 657.6±86.0 b 177.1±8.3 d
0 0 143.4±24.3 43.0±2.9
126.6±12.6 d 327.1±9.4 c 525.5±19.0 a 432.1±27.6 b
143.1±3.0 c 443.4±38.1 a 32.7±49 d 209.5±33 b
174.3±5.1 a 156.3±16.2 a 109.1±43 b 107.8±97 b
164.5±4.3 d 219.5±13.1 c 433.6±22.5 b 928.8±98 a
0 206.9±3.5 112.8±12.1 0
251.1±21.0 c 683.6±2.2 a 662.4±13.4 a 382.3±4.0 b
111.0±3.8 c 392.5±4.7 a 52.0±2.3 d 212.7±35.4 b
194.8±11.3 a 133.9±9.1 b 127.4±1.9 b 98.1±9.1 c
184.4±12.5 c 110.9±11.2 d 734.9±7.1 b 1 630.1±76.8 a
23.2±2.2 0 0 0
466.9±16.2 b 471.6±77.8b 661.7±9.1 a 336.7±19.02 c
Different small letters within the same column indicate significant differences (P<0.05). Data are means±SD (n=2).
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Use of the N-alkanes to Estimate Intake, Apparent Digestibility and Diet Composition in Sheep Grazing on Stipa breviflora
of C33 alkane than other species (P<0.05). In autumn pasture, C. ammannii Desr had highest concentration of C31 alkane, the highest concentrations of C33 alkane were found in S. breviflora Griseb and C. songorica Ohwi (P<0.05). In winter pasture, the highest concentration of C31 alkane was found in C. ammannii Desr with the lowest concentration of C33 alkane (P<0.05). The highest concentrations of C27 and C29 alkane were found in S. breviflora Griseb and Artemisia frigida Willd in three grazing seasons, respectively. Fecal n-alkanes concentrations (mg kg -1 DM) in
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different grazing seasons were presented in Table 2. Fecal concentrations of n-alkanes were corrected using published recovery rates reported by Newman et al. (1995). Fecal concentrations of odd-chain n-alkanes (C27-C33) tended to increase with carbon chain length. Fecal concentration of C33 alkane was highest (P<0.05). With season advance, C 27, C29, C31 and C32 alkanes concentrations in fecal samples decreased, whereas C33 alkane concentration increased. The mean fecal concentrations for most n-alkanes were the lowest in winter except for C33 alkane.
Table 2 N-Alkanes pattern of fecal samples from sheep in summer, autumn and winter (mg kg-1 DM) Peroid Summer Autumn Winter
C27 291.43±7.63 d 199.62±5.18 d 136.08±5.94 d
C29 629.75±13.69 b 243.68±3.64 c 194.42±11.48 c
C31 768.58±16.10 a 462.03±5.46 b 315.72±8.54 b
C32 264.27±5.49 d 208.88±2.17 d 200.67±6.38 c
C33 525.10±5.10 c 699.57±8.34 a 812.98±10.11 a
Diet composition obtained using n-alkanes profiles in fecal and pasture samples in each experimental period were presented in Figs. 1-3. In present study, A. frigida Willd was the most important dietary component of sheep in three seasons. A. frigida Willd constituted 79.68, 68.12 and 86.26% of the sheep’s diet in summer, autumn and winter, respectively. S. breviflora Griseb was minor component of sheep diet in three seasons. In summer, the sheep showed major preference for A. frigida Willd, which together with C. songorica Ohwi and Convolvulus ammannii Desr, comprised more than 98% of the diet. In autumn, the sheep consumed mostly A. frigida Willd (68.12%) and C. songorica Ohwi (23.57%), following similar amounts of S. breviflora Griseb (3.81%) and C. ammannii Desr (4.50%). In winter, the sheep’s diet was dominated by A. frigida Willd (86.26%) along with C. songorica Ohwi (8.33%) and C. ammannii Desr (5.41%), while no S. breviflora Griseb was detected in the sheep’s diet. The estimates of dry matter intake and apparent digestibility of herbage consumed by the grazing sheep using the pair C33:C32 and C33 alkane, respectively, were presented in Table 3. There were significant effects of grazing season on dry matter intake
90 80 70 60 50 40 30 20 10 0
Artemisia frigida Willd
Stipa breviflora Cleistogenes Griseb songorica Ohwi
Convolvulus ammannii Desr
Fig. 1 Diet composition of sheep in summer pasture. Vertical line on each bar represents standard error of the mean (n=6). The same as below. 80 70 Propotion in diet (%)
Estimations of diet composition, intake and apparent digestibility
Propotion in diet (%)
Different small letters within the same row indicate significant differences (P<0.05). Data are means±SE (n=6). The same as below.
60 50 40 30 20 10 0 Artemisia frigida Willd
Stipa breviflora Cleistogenes Convolvulus Griseb songorica Ohwi ammannii Desr
Fig. 2 Diet composition of sheep in autumn pasture.
(DMI) (P<0.01) and apparent DM digestibility of the diet selected (P<0.01). The daily dry matter intake of sheep was higher in summer and autumn (1.77 and 1.61 kg d-1, respectively) and lower in winter (1.18 kg d-1) (P<0.01). There was no difference in © 2014, CAAS. All rights reserved. Published by Elsevier Ltd.
HU Hong-lian et al.
Propotion in diet (%)
1068 100 90 80 70 60 50 40 30 20 10 0
DISCUSSION
Artemisia frigida Willd
Cleistogenes songorica Ohwi
Convolvulus ammannii Desr
Fig. 3 Diet composition of sheep in winter pasture. Table 3 Dry matter intake and apparent dry matter digestibility of diets consumed by sheep in summer, autumn and winter Period Summer Autumn Winter
DMI (g d-1) 1 769.99±61.45 A 1 610.19±61.47 A 1 178.32±48.90 B
DMD (g kg-1 DM) 661.80±4.34 A 527.02±5.51 B 467.8±7.21 C
DMI, dry matter intake; DMD, dry matter digestibility. Different capital letters within the same column indicate significant differences (P<0.01). The same as below.
DMI per sheep between summer and autumn. The apparent DM digestibility of the diet consumed by sheep was the highest in summer and the lowest in winter (P<0.01). Nutrient intake calculated using the estimates of DMI and diet apparent digestibility obtained using the n-alkane technique was presented in Table 4. The experimental period had a significant effect on the crude protein (CP), metabolizable energy (ME) and neutral detergent fiber (NDF) intake of sheep, no seasonal differences were observed in acid detergent fiber (ADF) and acid detergent lignin (ADL) intake of sheep. With season advance, CP and ME intake of sheep decreased significantly (P<0.01) from 286.74 g d-1 or 14.97 MJ d-1 in summer to 97.53 g d-1 or 6.42 MJ d-1 in winter, respectively. NDF intake was the lowest in winter (P<0.01), differences in NDF intake were not observed between the summer and autumn. It was clear that the CP and ME intake of sheep appeared seasonal fluctuations.
Knowledge of dry matter intake (DMI) and digestibility is usefully in assessing the nutrient status of grazing animals. The double n-alkane marker technique has been developed to allow estimation of DMI, apparent digestibility and botanical composition of diet consumed by animals. An important assumption when using the double n-alkane marker technique is that adjacent n-alkane used in the estimates of intake (dosed even-chain alkane and natural odd-chain alkane) has similar faecal recovery. So many studies have evaluated the suitability of different odd- and even-chain n-alkane pairs for estimating intake (Dove et al. 2002; Oliván et al. 2007). And in most of these studies, n-alkane pair C32:C31 and C32:C33 had the lowest discrepancy in feacal recovery, and hence were proposed to estimate DMI (Dove et al. 2002). Estimates of DMI using the ratio C32:C33 was more reliable than the ratio C32:C31 (Smit et al. 2005; Chopa et al. 2012) and more close to the measured intakes (Molina et al. 2004). For this reason, in the present study, the alkane pair C32:C33 and C33 as internal marker was used to estimate DM intake apparent DM digestibility. Accurate assessment of the concentration of the natural and dosed alkane in the feces to estimate DMI is critical to the validity of the alkane technique. Daily cyclic variation in fecal alkane concentration could lead to inaccurate intake estimate (Molina et al. 2004). In indoor experiments, total faeces would clearly provide the most representative samples of faces, but under grazing conditions, grab sampling is required. For this reason, in the present study, representative samples of faces were obtained by collecting rectal grab samples at 2 h interval from day 11 to 15. Furthermore, fecal n-alkane concentrations were corrected using published recovery rates reported by Newman et al. (1995). However, for accurate estimation of herbage DMI, apparent digestibility and diet composition, actual n-alkane fecal
Table 4 Estimated nutrient intake of diets consumed by sheep when intake and digestibility were derived from diet composition estimates Period Summer Autumn Winter
CP (g d-1) 286.74±0.71 A 210.43±2.09 B 97.53±7.38 C
ME (MJ d-1) 14.97±0.13 A 10.93±0.27 B 6.42±0.67 C
NDF (g d-1) 832.61±6.90 A 835.03±5.07 A 664.16±21.65 B
ADF (g d-1) 590.56±1.86 529.37±2.25 547.34±42.54
ADL (g d-1) 110.27±1.06 118.74±0.72 115.11±4.89
CP, crude protein; ME, metabolizable energy; NDF, neutral detergent fiber; ADF, acid detergent fiber; ADL, acid detergent lignin.
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Use of the N-alkanes to Estimate Intake, Apparent Digestibility and Diet Composition in Sheep Grazing on Stipa breviflora
recoveries rates should be used, and obtaining a representative fecal and herbage sample is essential. In this study area, A. frigida Willd was one of the most abundant browse species and important dietary component of sheep, contributed 79.68, 68.12 and 86.26% of sheep’s diet in summer, autumn and winter, respectively. Similar results were obtained by Biligetu (2003) and Wang (2009) who performed in moderate grazing intensity from same experiment area. The dominant specie, C. songorica Ohwi was larger proportion of the diet compared with C. ammannii Desr in whole experiment periods. The reason was that C. songorica Ohwi provided the greater cover and the height of C. ammannii Desr was lower (Liu 2005). Although S. breviflora Griseb was one of the main plant species in the study area, the sheep rarely chose it when good palatability or other nutritious species were available. These findings could be associated with plant prehensibility, particularly plant height and the presence of secondary compounds in these species. In the present study, CP, NDF, ADF and ADL contents in the diets were estimated by the sum of the product of the proportion that each plant species in the diet and their nutrient concentrations. ME concentration in the diet was calculated from apparent DM digestibility estimated using C33 alkane. With the decrease of herbage availability and nutritional value from summer to winter, the sheep decreased their DMI and intakes of both CP and ME. The intakes of both CP and ME for sheep were adequate to meet their nutritional demands for growth only from grazing in summer and autumn, according to the established requirement of sheep (NRC 1981). While in winter, both CP and ME intakes were deficient causing body weight gain loss (Hu 2005). These results were agreement with previous reports (Wang et al. 1997), which reported the lack of both CP and energy intakes were main nutritional constraints of grazing sheep in winter and spring in northern China. Other experiment also showed the CP content of forages may fall to less than 70 g kg-1 DM in long dry season (Tefera et al. 2009), which is considered to be limiting for an optimum rumen microbial growth and fermentation. In this study area, green herbage is available for only 5 mon (approximately mid May to mid October), with severe shortages of herbage during remaining 7
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mon. The nutrient intake is not able to meet nutrient requirements of grazing animals only from grazing in winter and spring. Therefore, it was important that appropriate feed supplementation should be considered to ensure productivity and health of grazing sheep during this period.
CONCLUSION The results from the present study showed that the n-alkane technology could be used to estimate simultaneously DM intake, apparent DM digestibility and diet selection of sheep under the particular conditions of this study. However, for accurate estimation of these parameters, actual alkane fecal recoveries should be used, and obtaining a representative fecal and herbage sample is essential. There was significant effect of grazing season on DM intake and apparent DM digestibility of the diet selected by sheep. Diet composition indicated A. frigida Willd and Cleistogenes songorica Ohwi were the most dominant diet component, but in different proportions at different seasons, Stipa breviflora Griseb was selected rarely. It was clear that the CP and ME intake of sheep appeared seasonal fluctuations. Both CP and ME intakes were deficient in winter, thus feed supplementation should be required.
MATERIALS AND METHODS Study site This study was conducted in the desert steppe in Siziwang Banner, Inner Mongolia (41°10´N longitude, 110°20´E latitude, about 1 430-1 506 m in elevation), where the longterm averages of annual mean air temperature and annual rainfall are 2.9°C and 200 mm, respectively. It has typical geographical representation of the desert steppe and farmingpastoral zone. The soil of the area around study site is sand chestnut. The herbage vegetation of the experimental plot consisted mainly Stipa breviflora Griseb, Artemisia frigida Willd, Cleistogenes songorica Ohw, Convolvulus ammannij Desr, Hetropappus altaicus Novepokr, Kochia prostrata Schrad, Caragana stenophylla Pojark, and Leymus chinensis Tzvel. S. breviflora Griseb, A. frigida Willd, C. songorica Ohwi and C. ammannij Desr are the dominant plant species in this area. Their percentage of plant production are 66.0, 8.2,
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6.9 and 10.4%, respectively. And these contributed more than 90% to total biomass yield (Liu 2005). The height and coverage of herbage community are about 8 cm, 17 to 20%, respectively. Due to grazing pressure, there has been some deterioration of the natural vegetation in the last decades.
according to Association of Official Analytical Chemists (1990). Neutral detergent fiber (NDF), acid detergent fiber (ADF) and acid detergent lignin (ADL) following van Soest et al. (1991). Concentrations of n-alkanes were determined by gas chromatography (Mayes et al. 1986).
Animals
N-alkane analysis
Six local Mongolian sheep, aged two-year old and weighing between 30 and 35 kg were used. Sheep were dewormed prior to the experiment. Sheep were grazed for 8 h per day in an area of 1.2 ha from summer to winter in three experimental periods: Summer (from middle June to early July), autumn (September) and winter (November). No supplementary feed and no minerals were supplied to all sheep throughout the grazing periods.
Samples were analyzed for n-alkanes C27-C33 concentrations by means of gas chromatography by the method of Mayes et al. (1986). Dried and milled herbage (1.2 g) and fecal samples (0.6 g) were weighted into screw-capped flasks prior to the addition of 50 μL of C34 internal standard (Sigma Chemical, St. Louis, Mo, USA) and 10 mL alcoholic KOH, overnight. The samples were heated for 2 h at 90°C. After cooling of samples, the alkanes were extracted by adding 3 mL of heptane and 2 mL of distilled water, the tubes were shaken vigorously and placed in a water bath at 45°C for 5 min. The nonaqueous top liquid layer was transferred to a glass vial and further extracted with 3 mL of heptane. The samples were evaporated to dryness, redissolved in 2 mL of heptanes and applied to the small column containing silica gel (200-400 mesh, Merck, China) with a 3-cm bed volume and glass fiber (0.5 cm). The hydrocarbons were eluted from the columns with heptane, evaporated and redissolved in 1 mL of heptane. 1 μL of each final redissolved n-alknae solution was injected into GC-9A gas chromatograph (Shimadzu Company, Japan) fitted with flame ionization detector. The column was a 30 m×0.53 mm insidediameter capillary column type SPB1 with 0.5 μmol L -1 film thickness. The carrier gas was nitrogen at a flow of 30 mL min-1. Temperature for the injection and detector ports were maintained at 275°C throughout the whole process. The ratio of the peak areas of the analyzed n-alkanes to that of internal standard (C34) was used to calculate n-alkane amounts in the sample. Identification of the different n-alkanes was made based on the relative retention times of known standards.
Preparation of gelatin capsules The content of the hard-shell gelatin capsules supplied to the animals was prepared as described by Vulich et al. (1991),
with some modifications. Briefly, the procedure involved dissolving the C32-alkane (97% purity, Acros Organics, NJ, USA) in a given volume of heptane, and mixing this solution with cellulose, at a ratio of 1 200 mg C32-alkane per 12 g powdered cellulose. The resulting suspension was easily homogenized with a glass rod at room temperature and evaporated in a water bath (40°C). Once the mixture was dry, remaining lumps were disaggregated using a mortar, and mixture was sieved through a 1-mm mesh. The powder was weighted and the capsules filled by hand. The average content of the C32-alkane in each capsule was 60 mg.
Dosing and sample collection procedures Following the 7 d adaptation period, the sheep were orally dosed twice daily for 15 d with a gelatin capsule
containing synthetic dotriacontane (C32-alkane), using a length of rubber tubing. Dosing was done at 7:00 and 18:00, in order to dose the animals at approximate 12 h intervals. The fecal samples were quantitatively collected directly from the rectum at 2-h intervals from day 11 to 15 within each period. Subsequently, samples were mixed and pooled per animal per day. All samples were air-dried, ground to
pass a 1-mm sieve and stored at -20°C for subsequent n-alkanes and other analyses. Representative herbage
samples were taken to a height similar to that consumed by the grazing sheep and were processed in same manner as the fecal samples.
Chemical analysis Ground samples were analyzed for crude protein (CP)
Calculations The dry matter intake was calculated from the pair of alkane C33 (naturally present in the herbage) and C32 alkane (dosed), according to the equation proposed by Mayes et al. (1986):
DMI=
D32× H33-
F33 F32
F33 F32 × H32
Where, DMI is daily dry matter intake (kg d-1), D32 is amount of C32 alkane dosed daily (mg d-1). F33 and F32 are fecal concentrations of C33 and C32 alkanes (mg kg-1 DM), H33 and H32 are the herbage concentrations of C33 and C32 alkanes (mg kg-1 DM), respectively. In this equation, it is assumed that C33 and C32 alkanes with nearly similar chain lengths have a similar fecal recovery, independent of their © 2014, CAAS. All rights reserved. Published by Elsevier Ltd.
Use of the N-alkanes to Estimate Intake, Apparent Digestibility and Diet Composition in Sheep Grazing on Stipa breviflora
source (natural occurrence in herbage or dosed). Herbage concentrations of C32 and C33 were calculated by the sum of the products of the estimated products of the estimated proportions of each plant species in the diet (using the n-alkane markers) and their alkane concentration. Apparent dry matter digestibility (DMD) was calculated using C33 as internal marker using the following formula:
DMD=1-
I33× FR33 F33
Where, FR33 represents the faecal recovery of C33 alkane, and I33 is the dietary C33 concentration. The botanical composition of diet consumed was estimated from the concentrations of the odd-chain n-alkanes, C 27-C 33 in feces and herbage using the nonnegative least-squares algorithm implemented in the software EATWHAT (Dove and Moore 1995), the recoveries of alkanes applied were 71.4, 74.5, 84.8 and 89.4 for C27-, C29-, C31- and C33-alkane, respectively, according to values from Newman et al. (1995).
Statistical analysis Statistical analyses were performed using SPSS (SPSS Inc., Ireland) ver. 11.5 (2002). Effect of season on DM intake, apparent dry matter digestibility and diet composition was analyzed by means of one-way analysis of variance (ANOVA). Significance levels ranged from 0.05 to 0.01. Multiple comparisons between means used Duncan’s multiple range test.
Acknowledgements The authors are particularly grateful to Dr. Hugh Dove, CSIRO, Australia, for his advices on the n-alkanes technique and kindly supplying the EATWHAT software. This study
was financially supported in part by China Agriculture Research System (CARS-37).
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Morais J A S, Berchielli T T, Vega A D, Queiroz M F S, Keli A, Reis R A, Bertipaglia L M A, Reis R A. 2011. The validity of n-alkanes to estimate intake and digestibility in Nellore beef cattle fed a tropical grass (Brachiaria brizantha cv. Maraandu). Livestock
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Newman J A, Thompson W A, Penning P D, Mayes R W. 1995. Least squares estimation of diet composition from n-alkanes in herbage and faeces using matrix mathematics. Australian Journal of Agricultural Research, 46, 793-805. NRC. 1981. Nutrient Requirements of Goats. National Academy Press, Washington, D.C. pp. 1-2. Oliván M, Ferreira L M M, Celaya R, Osoro K. 2007. Accuracy of the n-alkane technique for intake estimates in beef cattle using different sampling procedures and feeding levels. Livestock Science, 106, 28-40. Peiretti P G, Meineri G, Miraglia N, Mucciarelli M, Bergero D. 2006. Intake and apparent digesitibility of hay or hay plus concentrate diets determined in horses by the taotal collection of feces and n-alkanes as internal markers. Livestock Science, 100, 189-194. Premaratne S, Fontenot J P, Sharnklin R K. 2005. Use of n-alkane to estimate intake and digestibility by beef steers. Asian-Australasian Journal of Animal Sciences, 18, 1564-1568.
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for dietary fiber, neutral detergent fiber, and non-starch polysaccharides in relation to animal nutrition. Journal of Dairy Science, 74, 3583-3597. Sun Z W, Zhou D W, Ferreira L M M, Zhong Q Z, Lou Y J. 2008. Diet composition, herbage intake and digestibility in Inner Mongolian cashmere goats grazing on native Leymus chinensis plant communities. Livestock Science, 116, 146-155. Tefera S, Mlambo V, Dlamini A M, Koralagama K D N, Mould F L. 2009. Chemical composition and in vitro ruminal fermentation of selected grasses in the semiarid savannas of Swaziland. African Journal of Range and Forage Science, 26, 9-17. Valiente O L, Delgado P, Vega A D , Guada J A.2003. Validation of the n-alkane technique to estimate intake, digestibility, and diet composition in sheep consuming mixed grain: roughage diets. Australian Journal of Agricultural Research, 54, 693-702.
Vulich S A, O’Riordan E G, Hanrahan J P. 1991. Use of n-alkanes for the estimation of herbage intake in sheep: Accuracy and precision of the estimates. Journal of Agricultural Science, 116, 319-323. Wang H R, Feng Z C, Lu D X. 1997. Influences of seasonal changes of nutritive values of native pasture on feed intake and performance of grazing sheep. Inner Mongolian Academy of Animal Sciences, (Suppl.), 143149. (in Chinese) Wang X L. 2009. A study on sheep diet of different stocking rates on Stipa breviflora Desert Steppe. MSc thesis,
University of Inner Mongolian Agricultural, Huhhot. (in Chinese)
(Managing editor ZHANG Juan)
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May 2014
RESEARCH ARTICLE
Use of the N-alkanes to Estimate Intake, Apparent Digestibility and Diet Composition in Sheep Grazing on Stipa breviflora Desert Steppe HU Hong-lian1*, LIU Yong-zhi2, 3*, LI Ya-kui4, LU De-xun1 and GAO Min1 1
Institute of Animal Nutrition and Feed, Inner Mongolia Academy of Agricultural & Animal Husbandry Sciences, Hohhot 010031, P.R.China Inner Mongolia Prataculture Research Center, Chinese Academy of Sciences, Huhhot 010031, P.R.China 3 Institute of Botany, Chinese Academy of Sciences, Beijing 100093, P.R.China 4 College of Animal and Technology, Hebei North University, Zhangjiakou 075000, P.R.China 2
Abstract The application of n-alkane as markers to estimate herbage intake, apparent digestibility and species composition of diet consumed by grazing sheep was studied. Six local Mongolian sheep were used to determine dry matter (DM) intake, apparent DM digestibility and species composition of diet during summer, autumn and winter. Animals were orally dosed twice daily with n-alkane gelatin capsules containing 60 mg C32-alkane as an external marker. Diet composition was estimated by comparing the odd-chain n-alkanes pattern profile (C27-C31) of the consumed plant species with the n-alkanes fecal concentrations of grazing animals, using a non-negative least squares algorithm called EATWHAT software package. The alkane pair C32:C33 and C33 alkane were used to estimate DM intake and diet apparent DM digestibility, respectively. The results showed that daily dry matter intake of the sheep were 1.77, 1.61 and 1.18 kg d-1 in summer, autumn and winter, respectively. Apparent DM digestibility, crude protein (CP), metabolizable energy (ME) and neutral detergent fiber (NDF) intake of diet consumed by sheep decreased significantly (P<0.01) from summer to winter, with no evident changes in ADF and ADL intake. Diet composition indicated Artemisia frigida Willd was the most dominant diet component, contributed 79.68, 68.12 and 86.26% of sheep’s diets in summer, autumn and winter, respectively. Cleistogenes songorica Ohwi and Convolvulus ammannii Desr were the important components of the diet. Although Stipa breviflora Griseb is one of the main plant species in the study area, the sheep rarely choosed it. The study indicated that CP and ME in diet consumed by sheep were deficient in winter. Therefore, appropriate supplementation strategies should be indispensable during this period. Key words: n-alkanes, diet composition, intake, apparent digestibility, sheep
INTRODUCTION China belongs to one of the largest grassland regions of the world (Glindemann et al. 2009). In northern China, there are vast areas of grasslands in both arid and alpine environments, and used for grazing and supporting more than 55% of the sheep population
in China. Grazing is a nutritional necessity for most of the China’s sheep, but information about what and which plants grazing animals consumed are limited, which has restricted progress towards the development of sustainable grazing systems. The nutrient status of the grazing animals mainly depends on the nutritive value of the plants available, botanical composition of the consumed diet and the intake of animal (Dove
Received 27 December, 2012 Accepted 25 April, 2013 HU Hong-lian, Mobile: 13947198168, E-mail: [email protected]; Correspondence LIU Yong-zhi, Tel: +86-471-5295028, E-mail: [email protected] * These authors contributed equally to this study. © 2014, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S2095-3119(13)60502-X
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et al. 1996). Accurate measurement of feed intake and digestibility are important to meet nutritional requirements of the animal and optimize production (Bezabih et al. 2012). However, the estimation of those parameters is very difficult, complicated and laborious in grazing conditions, with errors often unacceptable due to limitations of available methods of measurement (Keli et al. 2008). Fortunately, n-alkanes as markers has been widespread for the last two decades and successfully applied to estimate intake, digestibility and diet composition in ruminants, such as sheep (Lewis et al. 2003; Valiente et al. 2003; Keli et al. 2008), goat (Sun et al. 2008), yak (Ding and Long 2010), beef steer (Premaratne et al. 2005; Chopa et al. 2012) and cattle (Morais et al. 2011; Bezabih et al. 2012). N-alkanes are saturated hydrocarbons with chain length varying from 21 to 35 carbon atoms, which can be found in the wax cuticle of most plants (Dove and Mayes 1996). The n-alkanes with odd-number carbon chains predominate and the relative levels and the patterns of n-alkane components differ among the plant species (Peiretti et al. 2006). As n-alkanes are mainly indigestible and have high feacal recovery rates (Ferreira et al. 2009), they are suitable markers to estimate diet selection and nutrient intake. The advantages of n-alkanes as marker include low invasiveness, accuracy and the possibility of taking into account diet-animal interactions (Bezabih et al. 2012). In addition, if the animals are dosed with even-chain alkane, estimates of total intake and the proportions of plant species in the diet of grazing animals can be obtained simultaneously using only one analytical procedure (Dove and Mayes 1996, 2005).
Although the n-alkane technique is widely applied in other parts of the world, there is limited information on its applications in China desert steppe ecosystems. Therefore, the objective of this study was to use the n-alkane technique to estimate simultaneously dry matter intake, apparent dry matter digestibility and diet composition selected by sheep grazing on desert steppe, and evaluate the quality of the diet during the year. This information is required in order to know aspects of selectivity and optimal timing of grazing, as well as to assure adequate livestock production.
RESULTS Herbage and fecal n-alkanes concentration Herbage n-alkane concentrations (mg kg -1 dry matter, DM) in different grazing seasons were showed in Table 1. As expected, concentration of C32 alkane in the most species was very low and almost zero, which was the reason for using C32 alkane as external marker as its presence in plants was almost negligible. Concentrations of C31 and C33 alkanes were the highest in herbage species, while C27 and C29 alkanes had very low concentrations, which could limit their use for intake calculation. The different herbage species differed in their alkanes patterns. In summer pasture, Stipa breviflora Griseb and Cleistogenes songorica Ohwi had the highest concentration of C 31 alkane (P<0.05),whist Cleistogenes songorica Ohwi and Convolvulus ammannii Desr had higher concentration
Table 1 N-alkanes concentration of diet components consumed by sheep in summer, autumn and winter pasture (mg kg-1 dry matter, DM) Species In summer pasture Artemisia frigida Willd Stipa breviflora Griseb Cleistogenes songorica Ohwi Convolvulus ammannii Desr In autumn pasture A. frigida Willd S. breviflora Griseb C. songorica Ohwi C. ammannii Desr In winter pasture A. frigida Willd S. breviflora Griseb C. songorica Ohwi C. ammannii Desr
C27
C29
C31
C32
C33
148.5±18.0 c 342.3±48.3 a 39.1±4.2 d 221.3±23.2 b
326.4±25.1 a 108.9±16.2 c 137.9±12.5 b 121.9±6.1 b
254.9±19.2 c 1 664.1±26.6 a 657.6±86.0 b 177.1±8.3 d
0 0 143.4±24.3 43.0±2.9
126.6±12.6 d 327.1±9.4 c 525.5±19.0 a 432.1±27.6 b
143.1±3.0 c 443.4±38.1 a 32.7±49 d 209.5±33 b
174.3±5.1 a 156.3±16.2 a 109.1±43 b 107.8±97 b
164.5±4.3 d 219.5±13.1 c 433.6±22.5 b 928.8±98 a
0 206.9±3.5 112.8±12.1 0
251.1±21.0 c 683.6±2.2 a 662.4±13.4 a 382.3±4.0 b
111.0±3.8 c 392.5±4.7 a 52.0±2.3 d 212.7±35.4 b
194.8±11.3 a 133.9±9.1 b 127.4±1.9 b 98.1±9.1 c
184.4±12.5 c 110.9±11.2 d 734.9±7.1 b 1 630.1±76.8 a
23.2±2.2 0 0 0
466.9±16.2 b 471.6±77.8b 661.7±9.1 a 336.7±19.02 c
Different small letters within the same column indicate significant differences (P<0.05). Data are means±SD (n=2).
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Use of the N-alkanes to Estimate Intake, Apparent Digestibility and Diet Composition in Sheep Grazing on Stipa breviflora
of C33 alkane than other species (P<0.05). In autumn pasture, C. ammannii Desr had highest concentration of C31 alkane, the highest concentrations of C33 alkane were found in S. breviflora Griseb and C. songorica Ohwi (P<0.05). In winter pasture, the highest concentration of C31 alkane was found in C. ammannii Desr with the lowest concentration of C33 alkane (P<0.05). The highest concentrations of C27 and C29 alkane were found in S. breviflora Griseb and Artemisia frigida Willd in three grazing seasons, respectively. Fecal n-alkanes concentrations (mg kg -1 DM) in
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different grazing seasons were presented in Table 2. Fecal concentrations of n-alkanes were corrected using published recovery rates reported by Newman et al. (1995). Fecal concentrations of odd-chain n-alkanes (C27-C33) tended to increase with carbon chain length. Fecal concentration of C33 alkane was highest (P<0.05). With season advance, C 27, C29, C31 and C32 alkanes concentrations in fecal samples decreased, whereas C33 alkane concentration increased. The mean fecal concentrations for most n-alkanes were the lowest in winter except for C33 alkane.
Table 2 N-Alkanes pattern of fecal samples from sheep in summer, autumn and winter (mg kg-1 DM) Peroid Summer Autumn Winter
C27 291.43±7.63 d 199.62±5.18 d 136.08±5.94 d
C29 629.75±13.69 b 243.68±3.64 c 194.42±11.48 c
C31 768.58±16.10 a 462.03±5.46 b 315.72±8.54 b
C32 264.27±5.49 d 208.88±2.17 d 200.67±6.38 c
C33 525.10±5.10 c 699.57±8.34 a 812.98±10.11 a
Diet composition obtained using n-alkanes profiles in fecal and pasture samples in each experimental period were presented in Figs. 1-3. In present study, A. frigida Willd was the most important dietary component of sheep in three seasons. A. frigida Willd constituted 79.68, 68.12 and 86.26% of the sheep’s diet in summer, autumn and winter, respectively. S. breviflora Griseb was minor component of sheep diet in three seasons. In summer, the sheep showed major preference for A. frigida Willd, which together with C. songorica Ohwi and Convolvulus ammannii Desr, comprised more than 98% of the diet. In autumn, the sheep consumed mostly A. frigida Willd (68.12%) and C. songorica Ohwi (23.57%), following similar amounts of S. breviflora Griseb (3.81%) and C. ammannii Desr (4.50%). In winter, the sheep’s diet was dominated by A. frigida Willd (86.26%) along with C. songorica Ohwi (8.33%) and C. ammannii Desr (5.41%), while no S. breviflora Griseb was detected in the sheep’s diet. The estimates of dry matter intake and apparent digestibility of herbage consumed by the grazing sheep using the pair C33:C32 and C33 alkane, respectively, were presented in Table 3. There were significant effects of grazing season on dry matter intake
90 80 70 60 50 40 30 20 10 0
Artemisia frigida Willd
Stipa breviflora Cleistogenes Griseb songorica Ohwi
Convolvulus ammannii Desr
Fig. 1 Diet composition of sheep in summer pasture. Vertical line on each bar represents standard error of the mean (n=6). The same as below. 80 70 Propotion in diet (%)
Estimations of diet composition, intake and apparent digestibility
Propotion in diet (%)
Different small letters within the same row indicate significant differences (P<0.05). Data are means±SE (n=6). The same as below.
60 50 40 30 20 10 0 Artemisia frigida Willd
Stipa breviflora Cleistogenes Convolvulus Griseb songorica Ohwi ammannii Desr
Fig. 2 Diet composition of sheep in autumn pasture.
(DMI) (P<0.01) and apparent DM digestibility of the diet selected (P<0.01). The daily dry matter intake of sheep was higher in summer and autumn (1.77 and 1.61 kg d-1, respectively) and lower in winter (1.18 kg d-1) (P<0.01). There was no difference in © 2014, CAAS. All rights reserved. Published by Elsevier Ltd.
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Propotion in diet (%)
1068 100 90 80 70 60 50 40 30 20 10 0
DISCUSSION
Artemisia frigida Willd
Cleistogenes songorica Ohwi
Convolvulus ammannii Desr
Fig. 3 Diet composition of sheep in winter pasture. Table 3 Dry matter intake and apparent dry matter digestibility of diets consumed by sheep in summer, autumn and winter Period Summer Autumn Winter
DMI (g d-1) 1 769.99±61.45 A 1 610.19±61.47 A 1 178.32±48.90 B
DMD (g kg-1 DM) 661.80±4.34 A 527.02±5.51 B 467.8±7.21 C
DMI, dry matter intake; DMD, dry matter digestibility. Different capital letters within the same column indicate significant differences (P<0.01). The same as below.
DMI per sheep between summer and autumn. The apparent DM digestibility of the diet consumed by sheep was the highest in summer and the lowest in winter (P<0.01). Nutrient intake calculated using the estimates of DMI and diet apparent digestibility obtained using the n-alkane technique was presented in Table 4. The experimental period had a significant effect on the crude protein (CP), metabolizable energy (ME) and neutral detergent fiber (NDF) intake of sheep, no seasonal differences were observed in acid detergent fiber (ADF) and acid detergent lignin (ADL) intake of sheep. With season advance, CP and ME intake of sheep decreased significantly (P<0.01) from 286.74 g d-1 or 14.97 MJ d-1 in summer to 97.53 g d-1 or 6.42 MJ d-1 in winter, respectively. NDF intake was the lowest in winter (P<0.01), differences in NDF intake were not observed between the summer and autumn. It was clear that the CP and ME intake of sheep appeared seasonal fluctuations.
Knowledge of dry matter intake (DMI) and digestibility is usefully in assessing the nutrient status of grazing animals. The double n-alkane marker technique has been developed to allow estimation of DMI, apparent digestibility and botanical composition of diet consumed by animals. An important assumption when using the double n-alkane marker technique is that adjacent n-alkane used in the estimates of intake (dosed even-chain alkane and natural odd-chain alkane) has similar faecal recovery. So many studies have evaluated the suitability of different odd- and even-chain n-alkane pairs for estimating intake (Dove et al. 2002; Oliván et al. 2007). And in most of these studies, n-alkane pair C32:C31 and C32:C33 had the lowest discrepancy in feacal recovery, and hence were proposed to estimate DMI (Dove et al. 2002). Estimates of DMI using the ratio C32:C33 was more reliable than the ratio C32:C31 (Smit et al. 2005; Chopa et al. 2012) and more close to the measured intakes (Molina et al. 2004). For this reason, in the present study, the alkane pair C32:C33 and C33 as internal marker was used to estimate DM intake apparent DM digestibility. Accurate assessment of the concentration of the natural and dosed alkane in the feces to estimate DMI is critical to the validity of the alkane technique. Daily cyclic variation in fecal alkane concentration could lead to inaccurate intake estimate (Molina et al. 2004). In indoor experiments, total faeces would clearly provide the most representative samples of faces, but under grazing conditions, grab sampling is required. For this reason, in the present study, representative samples of faces were obtained by collecting rectal grab samples at 2 h interval from day 11 to 15. Furthermore, fecal n-alkane concentrations were corrected using published recovery rates reported by Newman et al. (1995). However, for accurate estimation of herbage DMI, apparent digestibility and diet composition, actual n-alkane fecal
Table 4 Estimated nutrient intake of diets consumed by sheep when intake and digestibility were derived from diet composition estimates Period Summer Autumn Winter
CP (g d-1) 286.74±0.71 A 210.43±2.09 B 97.53±7.38 C
ME (MJ d-1) 14.97±0.13 A 10.93±0.27 B 6.42±0.67 C
NDF (g d-1) 832.61±6.90 A 835.03±5.07 A 664.16±21.65 B
ADF (g d-1) 590.56±1.86 529.37±2.25 547.34±42.54
ADL (g d-1) 110.27±1.06 118.74±0.72 115.11±4.89
CP, crude protein; ME, metabolizable energy; NDF, neutral detergent fiber; ADF, acid detergent fiber; ADL, acid detergent lignin.
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Use of the N-alkanes to Estimate Intake, Apparent Digestibility and Diet Composition in Sheep Grazing on Stipa breviflora
recoveries rates should be used, and obtaining a representative fecal and herbage sample is essential. In this study area, A. frigida Willd was one of the most abundant browse species and important dietary component of sheep, contributed 79.68, 68.12 and 86.26% of sheep’s diet in summer, autumn and winter, respectively. Similar results were obtained by Biligetu (2003) and Wang (2009) who performed in moderate grazing intensity from same experiment area. The dominant specie, C. songorica Ohwi was larger proportion of the diet compared with C. ammannii Desr in whole experiment periods. The reason was that C. songorica Ohwi provided the greater cover and the height of C. ammannii Desr was lower (Liu 2005). Although S. breviflora Griseb was one of the main plant species in the study area, the sheep rarely chose it when good palatability or other nutritious species were available. These findings could be associated with plant prehensibility, particularly plant height and the presence of secondary compounds in these species. In the present study, CP, NDF, ADF and ADL contents in the diets were estimated by the sum of the product of the proportion that each plant species in the diet and their nutrient concentrations. ME concentration in the diet was calculated from apparent DM digestibility estimated using C33 alkane. With the decrease of herbage availability and nutritional value from summer to winter, the sheep decreased their DMI and intakes of both CP and ME. The intakes of both CP and ME for sheep were adequate to meet their nutritional demands for growth only from grazing in summer and autumn, according to the established requirement of sheep (NRC 1981). While in winter, both CP and ME intakes were deficient causing body weight gain loss (Hu 2005). These results were agreement with previous reports (Wang et al. 1997), which reported the lack of both CP and energy intakes were main nutritional constraints of grazing sheep in winter and spring in northern China. Other experiment also showed the CP content of forages may fall to less than 70 g kg-1 DM in long dry season (Tefera et al. 2009), which is considered to be limiting for an optimum rumen microbial growth and fermentation. In this study area, green herbage is available for only 5 mon (approximately mid May to mid October), with severe shortages of herbage during remaining 7
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mon. The nutrient intake is not able to meet nutrient requirements of grazing animals only from grazing in winter and spring. Therefore, it was important that appropriate feed supplementation should be considered to ensure productivity and health of grazing sheep during this period.
CONCLUSION The results from the present study showed that the n-alkane technology could be used to estimate simultaneously DM intake, apparent DM digestibility and diet selection of sheep under the particular conditions of this study. However, for accurate estimation of these parameters, actual alkane fecal recoveries should be used, and obtaining a representative fecal and herbage sample is essential. There was significant effect of grazing season on DM intake and apparent DM digestibility of the diet selected by sheep. Diet composition indicated A. frigida Willd and Cleistogenes songorica Ohwi were the most dominant diet component, but in different proportions at different seasons, Stipa breviflora Griseb was selected rarely. It was clear that the CP and ME intake of sheep appeared seasonal fluctuations. Both CP and ME intakes were deficient in winter, thus feed supplementation should be required.
MATERIALS AND METHODS Study site This study was conducted in the desert steppe in Siziwang Banner, Inner Mongolia (41°10´N longitude, 110°20´E latitude, about 1 430-1 506 m in elevation), where the longterm averages of annual mean air temperature and annual rainfall are 2.9°C and 200 mm, respectively. It has typical geographical representation of the desert steppe and farmingpastoral zone. The soil of the area around study site is sand chestnut. The herbage vegetation of the experimental plot consisted mainly Stipa breviflora Griseb, Artemisia frigida Willd, Cleistogenes songorica Ohw, Convolvulus ammannij Desr, Hetropappus altaicus Novepokr, Kochia prostrata Schrad, Caragana stenophylla Pojark, and Leymus chinensis Tzvel. S. breviflora Griseb, A. frigida Willd, C. songorica Ohwi and C. ammannij Desr are the dominant plant species in this area. Their percentage of plant production are 66.0, 8.2,
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6.9 and 10.4%, respectively. And these contributed more than 90% to total biomass yield (Liu 2005). The height and coverage of herbage community are about 8 cm, 17 to 20%, respectively. Due to grazing pressure, there has been some deterioration of the natural vegetation in the last decades.
according to Association of Official Analytical Chemists (1990). Neutral detergent fiber (NDF), acid detergent fiber (ADF) and acid detergent lignin (ADL) following van Soest et al. (1991). Concentrations of n-alkanes were determined by gas chromatography (Mayes et al. 1986).
Animals
N-alkane analysis
Six local Mongolian sheep, aged two-year old and weighing between 30 and 35 kg were used. Sheep were dewormed prior to the experiment. Sheep were grazed for 8 h per day in an area of 1.2 ha from summer to winter in three experimental periods: Summer (from middle June to early July), autumn (September) and winter (November). No supplementary feed and no minerals were supplied to all sheep throughout the grazing periods.
Samples were analyzed for n-alkanes C27-C33 concentrations by means of gas chromatography by the method of Mayes et al. (1986). Dried and milled herbage (1.2 g) and fecal samples (0.6 g) were weighted into screw-capped flasks prior to the addition of 50 μL of C34 internal standard (Sigma Chemical, St. Louis, Mo, USA) and 10 mL alcoholic KOH, overnight. The samples were heated for 2 h at 90°C. After cooling of samples, the alkanes were extracted by adding 3 mL of heptane and 2 mL of distilled water, the tubes were shaken vigorously and placed in a water bath at 45°C for 5 min. The nonaqueous top liquid layer was transferred to a glass vial and further extracted with 3 mL of heptane. The samples were evaporated to dryness, redissolved in 2 mL of heptanes and applied to the small column containing silica gel (200-400 mesh, Merck, China) with a 3-cm bed volume and glass fiber (0.5 cm). The hydrocarbons were eluted from the columns with heptane, evaporated and redissolved in 1 mL of heptane. 1 μL of each final redissolved n-alknae solution was injected into GC-9A gas chromatograph (Shimadzu Company, Japan) fitted with flame ionization detector. The column was a 30 m×0.53 mm insidediameter capillary column type SPB1 with 0.5 μmol L -1 film thickness. The carrier gas was nitrogen at a flow of 30 mL min-1. Temperature for the injection and detector ports were maintained at 275°C throughout the whole process. The ratio of the peak areas of the analyzed n-alkanes to that of internal standard (C34) was used to calculate n-alkane amounts in the sample. Identification of the different n-alkanes was made based on the relative retention times of known standards.
Preparation of gelatin capsules The content of the hard-shell gelatin capsules supplied to the animals was prepared as described by Vulich et al. (1991),
with some modifications. Briefly, the procedure involved dissolving the C32-alkane (97% purity, Acros Organics, NJ, USA) in a given volume of heptane, and mixing this solution with cellulose, at a ratio of 1 200 mg C32-alkane per 12 g powdered cellulose. The resulting suspension was easily homogenized with a glass rod at room temperature and evaporated in a water bath (40°C). Once the mixture was dry, remaining lumps were disaggregated using a mortar, and mixture was sieved through a 1-mm mesh. The powder was weighted and the capsules filled by hand. The average content of the C32-alkane in each capsule was 60 mg.
Dosing and sample collection procedures Following the 7 d adaptation period, the sheep were orally dosed twice daily for 15 d with a gelatin capsule
containing synthetic dotriacontane (C32-alkane), using a length of rubber tubing. Dosing was done at 7:00 and 18:00, in order to dose the animals at approximate 12 h intervals. The fecal samples were quantitatively collected directly from the rectum at 2-h intervals from day 11 to 15 within each period. Subsequently, samples were mixed and pooled per animal per day. All samples were air-dried, ground to
pass a 1-mm sieve and stored at -20°C for subsequent n-alkanes and other analyses. Representative herbage
samples were taken to a height similar to that consumed by the grazing sheep and were processed in same manner as the fecal samples.
Chemical analysis Ground samples were analyzed for crude protein (CP)
Calculations The dry matter intake was calculated from the pair of alkane C33 (naturally present in the herbage) and C32 alkane (dosed), according to the equation proposed by Mayes et al. (1986):
DMI=
D32× H33-
F33 F32
F33 F32 × H32
Where, DMI is daily dry matter intake (kg d-1), D32 is amount of C32 alkane dosed daily (mg d-1). F33 and F32 are fecal concentrations of C33 and C32 alkanes (mg kg-1 DM), H33 and H32 are the herbage concentrations of C33 and C32 alkanes (mg kg-1 DM), respectively. In this equation, it is assumed that C33 and C32 alkanes with nearly similar chain lengths have a similar fecal recovery, independent of their © 2014, CAAS. All rights reserved. Published by Elsevier Ltd.
Use of the N-alkanes to Estimate Intake, Apparent Digestibility and Diet Composition in Sheep Grazing on Stipa breviflora
source (natural occurrence in herbage or dosed). Herbage concentrations of C32 and C33 were calculated by the sum of the products of the estimated products of the estimated proportions of each plant species in the diet (using the n-alkane markers) and their alkane concentration. Apparent dry matter digestibility (DMD) was calculated using C33 as internal marker using the following formula:
DMD=1-
I33× FR33 F33
Where, FR33 represents the faecal recovery of C33 alkane, and I33 is the dietary C33 concentration. The botanical composition of diet consumed was estimated from the concentrations of the odd-chain n-alkanes, C 27-C 33 in feces and herbage using the nonnegative least-squares algorithm implemented in the software EATWHAT (Dove and Moore 1995), the recoveries of alkanes applied were 71.4, 74.5, 84.8 and 89.4 for C27-, C29-, C31- and C33-alkane, respectively, according to values from Newman et al. (1995).
Statistical analysis Statistical analyses were performed using SPSS (SPSS Inc., Ireland) ver. 11.5 (2002). Effect of season on DM intake, apparent dry matter digestibility and diet composition was analyzed by means of one-way analysis of variance (ANOVA). Significance levels ranged from 0.05 to 0.01. Multiple comparisons between means used Duncan’s multiple range test.
Acknowledgements The authors are particularly grateful to Dr. Hugh Dove, CSIRO, Australia, for his advices on the n-alkanes technique and kindly supplying the EATWHAT software. This study
was financially supported in part by China Agriculture Research System (CARS-37).
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