Scholarly article on topic 'Impacts of the unsaturation degree of long-chain fatty acids on the volatile fatty acid profiles of rumen microbial fermentation in goats in vitro'

Impacts of the unsaturation degree of long-chain fatty acids on the volatile fatty acid profiles of rumen microbial fermentation in goats in vitro Academic research paper on "Animal and dairy science"

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Abstract of research paper on Animal and dairy science, author of scientific article — Jian GAO, Meng-zhi WANG, Yu-jia JING, Xue-zhao SUN, Tian-yi WU, et al.

Abstract This study investigated the impacts of the degree of unsaturation (unsaturity) of long-chain fatty acids on volatile fatty acid (VFA) profiles of rumen fermentation in vitro. Six types of long-chain fatty acids, including stearic acid (C18:0, control group), oleic acid (C18:1, n-9), linoleic acid (C18:2, n-6), α-linolenic acid (C18:3, n-3), arachidonic acid (C20:4, n-6) and eicosapentaenoic acid (C20:5, n-3), were tested. Rumen fluid from three goats fitted with ruminal fistulae was used as inoculum and the inclusion rate of long-chain fatty acid was at 3% (w/w) of substrate. Samples were taken for VFA analysis at 0, 3, 6, 9, 12, 18 and 24 h of incubation, respectively. The analysis showed that there were significant differences in the total VFA among treatments, sampling time points, and treatment×time point interactions (P<0.01). α-Linolenic acid had the highest total VFA (P<0.01) among different long-chain fatty acids tested. The molar proportion of acetate in total VFA significantly differed among treatments (P<0.01) and sampling time points (P<0.01), but not treatment×time point interactions (P>0.05). In contrast, the molar proportion of propionate did not differ among treatments during the whole incubation (P>0.05). However, for butyrate molar proportions, significant differences were found not only among sampling time points but also among treatments and treatment×time point interactions (P<0.01), with eicosapentaenoic acid having the highest value (P<0.01). Additionally, no statistically significant differences were found in the acetate to propionate ratios among treatments groups (P>0.05), even the treatments stearic acid and α-linolenic acid were numerically higher than the others. The inclusion of 3% long-chain unsaturated fatty acids differing in the degree of unsaturation brought out a significant quadratic regression relation between the total VFA concentration and the double bond number of fatty acid. In conclusion, the α-linolenic acid with 3 double bonds appeared better for improving rumen microbial fermentation and the total VFA concentration.

Academic research paper on topic "Impacts of the unsaturation degree of long-chain fatty acids on the volatile fatty acid profiles of rumen microbial fermentation in goats in vitro"

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Impacts of the unsaturation degree of long-chain fatty acids on the volatile fatty acid profiles of rumen microbial fermentation in goats in vitro ^

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GAO Jian1*, WANG Meng-zhi1*, JING Yu-jia1, SUN Xue-zhao2, WU Tian-yi1, SHI Liang-feng1

1 College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, P.R.China

2 Grasslands Research Centre, AgResearch Ltd., Private Bag 11008, Palmerston North, New Zealand

Abstract

This study investigated the impacts of the degree of unsaturation (unsaturity) of long-chain fatty acids on volatile fatty acid (VFA) profiles of rumen fermentation in vitro. Six types of long-chain fatty acids, including stearic acid (C18:0, control group), oleic acid (C18:1, n-9), linoleic acid (C18:2, n-6), a-linolenic acid (C18:3, n-3), arachidonic acid (C20:4, n-6) and eicosapen-taenoic acid (C20:5, n-3), were tested. Rumen fluid from three goats fitted with ruminal fistulae was used as inoculum and the inclusion rate of long-chain fatty acid was at 3% (w/w) of substrate. Samples were taken for VFA analysis at 0, 3, 6, 9, 12, 18 and 24 h of incubation, respectively. The analysis showed that there were significant differences in the total VFA among treatments, sampling time points, and treatment*time point interactions (P<0.01). a-Linolenic acid had the highest total VFA (P<0.01) among different long-chain fatty acids tested. The molar proportion of acetate in total VFA significantly differed among treatments (P<0.01) and sampling time points (P<0.01), but not treatment*time point interactions (P>0.05). In contrast, the molar proportion of propionate did not differ among treatments during the whole incubation (P>0.05). However, for butyrate molar proportions, significant differences were found not only among sampling time points but also among treatments and treatment*time point interactions (P<0.01), with eicosapentaenoic acid having the highest value (P<0.01). Additionally, no statistically significant differences were found in the acetate to propionate ratios among treatments groups (P>0.05), even the treatments stearic acid and a-linolenic acid were numerically higher than the others. The inclusion of 3% long-chain unsaturated fatty acids differing in the degree of unsaturation brought out a significant quadratic regression relation between the total VFA concentration and the double bond number of fatty acid. In conclusion, the a-linolenic acid with 3 double bonds appeared better for improving rumen microbial fermentation and the total VFA concentration.

Keywords: volatile fatty acid, unsaturation degree, long-chain fatty acid, in vitro fermentation

Received 2 December, 2015 Accepted 16 May, 2016 GAO Jian, E-mail: gaojianyzu@126.com; Correspondence WANG Meng-zhi, Tel/Fax: +86-514-87997196, E-mail: mengzhiwangyz @126.com

* These authors contributed equally to this study.

© 2016, CAAS. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/) doi: 10.1016/S2095-3119(16)61418-1

1. Introduction

Volatile fatty acids (VFA) produced in the rumen have a great influence on ruminant metabolism as they provide 70-80% of digestible energy required by the host animal (Van 1996; Feng 2004; Bannink et al. 2008). Different types of VFA have different impacts on the physiological function of ruminants. The VFA can also help maintain an

ideal environment in the rumen, which is beneficial to rumen microbial growth (Bensadoun et al. 1962).

Rumen microorganisms could rapidly degrade fats in the diet to glycerine and free fatty acids, while free fatty acids with a free carboxyl, whether saturated or not, have some toxic actions on rumen microorganisms (Jenkins 1993). The unsaturated fatty acids in the rumen can inhibit the growth of protozoa (Dijkstra et al. 2000), some types of bacterial genera (Zhao et al. 2005; Liu et al. 2011) and the engulfment activity of protozoa on bacteria (Wang et al. 2010). The rumen microbes can only adapt to low levels of fats while high levels of fats in the rumen may affect their normal metabolism and growth, particularly for ruminal protozoa lacking cell walls. Therefore, we could manage ruminal protozoa family through adding oils or fatty acids and consequently manipulate rumen flora and their fermentation patterns (McGinn et al. 2004).

Previous studies suggest that unsaturated fatty acids in oils or fats may modify ruminal microbes and ecosystem, and rumen fermentation as well. Some kinds of plant oil richly containing unsaturated fatty acid are able to change rumen fermentation pattern by enhancing the molar percentage of propionate (Machmuller et al. 1998; Jalc and Ceresnakova 2002). Zhang et al. (2008) have even demonstrated that the acetate to propionate ratio decreased significantly by adding octadeca carbon fatty acids in vitro. Potu et al. (2011) have shown that the supplements of lipid with different long-chain fatty acid compositions have some discrepancies when acetic acid and propionic acid were produced in the rumen fermentation process. Acetate, butyrate and total VFA decrease with the increasing degree of unsaturation of C18 fatty acids (oleic acid, linoleic and linolenic acids) in vitro (Li et al. 2012). These reports suggest that the effects of long-chain fatty acids on rumen fermentation have differences and may be related to the unsaturation of long-chain fatty acids.

Our previous study found that the inclusion of 3% of long-chain unsaturated fatty acids increased bacterial protein content, whereas reduced protozoal protein content and enhanced dehydrogenase activity of rumen microorganism (Gao et al. 2016), which may result in the change of the pattern of rumen fermentation. Thus, this study was to examine the effects of six types of long-chain fatty acids varying in the degrees of unsaturation on the VFA profiles and their concentrations of rumen fermentation in vitro.

mental Farm of Yangzhou University, China were used to provide rumen liquor as culture inoculum. These animals were fed at 07:00 and 19:00 in equal amounts of a diet containing 28% corn grains, 2% soybean and 70% Leymus chinensis hay. The daily feed allowance on the basis of dry matter was 2.5% of animal live weight. The animals had free access to clean drinking water at all the times. The use of animals and the experimental procedures were approved by the Animal Care and Use Committee of Yangzhou University, Jiangsu Province, China.

2.2. Experimental design, procedures and sampling

This work was part of a larger study. The experimental design and the in vitro substrates were the same as the work described by Gao et al. (2016). In brief, six kinds of long-chain fatty acids with different numbers of double bonds, including stearic acid (C18:0, control group), oleic acid (C18:1, n-9), linoleic acid (C18:2, n-6), a-linolenic acid (C18:3, n-3), arachidonic acid (C20:4, n-6), and eicosapen-taenoic acid (C20:5, n-3), were added at 3% of substrate weight for in vitro incubation. Since stearic acid did not affect ruminal fermentation or slightly increased rumen microbes in vitro (Chalupa et al. 1984; Zhang et al. 2008), stearic acid containing nil double bond was used as a control in this experiment. Incubations were run in triplicate, and a set of appropriate blank (without substrates) was included.

Around 300 mL of rumen fluid per goat were obtained using a vacuum pump through the rumen fistula and mixed as inoculum before morning feeding. The rumen fluid was filtered through four layers of gauze into an aseptic saline bottle which was aerated with CO2 and preheated at 39°C. Artificial saliva salt was made according to Menke and Steingass (1988) before the in vitro culture. The in vitro culture medium was prepared by mixing artificial saliva and rumen fluid in a ratio of 2:1 (v/v). The artificial saliva was used to neutralize fatty acids produced during fermentation in order to maintain pH within a normal range. A glass culture bottle used for incubation was added with 1.50 g of the substrate and 150 mL of the culture medium, flushed with CO2 and then sealed. The bottles were placed in a water bath (39°C) and constantly shaken at 50 r min-1 for 24 h.

A sample of the fermentation fluid (2 mL each) was taken from each bottle at 0, 3, 6, 9, 12, 18 and 24 h of incubation and stored at -20°C for VFA analysis.

2. Materials and methods

2.1. Animals and management

Three fistulated Xuhuai White goats with similar age (1.5 years old) and live weight, (29.4±2.7) kg, from the Experi-

2.3. Laboratory analysis

The concentrations of VFAs in fermentation fluid were determined using a GC-14B gas chromatograph (Shimadzu Corp., Kyoto) using the method as described by Xiong et al. (1999). The testing condition was listed as follow, capillary

GAO Jian et al. / Journal of Integrative Agriculture 2016, 15(12): 2827-2833

column CP-WAX (30 m long, 0.53 mm inner diameter, 1 pm film thickness), 200°C gasification chamber temperature, 200°C flame ionization detector temperature. The temperature of the column was controlled using temperature-programmed methods. The initial temperature was 100°C, the final temperature was 150°C, the warming rate was 3°C min-1, the sensitivity was 101, the attenuation was 25 and crotonic acid was used as the inner standard. The fermentation fluid was centrifuged at 15 000*g for 10 min and then 1 mL supernatant was added to 0.2 mL metaphosphoric acid (20%, w/w) containing 60 mmol L-1 crotonic acid. After vigorous shaking, the mixture was centrifuged and 0.4 pL supernatant gathered for gas chromatography (GC) analysis.

2.4. Statistical analysis

Analysis of variance (ANOVA) for repeated measures was used to examine the effects of treatment (i.e., long-chain fatty acids), sampling time (i.e., hours of the incubation) and the potential interaction between treatment and sampling time. The procedure of Univariate of General Linear Model in SPSS (ver. 22) was used to analyze the data with treatment, sampling time and their interaction having fixed effects and incubation run having random effects. The differences among the long-chain fatty acid treatments were examined using the Duncan test for multiple comparison across all the six sampling times. Statistical significance was declared at P<0.05.

3. Results

3.1. Effects on concentration of total VFA

The mean concentrations of total VFA increased with incubation time from 39 (0 h) to 119 mmol L-1 (24 h) for all treatments, with a-linolenic acid fluctuating at a high level (Table 1). Furthermore, significant differences were observed among treatments, sampling time points (P<0.01), and treatment*time point as well (P<0.05). The a-linolenic acid was the highest among all treatments (P<0.01).

The total VFA concentration increased with the unsaturation degree when the double bond increases from 0 to 3, and reached its peak (91 mmol L-1) with a-lin-olenic acid containing 3 double bonds; however, decreased after, and fell to 83 mmol L-1 with eicosapentaenoic acid containing 5 double bonds. The regression analysis between the mean total VFA concentration of 6 sampling times (mmol L-1) and the double bond number of long-chain fatty acid were further performed and the results are shown in Fig. 1, indicating that there was a significant quadratic regression relation between total VFA concentration and double bond number and the quadratic equation is y=6.726x-1.172x2+78.033 (R2=0.566, P=0.002). In the equation, y represents the mean total VFA concentrations of 6 sampling times (mmol L-1); x represents the double bond number of long-chain fatty acid.

3.2. Effects on the molar proportions of VFAs in total VFA

The molar proportion of acetate in total VFA generally presented a downward trend, and changed in a range between 63 and 70 mol per 100 mol (Table 2). Moreover, the molar proportion of acetate in total VFA had significant differences among treatments (P<0.05), and also among time points (P<0.01), but not treat-ment*time point. In contrast, the proportion of propionate in total VFA was low at 0 h and then increased rapidly within 3 h of incubation. However, there were

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Fig. 1 The regression analysis and curve fitting between the concentration of total volatile fatty acid and the double-bond number of fatty acid. y=6.726x-1.172x2+78.033 (R2=0.566, P=0.002). In the equation, y represents the mean total volatile fatty acid (VFA) concentration of 6 sampling times (mmol L-1); x represents the double-bond number of long-chain fatty acid.

little changes, fluctuating between 21 and 26 mol per 100 mol, from 3 to 24 h of incubation. No significant differences in propionate were found among treatments. However, the molar proportion of butyrate in total VFA varied greatly with the values ranging from 8 to 13 mol per 100 mol, and was significantly affected not only by sampling time but also by treatment and treatment*time interaction (P<0.01). The eicosapentaenoic acid treatment was recorded the highest value (P<0.01).

3.3. Effects on acetate/propionate

The ratio of acetate to propionate ranged from 2.48 to 3.34 (Table 3). The mean value of the ratio was the highest at 0 h (3.09) among all time points, however, rapidly dropped to 2.65 at 3 h and fluctuated between 2.66 and 2.76 thereafter. Significant differences were detected among time points (P<0.01), whereas no significant differences were found among treatments, although the treatments stearic acid and a-linolenic acid were numerically higher than the other treatments.

4. Discussion

4.1. Effects on the concentration of total VFA

In this study, the concentrations of total VFA in treatment groups were higher than the control. Furthermore, the a-lin-olenic acid treatment remained a higher level of total VFA

at all the time points (the mean at 91 mmol L-1) than other treatment, indicating that a-linolenic acid induced better effects on promoting rumen fermentation. The better result might result from the unsaturated bonds of free fatty acids inhibiting the growth of rumen protozoa (Jalc et al. 2009), especially for the rumen ciliate protozoa (Girard and Hawke 1978; Newbold and Chamberlain 1988; Williams 1989). As a result, the engulfing activity of rumen protozoa on bacteria would decrease, and the total microbial biomass (Wang et al. 2010) increases and promotes rumen fermentation. In our previous study, we found that adding 3% a-linolenic acid can increase the concentration of rumen bacterial protein representing the biomass of rumen bacteria and the protein of total microorganism was higher than other long-chain fatty acids (Gao et al. 2016). This may result in the highest concentration of total VFA in the present study.

Regarding the differences of the effects on fermentation among treatments, a possible reason might be attributed to the degree of unsaturation of the free fatty acids. Li et al. (2009) reported that the propionate increased and other VFAs decreased with the increase of unsaturation degree when malic acid or unsaturated fatty acid was added in vitro. In the present study, 3% of oleic acid, linoleic acid, a-linolenic acid, arachidonic acid and eicosapentaenoic acid were equal to 0.212, 0.214, 0.216, 0.198, 0.198 mmol of the corresponding fatty acids in their individual culture systems. The degrees of unsaturation also increased accordingly, i.e., 1, 2, 3, 4, and 5 unsaturated bonds. It is noteworthy, however, that the concentration of total VFA with the moderate dose of a-linolenic acid was higher than that of the other long-chain fatty acids with a higher degree of unsaturation such as arachidonic acid or eicosapentaenoic acid. The regression analysis results subsequently confirmed that a quadratic relationship existed between the total VFA concentration and the double bond number of fatty acid.

In conclusion, the results of the this study indicated that a proper level of unsaturation of long-chain fatty acids can improve the microbial fermentation through regulating microorganisms, while the negative effects on microorganisms of arachidonic acid or eicosapentaenoic acid which have more unsaturated bonds might be greater than the positive effects which unsaturated fatty acids inhibited the protozoa to induce any positive effects on bacterial biomass. The results above demonstrated that the 3% of arachidonic acid or eicosapentaenoic acid in culture had greater negative effects on microorganisms. The optimal levels of these long-chain fatty acids for a positive microbial biomass effect need to be further studied. When adding hard fat to an in vitro rumen fermenter, no differences on total VFA production were observed (Ferguson et al. 1990). Our results are consistent with this finding.

Table 2 Effects of long-chain fatty acids on the molar proportions of acetate, propionate and butyrate in the total VFA

Molar proportions of Incubation individual VFAs in total VFA (mol per 100 mol)

Treatment

P-value

time (h) Stearic acid (C18:0) Oleic acid (C18:1, /7-9) Linoleic acid (C18:2, /7-6) Linolenic acid (C18:3, /7-3) Arachidonic acid (C20:4, /7-6) Eicosapentaenoic acid (C20:5, /7-3) Mean

0 69.7 68.6 68.8 68.4 69.4 68.9 69.0 a

3 66.5 65.7 66.5 67.7 66.6 66.3 66.6 b

6 67.1 65.8 65.9 67.1 66.2 66.8 66.5 b

9 65.3 64.2 66.3 67.4 65.1 64.5 65.5 c

12 64.9 64.0 65.7 66.6 65.3 64.7 65.2 c

18 65.3 65.8 65.7 66.3 65.1 62.9 65.2 c

24 67.8 66.1 66.1 65.6 65.3 64.4 65.9 be

Mean 66.7 ab 65.8 cd 66.4 abc 67.0 a 66.1 bed 65.5 d

0 20.9 21.9 21.9 22.5 21.3 21.9 21.7 d

3 24.7 25.8 24.9 24.7 25.4 25.5 25.2 a

6 23.3 25.3 25.4 24.4 25.0 24.5 24.6 ab

9 24.1 24.3 24.8 23.9 25.0 25.3 24.6 ab

12 24.5 25.8 24.7 23.9 24.9 24.4 24.7 ab

18 23.0 23.7 23.3 23.2 24.1 24.1 23.6 c

24 24.0 23.9 23.3 23.2 23.3 24.3 23.7 c

Mean 23.5 24.4 24.0 23.7 24.1 24.3

0 9.4 9.5 9.4 9.0 9.3 9.3 9.3 be

3 8.8 8.5 8.6 7.6 8.1 8.2 8.3 d

6 9.6 8.9 8.7 8.5 8.9 8.7 8.9 cd

9 10.6 11.4 9.0 8.7 9.9 10.2 10.0 be

12 10.6 10.2 9.7 9.5 9.9 11.0 10.1 b

18 11.7 10.5 11.0 10.4 10.9 13.1 11.3a

24 8.2 10.1 10.6 11.2 11.4 11.3 10.5 b

Mean 9.8 ab 9.9 ab 9.6 b 9.3 b 9.8 ab 10.3 a

SEM Treatment Incubation

time (I)

Acetate

Propionate

Butyrate

<0.001 0.508

<0.001 0.991

<0.001 <0.001

Table 3 Effects of long-chain fatty acids on the acetate/propionate ratio

Treatment P-value

M lUUUCILIUI 1 time (h) Stearic acid Oleic acid Linoleic acid Linolenic acid Arachidonic acid Eicosapentaenoic acid Mean SEM Treatment Incubation T*l

(C18:0) (C18:1, /7-9) (C18:2, /7-6) (C18:3, /7-3) (C20:4, /7-6) (C20:5, /7-3) (T) time (I)

0 3.34 3.13 3.14 3.12 3.29 3.15 3.09 a 0.064 0.103 <0.001 0.999

3 2.69 2.56 2.70 2.75 2.64 2.61 2.65 b

6 2.87 2.61 2.60 2.75 2.66 2.73 2.67 b

9 2.71 2.64 2.68 2.83 2.62 2.55 2.66 b

12 2.65 2.48 2.66 2.79 2.63 2.66 2.65 b

18 2.84 2.78 2.82 2.85 2.71 2.62 2.74 b

24 2.84 2.77 2.83 2.84 2.80 2.66 2.76 b

Mean 2.85 2.71 2.78 2.85 2.76 2.71

4.2. Effects on the molar proportion of VFAs in total VFA

The molar proportion of acetate slightly changed with the degree of unsaturation of the long-chain fatty acids, with a-linolenic acid being the highest, while the molar proportion of propionate did not change significantly. These results for linoleic acid and linolenic acid differ from the report by Ivan et al. (2013) who compared the effects of individual C-18:2 and C-18:3 fatty acids on rumen fermentation of cows. In our study the molar proportion of propionate in total VFA seemed no significant differences. This may be because the in vitro experiment differs from the in vivo test. Additionally in the present study, the molar proportion of butyrate in total VFA in the a-linolenic acid treatment was lower than those of the other treatments. Our results suggest that the 3% level of a-linolenic acid had an inhibitory effect to some extent on butyrate-producing microbe in the rumen. It agrees with the results mentioned above, i.e., the final product of protozoa metabolism is acetate and butyrate and adding a-linoleic acid might have had greater inhibitory effects on protozoa.

Combined with the higher concentration of total VFA and the lower butyrate proportion, it is indicated that 3% of a-lin-olenic acid had a stronger impact on rumen fermentation. The possible reason might be that 3% of a-linolenic acid inhibits the growth and predation activity of protozoa, and promotes the growth of bacteria, and consequently results in the increase of rumen microbial biomass. This agrees with another report from our research team about rumen microbial biomass, which showed that microbial concentration was highest with a-linolenic acid supplementation (Gao et al. 2016). The exact mechanism for the effects is still unclear.

4.3. Effects on acetate to propionate ratio

Other studies have shown that the main final products of protozoa fermentation are acetate and butyrate (Williams and Coleman 1992). The findings in current study that the acetate to propionate ratio sharply decreased from 0 (3.09) to 3 h (2.65) might be partly due to the protozoa inhibition (Gao et al. 2016) by long-chain fatty acids no matter unsaturated or not (Firkins et al. 2007; Faciola and Broderick 2014). The unsaturated long-chain fatty acids can inhibit rumen protozoa growth and decrease protozoal quantity (Fujihara et al. 1996; Tackett et al. 1996), which may result in the decline of the production of acetate and the ratio of acetate to propionate observed in our study. However, the a-linolenic acid treatment was similar to stearic acid (the control), and just higher than other long-chain fatty acids in the numeric value in this study. The results indicated that a-linolenic acid at 3% level might increase the total volatile fatty acid concentration (the results mentioned above) but

had no obvious effect on acetate to propionate ratio. Chalupa et al. (1986) reported adding 10% long-chain fatty acids to the diet decreased the acetate to propionate ratio by more than 20%, which differs with our results. This may be because 10% fatty acids of the diet are excessive.

5. Conclusion

In vitro supplementation with 3% of different types of long-chain fatty acids had different regulatory effects on the total concentration and composition of volatile fatty acids. The effects of a-linolenic acid appeared better for improving rumen microbial fermentation and the concentration of total volatile fatty acids. For the first time, we reported that there was a significant quadratic relation between the total VFA concentration and the double bond number of long-chain fatty acid. However, more research is needed to elucidate the exact mechanisms of the different long-chain fatty acids on rumen fermentation, such as manipulating rumen microbial community.

Acknowledgements

The study was financially supported by the Graduate Student Innovation Project of Jiangsu Province, China (KYLX15_1377), the Natural Science Foundation of Jiangsu Province, China (BK20151312), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), China.

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(Managing editor ZHANG Juan)