Seasonal heat production and energy balance of grazing yaks on the Qinghai-Tibetan plateau

Animal Feed Science and Technology 198 (2014) 83–93

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Seasonal heat production and energy balance of grazing yaks on the Qinghai-Tibetan plateau L.M. Ding a,∗ , Y.P. Wang a , A. Brosh b , J.Q. Chen c , M.J. Gibb d , Z.H. Shang c , X.S. Guo a , J.D. Mi a , J.W. Zhou c , H.C. Wang c , Q. Qiu a , R.J. Long a,∗ a State Key Laboratory of Grassland Agro-Ecosystem, School of Life Sciences, International Centre for Tibetan Ecosystem Management, Lanzhou University, Lanzhou 730000, China b Beef Cattle Section, ARO, Newe Yaar Research Center, P.O. Box 1021, Ramat Yishay 30095, Israel c State Key Laboratory of Grassland Agro-Ecosystem, International Centre for Tibetan Ecosystem Management, College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou 730000, China d Formerly of the Institute of Grassland and Environmental Research, North Wyke, Devon EX20 2SB, UK

a r t i c l e

i n f o

Article history: Received 10 January 2014 Received in revised form 19 September 2014 Accepted 23 September 2014 Keywords: Energy metabolism Heart rate Tibetan plateau Yak

a b s t r a c t A study was conducted to measure the energy balance of free-ranging yak during the four annual seasons in order to elucidate the factors constraining energy utilization by grazing yak. The heat production (HP, kJ/day) of grazing non-lactating female yaks was calculated as the product of heart rate (HR, beats/min) and the amount of O2 delivered to the body at every heartbeat (O2 P, ␮l), and by the constant value of 20.47 kJ/l of O2 consumed. Heart rates were recorded continuously over 4 days, using modified heart rate monitors. Individual daily fecal output was measured using Cr2 O3 as an external marker. Daily herbage dry matter (DM) intake was calculated from fecal output and digestibility of the forage determined in vitro. The greatest herbage mass was measured in August (496 kg DM/ha), and the least in December and May (208 and 226 kg DM/ha). However, the herbage present in both May and August had higher crude protein contents and lower NDF contents than those sampled in October and December. Daily average HR (beats/min) was greater in summer (August) than during the other three seasons (78 vs. 49–52). The greatest O2 P was recorded in May. The highest metabolizable energy intake (MEI) (1120 kJ/kg BW0.75 per day) was measured in August when yaks grazed on lush green forage. HP was higher in August than in October and December (715, 548 and 400 kJ/kg BW0.75 per day, respectively), but did not differ significantly from that measured in May (640 kJ/kg BW0.75 per day). The animals were in positive energy balance only during August (energy retention (ER) = 405 kJ/kg BW0.75 per day). Energy balance did not differ between the other seasons: −111 (October), −91 (December) and −13 (May) kJ/kg BW0.75 per day, respectively. HP and ER were highly correlated with MEI (R2 = 0.73 and 0.88, respectively). The formulas calculated through the regression of HP and ER on MEI were used to estimate fasting heat production (FHP = 341 kJ/kg BW0.75 per day) and maintenance ME requirements (MEm , 545 kJ/kg BW0.75 per day) of the free grazing yaks. The results showed that free-ranging yaks expended much more energy to resist harsh environmental and sward conditions compared with confined yak or cattle and grazing cattle in low land area. © 2014 Elsevier B.V. All rights reserved.

Abbreviations: HP, heat production; HR, heart rate; DM, dry matter; OM, organic matter; NDF, neutral detergent fiber; O2 P, oxygen pulse; MEI, metabolizable energy intake; BW, body weight; ER, energy retention; FHP, fasting heat production; MEm , maintenance metabolizable energy requirement; CP, crude protein; DE, digestible energy; VO2 , oxygen consumption; RH, relative humidity; DMI, dry matter intake; BW, body weight. ∗ Corresponding authors. Tel.: +86 931 8915650; fax: +86 931 8915650. E-mail addresses: [email protected] (L.M. Ding), [email protected] (R.J. Long). http://dx.doi.org/10.1016/j.anifeedsci.2014.09.022 0377-8401/© 2014 Elsevier B.V. All rights reserved.

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1. Introduction The yak is a unique bovine that has survived on the Tibetan plateau for centuries under harsh alpine environmental conditions and under extensive grazing management all the year round. Because of the long period of negligible or zero plant growth each year (7–8 months) and year-round grazing management, the winter dormant period is a harsh period for yaks (Ding et al., 2007). This situation has been exacerbated in recent years because of rangeland degradation, which has reduced the amount and quality of the herbage available, especially in the winter. Deterioration of pasture can affect yak productivity throughout the year because of the expected high energy requirement for maintenance due to severe climatic conditions (long-term extreme cold in winter, and increased temperature in summer), and the requirement to walk long distances in steep terrain to reach their pastures. Various adaptive features regarding yak energy utilization have been studied and reported, such as high hemoglobin concentrations, a short trachea with a large cross-sectional area, and thick hair with a dense undercoat in winter (Zhang, 1989). So far no studies have been conducted on the energy metabolism of free-range yak. Previous studies of yak energy balance have been based on confined animals (Han et al., 1992a, 1992b, 1993; Hu et al., 1992a, 1992b). A method for measuring heat production (HP) in grazing animals has been presented and evaluated in beef cows by Brosh et al. (1998, 2002, 2004), and (Brosh, 2007). This method was also used in this study of grazing yaks on the Qinghai-Tibetan plateau. A better understanding and quantification of energy balance of yaks grazing on the Qinghai-Tibetan plateau is required for further studies of yak nutrition and developing optimal grazing practices. The present study was designed to assess the energy components of free-ranging yak during the different grazing seasons, and to explain the factors affecting and restricting energy utilization in grazing yak. The hypothesis is that harsh environmental factors and grazing activities play a great role in yak energy expenditure. The objective of present study is to reveal the energy utilization and balance of grazing yaks in different seasons. 2. Materials and methods 2.1. Study site Measurements were conducted during four periods in 2010 and 2011: August (summer), December (winter), May (spring) and October (autumn). The study area was situated in an alpine rangeland, at Wushaoling (N 37◦ 12.48 and E 102◦ 51.70 ), 50 km north of Tianzhu Tibetan Autonomous County, Gansu Province, northwest China. The pasture was under communal village grazing management and is typical of alpine meadow without any artificial improvement (irrigation or fertilization), being predominantly composed of sedge species (Carex qinghaiensis and Kobresia pygmaea), which made up >85% of the total vegetation (Ding and Long, 2010) with small seasonal changes of the botanical composition. Grazing management was seasonal rotation system, normally with summer pasture, autumn pasture, winter–spring pasture, where the animals moved in the different pastures throughout the year (Long et al., 1999). The climate is dominated by the southeast monsoon and high atmospheric pressure from Siberia, with severe, long winters and short, cool summers, which is referred to alpine climate in the Köppen climate classification (Wong et al., 2012). The climate becomes colder at high elevations. And with the increase of altitude, the main form of precipitation becomes snow, and the winds increase. The mean annual temperature was −0.1 ◦ C, and mean annual precipitation is 416 mm. 2.2. Animals and management Twelve 4- to 8-year-old non-lactating yaks were used throughout the experiment selected from a farmer’s large yak herd (80 yaks). The experimental yaks were integrated within a larger yak herd and subjected to traditional management, which was carried out on open pastures without fencing. The yak herd was grazing at pasture continually from June to October, except when the lactating yaks were corralled between 0600 and 1030 h for milking, and between 1730 and 2000 h to allow the calves to be separated from the cows. From November to May, the yak herd was corralled at night between about 2000–0700 h from November to April, and 2100–0500 h in May. No supplements were provided for the experimental yaks. 2.3. ME and MEI calculation Six yaks of the twelve were used as a group in each period to measure daily herbage intake. After collection of fecal samples for determination of background content of Cr2 O3 , each yak was dosed every morning for 10 days with a capsule containing 20 g Cr2 O3 administered using a dosing gun. During the last three days of dosing period, fecal samples were collected from the individual yaks by following them during the morning from 0800 to 1100 and afternoon from 1400 to 1830. The fresh feces were first air dried in a room avoiding direct sunshine before being dried in a ventilated oven at 60 ◦ C for 72 h, then ground through a 1 mm screen for analyzing DM by drying at 105 ◦ C for 48 h (AOAC, 1990; method 934.01). Chromium was determined by atomic absorption spectrophotometry following the protocol of Costigan and Ellis (1987). Neutral detergent fiber (NDF) content of feces was determined according to the method of Goering et al. (1970) and Van Soest et al. (1991) without adding heat stable amylase and expressed inclusive of residual ash. In addition, forage samples

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representative of that selected by the yaks were collected by hand while following and observing them grazing. Because the main herbage species the yak selected were grasses and sedges, and the alpine sward was homogenous, it was possible to observe yak grazing by telescope and to collect representative samples of the grazed forage by cutting herbage to mimic yak grazing. Twenty samples of 150 g each were collected every day for 6 days. The herbage mass present at grazing was measured by cutting all herbage to ground level within thirty randomly selected quadrats (50 cm × 50 cm) in the grazing area. Cutting at ground level was considered necessary because yaks graze down to very low heights using their incisors and lips like sheep (Ding et al., 2007). The herbage samples were dried at 60 ◦ C, and ground to pass through a 1 mm sieve. The ground samples were then dried at 105 ◦ C for a further 48 h, and then used for determination of crude protein (CP) according to a Kjeldahl procedure (AOAC, 1990; method 981.10) and NDF contents (Goering et al., 1970; Van Soest et al., 1991). The forage and fecal samples were incinerated at 550 ◦ C to determine the ash content (AOAC, 1990; method 923.03). Forage digestibility was measured using a modified two-stage in vitro digestion technique as described by Tilley and Terry (1963). Rumen fluid was withdrawn via the esophageal tube in the morning. Forage samples (0.5 g) in nylon bag (48 ␮m porosity) were incubated for 48 h with the ratio of 1:1 of yak rumen liquor and buffer solution (Menke and Steingass, 1988) in a 100 ml syringe at 39 ◦ C in a water bath. After 48 h incubation, the liquid was discarded, and a pepsin solution was added for another 48 h incubation at 39 ◦ C. Following incubation the samples were dried at 105 ◦ C for 24 h to determine the DM content of the remaining material. Metabolizable energy of the forage was calculated using the formula: ME (MJ/kg DM) = digestible OM (kg/kg DM) × 19 MJ/kg digestible OM × 0.82, implying to assume that the digestible OM of the forage contains 19 MJ of gross energy and that 18% of this energy is lost both as methane and urine (ARC, 1980). Digestible OM was calculated according to the following equations: [1000g DM − crude ash(g/kg DM)] × digestible OM (g/kg DM) + crude ash (g/kg DM) × [1000 − digestible OM (g/kg DM)] = 1000g DM × digestible DM (g/kg DM) Fecal

output

and

daily

intake

were estimated by the following output (kg/day) DM/day) = fecal 1−digestibility

weight of Cr given (g/day) Intake (kg mean concentration of Cr in feces (g/kg DM)

calculations:Fecal output (kg DM/day) =

2.4. Heat production measurements Heat production was measured by using the heart rate (HR) method as described by Brosh et al. (2004). This method is based on the measurement of HR and then relating it to the HP by calculation of O2 P through short simultaneous measurements of oxygen consumption (VO2 ) and HR. Twelve non-lactating yaks (4–8 years old) were used throughout the experiment, in two groups of six. The VO2 measurements were conducted in six yaks confined in a pen which was located in a valley at an altitude of ∼3000 m. These six yaks were also used for MEI measurements. After VO2 measurements, the experimental yaks with HR belts were released to the pasture, which is a typical alpine mountain and valley terrain (from 2900 to 3600 m above seal level). The O2 P of each yak was calculated on the first day of seasonal measurement starting around at 1000 h in the morning from the simultaneous measurements of HR and VO2 (Brosh et al., 2004). Heart rate was measured continuously using a modified heart rate monitor (Polar Electro, Kempele, Finland RS400), held in place against the thorax with belt behind the forelegs, at 1 min recording intervals for 4 days. The VO2 of each yak was measured over a period of approximately 20 min using an open-circuit system incorporating an O2 analyzer (Servomex Asia Pacific, Shanghai, China; Model 1440D). Air was sucked from the yak’s face using an open face mask. A low differential pressure transducer (Setra Systems Inc, Model 269) was used for calculating air flow rate. The HR of each yak was recorded at 1 s intervals and was combined with VO2 data to allow the calculation of O2 P. All VO2 data were recorded and integrated using a data logger (Datataker Pty Ltd., Model DT80). After the face mask and HR belt were fitted to each yak, data were only recorded after the yaks were calm down and displayed constant HR and VO2 values. The N2 recovery of this system was measured gravimetrically using the procedure described by McLean and Tobin (1990). Pure N2 was injected into the system through the face mask for about 9 min after O2 P measurement in order to measure N2 recovery by weight difference. After measuring O2 P, the HR monitors were adjusted to a recording interval of 1 min to continuously monitor the first group of six grazing yaks for 4 consecutive days (100 h), and then changed to the second group. All HR data were downloaded to a computer. Yak HP was measured during four periods, representative of the four annual seasons. In 2010, measurements were conducted during August when yaks were fed typical summer pasture, with a relatively large herbage mass of green plant material, and during December when yaks were fed typical winter pasture containing mainly senescent herbage. In 2011, HP was measured in May when yaks were fed typical spring pasture with a relatively small total herbage mass, a large proportion of which was senescent material but less consumed by yak, and in October when yaks were fed typical autumn pasture containing mainly senescent material. 2.5. Weather conditions and grazing yaks’ location Data were obtained from a meteorological station that was located nearby (1 km to the research site). During December 2010 and two measurement periods in 2011 (October and May), a separate group of 6 yaks were equipped with GPS location and motion recorders (Lotek Engineering Inc., Canada, GPS 3300) mounted on collars. The recorders were programmed to

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Table 1 Meteorological data of the four periods of measurements through the studya . 2010

Mean-Temp, ◦ C Max-Temp, ◦ C Min-Temp, ◦ C RH, % WS, m/s

2011

August (summer)

December (winter)

May (spring)

October (autumn)

11.6a 17.2a 7.1a 66.0a 5.3b

−10.6d −4.3d −15.3d 47.7c 5.3b

4.9b 10.1b 0.7b 56.6b 5.3b

1.7c 7.1c −2.1c 58.1ab 18.1a

SEM

P

0.7 0.8 0.6 3.0 0.6

<0.001 <0.001 <0.001 <0.001 <0.001

Mean-Temp: daily mean temperature; Max-Temp: mean daily maximum temperature; Min-Temp: mean daily minimum temperature; RH: relative humidity; WS: wind speed. Means within a row without a common letter (a−c) are different at P<0.05. a n = 31 at each period. Table 2 Biomass production and chemical composition of the herbage available and of feces voided by grazing yaks. 2010

2011

August (summer)

December (winter)

May (spring)

October (autumn)

Forage Biomass, kg DM/ha CP, g/kg DM NDF, g/kg DM Digestible OM, g/kg DM ME, MJ/kg OM Crude ash, g/kg DM

496a 166a 329b 665b 10.4b 106.1b

208c 57.9b 657a 218c 3.4c 163.3a

226c 179a 312b 819a 12.8a 124.1ab

351b 62.7b 615a 242c 3.8c 156.6a

Feces NDF, g/kg DM Crude ash, g/kg DM

452b 95c

489a 205b

464a 273a

477a 203b

SEM

P

21 2.3 1.7 20.9 0.3 1.4

<0.001 <0.001 <0.001 <0.001 <0.001 <0.001

1.8 1.0

0.04 <0.001

Means within a row without a common letter (a−c) are different at P<0.05.

store GPS co-ordinates and motion sensor counts at 5 min intervals. The yaks grazing locations were identified by overlaying the GPS coordinates on aerial photographs of the alpine pastures. The horizontal distance traveled during each 5 min interval was calculated using ArcMap 9.2 software. Because of the limited accuracy of the vertical component of GPS location, all coordinates were converted to horizontal coordinates. Therefore, the calculated traveling distances represent only the horizontal distance covered, and not the actual distance traveled by the yaks. GPS location data were used to track the yaks’ traveling activity. Unfortunately, some of the GPS recorder data in October were lost because of technical problems during data download and only location data were available in this month. 2.6. Statistical analysis All statistical analyses were conducted using SAS software (version 9.2, SAS Institute Inc., Cary, NY). A mixed model (Proc MIXED) was used with period (four periods) as fixed effects by least squares means, and yak as random effects. The Student–Newman–Keuls multiple-range test within one-way ANOVA was applied as multi-comparison between the four seasons with season as the main effect. The correlations of HP and ER with MEI were estimated using PROC REG and PROC CORR. The diurnal pattern of HP was determined by separating the 24 h of a day into 12 2-h sub periods, and calculating the average HP for each yak in each sub period. 3. Results 3.1. Meteorological conditions Meteorological recordings during the four measurement periods are presented in Table 1. Mean daily and maximum and minimum temperatures differed significantly between the four periods and showed the same pattern of differences: May < August > October > December. During December the temperature never rose above −4 ◦ C. Mean relative humidity (RH) was significantly greater in August than in May and December. The least mean RH was found in December. Mean wind speeds were similar in August, December and May, but were lower in October. 3.2. Herbage biomass and quality The vegetation biomass and chemical analyses of the representative samples selected by yaks and of feces are shown in Table 2. The aboveground herbage mass differed significantly between the four periods, with the greatest biomass occurring

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1200

Daily traveling distance (km/day, P < 0.05): 1000

Traveling distance (m)

December: 3.2± 0.21 May: 5.3± 0.13

800

600

400

200

9: 00 10 :0 0 11 :0 0 12 :0 0 13 :0 0 14 :0 0 15 :0 0 16 :0 0 17 :0 0 18 :0 0 19 :0 0 20 :0 0 21 :0 0 22 :0 0 23 :0 0

8: 00

7: 00

6: 00

5: 00

4: 00

3: 00

00 2: 00

1:

0: 00

0

Time Fig. 1. Mean horizontal distances traveled (±S.E.) by free-ranging yaks within each hour of the day measured 6 days during May (, n = 30) and December (, n = 22). Corralled time: December 2000–0700 h, May 2100–0500 h.

in August (summer) and the least mass in December. Analysis of samples representative of herbage selected by the yaks showed that CP concentration, digestible OM and ME were significantly higher in August and May, when vegetation was green, compared with December and October after the pasture had senesced. The digestible OM and ME of herbage were significantly greater in May than in August. Conversely, the NDF concentration of the herbage was significantly lower in August and May than in December and October. In addition analysis of fecal samples showed that NDF concentration was significantly lower in August compared with the other three sampling periods. 3.3. Traveling pattern The average traveling distances calculated from the GPS data in December and May is shown in Fig. 1. The distance traveled whilst grazing was included in the total traveling distance. The diurnal pattern of activity differed between periods. In December yaks traveled between 1000 and 1900 h, with a minor peak around 1200 h and a major peak around 1730 h. In May, yaks started to travel at about 0530 h and continued until 2400 h, but with two peaks of activity at around 0830 and 1830 h. The total daily distance traveled in December was lower than in May (3.2 vs. 5.3 km), this is a reflection of the distribution of grazing locations in the two periods (Fig. 2). Data for calculating the distances traveled in October were lost because of technical problems during downloading. Comparison of the overlays of the GPS coordinates of yak locations superimposed on an aerial photograph of the pastures illustrates the greater pasture area traversed by the yaks in October and May than in December. 3.4. Yak energy balance Calculated energy balance variables are presented in Table 3. Mean bodyweight (BW) and dry matter intake (DMI) of the experimental yaks did not differ significantly between August, December and October, but were significantly less in May. O2 P (ml/beat) was significantly greater in May than in December, with the values in August and October being intermediate. However, when expressed relative to BW, O2 P (␮l/beat/kg BW) was significantly greater in May than in the other three periods, which did not differ significantly. Mean MEI and HP per yak were significantly greater in August (summer) than during the other three periods of the year. The lowest MEI per yak was in December, and no significant differences were found between May and October. The HP per yak was greater in October than in December, but not different with in May. The energy retention (ER) per yak during August was significantly greater compared to October, December and May. The lowest ER per yak was in May. The term ER in the present study represents the sum of energy retained in the body (mainly fat accretion) and energy from tissues mobilization in support of maintenance, including physical activity and/or thermoregulation. When MEI, HP and ER were expressed relative to BW0.75 , the pattern of differences across the four seasons was the same as when values were expressed per yak except that no significant differences of MEI were found between December and October, no significant differences of ER among May, December and October, and no significant differences of HP between August and May. Otherwise, the lowest HP was in December. Calculation of HP from measurements of HR at 0230 h (from 0130 to 0330), when yaks were restrained in corrals, is also presented in Table 3, where HP is shown to be significantly lower during December than in May, August or October. The diurnal patterns of heat production in the four seasons are shown in Fig. 3. There is an increase of HP in the morning and decrease at night from 1830 to 0230 in four seasons.

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Fig. 2. Overlays of aerial photographs showing grazing locations of six individually monitored yaks recorded at 5-min intervals over 6 days during December, May and October. Table 3 Energy balance of grazing yaks in August, December, May and Octoberb . 2010 August (summer) LW (kg) DMI (kg/day) HR (beats/min) O2 P (ml/beat) O2 P-1 (␮l/beat per kg BW) Energy balance MJ/animal per day MEI HP ER kJ/kg BW0.75 per day MEI HP ER HP at 0230 ha

253a 6.7a 78a 19.7ab 75.7b

70.1a 44.8a 25.4a 1120a 715a 405a 679a

2011

SEM

P

December (winter)

May (spring)

October (autumn)

254a 5.7a 49b 18.0b 70.8b

187b 2.4b 52b 23.0a 119.3a

243a 7.1a 52b 20.4ab 91.2b

5 0.2 2 2.4 4.7

<0.001 <0.001 <0.001 0.03 <0.001

27.1b 33.9b −6.8b

2.8 2.1 1.6

<0.001 0.001 <0.001

19.4c 25.2c −5.8b 309c 400c −91b 362b

30.6b 31.3bc −0.7c 627b 640ab −13b 616a

437bc 548b −111b 556a

51 38 27 30

<0.001 <0.001 <0.001 <0.001

Abbreviations: BW – body weight, DMI – dry matter intake, HR – heart rate, O2 P – oxygen pulse, MEI – metabolizable energy intake, HP – heat production, ER – energy retention. Means within a row without a common letter (a−c) differ (P<0.05). a Average HP between 0130 and 0330, which represents HP of resting yaks in all period. b n = 6.

3.5. Correlation of HP and ER with MEI Yak HP dependency on MEI is presented in Fig. 4. Linear regressions of HP and of ER on MEI by yaks over the four periods of measurement were: HP = 342(±35) + 0.373(±0.051) × MEI (n = 21, R2 = 0.73, P < 0.001)

(1)

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Fig. 3. Diurnal pattern of mean heat production (HP) within each 2-h of the day, measured over 4 days in 10 free-ranging yaks (n = 40) during four seasons;  spring (May), ♦ summer (August), 䊉 autumn (October),  winter (December).

Fig. 4. Effect of MEI on heat production (HP, ) and energy retention (ER, ) of free-ranging yaks measured over the four seasons of the entire experiment (n = 22, 5 means in summer and winter, and 6 means in spring and autumn).

ER = −342(±35) + 0.627(±0.051) × MEI (n = 21, R2 = 0.88, P < 0.001)

(2)

HP, ER and MEI are presented as KJ/kg BW0.75 per day. The quadratic regression also showed significant effect (P=0.0084), but it is weaker than linear regression. The implication of the above regression equations are: (1) Yak fasting HP is 342 kJ/kg BW0.75 per day. (2) Calculated MEI for maintenance (MEm ; when ER = 0 and MEI = HP) of grazing yaks is 545 kJ/kg BW0.75 per day. (3) In the grazing yak, in which concomitant energy expenses for physical activity and thermoregulation are expected, the net efficiency of utilization of ME for gain is estimated as 0.627. 4. Discussion 4.1. Adaptation of yaks to harsh environment The experimental design and analysis were constrained by the numbers of suitable yaks available. The calculated MEI based on the use of Cr2 O3 as a marker was also constrained by the soil ingestion. It also required knowledge of the digestible OM content (g/kg DM) to obtain the ME content of the forage consumed, considering that the energy content of the digestible OM is 19 MJ/kg and that corresponding energy losses as methane and urine attains 18% of this energy. Yaks have evolved to survive in the harsh environment of the Tibetan plateau, and display various adaptations including: (1) their ability to withstand considerable gains and losses in live weight, mainly attributable to deposition and utilization of fat reserves (Xue et al., 2005); (2) morphological and behavioral adaptations for eating a wide variety of plant species when the herbage mass is very low (Shao et al., 2010; Weiner et al., 2003); (3) morphological adaptation of the trachea and

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lungs for cold, high altitudes (Zhang, 1989); (4) good insulation during winter, afforded by coverage of the whole body with a highly insulating double coat. The yak pelage comprises an outer coat that is thick, long and course and an under coat that is fine and dense (Weiner et al., 2003). The season of pasture growth on the Tibetan plateau is from June to September. The pastures are transitional in quality during May, when they consist of a mixture of dead and newly emerging herbage, and during October when the current season’s herbage is beginning to senesce. The protein concentration of the herbage usually rises rapidly to a maximum in June, declining slowly over the following four months. Herbage biomass usually reaches a maximum in August (Zhao and Zhou, 1999). Thus there are wide fluctuations in the availability and quality of the natural food supply, in addition to the environmental challenges of climate and altitude. This paper focuses for the first time on measuring the energy balance of free-grazing yaks on the Tibetan plateau during four seasons of the year. Herbage representative of that eaten by the yaks manifested the lowest NDF concentration and highest CP concentration in May and August. With the increasing senescence of the pasture during the autumn, NDF concentration rose and CP declined. Thus, by October and December NDF concentration was significantly greater and CP was significantly lower than in the previous months. These seasonal values are in accordance with those reported in a previous study at this site (Ding et al., 2007). Organic matter digestibility of the herbage representing that selected by the yaks was highest in May due to the contribution of newly emerging plants. For Friesian dairy cows, the major determinants of herbage DMI are usually considered to be the morphological characteristics of pastures, such as sward mass or sward surface height, rather than herbage quality (Gibb et al., 1997). However, in the present study, no significant relationship between herbage biomass and daily DMI was found. Although herbage biomasses were similar in May and December and significantly lower than in August and October, herbage DM intake in December was nearly twice of that in May. A possible explanation for this contrast undoubtedly lies in the quality of the herbage in the pastures. During the winter pastures consist entirely of dead plant material with low CP and high fiber concentrations. During this period yaks have to maintain high daily herbage intake simply in order to survive. Indeed, it is so vital to maintain these levels of intake, despite the requirement to dig through moderate coverings of snow (Weiner et al., 2003). Severe snowfall events can cause catastrophic losses in yak populations (Shang et al., 2012). In contrast, with similar herbage mass in May, but when young green plants were emerging, it appears that the yaks adopted a selective strategy of maximizing quality at the expense of quantity, as evident from their daily DM intakes and the CP and fiber concentrations of sampled herbage representative of that which was being eaten. The significantly higher fecal ash content in May is because of the great soil ingestion when the yaks hunted the new germinating herbage as reported by Aharoni et al. (2004). 4.2. Energy balance Live weights of yaks in the present experiment were lowest in May and equivalent to 75% of the mean live weight in the other three periods, similar to earlier findings (Ding et al., 2007). Such loss of live weight in winter and spring is due to the mobilization of body reserves, mainly fat, to provide the energy necessary for maintenance during the cold winter months when herbage quality is poor and frequently scarce due to overgrazing or snow cover. The differences in herbage intake and live weight during the different seasons showed seasonal balances between energy intake and animal requirements. Fat tissue has a low energy requirement for maintenance compared with muscle tissue (Chowdhury and Ørskov, 1997), and therefore a much lower maintenance HP. Thus the energy requirement for maintenance is largely related to the lean body mass, rather than live weight. Consequently, it may appear logical to compare the physiological energy variables measured in this experiment when expressed on a whole-animal basis, and not only relative to their current metabolic body weight. In addition, in a study covering a large range of BW, Shargal (2006) concluded that O2 P should be calculated per BW rather than per BW0.75 . Heat production was calculated in the yaks as the product of heart rate by oxygen consumption per heart beat (O2 P) and energy emitted by oxidation. With the relatively simple equipment currently available, reliable measurements of heart rate can easily be determined on free-ranging herbivores over periods of several days. However, in contrast, O2 P is calculated from measurements of the number of heart beats and volume of oxygen consumed (VO2 ) over periods of approximately 20 min. In a review of the technique to measure energy expenditure, Brosh (2007) cited evidence (Aharoni et al., 2004; Brosh et al., 2004) that O2 P measured in non-pregnant, non-lactating beef cows was approximately 20% greater when they were grazing compared to when they were confined. The O2 P of the above cited grazing and confined cows were measured in a squeeze chute. In the present experiment, measurements of O2 P were conducted, similarly to the above cited experiments, by restraining the yaks using a metal cage following adaptation to this means of restraint in order to minimize interference with their daily behavior and natural energy balance. The variation of O2 P in the four periods was possibly due to environmental factors, notably the variable temperature at the trial site which cover a wide range (>20 ◦ C) across seasons. However, much of the O2 P variation can be explained when O2 P is expressed relative to BW, as recommended by Shargal (2006). Thus the O2 P (␮l/beat/kg BW) did not differ significantly between August, October and December, but was significantly higher in May. We assumed that most of the reduction in yak BW at May was the result of body fat loss. Consequently the highest O2 P (␮l/beat/kg BW) in May is mainly explained by the greater proportion of the lean mass tissues in the BW being attributable to high HP of muscle tissue and visceral tissues compared with fat tissues. The greater daily traveling distance in May (spring) probably contributed to the increased O2 P in May. When the grazing yaks’ O2 P were calculated as ␮l/beat/kg BW recommended by Brosh et al. (2002) they were in the range of those they found in grazing cows (63–81 ␮L/beat/kg BW).

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The increased walking activity in May, in spite of the shortage in energy reserves of the yaks, was probably motivated by the combination of high quality forage and low herbage mass. Ding et al. (2008) reported that the duration of yaks’ daily grazing activity increased up to 11.25 h at this time of year, compared with 6–7 h in the winter and early spring (December–April). The greater dietary ME concentration during spring may compensate for the increased energy expenditure associated with increased grazing and walking activity. The significantly negative energy balance in October is mainly a consequence of the low quality of grazed forage due to seasonal senescence. In addition, the seasonal high wind speeds in October (Table 2), before growth of a full winter coat, may increase heat dissipation from the yaks’ bodies. The worst survival conditions for yaks in the present study were recorded in December. This was confirmed by measurement of the lowest ambient temperature, lowest standing biomass and lowest forage quality. In spite of these extremely harsh conditions, the yaks’ ER in December was not significantly different from the ER measured in May and October. This unusual result can be explained by the good insulation of the yak’s pelage (Zhang, 1989), by a reduction in the yaks’ activity during winter and by the yaks’ ability to reduce their HP to less than their maintenance level, especially when resting during extremely cold winter nights when HP recorded at 0230 was 362 ± 25 kJ/kg BW0.75 per day (Table 3). 4.3. Energy efficiency in yaks In the present study, FHP, MEm and kg have been estimated for yaks in the open-range. Therefore, the yaks were using part of the ingested ME to meet energy expenses for grazing and walking activities which is linked to the availability and nature of the pasture, and for thermoregulation provided that the environmental temperature stood below the thermoneutral zone. In the present study we used adult dairy yaks, whose BW changes were mainly changes of fat content and partly changes in rumen gut fill. Thus BW changes were mainly related to the changes in fat content (depletion and deposition) whose energy efficiency use for maintenance and for gain respectively was much more efficient (Old and Garrett, 1987) than for conventional gain (protein and fat deposition). According to NRC (2001), which cited Moe et al. (1971), the kg of non lactating dairy cows is 0.6, which is very close to the km value of 0.62 (NRC, 2001). Consequently, it is legitimate to use the regression equation of HP dependency on MEI for calculating yaks’ FHP, MEm and km,g . The heat increment fraction of 0.37 ± 0.051 (see Eq. (1)) means an energy efficiency of converting changes in MEI to ER of 0.63 ± 0.051 (see Eq. (2)), which is same with the value of 0.63 measured by Brosh et al. (2004) for grazing cows. According to Tolkamp (2010), under natural conditions, where ruminants were fed ad libitum, the overall energetic efficiencies (km , kg , kL ) are the same and close to 0.6. Tolkamp’s (2010) suggestions are in full agreement with the findings of Brosh et al. (2004), in which beef cows in all reproductive states, including lactation, were studied, while grazing forages with a large range of nutritive quality. These previously reported values for km and kg agree with the present measurements in grazing yaks consuming forages with a wide range of nutritive quality, during which intake was close to maintenance requirements or caused significant accretion or loss of body fat.

4.4. Fasting heat production (FHP) The significant linear regressions of the HP and ER on MEI (Fig. 4) in the four periods were similar to the results found by Brosh et al. (2002, 2004) in confined and grazing beef cows, respectively, with a wide range and distribution of diet ME. The high dependency of HP and ER on MEI was used to calculate fasting HP (FHP, regression intercept of HP on MEI), heat increment (regression slope), maintenance requirement under natural grazing conditions (MEm , when MEI = HP). Brosh et al. (2004) calculated the above energy parameters of grazing beef cows. The parameters obtained in the present study were very close to the widely accepted values for energy parameters of beef cattle published by NRC (1996) and AFRC (1993). In the present study, the recorded data were collected only during four periods. Thus, in spite of the statistical significance of the energy parameters determined in the present study, it is recommended that additional studies, representing a greater distribution of forage ME, are required to verify yaks’ MEI. In spite of the above mentioned limitation, this study is the first to measure yaks’ energy balance parameters in their natural grazing habitat in different seasons of the year. Energy balance and physiological parameters are frequently expressed relative to an animal’s metabolic body weight. In the present study, the predicted FHP of yaks was 342 kJ/kg BW0.75 per day, which is numerically greater than the FHP of beef cattle (322 kJ/kg BW0.75 per day) and grazing cows (328 kJ/kg BW0.75 per day), reported by NRC (1984) and by Brosh et al. (2004), respectively. The present value for FHP seems to be greater than the reported value of 3- to 3.5-year-old yaks in confinement in summer (304 kJ/kg BW0.75 per day) (Han et al., 2002).

4.5. Metabolic energy for maintenance Calculations from our data showed the maintenance ME requirements (MEm ) of grazing yaks (545 kJ/kg BW0.75 per day) to be numerically greater than the corresponding values of beef cattle (523 kJ/kg BW0.75 per day) reported by Brosh et al. (2004) and greater than values published for beef cows (402–527 kJ/kg BW0.75 per day; NRC, 1996). The MEm of grazing yaks in the present study was considerably greater than the value 458 kJ/kg BW0.75 per day calculated from measurements made

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on confined 3-year-old yaks by Han et al. (1993). The explanation of this difference is the greater physical activity of the free-ranging yaks compared to the confined conditions. 5. Conclusions This is the first study on yak energy utilization under free grazing conditions and it has demonstrated the application of HR and O2 P measurement techniques in such studies. The high dependence of HP and ER on MEI can be used to estimate FHP, energy efficiency and maintenance energy requirement of grazing yaks. We suggest that the ability of yaks saving energy by a significantly reduction of HP at night, when they are not grazing, greatly helps the yak to survive in a harsh habitat. It is concluded that the free-ranging yaks expended much more energy to resist the extremely harsh conditions imposed by low temperatures, very low forage quality and availability, compared with confined yaks or cattle, and the grazing cattle under less severe, climatic conditions. 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