Animal Feed Science and Technology 178 (2012) 115–119

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Animal Feed Science and Technology

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Short communication

Heat production determined by the RQ and CN methods, fasting heat

production and effect of the energy intake on substrates oxidation of
indigenous Manchega sheep
C. Fernández a,∗ , M.C. López a , M. Lachica b
a
Institute for Animal Science and Technology, Universidad Politécnica de Valencia, 46022 Valencia, Spain
b
Department of Physiology and Biochemistry of Animal Nutrition, Estación Experimental del Zaidín (CSIC), Camino del Jueves s/n, 18100 Armilla, Granada,
Spain

a r t i c l e i n f o a b s t r a c t

Article history: Heat production (HP) from CH4 and CO2 production, and O2 consumption was determined
Received 21 February 2012 by the respiratory quotient (RQ) method and by the C–N balance (CN method). Twelve dry
Received in revised form
Manchegas sheep were fed with a diet constituted by alfalfa hay and barley grain at three
19 September 2012
levels, approximately 1, 1.5 and 2× metabolizable energy for maintenance (MEm). Later,
Accepted 21 September 2012
the sheep were fed close to maintenance and HP determined by indirect calorimetry after
3 d fasting. The sheep were allocated to metabolism cages. After 10 d of adaptation, feed
Keywords: intake, and total fecal and urine output were recorded daily during 5 d, as well as the body
Fasting heat production weight (BW) at the beginning and end of the sampling period. Gas exchange measurements
Heat production were recorded by a mobile open-circuit respirometry system. Average HP measured by
Manchega sheep RQ method was in agreement with the HP determined by CN method accounting for 410
Retained energy and 407 kJ/kg0.75 BW/d, respectively. Fasting HP was 268 kJ/kg0.75 BW. Proportion of energy
Substrates oxidation associated to the oxidation of protein (OXP), carbohydrate (OXCHO) and fat (OXF) was
calculated. In fasting condition most of HP associated to substrates oxidation (HPx) was
due to OXF (0.931) and less to OXP (0.037) and OXCHO (0.032). OXP was increased with
the feeding level and was greater (0.193) than in fasting. OXCHO was increased with the
feeding level until a stable level (0.507).
© 2012 Elsevier B.V. All rights reserved.

1. Introduction

There is a lack of information about energy metabolism in Spanish breeds with only one published paper which deals
about fasting metabolism and energy requirements for maintenance of Segureña sheep (Aguilera et al., 1986). Manchega
breed is one of the most important in Spain; from a census of 1.63 million pure sheep more than 9% is Manchega (FEAGAS,
2009). It is mainly used for making cheese with and elevated productivity (250 L milk/sheep/yr; 4–4.5 L/kg of fresh cheese),
demand and profits.

Abbreviations: ADFom, acid detergent fiber exclusive of residual ash; aNDFom, neutral detergent fiber exclusive of residual ash; BW, body weight; CO2 x,
CO2 production from oxidation; CP, crude protein; DM, dry matter; GE, gross energy; HP, heat production; HPx, heat production associated to substrates
oxidation; Lignin(sa), lignin determined by solubilization of cellulose with sulfuric acid; MEI, metabolizable energy intake; MEm, metabolizable energy for
maintenance; OXCHO, oxidized carbohydrate; OXF, oxidized fat; OXP, oxidized protein; RE, retained energy; REfat , retained energy in fat; REprotein , retained
energy in protein; RQ, respiratory quotient.
∗ Corresponding author. Tel.: +34 963 877 007; fax: +34 963 877 439.
E-mail address: [email protected] (C. Fernández).

0377-8401/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
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116 C. Fernández et al. / Animal Feed Science and Technology 178 (2012) 115–119

Most studies to determine heat production (HP) have used indirect methods. One of them entails measurements of O2
consumption, CH4 and CO2 production and urine-N (RQ method); another uses the C–N balance (CN method) to calculate
the retained energy (RE), assuming that all energy is retained either as fat or protein. Then, HP is calculated as the difference
between metabolizable energy intake (MEI) and RE. Very similar values for HP have been obtained with both methods
(Blaxter, 1967), although lightly greater values were obtained with the RQ method.
Fasting HP reduces the effect of the diet on HP to a minimum. The agreement with published data can be considered as an
index of reliability and, tentatively, it could be used to estimate an approximate value of metabolizable energy requirement
for maintenance (MEm) from the published data of its efficiency.
The CO2 production is derived from nutrient oxidation and rumen fermentation. The separation between these two
components is necessary to calculate the substrates oxidation and its contribution to the total HP (HPx). Production of volatile
fatty acids from carbohydrate fermentation is followed by CO2 and CH4 production. Fahey and Berger (1988) demonstrated
a ratio CO2 /CH4 of 1.7/1 for high forage diets.
The objective of this study was to determine under different feeding levels the HP via RQ and CN methods and their
agreement, fasting HP and the contribution of protein (OXP), carbohydrates (OXCHO) and fat (OXF) oxidation to HPx in
indigenous Manchega breed.

2. Materials and methods

2.1. Animals and feeding

The experimental procedure was approved by the Committee on Animal Use and Care at the Politechnic University of
Valencia. Twelve mature Manchega female dry sheep of similar body weight (62.3 ± 2.3 kg BW) were used to determine gas
exchange and C-N balances. Diet was a mix of 130 and 870 g/kg of barley grain and alfalfa hay, respectively. Its chemical
composition was 897.9 g/kg for dry matter (DM); 310.5, 57.1, 471.0 and 145.9 g/kg DM for acid detergent fiber (ADFom),
lignin(sa), neutral detergent fiber (aNDFom) and crude protein (CP), respectively; and 18.6 MJ/kg DM for gross energy (GE).
Three levels of MEI were used with the same sheep corresponding approximately to 1, 1.5 and 2× MEm (374 kJ/kg0.75 BW/d,
Aguilera et al. (1986); treatments Low, Medium and High, respectively). Later, the sheep were fed close to maintenance level
(15 d) for determining after 3 d fasting the HP by indirect calorimetry. Half daily ration was offered at 08:00 and 16:00 h,
respectively. Sheep had free access to a mineral-vitamin block and water.

2.2. Experimental schedule and measurements

Sheep were allocated in individual metabolism cages. After 10 d of adaptation, feed intake, and fecal and urine output
were recorded daily during 5 d, as well as BW at the beginning and end of the period. Representative samples of diet, feces
and urine were collected daily, stored at −20 ◦ C, and pooled for chemical analysis. Within the balance trial, gas exchange was
measured for each treatment during 15 min/h per sheep and repeated each 3 h during 24 h (8 measures/d with 4 sheep/h)
using a mobile open-circuit respirometry system (Fernandez et al., 2012) attached to the sheep by a face mask. When the
gas exchange determinations were over, fasting HP was determined following identical schedule.

2.3. Calculations

MEI was calculated as the difference between GE intake and energy losses in feces, urine and CH4 (CH4 energy equiva-
lent = 39.54 kJ/L; Brouwer, 1965).
HP (RQ method) was calculated according to Brouwer (1965) as HP (kJ) = 16.18×O2 (L) + 5.02×CO2 (L) − 2.17×CH4
(L) − 5.99×urine N (Nur, g). RQ was determined as CO2 produced/O2 consumed. RE was calculated as MEI − HP.
In the CN method, C balance was the total amount of C retained in the body where the amount of C retained in fat was
calculated by subtracting the amount of C retained in protein determined by N balance. Assuming an energy equivalent
of 39.76 kJ/g and a content of 0.767 C for fat, and 23.86 kJ/g and 0.16 N and 0.52 C for protein, RE (kJ) in protein (REprotein )
and fat (REfat ) was calculated, respectively, as REprotein = N balance (g)×6.25×23.86, and REfat = (C balance (g) − N balance
(g)×6.25×0.52)×1.304×39.76. The RE (kJ) was calculated according to Brouwer (1965) as RE = REprotein + REfat .
The energy associated with the oxidation of protein (OXP), carbohydrate (OXCHO) and fat (OXF) was calculated by the
method followed by Chwalibog et al. (1997). CO2 production from oxidation (CO2 x) was calculated as CO2 − (1.7×CH4 )
(high forage diet; Fahey and Berger, 1988). The calculations were carried as following: OXP = 6.25×Nur×18.42 (kJ/g),
OXCHO = (−2.968×O2 + 4.174×CO2 x − 2.446×Nur)×17.58 (kJ/g), and OXF = (1.719×O2 − 1.719×CO2 x − 1.963×Nur)×39.76
(kJ/g). Then, HP from oxidation was HPx (kJ) = 16.18×O2 + 5.02×CO2 x − 5.99×Nur. Gases were expressed in L/d and Nur
in g/d.

2.4. Chemical analysis

It was conducted according to the methods of AOAC (2000) for DM (no. 934.01) and CP (no. 968.06). GE content was
determined in an adiabatic bomb calorimeter. Acid detergent fiber (ADFom) and neutral detergent fiber (aNDFom) were
 C. Fernández et al. / Animal Feed Science and Technology 178 (2012) 115–119 117

Table 1
Daily energy (kJ/kg0.75 BW) and C-N (g/kg0.75 BW) balances, heat production (HP), retained energy (RE) and respiratory quotient (RQ) of female dry Manchega
sheep (n = 12) under three metabolizable energy intake (MEI) levels (Low, Medium and High) calculated by the RQ and CN methods.

Low Medium High Pooled SEa P value

RQ method
Gross energy intake 1219.5 1330.6 1461.1 38.69
Energy in feces 704.3 662.7 617.6 31.87
Energy in urine 33.6 37.0 38.0 1.87
Energy in CH4 29.2 36.9 42.1 1.57
MEI 452.4x 593.9xy 763.3y 43.73 0.0545
HP 379.8x 404.4y 445.5z 19.07 0.0001
REb 72.6x 189.6y 317.9z 38.68 0.0009
RQ 0.88x 0.95y 0.96y 0.011 0.0318
CN method
N intake 1.60 1.65 1.81 0.035
N in feces 0.88 0.79 0.76 0.038
N in urine 0.63 0.69 0.71 0.039
N retained 0.09 0.16 0.33 0.051
C intake 26.75 31.12 33.78 0.796
C in feces 17.52 16.07 15.16 0.743
C in urine 0.59 0.75 0.66 0.039
C in CO2 6.59 10.00 11.06 0.520
C in CH4 0.53 0.67 0.76 0.028
C retained 1.52 3.63 6.14 0.848
REprotein c 13.0x 24.4y 50.0z 6.35 0.0034
REfat d 66.3x 165.7y 269.9z 35.13 0.0028
REe 79.3x 190.1y 319.9z 44.33 0.0025
HPf 373.1x 403.9y 443.4z 21.43 0.0023
x,y,z
For the comparisons made, values within a row with unlike superscript letter were significantly different (P<0.05).
a
Standard error.
b
Calculated as RE = MEI − HP.
c
REprotein , retained energy of protein.
d
REfat , retained energy of fat.
e
RE = REprotein [N balance (g)×6.25×23.86] + REfat [(C balance (g) − N balance (g)×6.25×0.52)×1.304×39.76].
f
Calculated as HP = MEI − RE.

measured in an ANKOM Fiber Analyzer (A220, ANKOM Technologies, Fairport, NY, USA) according to Van Soest et al. (1991),
both exclusive of residual ash. aNDFom was determined using sodium sulfite and alpha amylase. Lignin(sa) (lignin deter-
mined by solubilization of cellulose with sulfuric acid) was determined according to Robertson and Van Soest (1981). C and
N were analyzed by Dumas principle (TruSpec CN; Leco Corporation, St. Joseph, MI, USA).

2.5. Statistical analyses

Experimental data were analyzed by using repeated-measures analysis of variance with the animal as experimental unit
to determine the treatments effect. Tukey multiple range test was used to ascertain the statistical significance of differences.
One-way analysis of variance was used to establish differences between HP determined by RQ and CN method. Differences
were considered significant at P<0.05.

3. Results

Daily energy C–N balance, HP determined and RE estimated by RQ method, and calculated RE and estimated HP by CN
method are displayed in Table 1. MEI was different (P<0.05) between Low and High treatment. HP and RE were lower (P<0.05)
for Low vs. Medium vs. High treatment. REprotein and REfat were positive for the three treatments and both were increased
(P<0.05) with the MEI. HP was lower (P<0.05) for Low vs. Medium vs. High treatment. Average RQ for fasting and fed sheep
were 0.72 and 0.93, respectively.
Contribution to HPx due to oxidation of nutrients with the three feeding treatments and fasting is shown in Table 2.
Fasting HP was 268 kJ/kg0.75 BW/d (corresponding to RE = −268 kJ/kg0.75 BW/d). HPx from OXP was not different between
the feeding treatments (0.193 as average) and it was lower (P<0.05) under fasting conditions (0.037). The ratio OXCHO/HPx
was lower (P<0.05) for fasting (0.032) and it increased in the feeding treatments with the lower (P<0.05) value for Low (0.163)
vs. Medium and High (0.507 as average). OXF was lower (P<0.05) for Medium and High treatments (0.303 as average) vs.
fasting (0.931), and the Low treatment had an intermediate value (0.647).
118 C. Fernández et al. / Animal Feed Science and Technology 178 (2012) 115–119

Table 2
Daily oxidation (kJ/kg0.75 BW) of protein (OXP), carbohydrate (OXCHO) and fat (OXF) and their contribution to the heat production from substrates oxidation
(HPx) of female dry Manchega sheep (n = 12) under four metabolizable energy intake levels (Fasting, Low, Medium and High).

Fasting Low Medium High Pooled SEa P value

x y y y
OXP 9.8 72.3 79.6 81.9 4.80 0.0010
OXCHO 8.6x 60.9y 199.6z 224.6z 26.45 0.0004
OXF 249.6x 243.3x 120.1y 133.2y 18.89 0.0014
HPxb 268.1x 374.5y 398.5y 438.7z 18.22 0.0001
OXP/HPx 0.037x 0.193y 0.199y 0.187y 0.0231 0.0013
OXCHO/HPx 0.032x 0.163y 0.501z 0.512z 0.0473 0.0042
OXF/HPx 0.931x 0.647y 0.301z 0.304z 0.0563 0.0006
x,y,z
For the comparisons made, values within a row with unlike superscript letter were significantly different (P<0.05).
a
Standard error.
b
For fasting (CH4 production = 0), HPx = HP.

4. Discussion

Fasting HP value was similar than the obtained in Segureña sheep (272 kJ/kg0.75 BW/d; Aguilera et al., 1986). Olthoff
et al. (1989) found no differences for fasting HP between ewes of seven breeds differing in potential of production
(296 kJ/kg0.75 BW/d as average). Differences in fasting HP could be explained by the previous ME offered to the animals.
For no productive adult animals, the maintenance level of intake would be a normal occurrence (NRC, 2007) and fasting HP
is typically determined after 3 d fast. In absence of other experimental data, from fasting HP value and assuming that the
efficiency of utilization of MEm is accepted to be close to 0.7 (ARC, 1980), it could be established a tentative value for MEm of
383 kJ/kg0.75 BW/d for Mancheqa sheep. Aguilera et al. (1986) estimated for Segureña sheep the MEm at 374 kJ/kg0.75 BW/d.
Kearl (1982) using data from nine reports pointed out that the MEm ranged from 305 to 460 kJ/kg0.75 BW/d, and suggested
an average value of 385 kJ/kg0.75 BW/d.
RE determined with CN method was slightly greater than the calculated with RQ method and, thereafter, the HP lower.
However, both methods have given no significant different values accounting for 407 and 410 kJ/kg0.75 BW/d as average for
CN and RQ methods, respectively, in spite of the relatively short gas exchange periods (15 min) with respect to the usual 24 h.
Discrepancies averaged only 0.005 when expressed as percentage of MEI, a rather satisfactory value taking into account the
considerable amount of technical and analytical work involved. Aguilera et al. (1986) with wethers fed at about maintenance
level obtained an average discrepancy of 0.018.
Most of HPx derived from OXF and it was increased as the value of MEI decreased. This was in concordance with RE
and RQ values obtained for fasting and feeding treatments, its reduction was consistent with the switch between OXCHO
and OXF (less carbohydrate and more fat was oxidized). For the Medium and High treatment OXCHO/HPx was very close.
OXCHO and OXF were directly dependent on fermentation level. The increase in feeding level could produce higher glucose
fermentation (less glucose is oxidized as OXCHO) and short-chain fatty acids to be oxidized as OXF. Chwalibog et al. (1997) in
calves with positive REfat , as in the present study, pointed out that part of OXF should originate from ingested carbohydrate.
Also, those authors obtained that the OXP was about 0.16 of HPx on high feeding levels, being reduced to about 0.13 on low
feeding levels with a reverse increment for OXF from 0.15 to 0.78 of HPx.

5. Conclusions

The present paper offers pioneer data of fasting metabolism for Manchega sheep. HP determined by the RQ method was
in agreement with the estimated by CN method. Under the experimental conditions, most of HPx was derived from OXF,
with a lower and alike proportion from OXP and OXCHO.

Acknowledgments

This study was supported by Agroalimed Foundation and INIA Project (ref. RTA2011-00107-C02-02).

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