- Email: [email protected]
Urea kinetics and nitrogen balance and requirements for maintenance in Tibetan sheep when fed oat hay
Accepted Manuscript Title: Urea kinetics and nitrogen balance and requirements for maintenance in Tibetan sheep when fed oat hay Author: J.W. Zhou X.S. Guo A.A. Degen Y. Zhang H. Liu J.D. Mi L.M. Ding H.C. Wang Q. Qiu R.J. Long PII: DOI: Reference:
S0921-4488(15)00199-6 http://dx.doi.org/doi:10.1016/j.smallrumres.2015.05.009 RUMIN 4946
To appear in:
Small Ruminant Research
Received date: Revised date: Accepted date:
12-3-2015 6-5-2015 8-5-2015
Please cite this article as: Zhou, J.W., Guo, X.S., Degen, A.A., Zhang, Y., Liu, H., Mi, J.D., Ding, L.M., Wang, H.C., Qiu, Q., Long, R.J.,Urea kinetics and nitrogen balance and requirements for maintenance in Tibetan sheep when fed oat hay, Small Ruminant Research (2015), http://dx.doi.org/10.1016/j.smallrumres.2015.05.009 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.
Urea kinetics and nitrogen balance and requirements for maintenance in Tibetan sheep
2
when fed oat hay
3
J. W. Zhoua,b, X. S. Guob,c, A. A. Degend,*, Y. Zhanga,b, H. Liua,b, J. D. Mib,c, L. M. Dingb,c,
4
H. C. Wanga,b, Q. Qiuc and R. J. Longa,b,c
5
aState
6
Agriculture Science and Technology, Lanzhou University, Lanzhou 730000, PR China.
7
bInternational
8
Lanzhou 730000, PR China.
9
cSchool
of Life Sciences, Lanzhou University, Lanzhou 730000, PR China.
10
dDesert
Animal Adaptations and Husbandry, Wyler Department of Dryland Agriculture,
11
Blaustein Institutes for Desert Research, Ben-Gurion University of Negev, Beer Sheva
12
84105, Israel.
cr
an
us
Key Laboratory of Grassland and Agro-Ecosystems, College of Pastoral
te
d
M
Centre for Tibetan Plateau Ecosystem Management, Lanzhou University,
Ac ce p
13
ip t
1
14
*Corresponding
author: A. A. Degen; Email: [email protected]
15
Running head: Nitrogen metabolism and requirements in sheep
16
Highlights 1
Page 1 of 39
17 18 19 20 21
Tibetan sheep were offered oat hay at 0.3 to 0.9 times voluntary intake Estimated N requirements for maintenance were 0.50 g/kg BW0.75 per day This is 66% of the amount of N recommended by NRC for growing sheep of their size It was concluded that Tibetan sheep can cope with low N intake
ip t
22
ABSTRACT
24
Tibetan sheep inhabit the Qinghai-Tibetan Plateau, an area characterized by sparse
25
vegetation of low protein content much of the year. Consequently, we predicted that
26
their N requirements for maintenance would be low. To test our prediction, we
27
determined urea kinetics, N balance and requirements for maintenance in five growing
28
wethers. A 4 × 4 Latin square design was used with 4 levels of oat hay intakes (0.3, 0.5,
29
0.7, and 0.9 × voluntary intake). Urea kinetics were determined using a continuous
30
intrajugular infusion of
31
urinary N elimination and N retention (P < 0.01) with an increase in feed intake. From
32
the regression equation generated between N retention and N daily intake, the
33
estimated N requirements for maintenance were 0.50 g/kg BW0.75 per day, that is, only
34
66% of the amount recommended by NRC for growing sheep of its size. Urea-N entry
35
rate (UER), gastrointestinal tract (GIT) entry rate (GER), return to ornithine cycle (ROC)
Ac ce p
te
d
M
an
us
cr
23
15N15N-urea.
There was a linear increase in faecal N excretion,
2
Page 2 of 39
and faecal urea-N excretion all increased linearly (P < 0.01) with an increase in N intake.
37
The ratio of UER to apparent digestible N intake increased linearly from 1.53 to 2.99
38
with a decrease in feed intake. The proportion of UER that entered the gut did not differ
39
(P > 0.05) among intakes and ranged between 0.52 and 0.61. GER used for anabolism
40
decreased (P < 0.01) from 0.45 to 0.28, whereas GER to ROC increased (P < 0.01)
41
from 0.51 to 0.68 with increasing N intake. Glomerular filtration rate increased (P < 0.05)
42
with increasing N intake, but urinary creatinine excretion and plasma urea-N
43
concentrations remained constant (P > 0.05). The proportion of renal urea-N reabsorbed
44
increased linearly with a decrease in N intake (P < 0.05) while GIT urea-N clearance
45
was always higher than kidney urea-N clearance. The low nitrogen requirements
46
demonstrated by the Tibetan sheep supported our prediction. The increase in renal
47
urea-N absorption rate with a decrease in nitrogen intake, the greater ratios of UER to
48
apparent digestible N intake and the greater GIT urea-N clearance to kidney urea-N
49
clearance, regardless of N intake explain, at least in part, how Tibetan sheep cope with
50
low nitrogen intake.
Ac ce p
te
d
M
an
us
cr
ip t
36
51 3
Page 3 of 39
52
Keywords: Feeding level, Nitrogen balance, Urea kinetics, Nitrogen requirements for
53
maintenance, Tibetan sheep
1. Introduction
cr
55
ip t
54
The Qinghai-Tibetan Plateau is the highest and largest plateau in the world, with a
57
grassland area of 1.29 × 108 ha, which accounts for 1/3 of the total grassland in China
58
(Gerald et al., 2003; Long and Ma 1996). Due to the extremely harsh environment of the
59
plateau, namely, high altitude, severe cold, hypoxia, strong UV radiation and short
60
growing season, herbage and nutrients for livestock are deficient in the long cold season,
61
especially when raised under traditional grazing management (Gerald et al., 2003; Long
62
et al., 2004 and 2005; Xin et al., 2011). The indigenous yak has adapted well to these
63
conditions and today there are about 14 million yaks being raised on the plateau.
64
Although much less known and glamourous than the yak, Tibetan sheep are also
65
indigenous to the plateau. There are approximately 50 million Tibetan sheep being
66
raised at altitudes between 3000 and 5000 meters. Both of these indigenous ruminants
67
produce milk and meat for human consumption, fibre for clothing and dung for fuel, and
Ac ce p
te
d
M
an
us
56
4
Page 4 of 39
68
are vital for the livelihoods of the herders raising them (Long et al., 2008). Tibetan sheep and yaks only graze on natural pasture and are not offered
70
supplements. These livestock have to cope with poor pasture conditions, in particular,
71
the low N content of forage in winter. A number of studies have been made on yaks
72
including measurements on feed digestibility, rumen fermentation and nitrogen utilization
73
(Long et al., 1999, 2004 and 2005; Guo et al., 2012). Noteworthy among these
74
ruminants is their low nitrogen requirements for maintenance that ranged between 0.40
75
(Hu 2001) and 0.53 g N/kg0.75 per day (Long et al., 2004). We predicted that N
76
requirements for maintenance in Tibetan sheep would also be low. We tested our
77
prediction by determining N requirements for maintenance and by examining urea
78
kinetics in Tibetan sheep receiving rations that differed in N and energy intakes.
cr
us
an
M
d
te
Ac ce p
79
ip t
69
80
2. Materials and methods
81
2.1 Study site
82
The study was conducted at Wushaoling Yak Research Facility (37°12.4 N,
83
102°51.7 E, altitude 3,154 m, located in the northeast of the Qinghai-Tibetan Plateau) of 5
Page 5 of 39
the International Centre for Tibetan Plateau Ecosystem Management, Lanzhou
85
University, from November, 2012, to March, 2013. The experimental procedures were
86
approved by the Animal Ethic Committee of Gansu Province. Air temperature ranged
87
between 2 and 6°C and relative humidity ranged between 67 and 80% throughout the
88
experimental period.
us
cr
ip t
84
2.2 Animals and diets
M
90
an
89
Five wether Tibetan sheep (20 - 24 months old; 43.0 ± 2.3 kg live weight) were
92
purchased from nomadic herders. They were offered only oat hay, which was chopped
93
into 1 - 2 cm length before feeding. The oat hay contained: 885.0 g dry matter (DM)/kg;
94
15.3 g N/kg DM; 3.9 g Ca/kg DM; 1.8 g P/kg DM and yielded 8.5 MJ metabolizable
95
energy (ME)/kg DM.
Ac ce p
te
d
91
96
The sheep were penned individually in metabolic cages, and were provided with
97
fresh water ad libitum throughout the study. As these animals only grazed on the
98
rangeland previously, a long adaptation period of 45 days was allowed for the sheep to
99
familiar themselves with the feed, workers, facilities, and urine-collection apparatus. 6
Page 6 of 39
100
Then, the animals were fed ad libitum for 14 days to determine voluntary intake (VI), and
101
the lowest intake (1013 g/d DM) was used to determine the different feeding levels. All animals were offered 4 feeding levels (0.3VI, 0.5VI, 0.7VI and 0.9VI) in a 4 × 4
103
Latin square design with one sequence repeated. The intakes were 0.38, 0.64, 0.90 and
104
1.12 times estimated energy requirements for maintenance. The feeding levels from
105
0.9VI to 0.3VI were roughly equal to the daily DM intake of grazing Tibetan sheep from
106
start of winter to early spring (Li et al., 2009). The sheep were fed twice a day, receiving
107
half the portion at 0800 h and the other half at 1800 h.
108
2.3 Sample collections
Ac ce p
109
te
d
M
an
us
cr
ip t
102
110
The feeding trial consisted of 4 periods, each lasting 21 days: 15 days for diet
111
adaptation, 5 days for collection of outputs and the final day for blood sampling.
112
Experimental procedures followed previous urea kinetics studies using the
113
tracer method (Lobley et al., 2000; Huntington et al., 2009; Marini et al., 2003). Sheep
114
were weighed at the beginning and the end of each period before the morning feeding.
115
15N15N-urea
For urine collection, a latex bag was fixed around the penis of each wether using 7
Page 7 of 39
elastic belts. Urine flowed via a tube from the bag to a plastic container which contained
117
sufficient 50% (v/v) H2SO4 solution to keep the pH < 3. On day 15, in-dwelling catheters
118
were fixed in the sheep’s left and right jugular veins for isotope infusion and blood
119
sampling. Starting on day 18, animals were infused continuously for 56 h with15N15N-
120
urea (99.24 atom %
121
sterile 0.15 mol/l NaCl solution, and the infusion rate was adjusted to 30 ml/h using a
122
peristaltic pump (BT100-1L, Baoding constant-flow Pump Corporation, Baoding, China).
123
The concentration of the
124
the
125
(APE) at plateau. The calculation of
126
described by Guo et al. (2012).
Shanghai Chemical Research Institute, Shanghai, China) in
d
was 2.213 mmol/l, which was predicted to enrich
concentration in the 0.9VI fed sheep by 0.15-0.25 atom percent excess
te
15N15N-urea
15N15N-urea
M
an
us
15N,
cr
ip t
116
infusion was according to the method
Ac ce p
15N15N-urea
127
Faeces and urine samples were taken before infusion for background samples, and
128
then collected every 2 h between 48 to 56 h to calculate urea kinetics (assumed the
129
plateau was reached; Lobley et al., 2000). Urine samples were stored at -20°C for later
130
analysis of urea-N concentration and enrichment with
131
Total faeces and urine were collected during days 15 to 20. Before the morning feeding,
15N15N-urea
and
14N15N-urea.
8
Page 8 of 39
outputs were measured and subsamples (0.30 of daily urine and 0.40 of daily faeces)
133
were retained. Faecal subsamples were combined for each period and urine
134
subsamples were mixed daily and stored at -20°C. On day 21, jugular blood was
135
collected before the morning feeding and at 2-h intervals after feeding until 1600 h into
136
heparinized vacutainer tubes (Shanghai Kehua Bio-engineering Co. Ltd, Shanghai,
137
China). The blood was placed on ice, centrifuged at 1200 g for 15 min at 4°C within 1 h
138
after collection and the plasma was stored at -20°C for determination of urea-N and
139
creatinine concentrations.
d Ac ce p
2.4 Laboratory analyses
te
140
141
M
an
us
cr
ip t
132
142
Oat hay and faecal samples were freeze-dried for 1 week using a FreeZone-12L
143
freeze dryer (Labconco Corporation, Kansas City, Missouri, USA), ground to pass
144
through a 1-mm screen, and then stored in self-sealed plastic bags at room temperature.
145
The DM of oat hay and faeces was determined by drying in a forced-draught oven for 24
146
h at 105°C. N content of oat hay, faeces and urine was determined using the micro
147
Kjeldahl N method (AOAC, 1990). Creatinine concentration of the plasma and urine was 9
Page 9 of 39
determined using Agilent 1200 high-performance liquid chromatography (Agilent
149
Technologies Corporation, Santa Clara, California, USA) with the reversed-phase
150
column according to the procedure of Balcells et al. (1992). Plasma and urine urea-N
151
concentrations were determined using the spectrophotometer (U-2900, Hitachi, Tokyo,
152
Japan) in the diacetymonoxime method described by Marsh et al. (1957). Ammonia-N in
153
urine was determined colorimetrically as described by Broderick and Kang (1980).
cr
us
15N
an
Faecal
enrichment was analyzed in a continuous-flow mode using a stable
M
154
ip t
148
isotope elemental analysis (Flash EA 1112, Thermo Electron SPA; DELYAplus XP,
156
Thermo Finnigan, San Jose, CA, USA). Urinary ammonia-N was removed from the
157
isotope urine samples with a cation-exchange resin (AG 50W-X8, 100- 200 mesh
158
hydrogen form, Bio-Rad, Richmond, CA) according to the procedures described by
159
Wickersham et al. (2008). Final urea-N concentration of the retained effluent was
160
adjusted to 14 mmol/l. Gases of
161
degradation according to the methods described by Marini and Attene-Ramos (2006)
162
and the enrichment of these gases was analyzed using a stable isotope gas bench
163
(ThermoFinnigan Delta Plus).
Ac ce p
te
d
155
28N2, 29N2
and
30N2
were obtained from Hoffman
10
Page 10 of 39
164
166
2.5 Calculation of urea kinetics, glomerular filtration rate and urea pool size The calculation of urea kinetics followed the model generated by Lobley et al. (2000).
ip t
165
The glomerular filtration rate (GFR) was equated with creatinine clearance by the kidney
168
(Wang et al., 2009). The urea pool size was calculated from the urea space and urea
169
concentration in the urea pool according to Harmeyer and Martens (1980), assuming the
170
urea space to be 48.7% of BW (Bartle et al., 1988) and the concentration of the urea
171
pool to be equal to plasma urea-N (Marini et al., 2003).
172
2.6 Statistical analyses
Ac ce p
173
te
d
M
an
us
cr
167
174
Data were analyzed using the MIXED procedure of SAS (SAS Inc., Cary, NC, USA).
175
Feeding level was the fixed effect, with the period and sheep as random effects. Linear
176
and quadratic effects were tested using polynomial contrasts. Model terms for isotope
177
enrichment during infusion were feeding level, time, feeding level × time, and sheep
178
were included as a random effect. Differences were accepted as significant at P < 0.05.
179 11
Page 11 of 39
180
3. Results
181
3.1 N balance and utilization All rations were consumed completely by each sheep throughout the study. Daily
183
total N and digestible N intakes increased linearly with an increase in feeding level (P <
184
0.001), as did faecal N and urine N outputs and N retention (P < 0.01; Table 1). The
185
relationship between N retention (g/d) and N intake (g/d) was highly significant and took
186
the form: N retention = 0.547 N intake ‒ 4.566 (R2 = 0.998, n = 20, SE
187
Figure 1). From this equation, the estimated N requirements for maintenance of the
188
sheep were calculated as 0.50 g/kg BW0.75 per day.
y.x=
0.287y.x;
te
d
M
an
us
cr
ip t
182
N retention rate and urinary urea-N to total urine N ratio increased (P < 0.05) while
190
urinary urea-N elimination rate (either expressed as the proportion of total N or
191
digestible N intake) decreased linearly (P < 0.001) as N intake increased. Urea-N
192
composed 0.71 - 0.91 of total urine N, whereas ammonia-N was less than 1% of the
193
total. Urea-N and ammonia-N increased linearly (P < 0.05) with an increase in N and
194
feed intake. Daily weight gain increased linearly with DM intake (P < 0.001).
Ac ce p
189
195 12
Page 12 of 39
196
197
3.2 Urea kinetics characteristics Urinary
15N15N-urea
and
14N15N-urea
APE both reached plateau enrichments after
infusion of 48 h (Figure 2). Thus the ratio of 14N15N-urea:
199
over the sampling period. Faecal total
200
over the sampling period and, therefore, the faecal urea-N excretion was slightly
201
underestimated and, consequently, the urea-N for anabolism was slightly overestimated.
202
Urea-N entry rate (UER), urinary urea-N elimination (UUE), gastrointestinal tract
203
(GIT) entry rate (GER), return to ornithine cycle (ROC) and faecal urea-N excretion
204
(UFE) all increased linearly (P < 0.01; Table 2) while urea-N for anabolism (UUA)
205
increased quadratically (P < 0.05) as the N and feeding intake increased. UER (g/d) and
206
GER (g/d) related linearly and highly significantly to total N intake (g/d; Figure 3), and
207
could be expressed by the following equations:
208
UER = 0.597 N intake + 5.748 (R2 = 0.998, n = 20, .);
209
GER = 0.390 N intake + 3.177 (R2 = 0.894, n = 20, .).
210
UER was substantially greater than the digestible N intake (2.99, 2.14, 1.77 and 1.53
211
times for 0.3VI, 0.5VI, 0.7VI and 0.9VI diets, respectively), and decreased linearly (P <
was at plateau
cr
enrichment, however, did not reach a plateau
Ac ce p
te
d
M
an
us
15N
15N15N-urea
ip t
198
13
Page 13 of 39
0.001) with N intake. As N intake increased, ROC: GER increased (P < 0.01), UUA:
213
GER decreased (P < 0.01), while GER: UER remained relatively constant (0.52 to 0.61,
214
P > 0.05).
ip t
212
3.3 Plasma urea-N and creatinine concentrations, GFR and renal urea-N reabsorption
us
216
cr
215
GFR increased (P < 0.05, Table 3) and plasma creatinine concentration decreased
218
linearly (P < 0.01) as N intake increased, but urinary creatinine excretion was not
219
affected by intake (P > 0.05). With an increased feeding level, plasma urea-N
220
concentration and urea-N reabsorption did not change (P > 0.05), the urea-N tubular
221
load increased linearly (P < 0.05) but the proportion of renal urea-N reabsorbed
222
decreased linearly (P < 0.05).
224
M
d
te
Ac ce p
223
an
217
3.4 Urea-N pool size, urea-N clearance by the GIT and kidney
225
The urea-N pool size remained constant (P > 0.05; Table 4) when feed intake
226
increased from 0.3VI to 0.9VI, but the turnover time decreased linearly (P < 0.001).
227
Urea-N clearance by the GIT and kidney both increased linearly as feed intake 14
Page 14 of 39
228
increased (P < 0.001).
4. Discussion
231
4.1 N balance and requirements for maintenance
cr
230
ip t
229
Our prediction that N requirements for maintenance in Tibetan sheep would be low
233
was supported by this study. The requirements for maintenance, 0.50 g N/kg BW0.75 per
234
day, were only 66% of the amount recommended by NRC (1985) for a growing sheep of
235
its size and fell within the values of 0.40 to 0.53 g N/kg BW0.7 reported for yaks (Hu 2001;
236
Long et al., 2004). The Baluchi fat‒tailed sheep, a breed also adapted to harsh
237
conditions, required approximately 1.17 g N/kg BW0.75 per day for maintenance
238
(Kamalzadeh and Shabani, 2007).
Ac ce p
te
d
M
an
us
232
239
Absolute N retention increased in Tibetan sheep with an increase in apparent
240
digestible N intake, which was also reported in other sheep breeds, (Sarraseca et al.,
241
1998; Lobley et al., 2000; Marini et al., 2004; Kamalzadeh and Shabani, 2007), as well
242
as in yaks (Guo et al., 2012) and in cattle (Marini and Van Amburgh, 2003). In addition,
243
the ratio of N retention to apparent digestible N intake with an increase in apparent 15
Page 15 of 39
digestible N intake increased in Tibetan sheep. A similar response was found in 40-50
245
kg Suffolk wether sheep (Sarraseca et al., 1998), but, in contrast, a decrease with an
246
increase in apparent digestible N intake was reported in 21 kg Dorset-Finn ewe lambs
247
(Marini et al., 2004). In yaks, Guo et al. (2012) reported that apparent digestible N intake
248
did not affect the ratio of N retention to apparent digestible N intake.
us
cr
ip t
244
Tibetan sheep increased faecal N output linearly with increasing N intake. An
250
increase of faecal N was accompanied by an increase in N intake in some sheep breeds
251
(Sarraseca et al., 1998; Kamalzadeh and Shabani, 2007) but not in others (Lobley et al.,
252
2000; Marini et al., 2004) and, also not in heifers (Marini and Van Amburgh, 2003) nor in
253
yaks (Guo et al., 2012). Urine N also increased with an increase in N intake in Tibetan
254
sheep. This pattern was reported in a number of other sheep breeds (Sarraseca et al.,
255
1998; Marini et al., 2004; Sunny et al., 2007), and in heifers (Marini and Van Amburgh,
256
2003) and in yaks (Guo et al., 2012). In the Tibetan sheep, urine N was always higher
257
than faecal N but the urine N: faecal N ratio declined from 2.60 to 1.27 with an increase
258
in N intake. This is unlike the findings in yaks in which faecal N was greater than urinary
259
urea-N at low N intakes, with the urine N: faecal N ratio at 0.56 and 0.86. At higher N
Ac ce p
te
d
M
an
249
16
Page 16 of 39
intakes the ratios were 1.37 and 1.66 (Guo et al., 2012), that is, within the range for the
261
Tibetan sheep. However, in the Tibetan sheep the urine N: faecal N ratio decreased with
262
an increase in N intake whereas in the yaks it increased. The reason for this difference
263
is that Tibetan sheep increased faecal N excretion with increased N intake but this did
264
not occur in yaks. Faecal N is affected by DM intake regardless of N intake (Mariani et
265
al., 2004) and intake differed among treatments in Tibetan sheep but not in yaks.
an
us
cr
ip t
260
M
266
4.2 Urea metabolism
268
Among ruminants, it is not uncommon for UER to be greater than the apparent
269
digestible N intake (Lapierre and Lobley, 2001). Consequently, in order for such
270
ruminants to remain in positive N balance, a portion of the hepatic urea-N produced
271
must be recycled into the GIT and salvaged for microbial protein synthesis and then AA
272
absorption. In Tibetan sheep, the ratio of UER to apparent digestible N intake decreased
273
linearly from 2.99 to 1.53 with an increase in apparent digestible N and energy intakes.
274
At the two highest ratios, the sheep were in negative N balance and were losing body
275
mass. Sarraseca et al. (1998) also reported a decline in the ratio of UER to apparent
Ac ce p
te
d
267
17
Page 17 of 39
digestible N intake with an increase in apparent digestible N intake in 40-50 kg Suffolk
277
wether sheep. The ratio was 2.03 when the sheep lost 0.31 g N/d but was 1.19 and 1.29
278
when the sheep retained 4.66 and 7.34 g N/d, respectively. In contrast to these results,
279
21 kg Dorset-Finn ewe lambs had a ratio below 1.0 at all N intakes and increased the
280
ratio of UER to apparent digestible N intake from 0.62 to 0.96 with an increase in
281
digestible N intake from 3.9 to 20.0 g N/d (calculated from Marini et al., 2004). High
282
UER to apparent digestible N intakes, as found in the present study, were also reported
283
in semi-wild yak (1.38 to 2.06; Gou et al., 2012) and in wild elk (2.83 to 4.87; Mould and
284
Robbins, 1981), two ruminants that are well adapted to harsh conditions, including
285
periods of low N availability. The high ratios, in particular at the low N intakes, suggest
286
high urea recycling.
Ac ce p
te
d
M
an
us
cr
ip t
276
287
Urea-N recycled to the GIT increased linearly with an increase in N intake, as was
288
also found in a number of sheep breeds (Sarraseca et al., 1998; Lobley et al., 2000;
289
Sunny et al., 2007) and in the yak (Guo et al., 2012). GER as a ratio of UER ranged
290
between 0.52 and 0.61 in the Tibetan sheep, which fell within the 40% to 80% range
291
generally found for sheep (Lapierre and Lobley, 2001). It is expected that the ratio would 18
Page 18 of 39
increase with a decrease in apparent digestible N intake as the demands for N is higher
293
in low N intake animals (Marini and Van Amburgh, 2003). Indeed, this occurred in some
294
sheep such as Dorset-Finn ewe lambs where the ratio decreased from 0.75 to 0.30
295
(Marini et al., 2004) and in Suffolk wethers where it decreased from 0.70 to 0.61 (Lobley
296
et al., 2000) with an increase in apparent digestible N intake. A similar pattern of a
297
decrease in GER: UER ratio, from 0.87 to 0.73, with an increase in N intake was
298
observed in yaks (Guo et al., 2012). However, in Tibetan sheep, this did not occur as the
299
ratio was not affected by apparent digestible N intake. In the studies mentioned above,
300
all animals were in positive N balance. However, the Tibetan sheep were neither in N
301
balance nor energy balance in two of the dietary treatments, as they were below N and
302
energy requirements for maintenance. Urea-N recycling is affected by a number of
303
factors including plasma urea-N concentration (Sunny et al., 2007) and fermentable
304
carbohydrates in the gastrointestinal tract (Kennedy and Milligan, 1980; Lapierre and
305
Lobley, 2001). A fermentable source stimulates the bacterial population which utilizes
306
urea-N and increases GER and, consequently, GER: UER (Kennedy and Milligan, 1980;
307
Sarraseca et al., 1998). Plasma urea-N did not differ in the Tibetan sheep among
Ac ce p
te
d
M
an
us
cr
ip t
292
19
Page 19 of 39
treatments. However, there presumably was a lack of a fermentable source on the low
309
energy intakes, which limited the use of recycled urea-N and decreased GER and GER:
310
UER ratio. There is some evidence to support this. In another study in Tibetan sheep, in
311
which apparent digestible N intake was low but energy intake was above maintenance,
312
the GER: UER ratio was 0.88 (unpublished data). Also, in a study by Sarraseca et al.
313
(1998) in which sheep were offered three diets, one of which was below N and energy
314
requirements for maintenance, the decrease in GER: UER ratio with an increase in
315
apparent digestible N intake was not observed. The lowest GER: UER ratio occurred
316
with a dietary intake below N and energy requirements for maintenance. With an
317
insufficient energy supply, non-protein compounds are converted to ammonia in the
318
rumen which is eventually absorbed and converted to urea. Elevated levels of rumen
319
ammonia reduce GER (Rémond et al., 1993).
Ac ce p
te
d
M
an
us
cr
ip t
308
320
In the Tibetan sheep, UUA and ROC both increased with an increase in apparent
321
digestible N intake. This was also found in other sheep breeds (Lobley et al., 2000;
322
Sunny et al., 2007), heifers (Mariani and Van Amburgh, 2003) and yaks (Guo et al.,
323
2012). Furthermore, the UUA: GER ratio increased while the ROC: GER ratio decreased 20
Page 20 of 39
with a decrease in apparent digestible N intake in Tibetan sheep. That is, Tibetan sheep
325
on the lower N and energy intakes used a larger proportion of the GER for anabolic
326
purposes than sheep on higher N and energy intakes. An increase in UUA: GER ratio
327
with a decrease in apparent digestible N intake was also noted in Polypay × Dorsett
328
wether sheep while a decrease in ROC: GER ratio with a decrease in apparent
329
digestible N intake was also noted in Suffolk cross wether sheep (Lobley et al., 2000)
330
and in heifers (Marini and Van Amburgh, 2003). In Suffolk cross wether sheep (Lobley et
331
al., 2000) and in heifers (Marini and Van Amburgh, 2003), the UUA: GER ratio was not
332
affected by the apparent digestible N intake. Interestingly, a different pattern of that
333
found in the Tibetan sheep was reported for yaks, that is, the UUA: GER ratio increased
334
and ROC: GER ratio decreased with an increase in apparent digestible N intake (Guo et
335
al., 2012).
Ac ce p
te
d
M
an
us
cr
ip t
324
336
UFE was small and composed only 0.06 to 0.08 of the total faecal N excreted in
337
Tibetan sheep. It increased linearly with an increase in N intake, as was found in other
338
sheep breeds (Sarraseca et al., 1998; Sunny et al., 2007) and in the yak (Guo et al.,
339
2012), but not in heifers, that showed a decrease (Marini and Van Amburgh, 2003). The 21
Page 21 of 39
ratio of UFE: GER was also small in Tibetan sheep, ranging between 0.03 and 0.04. In
341
another sheep breed, the ratio decreased from 0.21 to 0.11 with an increase in N intake
342
(Sunny et al., 2007) and in heifers from 0.20 to 0.08 (Marini and Van Amburgh, 2003),
343
but increased from 0.02 to 0.12 in another breed (Sarraseca et al., 1998). The pattern in
344
yaks was similar to that in Tibetan sheep with the ratio ranging between 0.04 and 0.07
345
(Guo et al., 2012).The low values in the Tibetan sheep and yaks would indicate that very
346
little of the GER entered the caecum and colon, which has been suggested for sheep by
347
Dixon and Nolan (1986).
d
M
an
us
cr
ip t
340
UUE increased linearly with an increase in N intake and composed 0.71 to 0.91 of
349
urine N excretion, a ratio that was generally higher than in other ruminants. In Polypay ×
350
Dorsett wether sheep it ranged between 0.40 and 0.80 (Sunny et al., 2007); in Suffolk
351
cross-bred wether sheep between 0.50 and 0.67 (Sarraseca et al., 1998; Lobley et al.,
352
2000), in Dorsett-Finn ewe lambs between 0.29 and 0.81 (Marini et al., 2004), in heifers
353
between 0.18 and 0.74 and in yaks between 0.46 and 0.60 (Guo et al., 2012). Possible
354
reasons for these differences between the Tibetan sheep and the other ruminants could
355
be traced, at least in part, to the rates of UER to urine. UER to urine in Tibetan sheep
Ac ce p
te
348
22
Page 22 of 39
was unaffected by N intake and ranged between 0.39 and 0.48. In fact, the highest
357
value occurred at the lowest N intake. In general, in other ruminants, the proportion of
358
UER to urine was lower. In yaks the proportion was substantially lower than that in
359
Tibetan sheep, ranging between 0.13 and 0.27, and increased with an increase in N
360
intake (Guo et al., 2012). The proportion ranged between 0.19 and 0.37 in Polypay ×
361
Dorsett wether sheep (Sunny et al., 2007), between 0.26 and 0.39 in Suffolk cross-bred
362
wether sheep (Sarraseca et al., 1998; Lobley et al., 2000), between 0.25 and 0.69 in
363
Dorsett-Finn ewe lambs (Marini et al., 2004) and between 0.17 and 0.64 in heifers
364
(Marini and Van Amburgh, 2003).
te
d
M
an
us
cr
ip t
356
Urinary creatinine excretion was not affected by feed intake and, therefore,
366
creatinine was an appropriate internal marker to estimate GFR (Wang et al., 2009). GFR
367
increased with N intake, which was in agreement with previous studies in cattle (Liang et
368
al., 1999), buffaloes (Liang et al., 1999), yaks (Wang et al., 2009), goats (Jetana et al.,
369
2005) and sheep (Chen et al., 1995). The rate ranged from 5.41 to 6.91 l/kg BW0.75 per
370
day in the present study, which was similar to rates in Dorset-Finn lambs (6.21- 7.48 l/kg
371
BW0.75 per day; Marini et al., 2004) and Merino wethers (5.7- 6.8 l/kg BW0.75, Meintjes
Ac ce p
365
23
Page 23 of 39
372
and Engelbrecht, 2004) when receiving similar restricted diets. The urinary N tubular load increased linearly with an increase in N intake but renal
374
reabsorbed urea-N remained constant. This was because the proportion of renal urea-N
375
reabsorbed decreased with an increase in N intake. This would be expected as animals
376
with a lower N intake would need to conserve N to maintain N balance and one of the
377
ways would be to salvage urea from urinary excretion (Schmidt-Nielsen et al., 1958).
378
Similar findings of an increased proportion of renal urea-N reabsorption with a decrease
379
in N intake were reported in Dorsett-Finn ewe lambs (Marini et al., 2004) and in heifers
380
((Marini and Van Amburgh 2003). However, the proportion of renal urea-N reabsorbed in
381
the Tibetan sheep, 0.56 to 0.68, was not particularly high. For example, Bedouin and
382
Saanen goats consuming wheat straw reabsorbed 0.92 and 0.93 of the renal urea-N
383
(Silanikove, 1984). The findings in the Tibetan sheep were due, most likely, to the low
384
energy intake of the low N intake groups. In another study, in which Tibetan sheep
385
consumed a diet of low N intake but with 1.2 times maintenance energy requirements,
386
0.97 of renal urea-N was reabsorbed (unpublished data).
387
Ac ce p
te
d
M
an
us
cr
ip t
373
Urea-N pool size did not differ and N turnover time decreased with an increase in N 24
Page 24 of 39
intake in the Tibetan sheep. In contrast, urea-N pool size and N turnover rate increased
389
with an increase in N intake in yaks (Guo et al., 2012). Kidney and GIT urea clearance
390
increased with an increase in N intake in Tibetan sheep, but while renal urea-N
391
clearance also increased with N intake in yaks, GIT urea-N clearance decreased with an
392
increase in N intake. However, like in the yak, GIT urea-N clearance was always higher
393
than kidney urea-N clearance, the ratio ranging between 1.34 and 1.63. For the Tibetan
394
sheep, it was mainly due to the reduced urea-N excreted in the urine.
M
an
us
cr
ip t
388
In conclusion, Tibetan sheep, like yaks, demonstrated low N requirements for
396
maintenance. Although there were a number of differences between Tibetan sheep and
397
yaks in their nitrogen metabolism and recycling, there were also some common
398
responses. These included the greater ratios of UER to apparent digestible N intake and
399
the greater GIT urea-N clearance to renal urea-N clearance, regardless of N intake. Gou
400
et al. (2012) concluded that these two features “might demonstrate a special
401
characteristic of the yak’s N metabolism”. If so, it would also appear to be so for Tibetan
402
sheep.
Ac ce p
te
d
395
403 25
Page 25 of 39
404
405
Conflict of interest We do not have any conflict of interest.
Acknowledgements
cr
407
ip t
406
This work was supported by grants from the National Nature Science Foundation
409
of China project 31170378. Thanks to colleagues of the International Center for Tibetan
410
Plateau Ecosystem Management for valuable help during the experiment proceeding.
M
an
us
408
411
References
413
Association of Official Analytical Chemists, 1990. Official methods of analysis, 16th edition.
415
te
Ac ce p
414
d
412
AOAC, Arlington, VA, USA.
Barcells, J., Fondevila, M., Peiro, J.M., Parker, D.S., 1992. Simultaneous determination of
416
allantoin and oxypurines in biological fluids by high performance liquid chromatography. J.
417
Chromatog. A 575, 153‒157.
418
Bartle, S.J., Turgeon, O.A. Jr, Preston, R.L., Brink, D.R., 1988. Procedural and mathematical
419
considerations in urea dilution estimation of body composition in lambs. J. Anim. Sci. 66,
420
1920‒1927. 26
Page 26 of 39
421 422
Broderick, G.A., Kang, J.H., 1980. Automated simultaneous determination of ammonia and total amino acids in ruminal fluid and in vitro media. J. Dairy Sc. 63, 64‒75. Chen, X.B., Mejia, A.T., Kyle, D.J., Ørskov, E.R., 1995. Evaluation of the use of the purine
424
derivative: creatinine ratio in spot urine and plasma samples as an index of microbial
425
protein supply in ruminant: Studies in sheep. J. Agric. Sci., Camb. 125, 137-143.
429
cr
us
an
428
rumen in sheep given chopped Lucerne (Medicago sativa) hay. Br. J. Nutr. 55, 313-332. Gerald, W.N., Han, J.L., Long, R.J., 2003. The Yak, 2nd edition. Regional Office for Asia and the
M
427
Dixon, R.M., Nolan, J.V., 1986. Nitrogen and carbon flows between the caecum, blood and
Pacific, Food and Agriculture Organization of the United Nations, Bangkok, Thailand.
d
426
ip t
423
Guo, X.S., Zhang, Y., Zhou, J.W., Long, R.J., Xin, G.S., Qi, B., Ding, L.M., Wang, H.C., 2012.
431
Nitrogen metabolism and recycling in yaks (Bos grunniens) offered a forage-concentrate
432
diet differing in N concentration. Anim. Prod. Sci. 52, 287-296.
434 435 436 437
Ac ce p
433
te
430
Harmeyer, J., Martens, H., 1980. Aspects of urea metabolism in ruminants with reference to the goat. J. Dairy Sci. 63, 1707-1728. Hu, L.H., 2001. Chinese yak nutrition research advances. Qinghai Sci. Tech. 6, 37-39 (in Chinese). Huntington, G.B., Magee, K., Matthews, A., Poore, M., Burns, J., 2009. Urea metabolism in beef
27
Page 27 of 39
438
steers fed tall fescue , orchardgrass, or gamagrasshays. J. Anim. Sci. 87, 1346-1353. Jetana, T., 2005. Urinary purine derivatives as index for estimation of ruminal microbial nitrogen
440
production in sheep and goats. Thesis PhD, University Putra Malaysia, Kuala Lumpur,
441
Malaysia.
446 447
cr
us
an
445
Kennedy, P.M., Milligan, L.P., 1980. The degradation and utilization of endogenous urea in the gastrointestinal tract of ruminants: a review. Can. J. Anim. Sci. 60, 205-221.
M
444
nitrogen of Baluchi Sheep. Int. J. Agric. Biol. 9, 535-539.
Lapierre, H., Lobley, G.E., 2001. Nitrogen recycling in the ruminant: A review. J. Dairy Sci. 84, 223-236.
d
443
Kamalzadeh, A. and Shabani, A., 2007. Maintenance and growth requirements for energy and
te
442
ip t
439
Li, Y.X., Wang, J.Z., Li, L., Wang, H.H., Liu, S.Z., Qiangba, Y.Z., Bian, C., 2009. Research of
449
grazing sheep feed intake and digestibility in northern Tibet cold pastoral area in different
450
seasons. J. Domest. Anim. Ecol. 30, 41-45.
Ac ce p
448
451
Liang, J.B., Pimpa, O., Abdullah, N., Jelan, Z.A., 1999. Estimation of rumen microbial protein
452
production from urinary purine derivatives in zebu cattle and water buffalo. In Proceeding
453
of the Second Research Co-ordination Meeting of a Co-ordinated Research Project
454
(Phase 1), pp. 35-41. International Atomic Energy Agency, Vienna, Austria.
28
Page 28 of 39
Lobley, G.E., Bremmer, D.M., Zuur, G., 2000. Effects of diet quality on urea fates in sheep as
456
assessed by refined, non-invasive [15N15N] urea kinetics. Brit. J. Nutr. 84, 459-468.
457
Long, R.J., Ding, L.M., Shang, Z.H., Guo, X.S., 2008. The yak grazing system on the QinghaiTibetan Plateau and its status. The Rangeland J. 30, 241-246.
cr
458
ip t
455
Long, R.J., Dong, S.K., Hu, Z.Z., Shi, J.J., Dong, Q.M., Han, X.T., 2004. Digestibility, nutrient
460
balance and urinary purine derivative excretion in dry yak cows fed oat hay at different
461
levels of intake. Livest. Prod. Sci. 88, 27-32.
an
M
463
Long, R.J., Dong, S.K., Wei, X.H., Pu, X.P., 2005. The effect of supplementary feeds on the bodyweight of yaks in cold season. Livest. Prod. Sci. 93, 197-204.
d
462
us
459
Long, R.J., Ma, Y.S., 1996. Qinghai’s yak production system. In Proceedings of a workshop on
465
conservation and management of yak genetic diversity, 29- 31 October 1996,
466
Kathmandu, Nepal, pp. 105-115.
Ac ce p
te
464
467
Long, R.J., Zhang, D.G., Wang, X., Hu, Z.Z., Dong, S.K., 1999. Effect of strategic feed
468
supplementation on productive and reproductive performance in yak cows. Preventive
469
Veter. Med. 38, 195-206.
470
Marini, J.C., Attene-Ramos, M.S., 2006. An improved analytical method for the determination of
471
ureanitrogen isotopomers in biological samples utilizing continuous flow isotope ratio
29
Page 29 of 39
476 477 478
ip t
475
nitrogen recycling and urea transporter abundance in lambs. J. Anim. Sci. 82, 1157-1164. Marini, J.C., Van Amburgh, M.E., 2003. Nitrogen metabolism and recycling in Holstein heifers. J.
cr
474
Marini, J.C., Klein, J.D., Sands, J.M., Van Amburgh, M.E., 2004. Effect of nitrogen intake on
Anim. Sci. 81, 545-552.
us
473
mass spectrometry. Rapid Comm. Mass Spec. 20, 3736-3740.
Marsh, W.H., Fingerhut, B., Kirsch, E., 1957. Determination of urea N with the diacetyl method
an
472
and an automatic dialyzing apparatus. Amer. J. Clin. Path. 8, 681-688. Meintjes, R.A., Engelbrecht, H., 2004. Changes in the renal handling of urea in sheep on a low
480
protein diet exposed to saline drinking water. Onders. J. Veter. Res. 71, 165-170.
d
M
479
Mould, E.D., Robbins, C.T., 1981. Nitrogen metabolism in elk. J. Wildl. Manage. 45, 323-334.
482
National Research Council, 1985. Nutrient Requirements of Sheep. 6th revised edition. NRC,
484 485 486 487 488
Ac ce p
483
te
481
Washington, DC, USA.
Rémond, D., Chase, J.P., Delval, E., Poncet C., 1993. Net transfer of urea and ammonia across the ruminal wall of sheep. J. Anim. Sci. 71, 2785-2792. Sarraseca, A., Milne, E., Metcalf, M.J., Lobley, G.E., 1998. Urea recycling in sheep: effects of intake. Brit. J. Nutr. 79, 79-88. Schmidt-Nielsen, B., Osaki, H., Murdaugh, H.V., O’Dell, R., 1958. Renal regulation of urea
30
Page 30 of 39
489
excretion in sheep. Amer. J. Physiol. 194, 221-228. Silanikove, N., 1984. Renal excretion of urea in response to changes in nitrogen intake in desert
491
(black Bedouin goat) and non-desert (Swiss Saanen) goats. Comp. Biochem. Physiol.
492
79A, 651-654.
cr
ip t
490
Sunny, N.E., Owens, S.L., Baldwin IV, R.L., El-Kadi, S.W., Kohn, R.A., Bequette, B.J., 2007.
494
Salvage of blood urea nitrogen in sheep is highly dependent on plasma urea
495
concentration and the efficiency of capture within the digestive tract. J. Anim. Sci. 85,
496
1006-1013.
M
an
us
493
Wang, H., Long, R., Zhou, W., Li, X., Zhou, J., Guo, X., 2009. A comparative study on urinary
498
purine derivative excretion for yak, indigenous cattle and crossbred in the Qinghai-
499
Tibetan plateau, China. J. Anim. Sci. 87, 2355-2362.
Ac ce p
te
d
497
500
Wickersham, T.A., Titgemeyer, E.C., Cochran, R.C., Wickersham, E.E., Gnad, D.P., 2008. Effect
501
of rumen-degradable intake protein supplementation on urea kinetics and microbial use
502
of recycled urea in steers consuming low-quality forage. J. Anim. Sci. 86, 3079-3088.
503
Xin, G.S., Long, R.J., Guo, X.S., Irvine, J., Ding, L.M., Ding, L.L., Shang, Z.H., 2011. Blood
504
mineral status of grazing Tibetan sheep in Northeast of the Qinghai-Tibetan Plateau.
505
Livest. Sci. 136, 102-107.
31
Page 31 of 39
506
Table 1
507
Effect of oat hay feeding level on N balance and weight gain in Tibetan sheep
508
Oat hay feeding level 0.3VI
0.5VI
0.7VI 0.9VI s.e.m. Linear
Total N intake
4.64
7.74
10.81
13.93
0.02
Digestible N intake
2.80
5.13
7.10
9.16
0.08
Faecal N excretion
1.84
2.62
3.71
4.77
0.25
Urine N elimination
4.78
5.66
6.22
6.05
N retention
-1.98
-0.53
0.88
3.15
Urea-N (g/d)
3.63
4.03
4.78
Ammonia-N ( mg/d)
14.04
13.11
Dietary N utilization (g/d)
ns
0.19
**
ns
0.29
***
ns
0.22
an
***
ns
41.49
4.11
*
ns
205.6
16.4
***
ns
8.10
22.68
4.52
***
ns
51.99
44.25
39.52
3.83
***
**
71.53
77.11
91.28
2.66
*
ns
Ac ce p
509 510 511
M
-42.52 -6.79
Urinary urea-N:Total N intake 78.15 Urinary urea-N:Urine total N
5.51
29.18
d
te
N retention:Total N intake
cr ***
us
ns
-259.4 -118.6 47.3
Dietary N utilization efficiency (%)
ns
***
Urinary N composition
Weight gain (g/d)
***
Quadratic
ip t
Item
P-value
76.63
*P<0.001; **P<0.01; ***P<0.05
32
Page 32 of 39
511
Table 2
512
Effect of oat hay feeding level on urea kinetics in Tibetan sheep Oat hay feeding level
517
Item
0.3VI 0.5VI 0.7VI 0.9VI s.e.m. Linear Quadratic
UER (g/d)
8.11
10.93 12.30 13.81 0.54
***
UUE (g/d)
3.63
4.03
4.78
5.51
0.22
***
GER (g/d)
4.48
6.90
7.52
8.30
0.58
**
ROC (g/d)
2.22
3.92
4.05
5.54
0.54
UFE (g/d)
0.15
0.18
0.21
0.27
UUA (g/d)
2.11
2.79
3.52
2.48
UER: digestible N intake 2.99
2.14
1.77
UUE:UER (u)
0.48
0.39
0.40
GER:UER (1-u)
0.52
0.61
ROC:GER (r)
0.51
UFE:GER (f)
0.04
d
UUA:GER (a)
0.45
ip t
ns
ns
cr
ns
us
***
ns
***
ns
0.41
ns
*
1.53
0.16
***
ns
0.42
0.05
ns
ns
0.60
0.58
0.05
ns
ns
0.58
0.50
0.68
0.03
**
ns
0.03
0.03
0.04
0.01
ns
***
0.39
0.47
0.28
0.03
**
*
te
M
an
0.01
Ac ce p
513 514 515 516
P-value
UER = urea-N entry rate; GER = urea-N recycled to gastrointestinal tract (GIT); ROC = ureaN returned to ornithine cycle; UFE = urea-N excreted in faeces; UUA = urea-N utilized for anabolism; UUE = urinary urea-N elimination. **P<0.01; ***P<0.05
33
Page 33 of 39
517
Table 3
518
Effect of oat hay feeding level on urinary creatinine excretion, glomerular filtration rate,
519
plasma creatinine and urea-N concentrations, and renal urea-N reabsorption in Tibetan sheep Oat hay feeding level
0.3VI 0.5VI 0.7VI 0.9VI s.e.m. Linear
Urine creatinine excretion (mmol/day)
6.70
6.60
6.55
6.95
0.25
Plasma creatinine (μmol/l)
74.4
68.5
63.5
59.8
2.1
GFR (l/ day)
90.8
96.6
101.9
116.0
Plasma urea-N (mmol/l)
8.26
9.25
8.76
7.67
Urea-N tubular load (g/day)
10.2
10.6
11.4
Urea-N reabsorption (g/day)
6.72
6.55
6.72
Urea-N reabsorption rate (%)
65.3
68.0
62.0
ns
**
ns
cr
ns
*
ns
0.42
ns
ns
13.4
0.50
*
ns
8.06
0.34
ns
ns
55.8
1.91
*
ns
us
3.69
an
GFR = Glomerular filtration rate. *P<0.001; **P<0.01
Quadratic
ip t
Item
M
520 521
P-value
Ac ce p
te
d
522
34
Page 34 of 39
522
Table 4
523
Effect of feed intake on urea-N pool size, turnover time, urea-N clearance by the kidney and
524
gastrointestinal tract in Tibetan sheep Oat hay feeding level
ip t
Item
0.3VI 0.5VI 0.7VI 0.9VI s.e.m. Linear Quadratic
Urea-N pool size (g)
2.54
2.84
2.69
2.35
0.13
Turnover time (min)
451
374
315
245
23.2
Kidney
22.1
22.2
28.1
36.4
Gastrointestinal tract
29.7
36.3
44.1
cr
ns
us
***
ns
1.77
***
ns
***
ns
56.7
2.92
M
***P<0.05
ns
an
Urea-N clearance (ml/min)
525
P-value
Ac ce p
te
d
526
35
Page 35 of 39
Figure 1
527
N retention in response to different N intakes in Tibetan sheep,: SE y.x= 0.287.
an
us
cr
ip t
526
M
528
Ac ce p
te
d
529
36
Page 36 of 39
529
Figure 2.
531
Urinary
532
(APE) during a 56-h intravascular infusion of
533
different intakes.
534
(a)
14N15N-urea
(b), and faecal
15N
15N15N-urea
(c) atom percent excess
in Tibetan sheep receiving
cr
(a),
535 536
537
(b)
Ac ce p
te
d
M
an
us
15N15N-urea
ip t
530
37
Page 37 of 39
(c)
us
cr
ip t
538
an
539
Ac ce p
te
d
M
540
38
Page 38 of 39
540
Figure 3
542
UER (a) and GER (b) in response to different N intakes in Tibetan sheep; for (a): . : ..
543
(a)
M
an
us
cr
ip t
541
te
(b)
Ac ce p
545
d
544
546
39
Page 39 of 39
S0921-4488(15)00199-6 http://dx.doi.org/doi:10.1016/j.smallrumres.2015.05.009 RUMIN 4946
To appear in:
Small Ruminant Research
Received date: Revised date: Accepted date:
12-3-2015 6-5-2015 8-5-2015
Please cite this article as: Zhou, J.W., Guo, X.S., Degen, A.A., Zhang, Y., Liu, H., Mi, J.D., Ding, L.M., Wang, H.C., Qiu, Q., Long, R.J.,Urea kinetics and nitrogen balance and requirements for maintenance in Tibetan sheep when fed oat hay, Small Ruminant Research (2015), http://dx.doi.org/10.1016/j.smallrumres.2015.05.009 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.
Urea kinetics and nitrogen balance and requirements for maintenance in Tibetan sheep
2
when fed oat hay
3
J. W. Zhoua,b, X. S. Guob,c, A. A. Degend,*, Y. Zhanga,b, H. Liua,b, J. D. Mib,c, L. M. Dingb,c,
4
H. C. Wanga,b, Q. Qiuc and R. J. Longa,b,c
5
aState
6
Agriculture Science and Technology, Lanzhou University, Lanzhou 730000, PR China.
7
bInternational
8
Lanzhou 730000, PR China.
9
cSchool
of Life Sciences, Lanzhou University, Lanzhou 730000, PR China.
10
dDesert
Animal Adaptations and Husbandry, Wyler Department of Dryland Agriculture,
11
Blaustein Institutes for Desert Research, Ben-Gurion University of Negev, Beer Sheva
12
84105, Israel.
cr
an
us
Key Laboratory of Grassland and Agro-Ecosystems, College of Pastoral
te
d
M
Centre for Tibetan Plateau Ecosystem Management, Lanzhou University,
Ac ce p
13
ip t
1
14
*Corresponding
author: A. A. Degen; Email: [email protected]
15
Running head: Nitrogen metabolism and requirements in sheep
16
Highlights 1
Page 1 of 39
17 18 19 20 21
Tibetan sheep were offered oat hay at 0.3 to 0.9 times voluntary intake Estimated N requirements for maintenance were 0.50 g/kg BW0.75 per day This is 66% of the amount of N recommended by NRC for growing sheep of their size It was concluded that Tibetan sheep can cope with low N intake
ip t
22
ABSTRACT
24
Tibetan sheep inhabit the Qinghai-Tibetan Plateau, an area characterized by sparse
25
vegetation of low protein content much of the year. Consequently, we predicted that
26
their N requirements for maintenance would be low. To test our prediction, we
27
determined urea kinetics, N balance and requirements for maintenance in five growing
28
wethers. A 4 × 4 Latin square design was used with 4 levels of oat hay intakes (0.3, 0.5,
29
0.7, and 0.9 × voluntary intake). Urea kinetics were determined using a continuous
30
intrajugular infusion of
31
urinary N elimination and N retention (P < 0.01) with an increase in feed intake. From
32
the regression equation generated between N retention and N daily intake, the
33
estimated N requirements for maintenance were 0.50 g/kg BW0.75 per day, that is, only
34
66% of the amount recommended by NRC for growing sheep of its size. Urea-N entry
35
rate (UER), gastrointestinal tract (GIT) entry rate (GER), return to ornithine cycle (ROC)
Ac ce p
te
d
M
an
us
cr
23
15N15N-urea.
There was a linear increase in faecal N excretion,
2
Page 2 of 39
and faecal urea-N excretion all increased linearly (P < 0.01) with an increase in N intake.
37
The ratio of UER to apparent digestible N intake increased linearly from 1.53 to 2.99
38
with a decrease in feed intake. The proportion of UER that entered the gut did not differ
39
(P > 0.05) among intakes and ranged between 0.52 and 0.61. GER used for anabolism
40
decreased (P < 0.01) from 0.45 to 0.28, whereas GER to ROC increased (P < 0.01)
41
from 0.51 to 0.68 with increasing N intake. Glomerular filtration rate increased (P < 0.05)
42
with increasing N intake, but urinary creatinine excretion and plasma urea-N
43
concentrations remained constant (P > 0.05). The proportion of renal urea-N reabsorbed
44
increased linearly with a decrease in N intake (P < 0.05) while GIT urea-N clearance
45
was always higher than kidney urea-N clearance. The low nitrogen requirements
46
demonstrated by the Tibetan sheep supported our prediction. The increase in renal
47
urea-N absorption rate with a decrease in nitrogen intake, the greater ratios of UER to
48
apparent digestible N intake and the greater GIT urea-N clearance to kidney urea-N
49
clearance, regardless of N intake explain, at least in part, how Tibetan sheep cope with
50
low nitrogen intake.
Ac ce p
te
d
M
an
us
cr
ip t
36
51 3
Page 3 of 39
52
Keywords: Feeding level, Nitrogen balance, Urea kinetics, Nitrogen requirements for
53
maintenance, Tibetan sheep
1. Introduction
cr
55
ip t
54
The Qinghai-Tibetan Plateau is the highest and largest plateau in the world, with a
57
grassland area of 1.29 × 108 ha, which accounts for 1/3 of the total grassland in China
58
(Gerald et al., 2003; Long and Ma 1996). Due to the extremely harsh environment of the
59
plateau, namely, high altitude, severe cold, hypoxia, strong UV radiation and short
60
growing season, herbage and nutrients for livestock are deficient in the long cold season,
61
especially when raised under traditional grazing management (Gerald et al., 2003; Long
62
et al., 2004 and 2005; Xin et al., 2011). The indigenous yak has adapted well to these
63
conditions and today there are about 14 million yaks being raised on the plateau.
64
Although much less known and glamourous than the yak, Tibetan sheep are also
65
indigenous to the plateau. There are approximately 50 million Tibetan sheep being
66
raised at altitudes between 3000 and 5000 meters. Both of these indigenous ruminants
67
produce milk and meat for human consumption, fibre for clothing and dung for fuel, and
Ac ce p
te
d
M
an
us
56
4
Page 4 of 39
68
are vital for the livelihoods of the herders raising them (Long et al., 2008). Tibetan sheep and yaks only graze on natural pasture and are not offered
70
supplements. These livestock have to cope with poor pasture conditions, in particular,
71
the low N content of forage in winter. A number of studies have been made on yaks
72
including measurements on feed digestibility, rumen fermentation and nitrogen utilization
73
(Long et al., 1999, 2004 and 2005; Guo et al., 2012). Noteworthy among these
74
ruminants is their low nitrogen requirements for maintenance that ranged between 0.40
75
(Hu 2001) and 0.53 g N/kg0.75 per day (Long et al., 2004). We predicted that N
76
requirements for maintenance in Tibetan sheep would also be low. We tested our
77
prediction by determining N requirements for maintenance and by examining urea
78
kinetics in Tibetan sheep receiving rations that differed in N and energy intakes.
cr
us
an
M
d
te
Ac ce p
79
ip t
69
80
2. Materials and methods
81
2.1 Study site
82
The study was conducted at Wushaoling Yak Research Facility (37°12.4 N,
83
102°51.7 E, altitude 3,154 m, located in the northeast of the Qinghai-Tibetan Plateau) of 5
Page 5 of 39
the International Centre for Tibetan Plateau Ecosystem Management, Lanzhou
85
University, from November, 2012, to March, 2013. The experimental procedures were
86
approved by the Animal Ethic Committee of Gansu Province. Air temperature ranged
87
between 2 and 6°C and relative humidity ranged between 67 and 80% throughout the
88
experimental period.
us
cr
ip t
84
2.2 Animals and diets
M
90
an
89
Five wether Tibetan sheep (20 - 24 months old; 43.0 ± 2.3 kg live weight) were
92
purchased from nomadic herders. They were offered only oat hay, which was chopped
93
into 1 - 2 cm length before feeding. The oat hay contained: 885.0 g dry matter (DM)/kg;
94
15.3 g N/kg DM; 3.9 g Ca/kg DM; 1.8 g P/kg DM and yielded 8.5 MJ metabolizable
95
energy (ME)/kg DM.
Ac ce p
te
d
91
96
The sheep were penned individually in metabolic cages, and were provided with
97
fresh water ad libitum throughout the study. As these animals only grazed on the
98
rangeland previously, a long adaptation period of 45 days was allowed for the sheep to
99
familiar themselves with the feed, workers, facilities, and urine-collection apparatus. 6
Page 6 of 39
100
Then, the animals were fed ad libitum for 14 days to determine voluntary intake (VI), and
101
the lowest intake (1013 g/d DM) was used to determine the different feeding levels. All animals were offered 4 feeding levels (0.3VI, 0.5VI, 0.7VI and 0.9VI) in a 4 × 4
103
Latin square design with one sequence repeated. The intakes were 0.38, 0.64, 0.90 and
104
1.12 times estimated energy requirements for maintenance. The feeding levels from
105
0.9VI to 0.3VI were roughly equal to the daily DM intake of grazing Tibetan sheep from
106
start of winter to early spring (Li et al., 2009). The sheep were fed twice a day, receiving
107
half the portion at 0800 h and the other half at 1800 h.
108
2.3 Sample collections
Ac ce p
109
te
d
M
an
us
cr
ip t
102
110
The feeding trial consisted of 4 periods, each lasting 21 days: 15 days for diet
111
adaptation, 5 days for collection of outputs and the final day for blood sampling.
112
Experimental procedures followed previous urea kinetics studies using the
113
tracer method (Lobley et al., 2000; Huntington et al., 2009; Marini et al., 2003). Sheep
114
were weighed at the beginning and the end of each period before the morning feeding.
115
15N15N-urea
For urine collection, a latex bag was fixed around the penis of each wether using 7
Page 7 of 39
elastic belts. Urine flowed via a tube from the bag to a plastic container which contained
117
sufficient 50% (v/v) H2SO4 solution to keep the pH < 3. On day 15, in-dwelling catheters
118
were fixed in the sheep’s left and right jugular veins for isotope infusion and blood
119
sampling. Starting on day 18, animals were infused continuously for 56 h with15N15N-
120
urea (99.24 atom %
121
sterile 0.15 mol/l NaCl solution, and the infusion rate was adjusted to 30 ml/h using a
122
peristaltic pump (BT100-1L, Baoding constant-flow Pump Corporation, Baoding, China).
123
The concentration of the
124
the
125
(APE) at plateau. The calculation of
126
described by Guo et al. (2012).
Shanghai Chemical Research Institute, Shanghai, China) in
d
was 2.213 mmol/l, which was predicted to enrich
concentration in the 0.9VI fed sheep by 0.15-0.25 atom percent excess
te
15N15N-urea
15N15N-urea
M
an
us
15N,
cr
ip t
116
infusion was according to the method
Ac ce p
15N15N-urea
127
Faeces and urine samples were taken before infusion for background samples, and
128
then collected every 2 h between 48 to 56 h to calculate urea kinetics (assumed the
129
plateau was reached; Lobley et al., 2000). Urine samples were stored at -20°C for later
130
analysis of urea-N concentration and enrichment with
131
Total faeces and urine were collected during days 15 to 20. Before the morning feeding,
15N15N-urea
and
14N15N-urea.
8
Page 8 of 39
outputs were measured and subsamples (0.30 of daily urine and 0.40 of daily faeces)
133
were retained. Faecal subsamples were combined for each period and urine
134
subsamples were mixed daily and stored at -20°C. On day 21, jugular blood was
135
collected before the morning feeding and at 2-h intervals after feeding until 1600 h into
136
heparinized vacutainer tubes (Shanghai Kehua Bio-engineering Co. Ltd, Shanghai,
137
China). The blood was placed on ice, centrifuged at 1200 g for 15 min at 4°C within 1 h
138
after collection and the plasma was stored at -20°C for determination of urea-N and
139
creatinine concentrations.
d Ac ce p
2.4 Laboratory analyses
te
140
141
M
an
us
cr
ip t
132
142
Oat hay and faecal samples were freeze-dried for 1 week using a FreeZone-12L
143
freeze dryer (Labconco Corporation, Kansas City, Missouri, USA), ground to pass
144
through a 1-mm screen, and then stored in self-sealed plastic bags at room temperature.
145
The DM of oat hay and faeces was determined by drying in a forced-draught oven for 24
146
h at 105°C. N content of oat hay, faeces and urine was determined using the micro
147
Kjeldahl N method (AOAC, 1990). Creatinine concentration of the plasma and urine was 9
Page 9 of 39
determined using Agilent 1200 high-performance liquid chromatography (Agilent
149
Technologies Corporation, Santa Clara, California, USA) with the reversed-phase
150
column according to the procedure of Balcells et al. (1992). Plasma and urine urea-N
151
concentrations were determined using the spectrophotometer (U-2900, Hitachi, Tokyo,
152
Japan) in the diacetymonoxime method described by Marsh et al. (1957). Ammonia-N in
153
urine was determined colorimetrically as described by Broderick and Kang (1980).
cr
us
15N
an
Faecal
enrichment was analyzed in a continuous-flow mode using a stable
M
154
ip t
148
isotope elemental analysis (Flash EA 1112, Thermo Electron SPA; DELYAplus XP,
156
Thermo Finnigan, San Jose, CA, USA). Urinary ammonia-N was removed from the
157
isotope urine samples with a cation-exchange resin (AG 50W-X8, 100- 200 mesh
158
hydrogen form, Bio-Rad, Richmond, CA) according to the procedures described by
159
Wickersham et al. (2008). Final urea-N concentration of the retained effluent was
160
adjusted to 14 mmol/l. Gases of
161
degradation according to the methods described by Marini and Attene-Ramos (2006)
162
and the enrichment of these gases was analyzed using a stable isotope gas bench
163
(ThermoFinnigan Delta Plus).
Ac ce p
te
d
155
28N2, 29N2
and
30N2
were obtained from Hoffman
10
Page 10 of 39
164
166
2.5 Calculation of urea kinetics, glomerular filtration rate and urea pool size The calculation of urea kinetics followed the model generated by Lobley et al. (2000).
ip t
165
The glomerular filtration rate (GFR) was equated with creatinine clearance by the kidney
168
(Wang et al., 2009). The urea pool size was calculated from the urea space and urea
169
concentration in the urea pool according to Harmeyer and Martens (1980), assuming the
170
urea space to be 48.7% of BW (Bartle et al., 1988) and the concentration of the urea
171
pool to be equal to plasma urea-N (Marini et al., 2003).
172
2.6 Statistical analyses
Ac ce p
173
te
d
M
an
us
cr
167
174
Data were analyzed using the MIXED procedure of SAS (SAS Inc., Cary, NC, USA).
175
Feeding level was the fixed effect, with the period and sheep as random effects. Linear
176
and quadratic effects were tested using polynomial contrasts. Model terms for isotope
177
enrichment during infusion were feeding level, time, feeding level × time, and sheep
178
were included as a random effect. Differences were accepted as significant at P < 0.05.
179 11
Page 11 of 39
180
3. Results
181
3.1 N balance and utilization All rations were consumed completely by each sheep throughout the study. Daily
183
total N and digestible N intakes increased linearly with an increase in feeding level (P <
184
0.001), as did faecal N and urine N outputs and N retention (P < 0.01; Table 1). The
185
relationship between N retention (g/d) and N intake (g/d) was highly significant and took
186
the form: N retention = 0.547 N intake ‒ 4.566 (R2 = 0.998, n = 20, SE
187
Figure 1). From this equation, the estimated N requirements for maintenance of the
188
sheep were calculated as 0.50 g/kg BW0.75 per day.
y.x=
0.287y.x;
te
d
M
an
us
cr
ip t
182
N retention rate and urinary urea-N to total urine N ratio increased (P < 0.05) while
190
urinary urea-N elimination rate (either expressed as the proportion of total N or
191
digestible N intake) decreased linearly (P < 0.001) as N intake increased. Urea-N
192
composed 0.71 - 0.91 of total urine N, whereas ammonia-N was less than 1% of the
193
total. Urea-N and ammonia-N increased linearly (P < 0.05) with an increase in N and
194
feed intake. Daily weight gain increased linearly with DM intake (P < 0.001).
Ac ce p
189
195 12
Page 12 of 39
196
197
3.2 Urea kinetics characteristics Urinary
15N15N-urea
and
14N15N-urea
APE both reached plateau enrichments after
infusion of 48 h (Figure 2). Thus the ratio of 14N15N-urea:
199
over the sampling period. Faecal total
200
over the sampling period and, therefore, the faecal urea-N excretion was slightly
201
underestimated and, consequently, the urea-N for anabolism was slightly overestimated.
202
Urea-N entry rate (UER), urinary urea-N elimination (UUE), gastrointestinal tract
203
(GIT) entry rate (GER), return to ornithine cycle (ROC) and faecal urea-N excretion
204
(UFE) all increased linearly (P < 0.01; Table 2) while urea-N for anabolism (UUA)
205
increased quadratically (P < 0.05) as the N and feeding intake increased. UER (g/d) and
206
GER (g/d) related linearly and highly significantly to total N intake (g/d; Figure 3), and
207
could be expressed by the following equations:
208
UER = 0.597 N intake + 5.748 (R2 = 0.998, n = 20, .);
209
GER = 0.390 N intake + 3.177 (R2 = 0.894, n = 20, .).
210
UER was substantially greater than the digestible N intake (2.99, 2.14, 1.77 and 1.53
211
times for 0.3VI, 0.5VI, 0.7VI and 0.9VI diets, respectively), and decreased linearly (P <
was at plateau
cr
enrichment, however, did not reach a plateau
Ac ce p
te
d
M
an
us
15N
15N15N-urea
ip t
198
13
Page 13 of 39
0.001) with N intake. As N intake increased, ROC: GER increased (P < 0.01), UUA:
213
GER decreased (P < 0.01), while GER: UER remained relatively constant (0.52 to 0.61,
214
P > 0.05).
ip t
212
3.3 Plasma urea-N and creatinine concentrations, GFR and renal urea-N reabsorption
us
216
cr
215
GFR increased (P < 0.05, Table 3) and plasma creatinine concentration decreased
218
linearly (P < 0.01) as N intake increased, but urinary creatinine excretion was not
219
affected by intake (P > 0.05). With an increased feeding level, plasma urea-N
220
concentration and urea-N reabsorption did not change (P > 0.05), the urea-N tubular
221
load increased linearly (P < 0.05) but the proportion of renal urea-N reabsorbed
222
decreased linearly (P < 0.05).
224
M
d
te
Ac ce p
223
an
217
3.4 Urea-N pool size, urea-N clearance by the GIT and kidney
225
The urea-N pool size remained constant (P > 0.05; Table 4) when feed intake
226
increased from 0.3VI to 0.9VI, but the turnover time decreased linearly (P < 0.001).
227
Urea-N clearance by the GIT and kidney both increased linearly as feed intake 14
Page 14 of 39
228
increased (P < 0.001).
4. Discussion
231
4.1 N balance and requirements for maintenance
cr
230
ip t
229
Our prediction that N requirements for maintenance in Tibetan sheep would be low
233
was supported by this study. The requirements for maintenance, 0.50 g N/kg BW0.75 per
234
day, were only 66% of the amount recommended by NRC (1985) for a growing sheep of
235
its size and fell within the values of 0.40 to 0.53 g N/kg BW0.7 reported for yaks (Hu 2001;
236
Long et al., 2004). The Baluchi fat‒tailed sheep, a breed also adapted to harsh
237
conditions, required approximately 1.17 g N/kg BW0.75 per day for maintenance
238
(Kamalzadeh and Shabani, 2007).
Ac ce p
te
d
M
an
us
232
239
Absolute N retention increased in Tibetan sheep with an increase in apparent
240
digestible N intake, which was also reported in other sheep breeds, (Sarraseca et al.,
241
1998; Lobley et al., 2000; Marini et al., 2004; Kamalzadeh and Shabani, 2007), as well
242
as in yaks (Guo et al., 2012) and in cattle (Marini and Van Amburgh, 2003). In addition,
243
the ratio of N retention to apparent digestible N intake with an increase in apparent 15
Page 15 of 39
digestible N intake increased in Tibetan sheep. A similar response was found in 40-50
245
kg Suffolk wether sheep (Sarraseca et al., 1998), but, in contrast, a decrease with an
246
increase in apparent digestible N intake was reported in 21 kg Dorset-Finn ewe lambs
247
(Marini et al., 2004). In yaks, Guo et al. (2012) reported that apparent digestible N intake
248
did not affect the ratio of N retention to apparent digestible N intake.
us
cr
ip t
244
Tibetan sheep increased faecal N output linearly with increasing N intake. An
250
increase of faecal N was accompanied by an increase in N intake in some sheep breeds
251
(Sarraseca et al., 1998; Kamalzadeh and Shabani, 2007) but not in others (Lobley et al.,
252
2000; Marini et al., 2004) and, also not in heifers (Marini and Van Amburgh, 2003) nor in
253
yaks (Guo et al., 2012). Urine N also increased with an increase in N intake in Tibetan
254
sheep. This pattern was reported in a number of other sheep breeds (Sarraseca et al.,
255
1998; Marini et al., 2004; Sunny et al., 2007), and in heifers (Marini and Van Amburgh,
256
2003) and in yaks (Guo et al., 2012). In the Tibetan sheep, urine N was always higher
257
than faecal N but the urine N: faecal N ratio declined from 2.60 to 1.27 with an increase
258
in N intake. This is unlike the findings in yaks in which faecal N was greater than urinary
259
urea-N at low N intakes, with the urine N: faecal N ratio at 0.56 and 0.86. At higher N
Ac ce p
te
d
M
an
249
16
Page 16 of 39
intakes the ratios were 1.37 and 1.66 (Guo et al., 2012), that is, within the range for the
261
Tibetan sheep. However, in the Tibetan sheep the urine N: faecal N ratio decreased with
262
an increase in N intake whereas in the yaks it increased. The reason for this difference
263
is that Tibetan sheep increased faecal N excretion with increased N intake but this did
264
not occur in yaks. Faecal N is affected by DM intake regardless of N intake (Mariani et
265
al., 2004) and intake differed among treatments in Tibetan sheep but not in yaks.
an
us
cr
ip t
260
M
266
4.2 Urea metabolism
268
Among ruminants, it is not uncommon for UER to be greater than the apparent
269
digestible N intake (Lapierre and Lobley, 2001). Consequently, in order for such
270
ruminants to remain in positive N balance, a portion of the hepatic urea-N produced
271
must be recycled into the GIT and salvaged for microbial protein synthesis and then AA
272
absorption. In Tibetan sheep, the ratio of UER to apparent digestible N intake decreased
273
linearly from 2.99 to 1.53 with an increase in apparent digestible N and energy intakes.
274
At the two highest ratios, the sheep were in negative N balance and were losing body
275
mass. Sarraseca et al. (1998) also reported a decline in the ratio of UER to apparent
Ac ce p
te
d
267
17
Page 17 of 39
digestible N intake with an increase in apparent digestible N intake in 40-50 kg Suffolk
277
wether sheep. The ratio was 2.03 when the sheep lost 0.31 g N/d but was 1.19 and 1.29
278
when the sheep retained 4.66 and 7.34 g N/d, respectively. In contrast to these results,
279
21 kg Dorset-Finn ewe lambs had a ratio below 1.0 at all N intakes and increased the
280
ratio of UER to apparent digestible N intake from 0.62 to 0.96 with an increase in
281
digestible N intake from 3.9 to 20.0 g N/d (calculated from Marini et al., 2004). High
282
UER to apparent digestible N intakes, as found in the present study, were also reported
283
in semi-wild yak (1.38 to 2.06; Gou et al., 2012) and in wild elk (2.83 to 4.87; Mould and
284
Robbins, 1981), two ruminants that are well adapted to harsh conditions, including
285
periods of low N availability. The high ratios, in particular at the low N intakes, suggest
286
high urea recycling.
Ac ce p
te
d
M
an
us
cr
ip t
276
287
Urea-N recycled to the GIT increased linearly with an increase in N intake, as was
288
also found in a number of sheep breeds (Sarraseca et al., 1998; Lobley et al., 2000;
289
Sunny et al., 2007) and in the yak (Guo et al., 2012). GER as a ratio of UER ranged
290
between 0.52 and 0.61 in the Tibetan sheep, which fell within the 40% to 80% range
291
generally found for sheep (Lapierre and Lobley, 2001). It is expected that the ratio would 18
Page 18 of 39
increase with a decrease in apparent digestible N intake as the demands for N is higher
293
in low N intake animals (Marini and Van Amburgh, 2003). Indeed, this occurred in some
294
sheep such as Dorset-Finn ewe lambs where the ratio decreased from 0.75 to 0.30
295
(Marini et al., 2004) and in Suffolk wethers where it decreased from 0.70 to 0.61 (Lobley
296
et al., 2000) with an increase in apparent digestible N intake. A similar pattern of a
297
decrease in GER: UER ratio, from 0.87 to 0.73, with an increase in N intake was
298
observed in yaks (Guo et al., 2012). However, in Tibetan sheep, this did not occur as the
299
ratio was not affected by apparent digestible N intake. In the studies mentioned above,
300
all animals were in positive N balance. However, the Tibetan sheep were neither in N
301
balance nor energy balance in two of the dietary treatments, as they were below N and
302
energy requirements for maintenance. Urea-N recycling is affected by a number of
303
factors including plasma urea-N concentration (Sunny et al., 2007) and fermentable
304
carbohydrates in the gastrointestinal tract (Kennedy and Milligan, 1980; Lapierre and
305
Lobley, 2001). A fermentable source stimulates the bacterial population which utilizes
306
urea-N and increases GER and, consequently, GER: UER (Kennedy and Milligan, 1980;
307
Sarraseca et al., 1998). Plasma urea-N did not differ in the Tibetan sheep among
Ac ce p
te
d
M
an
us
cr
ip t
292
19
Page 19 of 39
treatments. However, there presumably was a lack of a fermentable source on the low
309
energy intakes, which limited the use of recycled urea-N and decreased GER and GER:
310
UER ratio. There is some evidence to support this. In another study in Tibetan sheep, in
311
which apparent digestible N intake was low but energy intake was above maintenance,
312
the GER: UER ratio was 0.88 (unpublished data). Also, in a study by Sarraseca et al.
313
(1998) in which sheep were offered three diets, one of which was below N and energy
314
requirements for maintenance, the decrease in GER: UER ratio with an increase in
315
apparent digestible N intake was not observed. The lowest GER: UER ratio occurred
316
with a dietary intake below N and energy requirements for maintenance. With an
317
insufficient energy supply, non-protein compounds are converted to ammonia in the
318
rumen which is eventually absorbed and converted to urea. Elevated levels of rumen
319
ammonia reduce GER (Rémond et al., 1993).
Ac ce p
te
d
M
an
us
cr
ip t
308
320
In the Tibetan sheep, UUA and ROC both increased with an increase in apparent
321
digestible N intake. This was also found in other sheep breeds (Lobley et al., 2000;
322
Sunny et al., 2007), heifers (Mariani and Van Amburgh, 2003) and yaks (Guo et al.,
323
2012). Furthermore, the UUA: GER ratio increased while the ROC: GER ratio decreased 20
Page 20 of 39
with a decrease in apparent digestible N intake in Tibetan sheep. That is, Tibetan sheep
325
on the lower N and energy intakes used a larger proportion of the GER for anabolic
326
purposes than sheep on higher N and energy intakes. An increase in UUA: GER ratio
327
with a decrease in apparent digestible N intake was also noted in Polypay × Dorsett
328
wether sheep while a decrease in ROC: GER ratio with a decrease in apparent
329
digestible N intake was also noted in Suffolk cross wether sheep (Lobley et al., 2000)
330
and in heifers (Marini and Van Amburgh, 2003). In Suffolk cross wether sheep (Lobley et
331
al., 2000) and in heifers (Marini and Van Amburgh, 2003), the UUA: GER ratio was not
332
affected by the apparent digestible N intake. Interestingly, a different pattern of that
333
found in the Tibetan sheep was reported for yaks, that is, the UUA: GER ratio increased
334
and ROC: GER ratio decreased with an increase in apparent digestible N intake (Guo et
335
al., 2012).
Ac ce p
te
d
M
an
us
cr
ip t
324
336
UFE was small and composed only 0.06 to 0.08 of the total faecal N excreted in
337
Tibetan sheep. It increased linearly with an increase in N intake, as was found in other
338
sheep breeds (Sarraseca et al., 1998; Sunny et al., 2007) and in the yak (Guo et al.,
339
2012), but not in heifers, that showed a decrease (Marini and Van Amburgh, 2003). The 21
Page 21 of 39
ratio of UFE: GER was also small in Tibetan sheep, ranging between 0.03 and 0.04. In
341
another sheep breed, the ratio decreased from 0.21 to 0.11 with an increase in N intake
342
(Sunny et al., 2007) and in heifers from 0.20 to 0.08 (Marini and Van Amburgh, 2003),
343
but increased from 0.02 to 0.12 in another breed (Sarraseca et al., 1998). The pattern in
344
yaks was similar to that in Tibetan sheep with the ratio ranging between 0.04 and 0.07
345
(Guo et al., 2012).The low values in the Tibetan sheep and yaks would indicate that very
346
little of the GER entered the caecum and colon, which has been suggested for sheep by
347
Dixon and Nolan (1986).
d
M
an
us
cr
ip t
340
UUE increased linearly with an increase in N intake and composed 0.71 to 0.91 of
349
urine N excretion, a ratio that was generally higher than in other ruminants. In Polypay ×
350
Dorsett wether sheep it ranged between 0.40 and 0.80 (Sunny et al., 2007); in Suffolk
351
cross-bred wether sheep between 0.50 and 0.67 (Sarraseca et al., 1998; Lobley et al.,
352
2000), in Dorsett-Finn ewe lambs between 0.29 and 0.81 (Marini et al., 2004), in heifers
353
between 0.18 and 0.74 and in yaks between 0.46 and 0.60 (Guo et al., 2012). Possible
354
reasons for these differences between the Tibetan sheep and the other ruminants could
355
be traced, at least in part, to the rates of UER to urine. UER to urine in Tibetan sheep
Ac ce p
te
348
22
Page 22 of 39
was unaffected by N intake and ranged between 0.39 and 0.48. In fact, the highest
357
value occurred at the lowest N intake. In general, in other ruminants, the proportion of
358
UER to urine was lower. In yaks the proportion was substantially lower than that in
359
Tibetan sheep, ranging between 0.13 and 0.27, and increased with an increase in N
360
intake (Guo et al., 2012). The proportion ranged between 0.19 and 0.37 in Polypay ×
361
Dorsett wether sheep (Sunny et al., 2007), between 0.26 and 0.39 in Suffolk cross-bred
362
wether sheep (Sarraseca et al., 1998; Lobley et al., 2000), between 0.25 and 0.69 in
363
Dorsett-Finn ewe lambs (Marini et al., 2004) and between 0.17 and 0.64 in heifers
364
(Marini and Van Amburgh, 2003).
te
d
M
an
us
cr
ip t
356
Urinary creatinine excretion was not affected by feed intake and, therefore,
366
creatinine was an appropriate internal marker to estimate GFR (Wang et al., 2009). GFR
367
increased with N intake, which was in agreement with previous studies in cattle (Liang et
368
al., 1999), buffaloes (Liang et al., 1999), yaks (Wang et al., 2009), goats (Jetana et al.,
369
2005) and sheep (Chen et al., 1995). The rate ranged from 5.41 to 6.91 l/kg BW0.75 per
370
day in the present study, which was similar to rates in Dorset-Finn lambs (6.21- 7.48 l/kg
371
BW0.75 per day; Marini et al., 2004) and Merino wethers (5.7- 6.8 l/kg BW0.75, Meintjes
Ac ce p
365
23
Page 23 of 39
372
and Engelbrecht, 2004) when receiving similar restricted diets. The urinary N tubular load increased linearly with an increase in N intake but renal
374
reabsorbed urea-N remained constant. This was because the proportion of renal urea-N
375
reabsorbed decreased with an increase in N intake. This would be expected as animals
376
with a lower N intake would need to conserve N to maintain N balance and one of the
377
ways would be to salvage urea from urinary excretion (Schmidt-Nielsen et al., 1958).
378
Similar findings of an increased proportion of renal urea-N reabsorption with a decrease
379
in N intake were reported in Dorsett-Finn ewe lambs (Marini et al., 2004) and in heifers
380
((Marini and Van Amburgh 2003). However, the proportion of renal urea-N reabsorbed in
381
the Tibetan sheep, 0.56 to 0.68, was not particularly high. For example, Bedouin and
382
Saanen goats consuming wheat straw reabsorbed 0.92 and 0.93 of the renal urea-N
383
(Silanikove, 1984). The findings in the Tibetan sheep were due, most likely, to the low
384
energy intake of the low N intake groups. In another study, in which Tibetan sheep
385
consumed a diet of low N intake but with 1.2 times maintenance energy requirements,
386
0.97 of renal urea-N was reabsorbed (unpublished data).
387
Ac ce p
te
d
M
an
us
cr
ip t
373
Urea-N pool size did not differ and N turnover time decreased with an increase in N 24
Page 24 of 39
intake in the Tibetan sheep. In contrast, urea-N pool size and N turnover rate increased
389
with an increase in N intake in yaks (Guo et al., 2012). Kidney and GIT urea clearance
390
increased with an increase in N intake in Tibetan sheep, but while renal urea-N
391
clearance also increased with N intake in yaks, GIT urea-N clearance decreased with an
392
increase in N intake. However, like in the yak, GIT urea-N clearance was always higher
393
than kidney urea-N clearance, the ratio ranging between 1.34 and 1.63. For the Tibetan
394
sheep, it was mainly due to the reduced urea-N excreted in the urine.
M
an
us
cr
ip t
388
In conclusion, Tibetan sheep, like yaks, demonstrated low N requirements for
396
maintenance. Although there were a number of differences between Tibetan sheep and
397
yaks in their nitrogen metabolism and recycling, there were also some common
398
responses. These included the greater ratios of UER to apparent digestible N intake and
399
the greater GIT urea-N clearance to renal urea-N clearance, regardless of N intake. Gou
400
et al. (2012) concluded that these two features “might demonstrate a special
401
characteristic of the yak’s N metabolism”. If so, it would also appear to be so for Tibetan
402
sheep.
Ac ce p
te
d
395
403 25
Page 25 of 39
404
405
Conflict of interest We do not have any conflict of interest.
Acknowledgements
cr
407
ip t
406
This work was supported by grants from the National Nature Science Foundation
409
of China project 31170378. Thanks to colleagues of the International Center for Tibetan
410
Plateau Ecosystem Management for valuable help during the experiment proceeding.
M
an
us
408
411
References
413
Association of Official Analytical Chemists, 1990. Official methods of analysis, 16th edition.
415
te
Ac ce p
414
d
412
AOAC, Arlington, VA, USA.
Barcells, J., Fondevila, M., Peiro, J.M., Parker, D.S., 1992. Simultaneous determination of
416
allantoin and oxypurines in biological fluids by high performance liquid chromatography. J.
417
Chromatog. A 575, 153‒157.
418
Bartle, S.J., Turgeon, O.A. Jr, Preston, R.L., Brink, D.R., 1988. Procedural and mathematical
419
considerations in urea dilution estimation of body composition in lambs. J. Anim. Sci. 66,
420
1920‒1927. 26
Page 26 of 39
421 422
Broderick, G.A., Kang, J.H., 1980. Automated simultaneous determination of ammonia and total amino acids in ruminal fluid and in vitro media. J. Dairy Sc. 63, 64‒75. Chen, X.B., Mejia, A.T., Kyle, D.J., Ørskov, E.R., 1995. Evaluation of the use of the purine
424
derivative: creatinine ratio in spot urine and plasma samples as an index of microbial
425
protein supply in ruminant: Studies in sheep. J. Agric. Sci., Camb. 125, 137-143.
429
cr
us
an
428
rumen in sheep given chopped Lucerne (Medicago sativa) hay. Br. J. Nutr. 55, 313-332. Gerald, W.N., Han, J.L., Long, R.J., 2003. The Yak, 2nd edition. Regional Office for Asia and the
M
427
Dixon, R.M., Nolan, J.V., 1986. Nitrogen and carbon flows between the caecum, blood and
Pacific, Food and Agriculture Organization of the United Nations, Bangkok, Thailand.
d
426
ip t
423
Guo, X.S., Zhang, Y., Zhou, J.W., Long, R.J., Xin, G.S., Qi, B., Ding, L.M., Wang, H.C., 2012.
431
Nitrogen metabolism and recycling in yaks (Bos grunniens) offered a forage-concentrate
432
diet differing in N concentration. Anim. Prod. Sci. 52, 287-296.
434 435 436 437
Ac ce p
433
te
430
Harmeyer, J., Martens, H., 1980. Aspects of urea metabolism in ruminants with reference to the goat. J. Dairy Sci. 63, 1707-1728. Hu, L.H., 2001. Chinese yak nutrition research advances. Qinghai Sci. Tech. 6, 37-39 (in Chinese). Huntington, G.B., Magee, K., Matthews, A., Poore, M., Burns, J., 2009. Urea metabolism in beef
27
Page 27 of 39
438
steers fed tall fescue , orchardgrass, or gamagrasshays. J. Anim. Sci. 87, 1346-1353. Jetana, T., 2005. Urinary purine derivatives as index for estimation of ruminal microbial nitrogen
440
production in sheep and goats. Thesis PhD, University Putra Malaysia, Kuala Lumpur,
441
Malaysia.
446 447
cr
us
an
445
Kennedy, P.M., Milligan, L.P., 1980. The degradation and utilization of endogenous urea in the gastrointestinal tract of ruminants: a review. Can. J. Anim. Sci. 60, 205-221.
M
444
nitrogen of Baluchi Sheep. Int. J. Agric. Biol. 9, 535-539.
Lapierre, H., Lobley, G.E., 2001. Nitrogen recycling in the ruminant: A review. J. Dairy Sci. 84, 223-236.
d
443
Kamalzadeh, A. and Shabani, A., 2007. Maintenance and growth requirements for energy and
te
442
ip t
439
Li, Y.X., Wang, J.Z., Li, L., Wang, H.H., Liu, S.Z., Qiangba, Y.Z., Bian, C., 2009. Research of
449
grazing sheep feed intake and digestibility in northern Tibet cold pastoral area in different
450
seasons. J. Domest. Anim. Ecol. 30, 41-45.
Ac ce p
448
451
Liang, J.B., Pimpa, O., Abdullah, N., Jelan, Z.A., 1999. Estimation of rumen microbial protein
452
production from urinary purine derivatives in zebu cattle and water buffalo. In Proceeding
453
of the Second Research Co-ordination Meeting of a Co-ordinated Research Project
454
(Phase 1), pp. 35-41. International Atomic Energy Agency, Vienna, Austria.
28
Page 28 of 39
Lobley, G.E., Bremmer, D.M., Zuur, G., 2000. Effects of diet quality on urea fates in sheep as
456
assessed by refined, non-invasive [15N15N] urea kinetics. Brit. J. Nutr. 84, 459-468.
457
Long, R.J., Ding, L.M., Shang, Z.H., Guo, X.S., 2008. The yak grazing system on the QinghaiTibetan Plateau and its status. The Rangeland J. 30, 241-246.
cr
458
ip t
455
Long, R.J., Dong, S.K., Hu, Z.Z., Shi, J.J., Dong, Q.M., Han, X.T., 2004. Digestibility, nutrient
460
balance and urinary purine derivative excretion in dry yak cows fed oat hay at different
461
levels of intake. Livest. Prod. Sci. 88, 27-32.
an
M
463
Long, R.J., Dong, S.K., Wei, X.H., Pu, X.P., 2005. The effect of supplementary feeds on the bodyweight of yaks in cold season. Livest. Prod. Sci. 93, 197-204.
d
462
us
459
Long, R.J., Ma, Y.S., 1996. Qinghai’s yak production system. In Proceedings of a workshop on
465
conservation and management of yak genetic diversity, 29- 31 October 1996,
466
Kathmandu, Nepal, pp. 105-115.
Ac ce p
te
464
467
Long, R.J., Zhang, D.G., Wang, X., Hu, Z.Z., Dong, S.K., 1999. Effect of strategic feed
468
supplementation on productive and reproductive performance in yak cows. Preventive
469
Veter. Med. 38, 195-206.
470
Marini, J.C., Attene-Ramos, M.S., 2006. An improved analytical method for the determination of
471
ureanitrogen isotopomers in biological samples utilizing continuous flow isotope ratio
29
Page 29 of 39
476 477 478
ip t
475
nitrogen recycling and urea transporter abundance in lambs. J. Anim. Sci. 82, 1157-1164. Marini, J.C., Van Amburgh, M.E., 2003. Nitrogen metabolism and recycling in Holstein heifers. J.
cr
474
Marini, J.C., Klein, J.D., Sands, J.M., Van Amburgh, M.E., 2004. Effect of nitrogen intake on
Anim. Sci. 81, 545-552.
us
473
mass spectrometry. Rapid Comm. Mass Spec. 20, 3736-3740.
Marsh, W.H., Fingerhut, B., Kirsch, E., 1957. Determination of urea N with the diacetyl method
an
472
and an automatic dialyzing apparatus. Amer. J. Clin. Path. 8, 681-688. Meintjes, R.A., Engelbrecht, H., 2004. Changes in the renal handling of urea in sheep on a low
480
protein diet exposed to saline drinking water. Onders. J. Veter. Res. 71, 165-170.
d
M
479
Mould, E.D., Robbins, C.T., 1981. Nitrogen metabolism in elk. J. Wildl. Manage. 45, 323-334.
482
National Research Council, 1985. Nutrient Requirements of Sheep. 6th revised edition. NRC,
484 485 486 487 488
Ac ce p
483
te
481
Washington, DC, USA.
Rémond, D., Chase, J.P., Delval, E., Poncet C., 1993. Net transfer of urea and ammonia across the ruminal wall of sheep. J. Anim. Sci. 71, 2785-2792. Sarraseca, A., Milne, E., Metcalf, M.J., Lobley, G.E., 1998. Urea recycling in sheep: effects of intake. Brit. J. Nutr. 79, 79-88. Schmidt-Nielsen, B., Osaki, H., Murdaugh, H.V., O’Dell, R., 1958. Renal regulation of urea
30
Page 30 of 39
489
excretion in sheep. Amer. J. Physiol. 194, 221-228. Silanikove, N., 1984. Renal excretion of urea in response to changes in nitrogen intake in desert
491
(black Bedouin goat) and non-desert (Swiss Saanen) goats. Comp. Biochem. Physiol.
492
79A, 651-654.
cr
ip t
490
Sunny, N.E., Owens, S.L., Baldwin IV, R.L., El-Kadi, S.W., Kohn, R.A., Bequette, B.J., 2007.
494
Salvage of blood urea nitrogen in sheep is highly dependent on plasma urea
495
concentration and the efficiency of capture within the digestive tract. J. Anim. Sci. 85,
496
1006-1013.
M
an
us
493
Wang, H., Long, R., Zhou, W., Li, X., Zhou, J., Guo, X., 2009. A comparative study on urinary
498
purine derivative excretion for yak, indigenous cattle and crossbred in the Qinghai-
499
Tibetan plateau, China. J. Anim. Sci. 87, 2355-2362.
Ac ce p
te
d
497
500
Wickersham, T.A., Titgemeyer, E.C., Cochran, R.C., Wickersham, E.E., Gnad, D.P., 2008. Effect
501
of rumen-degradable intake protein supplementation on urea kinetics and microbial use
502
of recycled urea in steers consuming low-quality forage. J. Anim. Sci. 86, 3079-3088.
503
Xin, G.S., Long, R.J., Guo, X.S., Irvine, J., Ding, L.M., Ding, L.L., Shang, Z.H., 2011. Blood
504
mineral status of grazing Tibetan sheep in Northeast of the Qinghai-Tibetan Plateau.
505
Livest. Sci. 136, 102-107.
31
Page 31 of 39
506
Table 1
507
Effect of oat hay feeding level on N balance and weight gain in Tibetan sheep
508
Oat hay feeding level 0.3VI
0.5VI
0.7VI 0.9VI s.e.m. Linear
Total N intake
4.64
7.74
10.81
13.93
0.02
Digestible N intake
2.80
5.13
7.10
9.16
0.08
Faecal N excretion
1.84
2.62
3.71
4.77
0.25
Urine N elimination
4.78
5.66
6.22
6.05
N retention
-1.98
-0.53
0.88
3.15
Urea-N (g/d)
3.63
4.03
4.78
Ammonia-N ( mg/d)
14.04
13.11
Dietary N utilization (g/d)
ns
0.19
**
ns
0.29
***
ns
0.22
an
***
ns
41.49
4.11
*
ns
205.6
16.4
***
ns
8.10
22.68
4.52
***
ns
51.99
44.25
39.52
3.83
***
**
71.53
77.11
91.28
2.66
*
ns
Ac ce p
509 510 511
M
-42.52 -6.79
Urinary urea-N:Total N intake 78.15 Urinary urea-N:Urine total N
5.51
29.18
d
te
N retention:Total N intake
cr ***
us
ns
-259.4 -118.6 47.3
Dietary N utilization efficiency (%)
ns
***
Urinary N composition
Weight gain (g/d)
***
Quadratic
ip t
Item
P-value
76.63
*P<0.001; **P<0.01; ***P<0.05
32
Page 32 of 39
511
Table 2
512
Effect of oat hay feeding level on urea kinetics in Tibetan sheep Oat hay feeding level
517
Item
0.3VI 0.5VI 0.7VI 0.9VI s.e.m. Linear Quadratic
UER (g/d)
8.11
10.93 12.30 13.81 0.54
***
UUE (g/d)
3.63
4.03
4.78
5.51
0.22
***
GER (g/d)
4.48
6.90
7.52
8.30
0.58
**
ROC (g/d)
2.22
3.92
4.05
5.54
0.54
UFE (g/d)
0.15
0.18
0.21
0.27
UUA (g/d)
2.11
2.79
3.52
2.48
UER: digestible N intake 2.99
2.14
1.77
UUE:UER (u)
0.48
0.39
0.40
GER:UER (1-u)
0.52
0.61
ROC:GER (r)
0.51
UFE:GER (f)
0.04
d
UUA:GER (a)
0.45
ip t
ns
ns
cr
ns
us
***
ns
***
ns
0.41
ns
*
1.53
0.16
***
ns
0.42
0.05
ns
ns
0.60
0.58
0.05
ns
ns
0.58
0.50
0.68
0.03
**
ns
0.03
0.03
0.04
0.01
ns
***
0.39
0.47
0.28
0.03
**
*
te
M
an
0.01
Ac ce p
513 514 515 516
P-value
UER = urea-N entry rate; GER = urea-N recycled to gastrointestinal tract (GIT); ROC = ureaN returned to ornithine cycle; UFE = urea-N excreted in faeces; UUA = urea-N utilized for anabolism; UUE = urinary urea-N elimination. **P<0.01; ***P<0.05
33
Page 33 of 39
517
Table 3
518
Effect of oat hay feeding level on urinary creatinine excretion, glomerular filtration rate,
519
plasma creatinine and urea-N concentrations, and renal urea-N reabsorption in Tibetan sheep Oat hay feeding level
0.3VI 0.5VI 0.7VI 0.9VI s.e.m. Linear
Urine creatinine excretion (mmol/day)
6.70
6.60
6.55
6.95
0.25
Plasma creatinine (μmol/l)
74.4
68.5
63.5
59.8
2.1
GFR (l/ day)
90.8
96.6
101.9
116.0
Plasma urea-N (mmol/l)
8.26
9.25
8.76
7.67
Urea-N tubular load (g/day)
10.2
10.6
11.4
Urea-N reabsorption (g/day)
6.72
6.55
6.72
Urea-N reabsorption rate (%)
65.3
68.0
62.0
ns
**
ns
cr
ns
*
ns
0.42
ns
ns
13.4
0.50
*
ns
8.06
0.34
ns
ns
55.8
1.91
*
ns
us
3.69
an
GFR = Glomerular filtration rate. *P<0.001; **P<0.01
Quadratic
ip t
Item
M
520 521
P-value
Ac ce p
te
d
522
34
Page 34 of 39
522
Table 4
523
Effect of feed intake on urea-N pool size, turnover time, urea-N clearance by the kidney and
524
gastrointestinal tract in Tibetan sheep Oat hay feeding level
ip t
Item
0.3VI 0.5VI 0.7VI 0.9VI s.e.m. Linear Quadratic
Urea-N pool size (g)
2.54
2.84
2.69
2.35
0.13
Turnover time (min)
451
374
315
245
23.2
Kidney
22.1
22.2
28.1
36.4
Gastrointestinal tract
29.7
36.3
44.1
cr
ns
us
***
ns
1.77
***
ns
***
ns
56.7
2.92
M
***P<0.05
ns
an
Urea-N clearance (ml/min)
525
P-value
Ac ce p
te
d
526
35
Page 35 of 39
Figure 1
527
N retention in response to different N intakes in Tibetan sheep,: SE y.x= 0.287.
an
us
cr
ip t
526
M
528
Ac ce p
te
d
529
36
Page 36 of 39
529
Figure 2.
531
Urinary
532
(APE) during a 56-h intravascular infusion of
533
different intakes.
534
(a)
14N15N-urea
(b), and faecal
15N
15N15N-urea
(c) atom percent excess
in Tibetan sheep receiving
cr
(a),
535 536
537
(b)
Ac ce p
te
d
M
an
us
15N15N-urea
ip t
530
37
Page 37 of 39
(c)
us
cr
ip t
538
an
539
Ac ce p
te
d
M
540
38
Page 38 of 39
540
Figure 3
542
UER (a) and GER (b) in response to different N intakes in Tibetan sheep; for (a): . : ..
543
(a)
M
an
us
cr
ip t
541
te
(b)
Ac ce p
545
d
544
546
39
Page 39 of 39