Accepted Manuscript Estimation of herbage intake and digestibility of grazing sheep in Zhenglan Banner of Inner Mongolia by using n-alkanes Changqing Li, Alatengdalai, Shuyuan Xue, Atsushi Tajima, Naoto Ishikawa PII: S2405-6545(15)30036-6 DOI: 10.1016/j.aninu.2015.11.004 Reference: ANINU 45 To appear in: Animal Nutrition Journal Received Date: 26 August 2015 Revised Date: 29 October 2015 Accepted Date: 1 November 2015 Please cite this article as: Li C, Alatengdalai Xue S, Tajima A, Ishikawa N, Estimation of herbage intake and digestibility of grazing sheep in Zhenglan Banner of Inner Mongolia by using n-alkanes, Animal Nutrition Journal (2016), doi: 10.1016/j.aninu.2015.11.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ACCEPTED MANUSCRIPT 1 2 Estimation of herbage intake and digestibility of grazing sheep in 3 Zhenglan Banner of Inner Mongolia by using n-alkanes 4 5 Changqing Li a, b, Alatengdalai b, Shuyuan Xue b, Atsushi Tajima a, Naoto Ishikawa a* 6 7 aFaculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, 8 305-8577,Japan 9 bInner Mongolia Academy of Agricultural and Animal Husbandry Sciences, Hohhot, 10 010031,China 11 12 13 MANUSCRIPT 14 15 16 17 18 19 20 *Corresponding author 21 E-mail address:ACCEPTED [email protected] (N. Ishikawa). ACCEPTED MANUSCRIPT 22 23 Abstract 24 To reveal the seasonal dynamics of herbage intake, diet composition and 25 digestibility and clarify the relationship of those with herbage nutrient and botanical 26 composition of grazing sheep in Zhenglan Banner of Inner Mongolia, the n-alkane 27 technique was used to test in sheep grazed during June, August and December. The 28 results showed that the sheep mainly ate fringed sagebrush , stipa krylovii and carex in 29 proportions of 33.5, 17.9 and 21.2%, respectively, in spring. In summer, the sheep 30 consumed cleistogenes, potentilla tanacetifolia , thyme , etc; the intake of fringed 31 sagebrush, carex and stipa declined. In winter, fringed sagebrush accounted for 50.1% 32 of herbage intake, and the intakes of cleistogenes and stipa krylovii increased to 15.3 33 and 18.4%, respectively. Herbage intake by the sheep in spring was 1.8 kg DM/d, and 34 digestibility was 71.4%. Herbage intake and digestiMANUSCRIPTbility decreased slightly to 1.7 kg 35 DM/d and 68.4% during the summer, respectively and decreased significantly to 1.2 kg 36 DM/d and 36.4% in winter. There were significant correlations between diet 37 composition and CP content in winter diet composition and botanical composition in 38 summer. A highly positive correlation between herbage intake and digestibility was 39 observed in grazing sheep. 40 41 Key words 42 N-alkane technique;ACCEPTED Diet composition; Grazing sheep; Herbage intake 43 ACCEPTED MANUSCRIPT 44 1 Introduction 45 The malnutrition of sheep during autumn to winter season is often observed in the 46 northern area of China because the animals are generally raised under the condition of 47 the natural grassland throughout the year. In order to improve the performance of 48 sheep production in this region, it is essential to know the nutritional level in sheep 49 throughout a year. Nutrient intake depends on the plant species, herbage intake and 50 digestibility of grazing sheep. However, it is very hard to estimate the herbage intake, 51 diet composition and digestibility of livestock in grazing condition. Although many 52 scientists performed plenty of research on many proposed estimating methods, most 53 methods have shown numerous limitations, such as low accuracy, complicated 54 procedure, tedious and expensive (Wang 1995). The n-alkane technique, on the other 55 hand, has proven to be effective and accurate for estimating herbage intake, diet 56 composition and digestibility of grazing sheepMANUSCRIPT (Mayes et al.1986, Dove 1992, Mayes 57 and Dove 2000). In previous experiments, estimates of herbage intake using the radio 58 C33:C32 were the most accurate among the other combination of C31:C32 (Zhang 59 2002). So in the present study, the alkane pair C32:C33 with C33 as internal marker 60 was used to estimate herbage intake, diet composition and digestibility in grazing 61 sheep. 62 It was well documented herbage intake is influenced by many factors, including the 63 species of grass, location, growth stage and region, environmental condition, and 64 analytical ACCEPTEDmethod (Laredo et al.1991, Olivan and Osoro 1999, Kelman et al. 2003, 65 Dove et al.1996). Considering the alkane recovery rate in the feces, the sampling for 66 multiple plants and the growing period of herbage (Liu et al.2006), only 8 species of 67 herbage were chosen in this study. Herbage intake, diet composition and digestibility ACCEPTED MANUSCRIPT 68 of sheep grazing on the arid grassland were determined in a recent study in Siziwang 69 Banner, in western Inner Mongolia (Hu, 2014). The results of n-alkane patterns were 70 consistent with those reported by other researchers (Wang 2000), proving the 71 feasibility of detecting diet composition of grazing sheep using the n-alkane technique 72 in Inner Mongolia. The results of Hu et al. (2014) showed that daily dry matter intake 73 and DM digestibility of sheep decreased significantly from summer to winter, and a 74 diet composition analysis indicated that Artemisia frigida Willd was the most dominant 75 diet component in the arid steppe of Siziwang Banner. However, information on 76 seasonal changes of herbage intake and digestibility in sheep is not available in 77 Xilingol Leagure which is the center of livestock production in Inner Mongolia. 78 Traditionally, Zhenglan Banner, located in the grassland of Xilingol, is considered to 79 be the typical steppe (Hu, 1995). However, the herbage species are complex. There is 80 no study that has estimated the intake and digestibMANUSCRIPTility of herbage in sheep grazed in 81 Xilingol. 82 So the aim of this study was to verify the seasonal changes of herbage intake, diet 83 composition and digestibility of grazing sheep, and clarify the relationship of those 84 with herbage nutrient and botanical composition in the grassland of Xilingol League. 85 2 Materials and methods 86 Experimental site 87 The experimental site, Zhenglan Banner (E 116.02°, N 42.25°), is located in the 88 central of ACCEPTEDInner Mongolia, China, is a typical steppe. The dominant species there 89 include stipa krylovii , fringed sagebrush , leymus chinensis , cleistogenes , and carex ; 90 and the major accompanying species are thyme , potential bifurca , potentilla 91 tanacetifolia , agropyron cristatum , etc. The plants are generally 6 to 25 cm in height ACCEPTED MANUSCRIPT 92 with average vegetation coverage of 25 to 35%. 93 Application of the n-alkane technique 94 Six grazing Mongolian female sheep (one year old with an average body weight of 95 45.0 ±1.0 kg) were on pasture from June to early December on the experimental site. 96 The sheep were free to roam all day long from summer to winter in three experimental 97 periods: spring (from middle May to early June), summer (August) and winter 98 (December). No supplementary feed was supplied throughout the experiment periods. 99 In each experimental period, all sheep were given one n-alkane capsule (97% purity, 100 Acros Organics, NJ, USA; including 60 mg C32) every 15 d from the start of 101 experiment to the end of experiment. Digestibility and diet composition in the grazing 102 sheep were determined using the n-alkane technique as described by Hu et al. (2014). 103 Collection of herbage and fecal samples 104 Herbage samples were collected manually. MANUSCRIPTThe species, positions and heights of 105 the herbage that the sheep ate were observed for 5 min every 2 h in grazing for 5 d. 106 Meanwhile, the herbage samples mimicking what the sheep were grazing on were 107 collected, weighed, mixed and reserved at -20 . Fecal samples were taken from the 108 rectums of the sheep. Fecal sample (10 g) of each tested sheep was collected 3 times a 109 day for 5 d in 12 time points, with an interval of 2 h. The samples were reserved in 110 sealed bags at -20 . 111 The herbage and fecal samples were analyzed for crude protein (CP),dry matter 112 (DM) ,Ca ACCEPTEDand P according to Association of Official Analytical Chemists (1990). 113 Neutral detergent fiber (NDF), acid detergent fiber (ADF) and ether extract (EE) 114 following van Soest et al. (1991). ME concentration in the diet was calculated from 115 apparent DM digestibility estimated using C33 alkane. ACCEPTED MANUSCRIPT 116 N-alkane determination and calculations 117 The samples were pretreated using the method proposed by Dove and Mayes (2005) 118 and assayed using a Shimadzu GC-9A gas chromatograph. 119 The herbage composition was calculated using the suggested program (Eat What) 120 proposed by Dove and Moore (1995).The dry matter intake was calculated from the 121 pair of alkane C33 (naturally present in the herbage) and C32 alkane(dosed): 122 DMI=D32*F33/(F32*H33-F33*H32). 123 Where, DMI is daily dry matter intake (kg/d), D32 is amount of C32 alkane dosed 124 daily (mg/d). F33 and F32 are fecal concentrations of C33 and C32 alkanes (mg/kg 125 DM), H33 and H32 are the herbage concentrations of C33 and C32 alkanes (mg/kg 126 DM), respectively.