Energy and protein requirements for maintenance of Hu sheep during pregnancy

ZHANG Hao, SUN Ling-wei WANG Zi-yu MA Tie-wei DENG Ming-tian WANG Feng ZHANG Yan-li

1 Jiangsu Engineering Technology Research Center of Mutton Sheep & Goat Industry, Nanjing Agricultural University, Nanjing 210095, P.R.China

2 Laboratory of Metabolic Manipulation of Herbivorous Animal Nutrition, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, P.R.China

3 Joint International Research Laboratory of Agriculture & Agri-product Safety, Yangzhou University, Yangzhou 225009, P.R.China

RESEARCH ARTICLE

Energy and protein requirements for maintenance of Hu sheep during pregnancy

ZHANG Hao1,2,3, SUN Ling-wei1, WANG Zi-yu1, MA Tie-wei1, DENG Ming-tian1, WANG Feng1, ZHANG Yan-li1

1 Jiangsu Engineering Technology Research Center of Mutton Sheep & Goat Industry, Nanjing Agricultural University, Nanjing 210095, P.R.China

2 Laboratory of Metabolic Manipulation of Herbivorous Animal Nutrition, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, P.R.China

3 Joint International Research Laboratory of Agriculture & Agri-product Safety, Yangzhou University, Yangzhou 225009, P.R.China

This study aimed to determine the effect of stage and level of feed intake on energy metabolism, carbon-nitrogen (C-N)balance, and methane emission to determine energy and protein requirements for maintenance of maternal body including pregnancy tissues during pregnancy using the method of C-N balance. Twenty-one ewes carrying twin fetuses were randomly divided into three groups of seven ewes each in the digestion and respirometry trial at d 40, 100, and 130 of gestation, respectively. Three groups were fed a mixed diet either for ad libitum intake, 70 or 50% of the ad libitum intake during pregnancy. The results showed that the apparent digestibility of C and N were increased as feeding levels decreased at each stage of gestation. The daily net energy requirements for maintenance (NEm) were 295.80, 310.09, and 323.59 kJ kg–1BW0.75(metabolic body weight) with a partial ef ficiency of metabolisable energy utilization for maintenance of 0.664, 0.644,and 0.620 at d 40, 100, and 130 of gestation, respectively. The daily net protein requirements for maintenance were 1.99,2.35, and 2.99 g kg–1BW0.75at d 40, 100, and 130 of gestation, respectively. These results for the nutritional requirements of the net energy and protein may help to formulate more balanced diets for Hu sheep during pregnancy.

carbon and nitrogen balance, energy, methane emission, protein, pregnancy

1. Introduction

The Hu sheep is an indigenous Chinese sheep breed that is well adapted to the ecological conditions of high temperature and high humidity areas of China and is noted for its precociousness and proli ficacy (Yue 1996; Nie et al. 2015).In China, the Hu sheep has become one of the dominant breeds for lamb meat production.

The feeding management of sheep in China is largely based on a foreign nutritional system, such as NRC (2007).However, China is such a huge country with various forage resources and sheep breeds that it is practically unreasonable to cover all the situations with a foreign standard. Furthermore, in China, nutrient requirements of Hu sheep during pregnancy have not been well established, which limits the development of more ef ficient feeding systems. Therefore,an accurate assessment of the nutritional requirements of Hu sheep is critical to maximize performance and ensure ef ficient feed utilization.

This study indicates the energy and protein requirements for the maintenance of Hu ewes using carbon and nitrogen methods during pregnancy, which coincides with animal welfare, as well as the methane emissions as measured by an open-circuit respirometry system. The carbon and nitrogen(C-N) balance has been used to calculate the retained energy (RE), assuming that all energy is retained either as fat or protein (Fernández et al. 2012). Therefore, we will further investigate the effect of stage of gestation and level of feed intake on energy metabolism, C-N balance, and methane emission to determine energy and protein requirements for maintenance of Hu sheep during pregnancy using the method of C-N balance.

2. Materials and methods

The experiment was conducted at the Jiangyan Experimental Station of Taizhou, Taizhou City, Jiangsu Province of China. During the research period, a heated indoor facility was used to keep the temperature within the range of (15.50±1.32)–(26.54±1.61)°C. The average relative humidity was (61.25±2.76)%. All trials were conducted in accordance with the Guidelines for the Care and Use of Animals in the College of Animal Science and Technology,Nanjing Agricultural University, China.

2.1. Animals and treatments

Thirty-six multiparous Hu sheep (body weight (BW)=(40.1±1.2) kg) of similar age ((18.5±0.5) mon) and body condition score (BCS) (2.55±0.18; scale 0=emaciated to 5=obese; Russel et al. 1969) were selected. After being drenched with 0.2 mg ivermectin per kg of BW against endoparasites, all ewes were synchronized using intravaginal progestagen sponges (30 mg; Pharmp PTY, Herston City,Australia) for 12 d. Estrous behavior was monitored using 3 vasectomized rams at 0800 and 1600 h following the second day of pessary removal. The ewes were arti ficially inseminated using fresh semen of Hu sheep breed 48 h after sponge withdrawal (d 0 of gestation) and placed in individual pens (3.20 by 0.80 m). Each pen was equipped with feeders and automatic water suppliers. From d 0 to 35 of gestation, ewes were randomly assigned to three groups(n=12): the ad libitum (AL) group, a low nutrient restricted group (fed at 70% of AL), and a high nutrient restricted group (fed at 50% of AL). The number of fetuses carried by each ewe was determined by ultrasonography (Asonics Microimager 1000 Sector Scanning Instrument, Ausonics Pty Ltd., Sydney, Australia) at d 35 of gestation. On d 35 of pregnancy, each seven ewes carrying twin fetuses from these three groups were selected and assigned to the three previously corresponding to feed intake in this study.Details of the diets are reported in Table 1 to meet 100% of the NRC (1985) nutrient requirements for pregnant sheep.Ad libitum was also expected to meet 100% of the NRC(1985) nutrient requirements for pregnant sheep. Nutrient restriction (70 or 50% of AL) was achieved by feeding three quarters or one-half of the total complete diet calculated to meet 100% NRC requirements. A total of 50% of AL at least covered the existing recommendations for ewes at maintenance during gestation. The choice of a pelleted diet was to prevent possible selectivity and waste and to facilitate accurate measurements of feed intake.

The ewes with ad libitum intake were fed once daily at 0800 h and ensured a 10% of refusal. The ewes were all provided with free access to water. The amount of feed offered to the restricted feed intake groups was also adjusted daily and based on the dry matter intake (DMI) of the ALgroup from the previous day during pregnancy.

Table 1 Ingredient and nutrient composition of the experimental diets

2.2. Digestibility trial and gases metabolism

The digestibility of nutrient and in vivo methane production were measured by digestion trial integrated with a respirometry trial. The ME of the diet fed at three levels of feed intake (i.e., ad libitum or 70 or 50% of ad libitum intake) was evaluated based on those data, along with urinary energy.Twenty-one Hu sheep carrying twin fetuses at d 40, 100, and 130 of pregnancy, respectively, were housed in individual metabolism cages (1.5 m×0.9 m) and assigned to the three previously corresponding to feed intake.

The digestibility trial lasted 14 d after a 7-d adaptation period at d 40, 100, and 130 of pregnancy, respectively.Feed offered, refused and feces were weighed, individually homogenized, and a 10% subsample was collected daily in the morning during each 14 d collection period. These samples were eventually oven-dried at 55°C for 72 h, ground to pass through a 1-mm screen using a Willey mill (Arthur H. Thomas, Philadelphia, PA), and stored until analyses.To prevent loss of N as ammonia, urine was collected in containers containing 100 mL of sulfuric acid (10% H2SO4)and measured for volume and a sample daily (10 mL L–1of total volume) was stored at –20°C until analysis. Samples of feeds, orts, feces, and urine were pooled, within class, to form a composite sample for each animal and experimental period.

An open-circuit respirometry system (Sable Systems International, Las Vegas, NV) was used to measure methane(CH4) production. There are three metabolism cages each equipped with a polycarbonate head box. As only three measurement units were available at a time, measurements of CH4production were staggered. The pregnant ewes were divided into seven groups, with each group consisting of one pregnant ewe from each dietary treatment. On d 0, 2, 4, 6, 8, 10, and 12 of the 14 d collection period, each group of pregnant ewes was moved in sequence from their metabolism cages for the digestibility assays to the metabolism cages equipped with head boxes for the CH4output assessments at d 40, 100, and 130 of pregnancy, respectively. Individual CH4production was measured over a 24-h period after a 24-h adaptation period. For con finement in head boxes attached to metabolism cages, all animals had been trained previously.

The concentration of CH4, carbon dioxide (CO2) and oxygen (O2) was measured by following the procedures of Tovar-Luna et al. (2007), with minor modi fications. A fuel cell FC-1B O2analyzer (Sable Systems, Las Vegas, NV)was used to analyze the concentration of O2. The infrared analyzers (FC-1B for CO2and MA-1 for CH4; Sable Systems)were used to measure the concentrations of CO2and CH4.Air was first analyzed for CH4then for CO2and O2. Before the gas exchange measurements, validity and accuracy of expired CO2and inspired O2flows were checked with ethanol combustion with the same flow rates used during measurements. Before each test, analyzers were calibrated with reference gas mixtures (19.5 and 20.5% O2, 0.0 and 1.5% CO2, and 0.0 and 0.3% CH4). Temperature (20 to 23°C) in the calorimetry room was maintained with a window air conditioning/heating unit (Carrier, Farmington, CT),and humidity was 50 to 55% through use of a dehumidi fier(Whirlpool, Benton Harbor, MI). The natural photoperiod was mimicked by use of fluorescent lights.

2.3. Chemical analyses

Samples of feed, orts and feces were analyzed for DM(method number 930.15; AOAC 1990), ash content (method number 924.05; AOAC 1990), ether extract (EE; method number 920.39; AOAC 1990), and Ca and P (methods number 968.08 and 965.17, respectively, AOAC 1990). The neutral detergent fiber (NDF) and acid detergent fiber (ADF)concentrations were quanti fied as described by Van Soest et al. (1991). A bomb calorimeter (C200, IKA Works Inc.,Staufen, Germany) was used to measure the gross energy(GE) in dietary ingredients and feces. Analyses of GE for urine sample, was performed as described by Deng et al.(2012, 2014). Estimation of C and N contents of feed, orts,feces and urine was done by combustion in a C-N analyzer(Elementar Vario MAX CN, Elementar Americas Inc., Mt.Laurel, NJ).

2.4. Data calculation and analyses

Prediction of diet metabolisable energyThe data obtained from the digestibility trial were used to calculate the metabolisable energy (ME) content of the diet. Digestible energy (DE) was calculated as the difference between GE intake and fecal energy. The ME of the diet at the three levels of feed intake was calculated by subtracting urinary energy and in vivo CH4energy from DE. CH4energy equivalent=39.54 kJ L–1(Brouwer 1965).

Carbon and nitrogen balance (C-N method)In the C-N method, RE is based on measurements of the C-N balance,assuming that all energy is retained either as fat or as protein(López and Fernández 2013). The C-N balance depends on measurements of C and N intake and their losses as urine,feces and gases (CO2and CH4). 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–1and a content of 0.767 C for fat, and 23.86 kJ g–1and 0.16 N and 0.52 C for pro-tein, 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 (Fernández et al. 2012). The RE (kJ) was calculated according to Brouwer(1965) as RE=REprotein+REfat

Energy and protein requirements for maintenanceHeat production (HP, kJ kg–1BW0.75) was calculated as the difference between ME intake (MEI, kJ kg–1BW0.75) and RE (kJ kg–1BW0.75).The antilog of the intercept of the linear regression between the log of HP and MEI was used to estimate net energy requirement for maintenance of Hu sheep including pregnancy tissues (NEm,kJ kg–1BW0.75) according to the methodology described by Lofgreen and Garrett (1968). The ME requirement for maintenance of Hu sheep including pregnancy tissues (MEm, kJ kg–1BW0.75) was calculated by iteration of the semi-log linear regression equation until HP was equal to MEI (Galvani et al. 2008). The ef ficiency of ME utilization for maintenance (km) was computed as NEm/MEm.

A linear regression of daily retained N (RN; g kg–1BW0.75) on daily N intake (NI, g kg–1BW0.75; INRA 1988) was used to estimate the net protein requirement for maintenance. The intercept of this regression should represent endogenous and metabolic N losses, multiplied by the factor 6.25, this value was assumed to be the net protein requirement for maintenance (NPm, g kg–1BW0.75).

2.5. Statistical analysis

Effects of stage of gestation and feeding levels on energy metabolism, carbon-nitrogen balance, CH4and CO2emissions were analyzed by two way analysis of variance using SAS (SAS Inst.Inc., Cary, NC). Data were analyzed as repeated measures using the MIXED procedures of SAS. The models contained the main effects of maternal dietary intake, day of gestation, and all interactions. The linear regressions analyses were conducted with PROC MIXED. Residuals plotted against the predicted values were used to check the assumptions of the model for homoscedasticity, independency, and normality of the errors. A data point was deemed to be an outlier and removed from the database if and only if the Studentized residual was outside the±2.5 range values. The comparison of the means was performed using the Duncan test at P=0.05.

3. Results

3.1. DM intake and energy balance

Diet×Stage of gestation interactions were present for urinary energy (P=0.04) and DE (P<0.001) in Table 2. The values of DMI(kg d–1) and CH4energy/GE increased between d 40 and 100 of pregnancy but subsequently gradually decreased (P<0.05) until d 130 of gestation. There was no interaction between stage and diet for CH4energy (P=0.23). CH4energy, DE, MEI, DE/GE, ME/GE, and ME/DE were increased (P<0.05) whereas CH4energy/ GE was decreased (P<0.05) as feeding level increased at each stage of gestation. MEI was increased (P=0.009) as day of gestation increased.

3.2. Methane and carbon dioxide emissions

There was no interaction between stage and feeding level for CH4and CO2production (P>0.05) (Table 3). The CH4emission (L d–1; L kg–1BW0.75d–1) was increased (P<0.001) whereas the CH4emission(L kg–1DMI; L kg–1NDFI) was decreased (P<0.01) as feeding level increased at each stage of gestation. The CH4emission (L d–1; L kg–1DMI; L kg–1NDFI) increased between d 40 and 100 of pregnancy but subsequently gradually decreased (P<0.001) until d 130 of gestation.

The CO2emission (L d–1; L kg–1BW0.75d–1) was increased (P<0.05)whereas the CO2emission (L kg–1DMI) was decreased (P<0.01) as feeding level increased at each stage of gestation. The CO2emission(L d–1; L kg–1DMI) decreased between d 40 and 100 of pregnancy but subsequently gradually increased (P<0.01) until d 130 of gestation.

3.3. C-N balances, heat production and retained energy

Diet×Stage of gestation interactions were present for RE (P<0.001)in Table 4. The RE and HP were increased (P<0.003) whereas the apparent digestibility of C and N was decreased (P<0.001) as feeding level increased at each stage of gestation. There was no difference(P>0.05) between d 40 and 100 of gestation for RE, except for RE in 50% group. There was no difference (P>0.05) between d 100 and 130 of gestation for HP, except for HP in 50% group. No interaction was found between stage and feeding level for apparent C and N digestibility (P>0.05). Apparent C digestibility was increased (P<0.001) as day of gestation increased. Apparent N digestibility increased between d 40 and 100 of pregnancy but subsequently gradually decreased(P<0.001) until d 130 of gestation.

3.4. DM, OM, ADF and NDF intake, excretion and apparent digestibility

Diet×Stage of gestation interactions were present for ADF intake(P=0.04), apparent ADF and NDF digestibility (P=0.006 and P=0.008,respectively) in Table 5. The apparent digestibility of DM, organic matter (OM), ADF, and NDF was increased (P<0.001) as feeding level decreased at each stage of gestation. The apparent digestibility of DM and OM was increased (P<0.001) as day of gestation increased.The apparent digestibility of NDF and ADF decreased between d 40 and 100 of gestation but subsequently gradually increased (P<0.01)until d 130 of gestation.

3.5. Net energy requirements for maintenance

The linear relationships between logHP and MEI at d 40, 100, and 130 of gestation are shown in Table 6. The antilog of the intercept of these regressions, 295.80, 310.09, and 323.59 kJ kg–1BW0.75d–1,were the NEmfor the maternal body including pregnancy tissues at d 40, 100, and 130 of gestation, respectively. The MEmvalues for the maternal body including pregnancy tissues at d 40, 100, and 130 of gestation, calculated by iteration of the regression equation of log HP on MEI until HP is equal to MEI, were 445.52, 481.72, and 521.65 kJ kg–1BW0.75d–1, respectively. The km(NEm/MEm) was calculated as 0.664, 0.644,and 0.620 at d 40, 100, and 130 of gestation, respectively. The NEmand MEmwere increased whereas the kmvalues were decreased as day of gestation increased.

3.6. Net protein requirements for maintenance

Endogenous and metabolic loss of N, estimated as the intercepts of the linear regression between RN and NI (Table 7), were 317.8, 375.4, and 478.2 mg kg–1BW0.75, and it corresponds to NPmof 1.99, 2.35, and 2.99 g kg–1BW0.75for the maternal body including pregnancy tissues at d 40,100, and 130 of gestation, respectively. The data demonstrate that the net N requirement for maintenance (NNm)and NPmwere increased as day of gestation increased.

4. Discussion

CH4emissions rate, the proportion of GE intake that is released as enteric CH4energy (% GEI), is a critical factor used to assess the potential extent of global warming in national inventories and enteric CH4estimation (Gerber et al. 2013). The results in our study indicated that an increased intake level reduced the CH4emission rate of Hu sheep at each stage of gestation. Improving amount of feeding at above maintenance may be an important strategy to mitigate enteric CH4emissions(Chaokaur et al. 2015), which was in accordance with the results in our study. The CH4energy/GE was increased but DE/GE, ME/GE, and ME/DE were decreased as feeding level increased in the growing period of Dorper crossbred ram lambs (Deng et al. 2012). However, these were not in accordance with the results for pregnant Hu ewes in our study (Table 2).These findings in our study (Table 2)are in agreement with the reports by Kamalzadeh &Shabani (2007),who reported an elevation in the ME/GE of a moderate-concentrate diet(concentrate:roughage=54:46) as the intake of the diet increased from maintenance to ad libitum level in Iranian Baluchi sheep.Data available in the literature concerning the utilization of dietary energy during pregnancy are inconsistent.Flatt et al. (1969)found a decrease in DE (3%) and CH4energy (0.8%)and no change in urinary energy during pregnancy in cattle. In contrast, Ferrell et al.(1976) reported that any difference of heifers in digestibility of energy due to pregnancy was of little importance in relation to differences which may be attributed to the level of feeding. The present data for Hu sheep during pregnancy suggest that fecal energy, urinary energy,methane energy, and DE (kJ kg–1BW0.75d–1) were increased as feed intake level increased and duration of pregnancy increased, except for DE, which was increased between d 40 and 100 of gestation, and there was no difference between d 100 and 130 of gestation. The reasons for the similar DE between d 100 and 130 of gestation may be due to the same diet fed during last period of pregnancy but this will also apply to all the other energy variables reported in Table 2.The comparisons between days 100 and 130 are all valid, i.e.,they are not confounded by the change in diet composition at d 90, but comparisons between d 40 and the other days are confounded. The diets used changed at d 90, and so the effect of the different diet ingredient compositions (Table 1) is confounded with any effects of feed intake (Table 2) and stage of pregnancy.

Kishan et al. (1986) found that levels of energy in fluenced the excretion of C and N in the urine and urinary carbon was signi ficantly (P<0.01) correlated with DE intake. There is also a correlation between urinary C and N excretion (Blaxter and Wainman 1964; Kishan et al. 1986). These were not accordance with the results in our study (Table 4).In our study, the apparent C digestibility was between 58–68%,which was in accordance with the results reported by Blaxter and Wainman (1964). In the present study, there was no difference between d 100 and 130 of gestation for RN calculated by using carbon and nitrogen balance (Table 4). This is not in accordance with previous results reported by McCGraham(1964), who found that the RN calculated by using the comparative slaughter technique was decreased as day of gestation increased. In the present study, the RN and RE were increased as feeding levels increased at each stage of gestation. This is in agreement to the results of George et al. (2005) and Singh et al. (2008). This is also to be expected as the foetuses were growing. The expected effect of different feed intakes on foetal growth would explain the differences in RE and RN. As well, the kpvalues (a partial energy ef ficiency for pregnancy) can not be calculated; this is not a major problem because the reported kmvalues give suf ficient information about the practical energy requirements of these animals.

In the present study, the elevation in HP with increasing feed intake at each stage of gestation, which concurs with findings in goats (Fernandes et al. 2007), sheep (Deng et al. 2012) and cattle (Chizzotti et al. 2007). In the present study, HP was increased as MEI increased at each stage of gestation and the values of HP were greater at d 100 and 130 of gestation compared with the values of HP at d 40 of gestation. Ferrell (1988) reported that energy intake affects HP due to an increase on mass and metabolic activity of visceral organs. The results of Turner and Taylor (1983),using cattle, showed that HP is greater with increased plane of nutrition, mainly due to an elevation in metabolism associated with the energy retention. A comprehensive analysis of energy metabolism in growing cattle revealed that HP increased exponentially with increasing MEI (Chizzotti et al.2008). The nonlinear regression indicated that HP increased exponentially as MEI increased (Costa et al. 2013). A large increase in HP during gestation has been well documented(Rattray et al. 1974).

Digestibility in the rumen is the result of the competition between digestion and passage rates, and passage rate is positively correlated with DMI (Van Soest 1994). Therefore,the lesser DMI of sheep at each stage of gestation on restricted intake likely resulted in a slower passage rate and a greater digestibility of the diet. The apparent digestibility of C, N, DM, OM, ADF, and NDF was decreased as feeding levels increased at each stage of gestation in the present study. These findings are in agreement to the report above.The results in the present study showed that the value of DMI(kg d–1) increased between d 40 and 100 of pregnancy but subsequently gradually decreased until d 130 of gestation.This may be due to nutrient requirements increase due to fetal growth, which increases intake, yet capacity of the rumen is restricted due to fetal growth. This is in accordance with previous results reported by Forbes (1969), who found that there was a signi ficant negative relationship between the volume of rumen contents and the volume of uterus plus abdominal fat plus other abdominal organs.

Pregnancy tissue includes the fetus, fetal fluids and related tissues (the sum of which is the conceptus), uterus, and mammary gland (NRC 2007). In the present study, energy and protein requirements for maintenance of maternal body including pregnancy tissues were increased as day of gestation increased. This may be attributed to the growth of fetal,gravid uterus and its appendages in the early pregnancy, and the rapid growth of fetus and gland in the late pregnancy.The value of NEmat d 130 of gestation in our study, was 9.4 and 4.8% greater than the values determined at d 40 and 100 of gestation, respectively. Many researchers have con firmed that the value of MEmof non-pregnancy was similar to the value of MEmin the early pregnancy (ARC 1980;NRC 1985). The value of MEmat d 130 of gestation in the present study was 7.2 and 4.5% greater than the values determined at d 40 and 100 of gestation, respectively. The net energy maintenance requirement is in fluenced by the physiological conditions of age, gender, physical activity and temperature (NRC 2007), and is further in fluenced by body composition (Chizzotti et al. 2007), since metabolic activity is more intense in muscular tissue than in adipose tissue(Garrett 1980). It is estimated that 50% of the maintenance energy requirement is consumed in protein recycling and transporting ions through the membranes (Baldwin et al.1980; Regadas Filho et al. 2013). Therefore, in our study,net energy requirement for maintenance vary with the development of pregnancy tissues.

In the present study, the km(NEm/MEm) was calculated as 0.664, 0.644, and 0.620 at d 40, 100, and 130 of gestation,respectively. The kmvalue is also calculated by the ARC(1980) and AFRC (1993) from equation km=0.503+0.35×qm,where, qm=dietary ME divided by GE. Using this equation,the values of kmmeasured were 0.681, 0.713, and 0.742 at d 40, 100, and 130 of gestation, respectively, which were greater than the kmin our study. Therefore, acceptance of kmestimated by the AFRC (1993) would result in the underestimation of MEmrequirements. According to Luo et al. (2004), differences in MEmappear to be primarily dependent on variation in NEmrequirements rather than km,and it may explain differences in MEmrequirements among genotypes or as a result of selection, as in our study. A study with growing goats (Fernandes et al. 2007) reported a kmvalue less than kmestimated by the AFRC (1993). This was agreed with the result in our study.

The NPmwas assumed by the ARC (1984) to be equivalent to the amount of protein that will counterbalance the inevitable urinary, fecal and dermal N losses, except for growing lambs, for which no allowance for dermal losses is made (ARC 1980). Chizzotti et al. (2007) reported that the NPmestimated using the relationship between RN calculated by the comparative slaughter method and daily NI was greater than that determined based on the relationship between RN calculated using the N balance data and daily NI. The difference may be attributed to losses of N that are not accounted for by the N balance (e.g., scurf, hair, saliva N losses) and issues related to accurate measurements of urinary N based on creatinine as a marker (Chizzotti et al. 2007). The scurf protein represents about 20% of the maintenance requirement of the ARC system (ARC 1980). In our study, the results in Table 7 showed that the NPmvalues were increased as day of gestation increased,which was in accordance with the report that the NPmvalues of Dorper×thin-tailed Han crossbred ewes carrying single fetus were lower than the recommended value of 2.19 g kg–1BW0.75d–1for sheep during pregnancy by AFRC (1993), except for the NPmvalue in the late gestation (Luo et al. 2014).

5. Conclusion

The daily NEmwere 295.8, 310.1, and 323.6 kJ kg–1BW0.75,the corresponding MEmwere 445.5, 481.7, and 521.7 kJ kg–1BW0.75, and the kmwere 0.664, 0.644, and 0.620 at d 40, 100, and 130 of gestation, respectively. The daily net protein requirements for maintenance were 1.99, 2.35,and 2.99 g kg–1BW0.75at d 40, 100, and 130 of gestation,respectively. These results for the nutritional requirements of the net energy and protein may help to formulate more balanced diets for Hu sheep during pregnancy. Further studies are warranted to investigate the energy and protein requirements for growth of pregnant ewes by using comparative slaughter trials.

Acknowledgements

The project was supported by the earmarked fund for China Agriculture Research System (CARS-39) and the Agro-scienti fic Research in the Public Interest, China (201303143).We express our thanks to all members in Prof. Wang Fang’s laboratory (Nanjing Agricultural University, China) who contributed to the sample determination. We also acknowledge Zhang Guomin (Nanjing Agricultural University) and Associate Prof. Ying Shijia (Jiangsu Academy of Agricultural Sciences, China) for writing help.

AFRC (Agricultural and Food Research Council). 1993. Energy and Protein Requirements of Ruminants. An Advisory Manual Prepared by the Agricultural and Food Research Council Technical Committee on Responses to Nutrients.CAB International, Wallingford, UK.

ARC (Agricultural Research Council). 1980. The Nutrient Requirements of Ruminant Livestock. Commonwealth Agricultural Bureaux, Slough, UK.

ARC (Agricultural Research Council). 1984. The Nutrient Requirements of Ruminant Livestock, Supplement No. 1.Report of the Protein Group of the ARC Working Party.Commonwealth Agricultural Bureaux, Slough, UK.

AOAC (Association of Of ficial Analytical Chemists). 1990.Of ficial Methods of Analysis. 15th ed. AOAC, Arlington, VA.

Baldwin R L, Smith N E, Taylor J, Sharp M. 1980. Manipulating metabolic parameters to improve growth rate and milk secretion. Journal of Animal Science,51, 1416–1428.

Blaxter K L, Wainman F W. 1964. The utilization of the energy of different rations by sheep and cattle for maintenance and for fattening. Journal of Agricultural Science,63, 113–128.

Brouwer E. 1965. Report of sub-committee on constants and factors. In: Blaxter K L, ed., Energy Metabolism.Proceedings of the 3rd Symposium Held at Troon, Scotland,May 1964. Academic Press, London.

Chaokaur A, Nishida T, Phaowphaisal I, Sommart K. 2015.Effects of feeding level on methane emissions and energy utilization of Brahman cattle in the tropics. Agriculture Ecosystems & Environment,199, 225–230.

Chizzotti M L, Tedeschi L O, Valadares Filho S C. 2008. A meta-analysis of energy and protein requirements for maintenance and growth of Nellore cattle. Journal of Animal Science,86, 1588–1597.

Chizzotti M L, Valadares Filho S C, Tedeschi L O. 2007. Energy and protein requirements for growth and maintenance of F Nellore×Red Angus bulls, steers, and heifers. Journal of Animal Science,85, 1971–1981.

Costa M, Pereira E S, Silva A M A. 2013. Body composition and net energy and protein requirements of Morada Nova lambs. Small Ruminant Research,114, 206–213.

Deng K D, Diao Q Y, Jiang C G. 2012. Energy requirements for maintenance and growth of Dorper crossbred ram lambs.Livestock Science,150, 102–110.

Deng K D, Jiang C G, Tu Y, Zhang N F, Liu J, Ma T, Diao Q Y.2014. Energy requirements of Dorper crossbred ewe lambs.Journal of Animal Science,92, 2161–2169.

Fernandes M H, Resende K T, Tedeschi L O, Fernandes Jr J S, Silva H M, Carstens G E, Berchielli T T, Teixeira I A, Akinaga L. 2007. Energy and protein requirements for maintenance and growth of Boer crossbred kids. Journal of Animal Science,85, 1014–1023.

Fernández C, López M C, Lachica M. 2012. 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. Animal Feed Science and Technology,178, 115–119.

Ferrell C L. 1988. Contribution of visceral organs to animal energy expenditure. Journal of Animal Science,66(Suppl.3), 23–34.

Ferrell C L, Garrett W N, Hinman N, Grichting. 1976. Energy utilization by pregnant and non-pregnant heifers. Journal of Animal Science,42, 937–950.

Flatt W P, Moe P W, Munson A W, Cooper T. 1969. Energy utilization by high producing dairy cows. II. Summary of energy balance experiments with lactating Holstein cows.Energy Metabolism of Farm Animals,12, 235–251.

Forbes J M. 1969. The effect of pregnancy and fatness on the volume of rumen contents in the ewe. Journal of Agricultural Science,72, 119–121.

Galvani D B, Pires C C, Kozloski G V, Wommer T P. 2008.Energy requirements of Texel crossbred lambs. Journal of Animal Science,86, 3480–3490.

Garrett W N. 1980. Factors in fluencing energetic ef ficiency of beef production. Journal of Animal Science,51, 1434–1440.

George S K, Dipu M T, Mehra U R, Singh P, Verma A K. 2005.Growth of crossbred bulls affected by level of feed intake.Indian Journal of Animal Science,22, 81–84.

Gerber P J, Steinfeld H, Henderson B, Mottet A, Opio C, Dijkman J, Tempio G. 2013. Tackling Climate Change Through Livestock: A Global Assessment of Emissions and Mitigation Opportunities. Food and Agriculture Organization of the United Nations (FAO), Rome, Italy.

INRA (Institut National de la Recherche Agronomique). 1988.Alimentation des Bovins Ovins et Caprins. Institut National de la Recherche Agronomique, Paris, France.

Kamalzadeh A, Shabani A. 2007. Maintenance and growth requirements for energy and nitrogen of Baluchi sheep.International Journal of Agricultural Biology,9, 535–539.

Kishan J, Khan M Y, Lal M. 1986. In fluence of dietary energy levels on urinary nitrogen and carbon excretion in buffaloes.Indian Journal of Animal Sciences,3, 197–200.

Lofgreen G P, Garrett W N. 1968. A system for expressing net energy requirements and feed values for growing and finishing Beef cattle. Journal of Animal Science,27,793–806.

López M C, Fernández C. 2013. Changes in heat production by sheep of Guirra breed after increase in quantity of barley grain on the diet. Small Ruminant Research,109, 113–118.

Lou C, Jiang C G, Ma T, Diao Q Y. 2014. Study of nitrogen metabolism and net protein requirement for maintenance of Dorper×Thin-tail Han Crossbred breeding ewes. Acta Veterinaria et Zootechnica Sinica,45, 943–952.

Luo J, Goetsch A L, Nsahlai I V, Johnson Z B, Sahlu T,Moore J E, Ferrell C L, Galyean M L, Owens F N. 2004.Maintenance energy requirements of goats: Predictions based on observations of heat and recovered energy. Small Ruminant Research,53, 221–230.

McCGraham N. 1964. Energy exchanges of pregnant and lactating ewes. Crop and Pasture Science,15, 127–141.

Nie H T, Zhang H, You J H, Wang F. 2015. Determination of energy and protein requirement for maintenance and growth and evaluation for the effects of gender upon nutrient requirement in Dorper×Hu crossbred Lambs. Tropical Animal Health and Production,47, 841–853.

NRC (National Research Council). 1985. Nutrient Requirements of Small Ruminants: Sheep, Goats, Cervids, and New World Camelids. National Academy Press, Washington, D.C.

NRC (National Research Council). 2007. Nutrient Requirements of Small Ruminants: Sheep, Goats, Cervids and New World Camelids. National Academy Press, Washington, D.C.

Rattray P V, Garrett W N, East N E, Hinman N. 1974. Ef ficiency of utilization of metabolizable energy during pregnancy and the energy requirements for pregnancy in sheep. Journal of Animal Science,38, 383–393.

Regadas Filho J G L, Pereira E S, Pimentel P G, Villarroel A B S, Medeiros A N, Fontenele R M. 2013. Body composition and net energy requirements for Santa Ines lambs. Small Ruminant Research,109, 107–112.

Russel A J F, Doney J M, Gunn R G. 1969. Subjective assessment of body fat in live sheep. Journal of Agricultural Science,72, 451–454.

Singh P, Verma A K, Sahu D S, Mehra U R. 2008. Utilization of nutrients as in fluenced by different restriction levels of feed intake under sub-tropical conditions in crossbred calves.Livestock Science,117, 308–314.

Van Soest P J. 1994. Nutritional Ecology of the Ruminant.Cornell University Press, New York, USA.

Van Soest P J, Robertson J B, Lewis B A. 1991. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. Journal of Dairy Science,74, 3583–3597.

Tovar-Luna I, Goetsch A L, Puchala R, Sahlu T, Carstens G E,Freetly H C, Johnson Z B. 2007. Effects of moderate feed restriction on energy expenditure by 2-year-old crossbred Boer goats. Small Ruminant Research,72, 25–32.

Turner H G, Taylor C S. 1983. Dynamic factors in models of energy utilization with particular reference to maintenance requirement of cattle. World Review of Nutrition and Dietetics,42, 135–190.

Yue G H. 1996. Reproductive characteristics of Chinese Hu sheep. Animal Reproduction Science,44, 223–230.

8 February, 2017 Accepted 5 June, 2017

ZHANG Hao, E-mail: zhanghao_850220@126.com;Correspondence ZHANG Yan-li, Tel/Fax: +86-25-84395381,E-mail: zhangyanli@njau.edu.cn

© 2018 CAAS. Publishing services by Elsevier B.V. All rights reserved.

10.1016/S2095-3119(17)61691-5

Section editor LUO Xu-gang

Managing editor ZHANG Juan