DOI: 10.1111/jpn.12295
ORIGINAL ARTICLE
Effects of supplementing rare earth element cerium on rumen fermentation, nutrient digestibility, nitrogen balance and plasma biochemical parameters in beef cattle S. X. Lin, C. Wei, G. Y. Zhao, T. T. Zhang and K. Yang State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing, China
Summary The objectives of the trial were to investigate the effects of supplementing rare earth element (REE) cerium (Ce) on rumen fermentation, nutrient digestibility, methane (CH4) production, nitrogen (N) balance and plasma biochemical parameters in beef cattle. Four Simmental male cattle, aged at 14 months, with initial liveweight of 355 8 kg and fitted with permanent rumen cannulas, were used as experimental animals. The cattle were fed with a total mixed ration (TMR) composed of concentrate mixture and corn silage. Four levels of cerium chloride (CeCl37H2O, purity 99.9%), that is 0, 80, 160 and 240 mg CeCl3/kg DM, were added to basal ration in a 4 9 4 Latin square design. Each experimental period lasted 15 days, of which the first 12 days were for pre-treatment and the last 3 days were for sampling. The results showed that supplementing CeCl3 at 160 or 240 mg/kg DM increased neutral detergent fibre (NDF) digestibility (p < 0.05) and tended to increased acid detergent fibre (ADF) digestibility (p = 0.083). Supplementing CeCl3 at 80, 160 or 240 mg/kg DM decreased the molar ratio of rumen acetate to propionate linearly (p < 0.05). Supplementing CeCl3 at 160 or 240 mg/kg DM decreased total N excretion, urinary N excretion and increased N retention (p < 0.05), increased excretion of total urinary purine derivatives (PD) (p < 0.05) and decreased CH4/kg DMI (p < 0.05). In conclusion, supplementing CeCl3 at 160 or 240 mg/kg DM in the ration of beef cattle increased the digestibility of NDF, decreased the molar ratio of rumen acetate to propionate, increased N retention and microbial N flow and decreased CH4/kg DMI. Keywords cattle, cerium, methane, nutrient digestibility, rumen Correspondence G. Y. Zhao, State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, No. 2 Yuan Ming Yuan West Road, Beijing 100193, China. Tel: +86-10-62733379; Fax: +86-10-62733379; E-mail: [email protected]. Received: 15 September 2014; accepted: 15 January 2015
Introduction Rare earth elements (REE) include 17 transition elements of the III subgroup in the periodic system. Many studies showed that dietary supplementation of REE improved productive performance of cows, pigs and broilers (Shen et al., 1991; He et al., 2001, 2010). In beef cattle, supplementing 50, 100 and 200 mg LaCl3/kg dry matter (DM) linearly increased whole tract digestibility of organic matter (OM), neutral detergent fibre (NDF) and crude protein (CP) (Liu et al., 2008). In Aerbasi white cashmere goats, supplementing 76 mg REE nitrate/kg ration increased nitrogen (N) retention (Gui et al., 1995), and in Small Tail Han sheep, supplementing 216.5 mg REE nitrate/kg DM to ration also increased N retention (Yu et al., 2010). In continuous culture, it was also found that supplementing 400 and 800 mg REE mixture (38.0% LaCl36H2O, 52.1% CeCl36H2O, 3.0% PrCl36H2O
and 6.9% other REE chlorides)/kg DM to feed mixtures, linearly increased ruminal digestibility of OM, acid detergent fibre (ADF) and CP (Yang et al., 2009). From previous trials, it could be found that the REE supplemented to the rations of animals in different trials were different in composition and level and in most trials the REE supplemented was mixtures (Gui et al., 1995; Yang et al., 2009; Yu et al., 2010) even though in the trial of Liu et al. (2008) a single REE La (in the form of LaCl3) was used as a supplement. Cerium (Ce), which accounts for approximately 48% of the total REE on the earth, was one of the major REE in the REE mixtures supplemented in many previous trials. It could be hypothesized that Ce could possibly play important roles in effects on animal metabolism and growth. The objectives of the present trial were to study effects of supplementing CeCl3 on rumen fermentation, nutrient digestion, N balance and plasma biochemical parameters in beef cattle.
Journal of Animal Physiology and Animal Nutrition 99 (2015) 1047–1055 © 2015 Blackwell Verlag GmbH
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S. X. Lin et al.
Effects of Ce on digestion of beef cattle
Materials and method
Measurements and sampling
Animals, experimental design and management
The cattle were weighed on an electronic balance (V8-I; Cangzhou Fangzheng Electronic Weighing Instrument Co. Ltd, Hebei, China) at the beginning and the end of each experimental period. During the sampling period, the faeces and urine of each cattle were completely collected and weighed daily. Urine was collected using a urine collector connected to a bucket. Aliquots of 3% of faeces and 2% urine were sampled daily and immediately frozen at 20 °C for later analysis. The maize silage and concentrate mixture were also sampled daily. Faeces and feeds were weighed on a top-loading analytical balance (1.0 g readability; YP5002, Puchun Measure Instrument Co. Ltd, Shanghai, China). On the first day of sampling period, a suitable amount of rumen digesta was taken from the rumen of each cattle through rumen cannula 2 h after morning feeding and strained through four layers of cheese cloth to yield approximately 200 ml of rumen fluid. The pH of rumen fluid was measured immediately using a pH meter (pH-HJ 90; Aerospace Computer Company, Beijing, China). The rumen fluid samples were subsequently stored at 20 °C for later analysis. On the second day of sampling period, approximately 10-ml blood sample was taken using vacutainer tubes containing EDTA-2K (Greiner Bio-one, Frickenhausen, Germany) from jugular vein of each cattle 3 h after morning feeding. The blood samples were centrifuged at 2800 g for 15 min at room temperature of approximately 25 °C, and plasma samples were taken and frozen at 20 °C. On the third day of sampling period, a mobile open-circuit respirometry system was used to take gas samples from the cattle. The system consisted of a face mask connected to 2 PVC corrugated tubes and a nondiffusing Tedlar bag with a maximum volume of 500 l (Safelab Technology Co. Ltd., Beijing, China). Two valves were fixed on the ends of two PVC tubes connected to the face mask. The valve on the tube connected to the gas bag opened while cattle exhaling, while another valve on another tube closed and vice versa. During the sampling period, the face mask was fitted to each cattle airtight and the gas exhaled flowed into the Tedlar bag. The gas samples were collected at four time points, that is 06:00, 12:00, 20:00 and 24:00 h, respectively, and the duration of each sampling period was 10 min. After sampling, the gas collected in each Tedlar bag was mixed well, and 100 ml was sampled using a syringe into a Tedlar bag with a volume of 250 ml for later analysis. The total volume of the gas exhaled
The protocols of the trial were approved by the Laboratory Animal Welfare and Animal Experimental Ethical Committee at China Agricultural University (Beijing, China) (No. 20130611-2). Four Simmental male cattle, aged 14 months, with initial liveweight of 355 8 kg and fitted with permanent rumen cannulas, were used in a 4 9 4 Latin square design. Four levels of cerium chloride (CeCl37H2O, purity 99.9%, Aladdin Industrial Inc., Shanghai, China), that is 0, 80, 160 and 240 mg CeCl3/kg DM, were added to basal ration (Table 1), respectively, as experimental treatments. The CeCl3 levels used in the trial were referred to the supplementation level of LaCl3 (Liu et al., 2008). The cattle were housed in a stall and fed in individual pens (1.5 m 9 2.5 m) and were fed with a total mixed ration (TMR) composed of concentrate mixture and corn silage (Table 1), and the amount of TMR offered to each cattle was fixed to 5.0 kg DM daily, supplying major nutrients at the levels of approximately 1.1 times of maintenance requirements (Feng, 2000). The ration was divided into two equal meals and provided to the cattle twice daily at 07:00 and 17:00 h, respectively. Cerium chloride was added to concentrate mixture and mixed well before feeding. Clean drinking water was freely available during the experimental periods. Each period lasted 15 days, of which the first 12 days were for pre-treatment and the last 3 days were for sampling. The ration, treatment and management for each cattle were kept unchanged during each experimental period.
Table 1 Ingredients and chemical composition of basal ration, g/kg DM Items Ingredients Corn silage Corn Soybean meal Corn gluten meal Wheat bran NaCl Sodium bicarbonate Chemical composition Organic matter Crude protein Neutral detergent fibre Acid detergent fibre Ether extract
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457.6 202.7 92.2 210.6 21.1 5.3 10.5 914.1 136.1 436.6 227.9 28.5
Journal of Animal Physiology and Animal Nutrition 99 (2015) 1047–1055 © 2015 Blackwell Verlag GmbH
S. X. Lin et al.
from each cattle during the sampling period was measured on a rotameter (LML-1; Zhenanyu instrument Co. Ltd, Beijing, China) using a gas pump (GM-0.33A; Tianjin Jinteng Experiment Equipment Co. Ltd, Tianjin, China). Determinations and analysis
The faecal samples were dried at 65 °C for 48 h. The corn silage was dried on a freeze-drying machine (LGJ-12; Songyuan Huaxing Technology Development Co. Ltd, Beijing, China) for 72 h. The faecal and feed samples were then milled through a sieve with pore size of 1 mm. The DM, ether extract (EE) and ash of feed and faecal samples were determined according to AOAC (1990) using the methods of no. 934.01, 920.39 and 924.05 respectively. The N of feed, faeces and urine was analysed using the Kjeldahl method (AOAC, 1990). The NDF and ADF were determined on an ANKOM200 Fiber Analyzer (ANKOM Technology Corp., Macedon, NY, USA) using the method of Van Soest et al. (1991). The OM was calculated as the difference between DM and ash. The CH4 of gas samples was analysed using gas chromatography (TP-2060T, Beijing Beifen Tianpu Instrument Technology Co. Ltd, Beijing, China). The conditions for the analysis were as follows: TCD detector, TDX-01 column, size 1 m 9 2 mm 9 3 mm, column temperature 70 °C and detector temperature 100 °C. The carrying gas was argon with flowing rate of 30 ml/min. The volatile fatty acids (VFA) of rumen fluid samples were analysed using gas chromatography (TP-2060F, Beijing Beifen Tianpu Instrument Technology Co. Ltd). One millilitre of rumen fluid was mixed with 0.25 ml metaphosphoric acid (concentration 25 g/100 ml, w/v) and centrifuged at 15 000 g for 15 min at 4 °C, and the supernatant was used for analysis. The conditions for analysis were as follows: FID detector, PEG-20M + H3PO4 glass column, column temperature 120 °C and detector temperature 220 °C. The carrying gas was argon with flowing rate of 30 ml/min and the flowing rates of H2 and air were 30 ml/min and 300 ml/min, respectively. The ammonia N (NH3-N) of rumen fluid was determined using the steam distillation method described by Bremner and Keeney (1965). The urinary allantoin and uric acid were determined on a spectrophotometer (K5500, Beijing Kaiao Technology Development Co. Ltd., Beijing, China) using the method described by Young and Conway (1942) and Fujihara et al. (1987), respectively.
Effects of Ce on digestion of beef cattle
The plasma total protein (TP), albumin (ALB), glucose (GLU), triglyceride (TG), plasma urea nitrogen (PUN), cholesterol (CHO), aspartate aminotransferase (AST), alanine aminotransferase (ALT) were analysed on an automatic biochemical analyser (7160; Hitachi Ltd., Tokyo, Japan) using commercial colorimetric kits (BioSino Bio-technology and Science Co. Ltd, Beijing, China). The plasma malondialdehyde (MDA), glutathione peroxidase (GSH-PX), superoxide dismutase (SOD), total antioxidant capability (T-AOC) and catalase (CAT) were analysed on an automatic biochemistry analyser (7160; Hitachi Ltd.) using kits HY-50116, HY-60005, HY-60001, HY-60021 and HY-50121 respectively (SinoUK Institute of Biological Technology, Beijing, China). The plasma human growth hormone (hGH), insulin (INS) and insulin-like growth factor-1 (IGF-1) were determined on an automatic radioimmunoassay counter (r-911; University of Science and Technology of China Industrial Co. Ltd, Hefei, China) using competitive radioimmunoassay (RIA) kits HY-10035, HY-10069 and HY-082, respectively (Sino-UK Institute of Biological Technology). Calculations
N retention (g/day) ¼ N intake (g/day) Faecal N (g/day) Urinary N (g/day) N retention rateð%Þ ¼ N retention (g/day)/N intake (g/day) 100 Nutrient digestibilityð%Þ ¼ [Nutrient intake (g/day) Nutrient in faeces (g/day)]=Nutrient intake (g/day) 100 CH4 production (l) ¼ CH4 concentrationð%Þ gas volume (l) CH4 productionðl=dayÞ ¼½CH4 productionðl; 06 : 00 hÞ þ CH4 productionðl; 12 : 00 hÞ þ CH4 productionðl; 20 : 00 hÞ þ CH4 productionðl; 24 : 00 hÞ 36 where it was assumed that the composition of gas samples collected for 10 min at four periods during a day represented the composition of the gas exhaled by cattle during a whole day. Total urinary purine derivatives (PD) (mmol/day) = Allantoin (mmol/day) + Uric acid (mmol/day) The microbial N was calculated based on the amount of microbial purines absorbed using the equations of Chen and Gomes (1992):
Journal of Animal Physiology and Animal Nutrition 99 (2015) 1047–1055 © 2015 Blackwell Verlag GmbH
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Effects of Ce on digestion of beef cattle
Y ¼ 0:85X þ 0:385W0:75 where X refers to PD absorbed, mmol/day; Y, total urinary PD excreted, mmol/day; W0.75, metabolic body weight of cattle at the end of each experimental period, kg.
Microbial N (g/day) ¼
Xðmmol/dayÞ 70 0:116 0:83 1000
where 70 refers to N content of purines (70 mg N/ mmol); 0.116, ratio of purine N:total N in mixed rumen microbes; 0.83, estimated digestibility of microbial purines. Statistical analysis
Statistical analysis was conducted using the GLM procedures of SAS 9.0 (2002) using the model: Yijk = l + Ti + Pj + Ck + eijk, where Yijk refers to observation; l, overall mean; Ti, treatment (i = 1, 2, 3 and 4); Pj, period (j = 1, 2, 3 and 4); Ck, cattle (k = 1, 2, 3 and 4); and eijk, residual error. Differences between treatments were considered to be significant at p < 0.05 and a tendency to be significant at 0.05 < p < 0.10. Comparison of means was carried out using Duncan’s multiple range tests. The data between different treatments were subjected to the orthogonal polynomial contrasts to determine linear and quadratic effects of increasing level of CeCl3. Results The results of nutrient digestibility are shown in Table 2. No significant difference was found between treatments in digestibility of DM, OM or EE (p > 0.05). Supplementing CeCl3 at 160 or 240 mg/kg
DM increased NDF digestibility (p < 0.05) and tended to increase ADF digestibility (p = 0.083). The digestibility of CP, NDF and ADF increased in a linear manner (p < 0.05), and EE digestibility increased in a linear trend (p = 0.076) with increasing level of CeCl3. The results of rumen fermentation parameters are shown in Table 3. The rumen pH of all treatments ranged from 6.68 to 6.73. Supplementing CeCl3 up to 240 mg/kg DM did not affect rumen pH, total VFA, butyrate and NH3-N (p > 0.05) whereas decreased the molar proportion ratio of acetate to propionate (p < 0.01). Supplementing CeCl3 at 160 or 240 mg/kg DM decreased CH4 production DM intake (DMI) basis (p < 0.05). The CH4 production decreased in a linear manner with increasing level of CeCl3 (p < 0.05). The results of N metabolism are shown in Table 4. Supplementing CeCl3 160 or 240 mg/kg DM decreased urinary N (p < 0.05) and total N excretion (p < 0.05), increased N retention and N retention rate (p < 0.05). Supplementing CeCl3 tended to decrease faecal N excretion in a linear manner (p < 0.10). The results of urinary PD and microbial N flow are shown in Table 5. Supplementing CeCl3 at 160 or 240 mg/kg DM increased total urinary PD (p < 0.05) and estimated microbial N flow based on urinary PD (p < 0.05) and tended to increase allantoin (p = 0.058). The allantoin, uric acid, total PD and microbial N flow increased linearly with increasing level of CeCl3. The results of plasma parameters are shown in Table 6. Supplementing CeCl3 increased plasma concentration of ALB (p < 0.05), whereas did not affect plasma concentration of TP, GLU, TG, CHO, PUN, AST, ALT, MDA, GSH-PX, SOD, T-AOC, CAT, hGH, INS or IGF-1.
Table 2 Effect of supplementing CeCl3 on nutrient digestibility of beef cattle CeCl3 supplemented, mg/kg DM
p-Value
Nutrient digestibility, %
0
80
160
240
SEM
Treatment
Linear
Quadratic
DM OM CP EE NDF ADF
70.08 71.68 72.81 81.52 54.40b 52.72
70.03 71.69 72.86 82.27 54.22b 55.37
70.77 72.46 73.82 84.73 58.47a 58.25
71.82 73.23 75.02 85.01 58.10a 58.86
0.66 0.61 0.92 1.53 1.00 1.48
0.283 0.311 0.367 0.354 0.039 0.083
0.145 0.189 0.038 0.076 0.043 0.007
0.298 0.401 0.094 0.218 0.139 0.023
SEM, standard error of mean; DM, dry matter; OM, organic matter; CP, crude protein; EE, Ether extract; NDF, Neutral detergent fibre; ADF, acid detergent fibre. Means within the same row marked with different superscripts differ significantly (p < 0.05).
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Table 3 Effect of supplementing CeCl3 on rumen fermentation of beef cattle CeCl3 supplemented, mg/kg DM
p-Value
Items
0
80
160
240
SEM
Treatment
Linear
Quadratic
pH Total VFA, mM Molar, % Acetate Propionate Butyrate Acetate/propionate NH3-N, mg/100 ml CH4 production, l/day CH4 production, l/kg DMI
6.68 82.84
6.68 83.41
6.71 84.37
6.73 85.03
0.06 1.39
0.911 0.701
0.623 0.679
0.884 0.886
40.81a 39.25c 13.52 1.04a 18.18 34.60a 6.90a
38.61ab 41.02bc 13.68 0.94b 18.74 31.90ab 6.35ab
37.24b 41.33b 13.70 0.90b 19.55 28.36b 5.64b
36.82b 43.91a 13.72 0.84c 19.91 25.84b 5.13b
0.70 0.55 0.25 0.02 0.81 1.72 0.35
0.025 0.006 0.937 0.001 0.486 0.044 0.047
0.003 0.004 0.753 0.001 0.506 0.013 0.011
0.008 0.017 0.942 0.004 0.808 0.052 0.045
SEM, standard error of mean; NH3-N, ammonia nitrogen; VFA, total volatile fatty acids; CH4, methane; DMI, dry matter intake. Means within the same row marked with different superscripts differ significantly (p < 0.05).
Table 4 Effects of supplementing CeCl3 on N metabolism of beef cattle CeCl3 supplemented, mg/kg DM
p-Value
Items
0
80
160
240
SEM
Treatment
Linear
Quadratic
N intake, g/day Urinary N, g/day Faecal N, g/day Total N excretion, g/day N retention, g/day N retention rate, %
109.40 51.31a 29.76 81.07a 28.37c 26.02c
109.40 49.77ab 29.74 79.51ab 29.92bc 27.41bc
109.40 47.57bc 28.66 76.23bc 33.20ab 30.39ab
109.40 46.75c 27.31 74.06c 35.37a 32.35a
– 0.78 1.01 1.28 1.28 1.13
– 0.021 0.356 0.030 0.030 0.027
– 0.092 0.082 0.060
ORIGINAL ARTICLE
Effects of supplementing rare earth element cerium on rumen fermentation, nutrient digestibility, nitrogen balance and plasma biochemical parameters in beef cattle S. X. Lin, C. Wei, G. Y. Zhao, T. T. Zhang and K. Yang State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing, China
Summary The objectives of the trial were to investigate the effects of supplementing rare earth element (REE) cerium (Ce) on rumen fermentation, nutrient digestibility, methane (CH4) production, nitrogen (N) balance and plasma biochemical parameters in beef cattle. Four Simmental male cattle, aged at 14 months, with initial liveweight of 355 8 kg and fitted with permanent rumen cannulas, were used as experimental animals. The cattle were fed with a total mixed ration (TMR) composed of concentrate mixture and corn silage. Four levels of cerium chloride (CeCl37H2O, purity 99.9%), that is 0, 80, 160 and 240 mg CeCl3/kg DM, were added to basal ration in a 4 9 4 Latin square design. Each experimental period lasted 15 days, of which the first 12 days were for pre-treatment and the last 3 days were for sampling. The results showed that supplementing CeCl3 at 160 or 240 mg/kg DM increased neutral detergent fibre (NDF) digestibility (p < 0.05) and tended to increased acid detergent fibre (ADF) digestibility (p = 0.083). Supplementing CeCl3 at 80, 160 or 240 mg/kg DM decreased the molar ratio of rumen acetate to propionate linearly (p < 0.05). Supplementing CeCl3 at 160 or 240 mg/kg DM decreased total N excretion, urinary N excretion and increased N retention (p < 0.05), increased excretion of total urinary purine derivatives (PD) (p < 0.05) and decreased CH4/kg DMI (p < 0.05). In conclusion, supplementing CeCl3 at 160 or 240 mg/kg DM in the ration of beef cattle increased the digestibility of NDF, decreased the molar ratio of rumen acetate to propionate, increased N retention and microbial N flow and decreased CH4/kg DMI. Keywords cattle, cerium, methane, nutrient digestibility, rumen Correspondence G. Y. Zhao, State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, No. 2 Yuan Ming Yuan West Road, Beijing 100193, China. Tel: +86-10-62733379; Fax: +86-10-62733379; E-mail: [email protected]. Received: 15 September 2014; accepted: 15 January 2015
Introduction Rare earth elements (REE) include 17 transition elements of the III subgroup in the periodic system. Many studies showed that dietary supplementation of REE improved productive performance of cows, pigs and broilers (Shen et al., 1991; He et al., 2001, 2010). In beef cattle, supplementing 50, 100 and 200 mg LaCl3/kg dry matter (DM) linearly increased whole tract digestibility of organic matter (OM), neutral detergent fibre (NDF) and crude protein (CP) (Liu et al., 2008). In Aerbasi white cashmere goats, supplementing 76 mg REE nitrate/kg ration increased nitrogen (N) retention (Gui et al., 1995), and in Small Tail Han sheep, supplementing 216.5 mg REE nitrate/kg DM to ration also increased N retention (Yu et al., 2010). In continuous culture, it was also found that supplementing 400 and 800 mg REE mixture (38.0% LaCl36H2O, 52.1% CeCl36H2O, 3.0% PrCl36H2O
and 6.9% other REE chlorides)/kg DM to feed mixtures, linearly increased ruminal digestibility of OM, acid detergent fibre (ADF) and CP (Yang et al., 2009). From previous trials, it could be found that the REE supplemented to the rations of animals in different trials were different in composition and level and in most trials the REE supplemented was mixtures (Gui et al., 1995; Yang et al., 2009; Yu et al., 2010) even though in the trial of Liu et al. (2008) a single REE La (in the form of LaCl3) was used as a supplement. Cerium (Ce), which accounts for approximately 48% of the total REE on the earth, was one of the major REE in the REE mixtures supplemented in many previous trials. It could be hypothesized that Ce could possibly play important roles in effects on animal metabolism and growth. The objectives of the present trial were to study effects of supplementing CeCl3 on rumen fermentation, nutrient digestion, N balance and plasma biochemical parameters in beef cattle.
Journal of Animal Physiology and Animal Nutrition 99 (2015) 1047–1055 © 2015 Blackwell Verlag GmbH
1047
S. X. Lin et al.
Effects of Ce on digestion of beef cattle
Materials and method
Measurements and sampling
Animals, experimental design and management
The cattle were weighed on an electronic balance (V8-I; Cangzhou Fangzheng Electronic Weighing Instrument Co. Ltd, Hebei, China) at the beginning and the end of each experimental period. During the sampling period, the faeces and urine of each cattle were completely collected and weighed daily. Urine was collected using a urine collector connected to a bucket. Aliquots of 3% of faeces and 2% urine were sampled daily and immediately frozen at 20 °C for later analysis. The maize silage and concentrate mixture were also sampled daily. Faeces and feeds were weighed on a top-loading analytical balance (1.0 g readability; YP5002, Puchun Measure Instrument Co. Ltd, Shanghai, China). On the first day of sampling period, a suitable amount of rumen digesta was taken from the rumen of each cattle through rumen cannula 2 h after morning feeding and strained through four layers of cheese cloth to yield approximately 200 ml of rumen fluid. The pH of rumen fluid was measured immediately using a pH meter (pH-HJ 90; Aerospace Computer Company, Beijing, China). The rumen fluid samples were subsequently stored at 20 °C for later analysis. On the second day of sampling period, approximately 10-ml blood sample was taken using vacutainer tubes containing EDTA-2K (Greiner Bio-one, Frickenhausen, Germany) from jugular vein of each cattle 3 h after morning feeding. The blood samples were centrifuged at 2800 g for 15 min at room temperature of approximately 25 °C, and plasma samples were taken and frozen at 20 °C. On the third day of sampling period, a mobile open-circuit respirometry system was used to take gas samples from the cattle. The system consisted of a face mask connected to 2 PVC corrugated tubes and a nondiffusing Tedlar bag with a maximum volume of 500 l (Safelab Technology Co. Ltd., Beijing, China). Two valves were fixed on the ends of two PVC tubes connected to the face mask. The valve on the tube connected to the gas bag opened while cattle exhaling, while another valve on another tube closed and vice versa. During the sampling period, the face mask was fitted to each cattle airtight and the gas exhaled flowed into the Tedlar bag. The gas samples were collected at four time points, that is 06:00, 12:00, 20:00 and 24:00 h, respectively, and the duration of each sampling period was 10 min. After sampling, the gas collected in each Tedlar bag was mixed well, and 100 ml was sampled using a syringe into a Tedlar bag with a volume of 250 ml for later analysis. The total volume of the gas exhaled
The protocols of the trial were approved by the Laboratory Animal Welfare and Animal Experimental Ethical Committee at China Agricultural University (Beijing, China) (No. 20130611-2). Four Simmental male cattle, aged 14 months, with initial liveweight of 355 8 kg and fitted with permanent rumen cannulas, were used in a 4 9 4 Latin square design. Four levels of cerium chloride (CeCl37H2O, purity 99.9%, Aladdin Industrial Inc., Shanghai, China), that is 0, 80, 160 and 240 mg CeCl3/kg DM, were added to basal ration (Table 1), respectively, as experimental treatments. The CeCl3 levels used in the trial were referred to the supplementation level of LaCl3 (Liu et al., 2008). The cattle were housed in a stall and fed in individual pens (1.5 m 9 2.5 m) and were fed with a total mixed ration (TMR) composed of concentrate mixture and corn silage (Table 1), and the amount of TMR offered to each cattle was fixed to 5.0 kg DM daily, supplying major nutrients at the levels of approximately 1.1 times of maintenance requirements (Feng, 2000). The ration was divided into two equal meals and provided to the cattle twice daily at 07:00 and 17:00 h, respectively. Cerium chloride was added to concentrate mixture and mixed well before feeding. Clean drinking water was freely available during the experimental periods. Each period lasted 15 days, of which the first 12 days were for pre-treatment and the last 3 days were for sampling. The ration, treatment and management for each cattle were kept unchanged during each experimental period.
Table 1 Ingredients and chemical composition of basal ration, g/kg DM Items Ingredients Corn silage Corn Soybean meal Corn gluten meal Wheat bran NaCl Sodium bicarbonate Chemical composition Organic matter Crude protein Neutral detergent fibre Acid detergent fibre Ether extract
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Contents
457.6 202.7 92.2 210.6 21.1 5.3 10.5 914.1 136.1 436.6 227.9 28.5
Journal of Animal Physiology and Animal Nutrition 99 (2015) 1047–1055 © 2015 Blackwell Verlag GmbH
S. X. Lin et al.
from each cattle during the sampling period was measured on a rotameter (LML-1; Zhenanyu instrument Co. Ltd, Beijing, China) using a gas pump (GM-0.33A; Tianjin Jinteng Experiment Equipment Co. Ltd, Tianjin, China). Determinations and analysis
The faecal samples were dried at 65 °C for 48 h. The corn silage was dried on a freeze-drying machine (LGJ-12; Songyuan Huaxing Technology Development Co. Ltd, Beijing, China) for 72 h. The faecal and feed samples were then milled through a sieve with pore size of 1 mm. The DM, ether extract (EE) and ash of feed and faecal samples were determined according to AOAC (1990) using the methods of no. 934.01, 920.39 and 924.05 respectively. The N of feed, faeces and urine was analysed using the Kjeldahl method (AOAC, 1990). The NDF and ADF were determined on an ANKOM200 Fiber Analyzer (ANKOM Technology Corp., Macedon, NY, USA) using the method of Van Soest et al. (1991). The OM was calculated as the difference between DM and ash. The CH4 of gas samples was analysed using gas chromatography (TP-2060T, Beijing Beifen Tianpu Instrument Technology Co. Ltd, Beijing, China). The conditions for the analysis were as follows: TCD detector, TDX-01 column, size 1 m 9 2 mm 9 3 mm, column temperature 70 °C and detector temperature 100 °C. The carrying gas was argon with flowing rate of 30 ml/min. The volatile fatty acids (VFA) of rumen fluid samples were analysed using gas chromatography (TP-2060F, Beijing Beifen Tianpu Instrument Technology Co. Ltd). One millilitre of rumen fluid was mixed with 0.25 ml metaphosphoric acid (concentration 25 g/100 ml, w/v) and centrifuged at 15 000 g for 15 min at 4 °C, and the supernatant was used for analysis. The conditions for analysis were as follows: FID detector, PEG-20M + H3PO4 glass column, column temperature 120 °C and detector temperature 220 °C. The carrying gas was argon with flowing rate of 30 ml/min and the flowing rates of H2 and air were 30 ml/min and 300 ml/min, respectively. The ammonia N (NH3-N) of rumen fluid was determined using the steam distillation method described by Bremner and Keeney (1965). The urinary allantoin and uric acid were determined on a spectrophotometer (K5500, Beijing Kaiao Technology Development Co. Ltd., Beijing, China) using the method described by Young and Conway (1942) and Fujihara et al. (1987), respectively.
Effects of Ce on digestion of beef cattle
The plasma total protein (TP), albumin (ALB), glucose (GLU), triglyceride (TG), plasma urea nitrogen (PUN), cholesterol (CHO), aspartate aminotransferase (AST), alanine aminotransferase (ALT) were analysed on an automatic biochemical analyser (7160; Hitachi Ltd., Tokyo, Japan) using commercial colorimetric kits (BioSino Bio-technology and Science Co. Ltd, Beijing, China). The plasma malondialdehyde (MDA), glutathione peroxidase (GSH-PX), superoxide dismutase (SOD), total antioxidant capability (T-AOC) and catalase (CAT) were analysed on an automatic biochemistry analyser (7160; Hitachi Ltd.) using kits HY-50116, HY-60005, HY-60001, HY-60021 and HY-50121 respectively (SinoUK Institute of Biological Technology, Beijing, China). The plasma human growth hormone (hGH), insulin (INS) and insulin-like growth factor-1 (IGF-1) were determined on an automatic radioimmunoassay counter (r-911; University of Science and Technology of China Industrial Co. Ltd, Hefei, China) using competitive radioimmunoassay (RIA) kits HY-10035, HY-10069 and HY-082, respectively (Sino-UK Institute of Biological Technology). Calculations
N retention (g/day) ¼ N intake (g/day) Faecal N (g/day) Urinary N (g/day) N retention rateð%Þ ¼ N retention (g/day)/N intake (g/day) 100 Nutrient digestibilityð%Þ ¼ [Nutrient intake (g/day) Nutrient in faeces (g/day)]=Nutrient intake (g/day) 100 CH4 production (l) ¼ CH4 concentrationð%Þ gas volume (l) CH4 productionðl=dayÞ ¼½CH4 productionðl; 06 : 00 hÞ þ CH4 productionðl; 12 : 00 hÞ þ CH4 productionðl; 20 : 00 hÞ þ CH4 productionðl; 24 : 00 hÞ 36 where it was assumed that the composition of gas samples collected for 10 min at four periods during a day represented the composition of the gas exhaled by cattle during a whole day. Total urinary purine derivatives (PD) (mmol/day) = Allantoin (mmol/day) + Uric acid (mmol/day) The microbial N was calculated based on the amount of microbial purines absorbed using the equations of Chen and Gomes (1992):
Journal of Animal Physiology and Animal Nutrition 99 (2015) 1047–1055 © 2015 Blackwell Verlag GmbH
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Y ¼ 0:85X þ 0:385W0:75 where X refers to PD absorbed, mmol/day; Y, total urinary PD excreted, mmol/day; W0.75, metabolic body weight of cattle at the end of each experimental period, kg.
Microbial N (g/day) ¼
Xðmmol/dayÞ 70 0:116 0:83 1000
where 70 refers to N content of purines (70 mg N/ mmol); 0.116, ratio of purine N:total N in mixed rumen microbes; 0.83, estimated digestibility of microbial purines. Statistical analysis
Statistical analysis was conducted using the GLM procedures of SAS 9.0 (2002) using the model: Yijk = l + Ti + Pj + Ck + eijk, where Yijk refers to observation; l, overall mean; Ti, treatment (i = 1, 2, 3 and 4); Pj, period (j = 1, 2, 3 and 4); Ck, cattle (k = 1, 2, 3 and 4); and eijk, residual error. Differences between treatments were considered to be significant at p < 0.05 and a tendency to be significant at 0.05 < p < 0.10. Comparison of means was carried out using Duncan’s multiple range tests. The data between different treatments were subjected to the orthogonal polynomial contrasts to determine linear and quadratic effects of increasing level of CeCl3. Results The results of nutrient digestibility are shown in Table 2. No significant difference was found between treatments in digestibility of DM, OM or EE (p > 0.05). Supplementing CeCl3 at 160 or 240 mg/kg
DM increased NDF digestibility (p < 0.05) and tended to increase ADF digestibility (p = 0.083). The digestibility of CP, NDF and ADF increased in a linear manner (p < 0.05), and EE digestibility increased in a linear trend (p = 0.076) with increasing level of CeCl3. The results of rumen fermentation parameters are shown in Table 3. The rumen pH of all treatments ranged from 6.68 to 6.73. Supplementing CeCl3 up to 240 mg/kg DM did not affect rumen pH, total VFA, butyrate and NH3-N (p > 0.05) whereas decreased the molar proportion ratio of acetate to propionate (p < 0.01). Supplementing CeCl3 at 160 or 240 mg/kg DM decreased CH4 production DM intake (DMI) basis (p < 0.05). The CH4 production decreased in a linear manner with increasing level of CeCl3 (p < 0.05). The results of N metabolism are shown in Table 4. Supplementing CeCl3 160 or 240 mg/kg DM decreased urinary N (p < 0.05) and total N excretion (p < 0.05), increased N retention and N retention rate (p < 0.05). Supplementing CeCl3 tended to decrease faecal N excretion in a linear manner (p < 0.10). The results of urinary PD and microbial N flow are shown in Table 5. Supplementing CeCl3 at 160 or 240 mg/kg DM increased total urinary PD (p < 0.05) and estimated microbial N flow based on urinary PD (p < 0.05) and tended to increase allantoin (p = 0.058). The allantoin, uric acid, total PD and microbial N flow increased linearly with increasing level of CeCl3. The results of plasma parameters are shown in Table 6. Supplementing CeCl3 increased plasma concentration of ALB (p < 0.05), whereas did not affect plasma concentration of TP, GLU, TG, CHO, PUN, AST, ALT, MDA, GSH-PX, SOD, T-AOC, CAT, hGH, INS or IGF-1.
Table 2 Effect of supplementing CeCl3 on nutrient digestibility of beef cattle CeCl3 supplemented, mg/kg DM
p-Value
Nutrient digestibility, %
0
80
160
240
SEM
Treatment
Linear
Quadratic
DM OM CP EE NDF ADF
70.08 71.68 72.81 81.52 54.40b 52.72
70.03 71.69 72.86 82.27 54.22b 55.37
70.77 72.46 73.82 84.73 58.47a 58.25
71.82 73.23 75.02 85.01 58.10a 58.86
0.66 0.61 0.92 1.53 1.00 1.48
0.283 0.311 0.367 0.354 0.039 0.083
0.145 0.189 0.038 0.076 0.043 0.007
0.298 0.401 0.094 0.218 0.139 0.023
SEM, standard error of mean; DM, dry matter; OM, organic matter; CP, crude protein; EE, Ether extract; NDF, Neutral detergent fibre; ADF, acid detergent fibre. Means within the same row marked with different superscripts differ significantly (p < 0.05).
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Table 3 Effect of supplementing CeCl3 on rumen fermentation of beef cattle CeCl3 supplemented, mg/kg DM
p-Value
Items
0
80
160
240
SEM
Treatment
Linear
Quadratic
pH Total VFA, mM Molar, % Acetate Propionate Butyrate Acetate/propionate NH3-N, mg/100 ml CH4 production, l/day CH4 production, l/kg DMI
6.68 82.84
6.68 83.41
6.71 84.37
6.73 85.03
0.06 1.39
0.911 0.701
0.623 0.679
0.884 0.886
40.81a 39.25c 13.52 1.04a 18.18 34.60a 6.90a
38.61ab 41.02bc 13.68 0.94b 18.74 31.90ab 6.35ab
37.24b 41.33b 13.70 0.90b 19.55 28.36b 5.64b
36.82b 43.91a 13.72 0.84c 19.91 25.84b 5.13b
0.70 0.55 0.25 0.02 0.81 1.72 0.35
0.025 0.006 0.937 0.001 0.486 0.044 0.047
0.003 0.004 0.753 0.001 0.506 0.013 0.011
0.008 0.017 0.942 0.004 0.808 0.052 0.045
SEM, standard error of mean; NH3-N, ammonia nitrogen; VFA, total volatile fatty acids; CH4, methane; DMI, dry matter intake. Means within the same row marked with different superscripts differ significantly (p < 0.05).
Table 4 Effects of supplementing CeCl3 on N metabolism of beef cattle CeCl3 supplemented, mg/kg DM
p-Value
Items
0
80
160
240
SEM
Treatment
Linear
Quadratic
N intake, g/day Urinary N, g/day Faecal N, g/day Total N excretion, g/day N retention, g/day N retention rate, %
109.40 51.31a 29.76 81.07a 28.37c 26.02c
109.40 49.77ab 29.74 79.51ab 29.92bc 27.41bc
109.40 47.57bc 28.66 76.23bc 33.20ab 30.39ab
109.40 46.75c 27.31 74.06c 35.37a 32.35a
– 0.78 1.01 1.28 1.28 1.13
– 0.021 0.356 0.030 0.030 0.027
– 0.092 0.082 0.060
