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The effects of the unsaturated degree of long-chain fatty acids on the rumen microbial protein content and the activities of transaminases and dehydrogenase in vitro
Journal of Integrative Agriculture 2016, 15(2): 424–431 Available online at www.sciencedirect.com
ScienceDirect
RESEARCH ARTICLE
The effects of the unsaturated degree of long-chain fatty acids on the rumen microbial protein content and the activities of transaminases and dehydrogenase in vitro GAO Jian1*, JING Yu-jia1*, WANG Meng-zhi1, SHI Liang-feng1, LIU Shi-min2 1 2
College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, P.R.China Institute of Agriculture, The University of Western Australia, Crawley WA 6009, Australia
Abstract This study investigated the effects of the degree of unsaturation (unsaturity) of long-chain fatty acids on microbial protein content and the activities of transaminases and dehydrogenase in vitro using goat rumen fluid as the cultural medium. Six types of fatty acids, stearic acid (C18:0, group A, control group), oleic acid (C18:1, n-9, group B), linoleic acid (C18:2, n-6, group C), α-linolenic acid (C18:3, n-3, group D), arachidonic acid (C20:4, n-6, group E), and eicosapentaenoic acid (C20:5, n-3, group F), were tested, and the inclusion ratio of each fatty acid was 3% (w/w) in total of culture substrate. Samples were taken at 0, 3, 6, 9, 12, 18 and 24 h, respectively, during culture for analyses. Compared with stearic acid, linoleic acid, α-linolenic acid, and arachidonic acid increased the bacterial protein content, while oleic acid and eicosapentaenoic acid had no significant effects; the protozoal protein content was reduced for all the unsaturated fatty acids, and the magnitude of the reduction appeared to be associated with the degree of unsaturity of fatty acids. The total microbial protein content was dominantly accounted by the protozoal protein content (about 4–9 folds of the bacterial protein), and only increased by linoleic acid, but reduced by oleic acid, arachidonic acid and eicosapentaenoic acid. There were no significant effects in the activities of both glutamic oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) for all the other fatty acids, except for arachidonic acid which enhanced GOT activity and oleic acid which enhanced GPT activity. The total dehydrogenase activity was positively correlated with the degree of unsaturation of fatty acids. In conclusion, the inclusion of 3% of long-chain unsaturated fatty acids increased bacterial protein content, whereas reduced protozoal protein content and enhanced dehydrogenase activity. The fatty acids with more than three double bonds had detrimental effects on the microbial protein content. This work demonstrates for the first time the effect rule of the unsaturation degree of long-chain fatty acids on the rumen microbial protein in vitro. Keywords: long-chain fatty acid, rumen microbes, goats, protein concentration
Received 26 December, 2014 Accepted 18 May, 2015 GAO Jian, E-mail: [email protected]; JInG Yu-jia, E-mail: [email protected]; Correspondence WAnG Meng-zhi, E-mail: [email protected] * These authors contributed equally to this study. © 2016, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S2095-3119(15)61081-4
1. Introduction Oil and fat are composed of various fatty acids (FA), contain high levels of energy, and can be used as dietary supplements to ruminant animals when the energy demand is particularly high, for example, at the peak period of lactation, or highly ambient temperature associated with reduction of
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dietary intake. However, the magnitude of unsaturation of fatty acids is unfavour for microorganisms in the rumen, and detrimental effects differ among species and genus of the ruminal microorganisms, in turn the ruminal fermentation patterns are altered (nangla and Rila 1994; Dijkstra et al. 2000; Mcginn et al. 2004; Jalč et al. 2009). Fatty acids can be used to stabilize rumen fermentation, enhance the yield of microbial proteins (MCP) which accounts for 40–60% of proteins into the small intestines of ruminants (Welch and Hooper 1993) and increase the utilization efficiency of energy and nitrogen of host animals (Clark et al. 1992; Bach and Stern 1999). Rapeseed oil, sunflower oil and linseed oil rich in octadeca carbon fatty acids (C18 fatty acids) have been shown different effects on the rumen microbial ecological system including fermentation patterns, bacterial and protozoal populations, and microbial protein production from previous researches (Machmüller et al. 1998; Jalč and Čerešňáková 2002). Cieslak et al. (2009) also demonstrated that there were major impacts of linoleic acid (LA) supplement in the bacterial fractions of both ciliate cultures including Entodinium caudatum (EC) and Diploplastron affine (DA). These researches suggest that addition of fat or fatty acids can change rumen fermentation patterns. Our previous research showed that the addition of 4% oil, such as rapeseed oil, soybean oil, corn oil and peanut oil with different degrees of unsaturation (unsaturity) inhibited the predation activity of protozoa on bacteria and increased the bacterial biomass and the yield of MCP (Wang et al. 2010). Potu (2011) reported that supplementations with different lipids which containing different fatty acid compositions had some discrepancies on productions of acetic acid and propionic acid in the rumen. Zhang et al. (2008) proposed that unsaturated C18-fatty acids changed the rumen fermentation patterns and microbial population profiles. The other research from Zhang et al. (2008) reported that adding mixtures of linoleic acid and linolenic acid significantly reduced the total gas production and methan production at 24 h fermentation, while the treatment had no significant influence on the microbial biomass. All these reports suggested that the effects of fatty acids on rumen fermentation had certain differences and may be related to both the unsaturity of FAs as well as the length of carbon chain. Thus, this study was conducted to examine the effects of six kinds of long-chain fatty acids varying in the degrees of unsaturation on microbial protein content, total transaminase activity which reflects the degree of cell damage (Jenkins 1993; Jalč et al. 2002) and dehydrogenase activity that indicates the activity of total microbes (Liu and Li 2005) in vitro, aiming to identify appropriate fatty acids for microbial protein production in the rumen.
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2. Results 2.1. Effects on rumen bacterial protein content and protozoal protein content The rumen bacterial protein content is shown in Table 1. The means of bacterial protein contents across six sampling times were significantly higher (P<0.05) for linoleic acid (C18:2, n-6), α-linolenic acid (C18:3, n-3), and arachidonic acid (C20:4, n-6), while no significant differences (P>0.05) were found for oleic acid (C18:1, n-9) and eicosapentaenoic acid (C20:5, n-3) compared with stearic acid (C18:0). The responsive curve of the bacterial protein content with the unsaturity of fatty acids appeared to be quadratic, with the peak values for linoleic acid and α-linolenic acid. The rumen protozoal protein is shown in Table 1. Compared with stearic acid, oleic acid, linoleic acid, α-linolenic acid, arachidonic acid, and eicosapentaenoic acid all reduced the protozoal protein content (P<0.05). The reduction of the protozoal protein content appeared to be inversely related to the unsaturity of the fatty acids (Fig. 1).
2.2. Effects on rumen microbial protein content The rumen microbial protein content was calculated as the sum of the bacterial protein and the protozoal protein, and the result is shown in Table 2. The total microbial protein contents were significantly lower (P<0.05) for oleic acid, arachidonic acid, and eicosapentaenoic acid, whereas linoleic acid increased the microbial protein content (P<0.05) compared with that for stearic acid. There were no significant differences in the microbial protein content between α-linolenic acid and stearic acid (P>0.05).
2.3. Effects on transaminase activity and total dehydrogenase activity Both the glutamic oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) activities for all the unsaturated fatty acids were not changed significantly in all the unsaturated fatty acid groups (P>0.05), except for the enhanced GOT activity by arachidonic acid (P<0.05), and the enhanced GPT activity by oleic acid (P<0.05) (Table 3). The GOT activity declined substantially only at 24 h, and the GPT activity declined continuously with the incubation time. Total dehydrogenase activity is shown in Table 4. The dehydrogenase activity was reduced in oleic acid treatment (P<0.05), and there were no significant differences in linoleic acid, α-linolenic acid and arachidonic acid treatments (P>0.05), whereas eicosapentaenoic acid enhanced the enzyme activity (P<0.05), when compared with that in
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Table 1 Effects of unsaturity of fatty acids on the content of bacterial protein and protozoal protein in goat ruminal fermentation Item Bacterial protein content (mg mL–1)
Protozoal protein content (mg mL–1)
Treatment1) Sampling time A B C D E (h) (C18:0) (C18:1, n-9) (C18:2, n-6) (C18:3, n-3) (C20:4, n-6) 3 0.125 0.133 0.281 0.291 0.275 6 0.123 0.128 0.286 0.296 0.228 9 0.122 0.129 0.293 0.310 0.237 12 0.121 0.127 0.252 0.268 0.159 18 0.123 0.126 0.272 0.257 0.228 24 0.123 0.126 0.272 0.285 0.212 Mean 0.123 0.128 0.276 a 0.285 a 0.223 a SEM 0.0092 P-value 0.001 3 1.123 0.947 1.063 1.042 0.989 6 1.174 1.026 1.130 1.072 0.931 9 1.163 0.993 0.999 0.996 0.891 12 1.156 0.952 1.166 0.895 0.834 18 1.170 0.987 1.074 0.964 0.793 24 1.165 1.162 1.167 1.161 1.166 Mean 1.159 1.011 a 1.100 a 1.022 a 0.934 a SEM 0.0108 P-value 0.001
F (C20:5, n-3) 0.199 0.112 0.155 0.105 0.140 0.138 0.142
0.889 0.914 0.807 0.780 0.908 1.169 0.911 a
Mean
SEM2) P-value
0.213 0.183 0.200 0.166 0.175 0.183
0.0055
0.001
1.009 1.041 0.975 0.964 0.983 1.165
0.0103
0.001
1)
Rumen protozoal protein content (mg mL–1) Total dehydrogenase activity (U mL–1）
The treatments are respectively group A (stearic acid, C18:0, control group), group B (oleic acid, C18:1, n-9), group C (linoleic acid, C18:2, n-6), group D (α-linolenic acid, C18:3, n-3), group E (arachidonic acid, C20:4, n-6), and group F (eicosapentaenoic acid, C20:5, n-3). The same as below. 2) SEM, standard error of mean. The different letters within the row of the means for fatty acids indicate significant differences (P<0.05) between group A and group B, C, D, E, or F, respectively. The same as below.
the incubation time.
Protozoal protein content Total dehydrogenase activity
1.6
3. Discussion
1.4 1.2
3.1. Effects on rumen microbial protein content
1.0 0.8 0.6 0.4 0.2 0.0
0
1 2 3 4 The number of double bond in fatty acids
5
Fig. 1 The rumen protozoal protein content (mg mL–1, triangles) and the total dehydrogenase activity (U mL–1, squares) in goat rumen fermentation fluid in responses to inclusion of 3% of stearic acid (C18:0), oleic acid (C8:1, n-9), linoleic acid (C18:2, n-6), α-linolenic acid (C18:3, n-3), arachidonic acid (C20:4, n-6), or eicosapentaenoic acid (C20:5, n-3). The incubation lasted for 24 h. The fermentation fluid was sampled six times at 3, 6, 9, 12, 18 and 24 h, and the means across the six times are presented here.
stearic acid. The enhancement of the total dehydrogenase appeared to be positively correlated with the unsaturity of the fatty acids (Fig. 1). The time-related changes in the dehydrogenase activity showed a quadratic pattern with
We found in this study that the unsaturation of the fatty acids reduced the rumen protozoal protein content, and the reduction appeared to be correlated with the unsaturity of the fatty acids. It obviously suggests that unsaturation of long-chain fatty acids has detrimental effects on rumen protozoal protein content. Dijkstra et al. (2000) also reported that, no matter saturated or not, the unsaturated long-chain fatty acids had negative effects on both bacteria and protozoa, and the effects increased with the unsaturity of fatty acids. Mcginn et al. (2004) and Benchaar et al. (2007) also reported changes in the biomasses of ruminal fauna and flora. The unsaturated fatty acids can reduce protozoal biomass, change protozoal commune compositions and inhibit their engulfing activities on bacteria (Dijkstra et al. 2000). Our results were in agreement with these reports. A reduction of the protein content could be attributed to a reduced biomass of rumen protozoa. Therefore, it could be speculated that long-chain unsaturated fatty acids have an unfavourable effect on rumen protozoal population. In contrast, we found the rumen bacterial protein content
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Table 2 Effects of unsaturity of fatty acids on the protein content of total microorganisms in goat ruminal fermentation (mg mL–1) Sampling time (h) 3 6 9 12 18 24 Mean SEM P-value
A (C18:0) 1.257 1.293 1.282 1.283 1.295 1.285 1.283
B (C18:1, n-9) 1.085 1.151 1.121 1.081 1.115 1.285 1.140 b
Treatment C D (C18:2, n-6) (C18:3, n-3) 1.352 1.322 1.412 1.374 1.289 1.308 1.422 1.156 1.349 1.217 1.436 1.451 1.377 a 1.304 0.0177 0.001
E (C20:4, n-6) 1.255 1.163 1.131 0.987 1.019 1.381 1.156 b
F (C20:5, n-3) 1.078 1.030 0.966 0.879 1.046 1.310 1.051 b
Mean
SEM
P-value
1.225 1.237 1.183 1.135 1.174 1.358
0.0125
0.001
Table 3 Effects of unsaturity of fatty acids on activity of glutamic oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) of goat rumen microorganisms in fermentation fluid (IU L–1) Item GOT activity
GPT activity
Treatment Sampling time A B C D (h) (C18:0) (C18:1, n-9) (C18:2, n-6) (C18:3, n-3) 3 4.851 6.430 4.647 4.859 6 4.189 5.363 3.240 3.382 9 3.642 2.817 7.714 6.277 12 3.534 5.982 3.359 4.527 18 4.268 3.283 2.879 2.753 24 1.222 3.925 0.968 3.592 Mean 3.618 4.633 3.801 4.232 SEM 0.397 P-value 0.001 3 10.065 13.341 9.641 10.082 6 8.713 11.105 6.763 5.805 9 7.561 5.841 16.011 13.018 12 3.423 11.530 2.843 4.979 18 7.351 6.209 5.455 3.917 24 4.256 7.790 1.172 4.544 Mean 6.895 9.303 a 6.981 7.058 SEM 0.932 P-value 0.001
E (C20:4, n-6) 3.864 5.776 4.589 4.443 7.044 2.836 4.759 a
8.017 11.990 9.523 6.069 6.213 3.787 7.600
F (C20:5, n-3) 4.205 4.005 4.015 5.836 5.912 1.813 4.298
Mean
SEM P-value
5.005 4.259 4.675 4.469 4.329 2.330
0.419
0.001
8.724 8.314 8.330 3.103 7.036 2.813 6.387
10.383 8.665 9.659 5.086 6.163 4.028 10.383
0.708
0.001
Table 4 Effects of unsaturity of fatty acids on the activity of total dehydrogenase of goat rumen microorganisms in fermentation fluid (U mL–1) Sampling time (h) 3 6 9 12 18 24 Mean SEM P-value
A (C18:0) 0.427 0.638 1.090 0.645 0.098 0.083 0.497
B (C18:1, n-9) 0.098 0.347 0.212 0.552 0.157 0.069 0.239 b
Treatment C D (C18:2, n-6) (C18:3, n-3) 0.217 0.147 0.248 0.482 0.597 0.882 0.715 0.632 0.068 0.383 0.061 0.052 0.317 0.429 0.083 0.001
was increased with the increase of the unsaturity of the C18- and C20-fatty acids, whereas the further increase in the unsaturity up to five double bounds in eicosapentaenoic acid
E (C20:4, n-6) 0.797 0.737 0.777 0.652 0.586 0.120 0.611
F (C20:5, n-3) 0.867 0.862 1.117 1.652 0.562 0.097 0.859 a
Mean
SEM P-value
0.451 0.548 0.794 0.793 0.328 0.074
0.085
0.001
did not have such an effect. Zhang et al. (2008) found that adding four kinds of C18-fatty acids into the rumen resulted in high propionate production, which was correlated with the
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unsaturity as well as with the biomass of cellulolytic bacteria. The data in this experiment agreed with their results in some degree. It is noteworthy that compared with those for linoleic acid and α-linolenic acids, the bacterial protein content declined by arachidonic acid (C20:4, n-6), though still significantly higher than that for stearic acid, and further declined by eicosapentaenoic acid (C20:5, n-3) in this study. It may be because arachidonic acid and eicosapentaenoic acid have excessive unsaturated bonds. Our current study showed that the unsaturity of fatty acids had diversified effects on the rumen bacteria and protozoa, with the consistently detrimental effect on protozoa, but the beneficial effect (i.e., elevating protein content) on bacteria depending on the length and the number of double bound in fatty acids. Eicosapentaenoic acid and arachidonic acid share the same length of C20 chain, but the former has one more double bond. This extra double bond in eicosapentaenoic acid resulted in a significant reduction in the bacterial protein content compared with that for arachidonic acid, indicating the effective role of the number of double bond on rumen bacteria. On the other hand, the bacterial protein content increased with the number of double bond from 0 (stearic acid) up to 3 (α-linolenic acid) in the C18-fatty acids in this study. Since the differences in the length of the carbon chain and the number of double bound among those six fatty acids used in this study, we cannot distinguish, at this stage, the respective effects of the length of the carbon chain from the effect of the unsaturity of fatty acids. Based on the quadratic pattern of the bacterial protein content with the number of the double bond among the six fatty acids in our results, we could preliminarily propose that two, three or four double bonds in fatty acids seems to be in favour for rumen bacterial content, and the further increase in unsaturation have no benefit. This proposal warrants further investigations.
3.2. Effects on total dehydrogenase activity and transaminase activities In this study, the total dehydrogenase activity in the fermentation fluid increased with the unsaturity of the fatty acids (Fig. 1), suggesting that the total dehydrogenase was probably induced by the unsaturation of fatty acids. The dehydrogenase activity (DHA) can reflect the microbial activity in sludge or wastewater (Tarafdar 2003; Sun et al. 2014). Therefore, we determined the total dehydrogenase activity as an indicator to the activity of rumen microorganisms. A possible reason for the association between the enzyme activity and the unsaturity of the fatty acids found in this study might be that the double bond of free fatty acids could inhibit protozoa (Jalč et al. 2009) which decreases the microbial
community biomass and the predation activity of protozoa on bacteria (Wang et al. 2009), and subsequently increases the whole microbial biomass. The total dehydrogenase activity can indicate the capability of dehydrogenases to transfer hydrogen in microbial metabolisms or fermentation process, and present the activity of microbial cells to a certain degree (Calsamiglia et al. 2007). In this study, the total dehydrogenase activity showed a quadratic responsive pattern with the incubation time, increasing from 0 h up to 12 h and then dropped from 12 to 24 h. This pattern of the total dehydrogenase activity derived from the microbiol cell was in line with the growth pattern of microorganisms that the growth of microorganisms will enter the stationary phase and then into the decline phase after the log phase during culture process, and the microbial activity will drop correspondingly (Cheng et al. 2004). The activities of GOT and GPT are much higher in the intracellular space rather than the extracellular spaces, but the activity would increase if the cells or cytomembranes are damaged. Some studies have shown that unsaturated fatty acids had certain negative effects on rumen protozoa or some species of bacteria, and the magnitude of the effects depended on unsaturity of fatty acids (Jenkins 1993; Bach and Stern 1999; Jalč et al. 2002). In the present study, the GOT and GPT activities did not change significantly among the six fatty acid treatments, indicating that the unsaturity of the fatty acids did not result in substantial alternation in autolysis of the rumen microorganisms. In summary, the current study found that the unsaturity (from 1 up to 5 double bond) of the long-chain (C18 and C20) fatty acids had detrimental effects on the rumen protozoal protein content, but elevated the rumen bacterial protein concentration, likely due to reduced engulfing of the protozoa to the bacteria. Based on the quadratic pattern of the bacterial protein content with the number of the double bond among the six fatty acids, we could preliminarily propose that two, three or four double bonds in fatty acids seems to be in favour for rumen bacterial content, and the further increase in unsaturation have no benefit. In future, we will select several kinds of long-chain fatty acids which have effects on rumen fermentation in experiments and then investigate the optimal combination of these fatty acids for improving the rumen fermentation.
4. Conclusion The inclusion of 3% of long-chain unsaturated fatty acids increased bacterial protein content, whereas reduced protozoal protein content and enhanced dehydrogenase activity. The fatty acids with more than three double bonds had detrimental effects on the microbial protein content.
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5. Materials and methods 5.1. Animals and management The use of animals and the experimental procedures were approved by the Animal Care and Use Committee of Yangzhou University, Jiangsu Province, China. Three goats with average weight of 29.4 kg (SD 2.7 kg) at about 1.5 years old from the Experimental Station of Yangzhou University were used to provide the rumen liquor as the culture inoculum for the in vitro studies. The animals had free access to fresh drinking water and the mineral block. The diet was composed of 28% corn grains, 2% soybean meal and 70% Lymus chinensis hay, mixed and fed to the animals at 07:00 and 19:00 in equal amounts. The amount of dry matter of the feed offered to the animals was about 2.5% of body weight.
5.2. Experimental design, procedures and sampling In vitro fermentation was used to test the effects of the unsaturity of long chain fatty acids on the ruminal fermentation. Six fatty acids with different numbers of double bond as shown in Table 5, stearic acid (C18:0, group A, control group), oleic acid (C18:1, n-9, group B), linoleic acid (C18:2, n-6, group C), α-linolenic acid (C18:3, n-3, group D), arachidonic acid (C20:4, n-6, group E), and eicosapentaenoic acid (C20:5, n-3, group F) were tested and each fatty acid was included as 3% (w/w) of total substrate. Stearic acid contains nil double bond and was used as control in this experiment. Chalupa et al. (1984) supported that stearic acid did not affect ruminal fermentation. Zhang et al. (2008) also found that stearic acid slightly increased rumen microbes in vitro. All fatty acids were purchased and added as the solid form and the characteristics of them were shown in Table 5. In addition to the fatty acids, the culture substrate consisted of 25.50% starch, 4.25% xylan, 4.25% araban, 4.25% glucan, 4.25% mannan, 34.00% cellulose, 4.25% pectin, 4.25% lignin, 3.60% urea and 8.40% casein. Rumen fluid was collected from 3 goats as inoculum.
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Around 300 mL of rumen fluid was collected from each goat before morning feeding using a vacuum pump through the rumen fistula. The fluid from three goats was pooled, filtered through four layer gauze into an aseptic saline bottle. The bottle was flushed with CO2 and sealed. The fermentation fluid was prepared by mixing a portion of the rumen fluid with two portions of the artificial saliva prepared according to Menke and Steingass (1988). The artificial saliva was added to neutralize fatty acids produced during fermentation in order to maintain pH in a normal range. The fermentation was carried out in glass culture bottle, into which 1.50 g of the substrate and 150 mL of the fermentation fluid were added, flushed with CO2 and then sealed. The bottles were placed in water-bath with constantly shaking at 50 r min–1 at 39°C for 24 h. Samples of the fermentation fluid, 5 mL each, were drawn from the bottles using syringe respectively at 0, 3, 6, 9, 12, 18 and 24 h for the measurements as listed below. Each treatment (i.e., fatty acid) group had 3 replicates.
5.3. Analysis Activities of glutamic oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) were determined using commercial kits (nanjing Jiancheng Bioengineering Institute, nanjing, China). The sample preparation was performed according the procedures as described in the kits, and the enzyme activity was quantitated using an MD-SpectraMax M5 plate reader (Molecular Devices Corporation, USA). The total dehydrogenase activity in the fermentation fluid was determined using the triphenyltetrazolium chloride (TTC) method as described by Dror et al. (1969) with some modifications. The 2 mL fermentation fluid was transferred into a 10-mL glass tube, then 0.2 mL of 1.5% TTC was added. The tube was incubated in water bath at 38°C for 10 min. The reaction was stopped by an addition of 5 mL isopropanol into the tube. After vortexing, the tube was centrifuged at 3 220×g for 10 min. Then an aliquot of the supernatant was taken, and diluted 15 times with water,
Table 5 The characteristics of the long-chain fatty acids used in this experiment Group
name
A
Stearic acid
B C D E F
Oleic acid Linoleic acid α-Linolenic acid Arachidonic acid Eicosapentaenoic acid
Length of carbon chain 18
number of double bonds 0
18 18 18 20 20
1 2 3 4 5
Position of Chemical first double Abstracts Source bond Service (CAS) – 21-12-8 Beijing Beilingwei Scientific and Technical Co. Ltd., China 9 112-80-1 Beijing Beilingwei Scientific and Technical Co. Ltd. 6 60-33-3 Beijing Beilingwei Scientific and Technical Co. Ltd. 3 463-40-1 Beijing Beilingwei Scientific and Technical Co. Ltd. 6 506-32-1 Beijing Beilingwei Scientific and Technical Co. Ltd. 3 10 417-94-4 Shanghai Kewei Chemical Co. Ltd., China
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and its absorbency intensity at 485 nm was registered using a UV-7502C spectrophotometer (molecular devices, Shanghai Precision Instruments Co., Ltd., China). The unit of the enzyme activity was defined as the rate absorbency intensity per 0.1 mL per min. Protein contents of bacteria and protozoa in the fermentation fluid were determined using the method described by Wang et al. (2008). To isolate protozoa, the sample was diluted in equal volume saline, incubated in water bath constantly shaking at 125 r min–1 at 39°C for 1 h, then vortexed thoroughly and squeezed through four layer gauze. The mixture was then centrifuged at 150×g for 15 min. Sediment, regarded as the protozoal fraction, was collected, rinsed twice using saline and then re-suspended in saline and stored at –20°C until analysis. For bacteria, the suspension was collected from the above centrifugation, and then further centrifuged at 22 000×g for 15 min. Sediment, regarded as the bacterial fraction, was collected, rinsed twice using saline and then re-suspended in saline and stored at –20°C until analysis. Microbial true protein was estimated as microbial production using the method of trichloroacetic acid precipitation, and then calculated using the equation as followings: Protein (mg mL–1)=(1.45×OD280–0.740×OD260)×Dilution
5.4. Statistical analysis Analysis of variance (AnOVA) for repeated measures was used to examine the effects of the treatments (i.e., fatty acids), time (i.e., hours of the incubation) and potential interaction between treatment and time. The measurement at 0 h was considered as a co-variate in the model. The procedure of residual maximum likelihood (REML) for repeated measures in GenStat (ver. 16, VSn International Ltd.) was used to analyze the data, and treatment (i.e., fatty acid), time (hours) and their interactions were included as fixed effect in the model. To focus on the differences among the fatty acid groups, the multiple comparisons were performed for the means of the fatty acids across all the six sampling times, as well as for the means of the sampling times (hours) across all the fatty acids, and the corresponding standard error of means (SEM) are presented in the Tables respectively for fatty acid and time. P values less than 0.05 (P<0.05) were declared to be significant.
Acknowledgements The authors would like to give special thanks to MSc Yu Pi (Yangzhou University, China) for the help in this experiment. This work was financially supported by the Innovation Foundation for Undergraduate of Yangzhou University, China (201311117034), the Domestic Cooperative Innovation of
Industry-University-Research (XT20140012), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), China.
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ScienceDirect
RESEARCH ARTICLE
The effects of the unsaturated degree of long-chain fatty acids on the rumen microbial protein content and the activities of transaminases and dehydrogenase in vitro GAO Jian1*, JING Yu-jia1*, WANG Meng-zhi1, SHI Liang-feng1, LIU Shi-min2 1 2
College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, P.R.China Institute of Agriculture, The University of Western Australia, Crawley WA 6009, Australia
Abstract This study investigated the effects of the degree of unsaturation (unsaturity) of long-chain fatty acids on microbial protein content and the activities of transaminases and dehydrogenase in vitro using goat rumen fluid as the cultural medium. Six types of fatty acids, stearic acid (C18:0, group A, control group), oleic acid (C18:1, n-9, group B), linoleic acid (C18:2, n-6, group C), α-linolenic acid (C18:3, n-3, group D), arachidonic acid (C20:4, n-6, group E), and eicosapentaenoic acid (C20:5, n-3, group F), were tested, and the inclusion ratio of each fatty acid was 3% (w/w) in total of culture substrate. Samples were taken at 0, 3, 6, 9, 12, 18 and 24 h, respectively, during culture for analyses. Compared with stearic acid, linoleic acid, α-linolenic acid, and arachidonic acid increased the bacterial protein content, while oleic acid and eicosapentaenoic acid had no significant effects; the protozoal protein content was reduced for all the unsaturated fatty acids, and the magnitude of the reduction appeared to be associated with the degree of unsaturity of fatty acids. The total microbial protein content was dominantly accounted by the protozoal protein content (about 4–9 folds of the bacterial protein), and only increased by linoleic acid, but reduced by oleic acid, arachidonic acid and eicosapentaenoic acid. There were no significant effects in the activities of both glutamic oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) for all the other fatty acids, except for arachidonic acid which enhanced GOT activity and oleic acid which enhanced GPT activity. The total dehydrogenase activity was positively correlated with the degree of unsaturation of fatty acids. In conclusion, the inclusion of 3% of long-chain unsaturated fatty acids increased bacterial protein content, whereas reduced protozoal protein content and enhanced dehydrogenase activity. The fatty acids with more than three double bonds had detrimental effects on the microbial protein content. This work demonstrates for the first time the effect rule of the unsaturation degree of long-chain fatty acids on the rumen microbial protein in vitro. Keywords: long-chain fatty acid, rumen microbes, goats, protein concentration
Received 26 December, 2014 Accepted 18 May, 2015 GAO Jian, E-mail: [email protected]; JInG Yu-jia, E-mail: [email protected]; Correspondence WAnG Meng-zhi, E-mail: [email protected] * These authors contributed equally to this study. © 2016, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S2095-3119(15)61081-4
1. Introduction Oil and fat are composed of various fatty acids (FA), contain high levels of energy, and can be used as dietary supplements to ruminant animals when the energy demand is particularly high, for example, at the peak period of lactation, or highly ambient temperature associated with reduction of
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dietary intake. However, the magnitude of unsaturation of fatty acids is unfavour for microorganisms in the rumen, and detrimental effects differ among species and genus of the ruminal microorganisms, in turn the ruminal fermentation patterns are altered (nangla and Rila 1994; Dijkstra et al. 2000; Mcginn et al. 2004; Jalč et al. 2009). Fatty acids can be used to stabilize rumen fermentation, enhance the yield of microbial proteins (MCP) which accounts for 40–60% of proteins into the small intestines of ruminants (Welch and Hooper 1993) and increase the utilization efficiency of energy and nitrogen of host animals (Clark et al. 1992; Bach and Stern 1999). Rapeseed oil, sunflower oil and linseed oil rich in octadeca carbon fatty acids (C18 fatty acids) have been shown different effects on the rumen microbial ecological system including fermentation patterns, bacterial and protozoal populations, and microbial protein production from previous researches (Machmüller et al. 1998; Jalč and Čerešňáková 2002). Cieslak et al. (2009) also demonstrated that there were major impacts of linoleic acid (LA) supplement in the bacterial fractions of both ciliate cultures including Entodinium caudatum (EC) and Diploplastron affine (DA). These researches suggest that addition of fat or fatty acids can change rumen fermentation patterns. Our previous research showed that the addition of 4% oil, such as rapeseed oil, soybean oil, corn oil and peanut oil with different degrees of unsaturation (unsaturity) inhibited the predation activity of protozoa on bacteria and increased the bacterial biomass and the yield of MCP (Wang et al. 2010). Potu (2011) reported that supplementations with different lipids which containing different fatty acid compositions had some discrepancies on productions of acetic acid and propionic acid in the rumen. Zhang et al. (2008) proposed that unsaturated C18-fatty acids changed the rumen fermentation patterns and microbial population profiles. The other research from Zhang et al. (2008) reported that adding mixtures of linoleic acid and linolenic acid significantly reduced the total gas production and methan production at 24 h fermentation, while the treatment had no significant influence on the microbial biomass. All these reports suggested that the effects of fatty acids on rumen fermentation had certain differences and may be related to both the unsaturity of FAs as well as the length of carbon chain. Thus, this study was conducted to examine the effects of six kinds of long-chain fatty acids varying in the degrees of unsaturation on microbial protein content, total transaminase activity which reflects the degree of cell damage (Jenkins 1993; Jalč et al. 2002) and dehydrogenase activity that indicates the activity of total microbes (Liu and Li 2005) in vitro, aiming to identify appropriate fatty acids for microbial protein production in the rumen.
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2. Results 2.1. Effects on rumen bacterial protein content and protozoal protein content The rumen bacterial protein content is shown in Table 1. The means of bacterial protein contents across six sampling times were significantly higher (P<0.05) for linoleic acid (C18:2, n-6), α-linolenic acid (C18:3, n-3), and arachidonic acid (C20:4, n-6), while no significant differences (P>0.05) were found for oleic acid (C18:1, n-9) and eicosapentaenoic acid (C20:5, n-3) compared with stearic acid (C18:0). The responsive curve of the bacterial protein content with the unsaturity of fatty acids appeared to be quadratic, with the peak values for linoleic acid and α-linolenic acid. The rumen protozoal protein is shown in Table 1. Compared with stearic acid, oleic acid, linoleic acid, α-linolenic acid, arachidonic acid, and eicosapentaenoic acid all reduced the protozoal protein content (P<0.05). The reduction of the protozoal protein content appeared to be inversely related to the unsaturity of the fatty acids (Fig. 1).
2.2. Effects on rumen microbial protein content The rumen microbial protein content was calculated as the sum of the bacterial protein and the protozoal protein, and the result is shown in Table 2. The total microbial protein contents were significantly lower (P<0.05) for oleic acid, arachidonic acid, and eicosapentaenoic acid, whereas linoleic acid increased the microbial protein content (P<0.05) compared with that for stearic acid. There were no significant differences in the microbial protein content between α-linolenic acid and stearic acid (P>0.05).
2.3. Effects on transaminase activity and total dehydrogenase activity Both the glutamic oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) activities for all the unsaturated fatty acids were not changed significantly in all the unsaturated fatty acid groups (P>0.05), except for the enhanced GOT activity by arachidonic acid (P<0.05), and the enhanced GPT activity by oleic acid (P<0.05) (Table 3). The GOT activity declined substantially only at 24 h, and the GPT activity declined continuously with the incubation time. Total dehydrogenase activity is shown in Table 4. The dehydrogenase activity was reduced in oleic acid treatment (P<0.05), and there were no significant differences in linoleic acid, α-linolenic acid and arachidonic acid treatments (P>0.05), whereas eicosapentaenoic acid enhanced the enzyme activity (P<0.05), when compared with that in
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Table 1 Effects of unsaturity of fatty acids on the content of bacterial protein and protozoal protein in goat ruminal fermentation Item Bacterial protein content (mg mL–1)
Protozoal protein content (mg mL–1)
Treatment1) Sampling time A B C D E (h) (C18:0) (C18:1, n-9) (C18:2, n-6) (C18:3, n-3) (C20:4, n-6) 3 0.125 0.133 0.281 0.291 0.275 6 0.123 0.128 0.286 0.296 0.228 9 0.122 0.129 0.293 0.310 0.237 12 0.121 0.127 0.252 0.268 0.159 18 0.123 0.126 0.272 0.257 0.228 24 0.123 0.126 0.272 0.285 0.212 Mean 0.123 0.128 0.276 a 0.285 a 0.223 a SEM 0.0092 P-value 0.001 3 1.123 0.947 1.063 1.042 0.989 6 1.174 1.026 1.130 1.072 0.931 9 1.163 0.993 0.999 0.996 0.891 12 1.156 0.952 1.166 0.895 0.834 18 1.170 0.987 1.074 0.964 0.793 24 1.165 1.162 1.167 1.161 1.166 Mean 1.159 1.011 a 1.100 a 1.022 a 0.934 a SEM 0.0108 P-value 0.001
F (C20:5, n-3) 0.199 0.112 0.155 0.105 0.140 0.138 0.142
0.889 0.914 0.807 0.780 0.908 1.169 0.911 a
Mean
SEM2) P-value
0.213 0.183 0.200 0.166 0.175 0.183
0.0055
0.001
1.009 1.041 0.975 0.964 0.983 1.165
0.0103
0.001
1)
Rumen protozoal protein content (mg mL–1) Total dehydrogenase activity (U mL–1）
The treatments are respectively group A (stearic acid, C18:0, control group), group B (oleic acid, C18:1, n-9), group C (linoleic acid, C18:2, n-6), group D (α-linolenic acid, C18:3, n-3), group E (arachidonic acid, C20:4, n-6), and group F (eicosapentaenoic acid, C20:5, n-3). The same as below. 2) SEM, standard error of mean. The different letters within the row of the means for fatty acids indicate significant differences (P<0.05) between group A and group B, C, D, E, or F, respectively. The same as below.
the incubation time.
Protozoal protein content Total dehydrogenase activity
1.6
3. Discussion
1.4 1.2
3.1. Effects on rumen microbial protein content
1.0 0.8 0.6 0.4 0.2 0.0
0
1 2 3 4 The number of double bond in fatty acids
5
Fig. 1 The rumen protozoal protein content (mg mL–1, triangles) and the total dehydrogenase activity (U mL–1, squares) in goat rumen fermentation fluid in responses to inclusion of 3% of stearic acid (C18:0), oleic acid (C8:1, n-9), linoleic acid (C18:2, n-6), α-linolenic acid (C18:3, n-3), arachidonic acid (C20:4, n-6), or eicosapentaenoic acid (C20:5, n-3). The incubation lasted for 24 h. The fermentation fluid was sampled six times at 3, 6, 9, 12, 18 and 24 h, and the means across the six times are presented here.
stearic acid. The enhancement of the total dehydrogenase appeared to be positively correlated with the unsaturity of the fatty acids (Fig. 1). The time-related changes in the dehydrogenase activity showed a quadratic pattern with
We found in this study that the unsaturation of the fatty acids reduced the rumen protozoal protein content, and the reduction appeared to be correlated with the unsaturity of the fatty acids. It obviously suggests that unsaturation of long-chain fatty acids has detrimental effects on rumen protozoal protein content. Dijkstra et al. (2000) also reported that, no matter saturated or not, the unsaturated long-chain fatty acids had negative effects on both bacteria and protozoa, and the effects increased with the unsaturity of fatty acids. Mcginn et al. (2004) and Benchaar et al. (2007) also reported changes in the biomasses of ruminal fauna and flora. The unsaturated fatty acids can reduce protozoal biomass, change protozoal commune compositions and inhibit their engulfing activities on bacteria (Dijkstra et al. 2000). Our results were in agreement with these reports. A reduction of the protein content could be attributed to a reduced biomass of rumen protozoa. Therefore, it could be speculated that long-chain unsaturated fatty acids have an unfavourable effect on rumen protozoal population. In contrast, we found the rumen bacterial protein content
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Table 2 Effects of unsaturity of fatty acids on the protein content of total microorganisms in goat ruminal fermentation (mg mL–1) Sampling time (h) 3 6 9 12 18 24 Mean SEM P-value
A (C18:0) 1.257 1.293 1.282 1.283 1.295 1.285 1.283
B (C18:1, n-9) 1.085 1.151 1.121 1.081 1.115 1.285 1.140 b
Treatment C D (C18:2, n-6) (C18:3, n-3) 1.352 1.322 1.412 1.374 1.289 1.308 1.422 1.156 1.349 1.217 1.436 1.451 1.377 a 1.304 0.0177 0.001
E (C20:4, n-6) 1.255 1.163 1.131 0.987 1.019 1.381 1.156 b
F (C20:5, n-3) 1.078 1.030 0.966 0.879 1.046 1.310 1.051 b
Mean
SEM
P-value
1.225 1.237 1.183 1.135 1.174 1.358
0.0125
0.001
Table 3 Effects of unsaturity of fatty acids on activity of glutamic oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) of goat rumen microorganisms in fermentation fluid (IU L–1) Item GOT activity
GPT activity
Treatment Sampling time A B C D (h) (C18:0) (C18:1, n-9) (C18:2, n-6) (C18:3, n-3) 3 4.851 6.430 4.647 4.859 6 4.189 5.363 3.240 3.382 9 3.642 2.817 7.714 6.277 12 3.534 5.982 3.359 4.527 18 4.268 3.283 2.879 2.753 24 1.222 3.925 0.968 3.592 Mean 3.618 4.633 3.801 4.232 SEM 0.397 P-value 0.001 3 10.065 13.341 9.641 10.082 6 8.713 11.105 6.763 5.805 9 7.561 5.841 16.011 13.018 12 3.423 11.530 2.843 4.979 18 7.351 6.209 5.455 3.917 24 4.256 7.790 1.172 4.544 Mean 6.895 9.303 a 6.981 7.058 SEM 0.932 P-value 0.001
E (C20:4, n-6) 3.864 5.776 4.589 4.443 7.044 2.836 4.759 a
8.017 11.990 9.523 6.069 6.213 3.787 7.600
F (C20:5, n-3) 4.205 4.005 4.015 5.836 5.912 1.813 4.298
Mean
SEM P-value
5.005 4.259 4.675 4.469 4.329 2.330
0.419
0.001
8.724 8.314 8.330 3.103 7.036 2.813 6.387
10.383 8.665 9.659 5.086 6.163 4.028 10.383
0.708
0.001
Table 4 Effects of unsaturity of fatty acids on the activity of total dehydrogenase of goat rumen microorganisms in fermentation fluid (U mL–1) Sampling time (h) 3 6 9 12 18 24 Mean SEM P-value
A (C18:0) 0.427 0.638 1.090 0.645 0.098 0.083 0.497
B (C18:1, n-9) 0.098 0.347 0.212 0.552 0.157 0.069 0.239 b
Treatment C D (C18:2, n-6) (C18:3, n-3) 0.217 0.147 0.248 0.482 0.597 0.882 0.715 0.632 0.068 0.383 0.061 0.052 0.317 0.429 0.083 0.001
was increased with the increase of the unsaturity of the C18- and C20-fatty acids, whereas the further increase in the unsaturity up to five double bounds in eicosapentaenoic acid
E (C20:4, n-6) 0.797 0.737 0.777 0.652 0.586 0.120 0.611
F (C20:5, n-3) 0.867 0.862 1.117 1.652 0.562 0.097 0.859 a
Mean
SEM P-value
0.451 0.548 0.794 0.793 0.328 0.074
0.085
0.001
did not have such an effect. Zhang et al. (2008) found that adding four kinds of C18-fatty acids into the rumen resulted in high propionate production, which was correlated with the
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unsaturity as well as with the biomass of cellulolytic bacteria. The data in this experiment agreed with their results in some degree. It is noteworthy that compared with those for linoleic acid and α-linolenic acids, the bacterial protein content declined by arachidonic acid (C20:4, n-6), though still significantly higher than that for stearic acid, and further declined by eicosapentaenoic acid (C20:5, n-3) in this study. It may be because arachidonic acid and eicosapentaenoic acid have excessive unsaturated bonds. Our current study showed that the unsaturity of fatty acids had diversified effects on the rumen bacteria and protozoa, with the consistently detrimental effect on protozoa, but the beneficial effect (i.e., elevating protein content) on bacteria depending on the length and the number of double bound in fatty acids. Eicosapentaenoic acid and arachidonic acid share the same length of C20 chain, but the former has one more double bond. This extra double bond in eicosapentaenoic acid resulted in a significant reduction in the bacterial protein content compared with that for arachidonic acid, indicating the effective role of the number of double bond on rumen bacteria. On the other hand, the bacterial protein content increased with the number of double bond from 0 (stearic acid) up to 3 (α-linolenic acid) in the C18-fatty acids in this study. Since the differences in the length of the carbon chain and the number of double bound among those six fatty acids used in this study, we cannot distinguish, at this stage, the respective effects of the length of the carbon chain from the effect of the unsaturity of fatty acids. Based on the quadratic pattern of the bacterial protein content with the number of the double bond among the six fatty acids in our results, we could preliminarily propose that two, three or four double bonds in fatty acids seems to be in favour for rumen bacterial content, and the further increase in unsaturation have no benefit. This proposal warrants further investigations.
3.2. Effects on total dehydrogenase activity and transaminase activities In this study, the total dehydrogenase activity in the fermentation fluid increased with the unsaturity of the fatty acids (Fig. 1), suggesting that the total dehydrogenase was probably induced by the unsaturation of fatty acids. The dehydrogenase activity (DHA) can reflect the microbial activity in sludge or wastewater (Tarafdar 2003; Sun et al. 2014). Therefore, we determined the total dehydrogenase activity as an indicator to the activity of rumen microorganisms. A possible reason for the association between the enzyme activity and the unsaturity of the fatty acids found in this study might be that the double bond of free fatty acids could inhibit protozoa (Jalč et al. 2009) which decreases the microbial
community biomass and the predation activity of protozoa on bacteria (Wang et al. 2009), and subsequently increases the whole microbial biomass. The total dehydrogenase activity can indicate the capability of dehydrogenases to transfer hydrogen in microbial metabolisms or fermentation process, and present the activity of microbial cells to a certain degree (Calsamiglia et al. 2007). In this study, the total dehydrogenase activity showed a quadratic responsive pattern with the incubation time, increasing from 0 h up to 12 h and then dropped from 12 to 24 h. This pattern of the total dehydrogenase activity derived from the microbiol cell was in line with the growth pattern of microorganisms that the growth of microorganisms will enter the stationary phase and then into the decline phase after the log phase during culture process, and the microbial activity will drop correspondingly (Cheng et al. 2004). The activities of GOT and GPT are much higher in the intracellular space rather than the extracellular spaces, but the activity would increase if the cells or cytomembranes are damaged. Some studies have shown that unsaturated fatty acids had certain negative effects on rumen protozoa or some species of bacteria, and the magnitude of the effects depended on unsaturity of fatty acids (Jenkins 1993; Bach and Stern 1999; Jalč et al. 2002). In the present study, the GOT and GPT activities did not change significantly among the six fatty acid treatments, indicating that the unsaturity of the fatty acids did not result in substantial alternation in autolysis of the rumen microorganisms. In summary, the current study found that the unsaturity (from 1 up to 5 double bond) of the long-chain (C18 and C20) fatty acids had detrimental effects on the rumen protozoal protein content, but elevated the rumen bacterial protein concentration, likely due to reduced engulfing of the protozoa to the bacteria. Based on the quadratic pattern of the bacterial protein content with the number of the double bond among the six fatty acids, we could preliminarily propose that two, three or four double bonds in fatty acids seems to be in favour for rumen bacterial content, and the further increase in unsaturation have no benefit. In future, we will select several kinds of long-chain fatty acids which have effects on rumen fermentation in experiments and then investigate the optimal combination of these fatty acids for improving the rumen fermentation.
4. Conclusion The inclusion of 3% of long-chain unsaturated fatty acids increased bacterial protein content, whereas reduced protozoal protein content and enhanced dehydrogenase activity. The fatty acids with more than three double bonds had detrimental effects on the microbial protein content.
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5. Materials and methods 5.1. Animals and management The use of animals and the experimental procedures were approved by the Animal Care and Use Committee of Yangzhou University, Jiangsu Province, China. Three goats with average weight of 29.4 kg (SD 2.7 kg) at about 1.5 years old from the Experimental Station of Yangzhou University were used to provide the rumen liquor as the culture inoculum for the in vitro studies. The animals had free access to fresh drinking water and the mineral block. The diet was composed of 28% corn grains, 2% soybean meal and 70% Lymus chinensis hay, mixed and fed to the animals at 07:00 and 19:00 in equal amounts. The amount of dry matter of the feed offered to the animals was about 2.5% of body weight.
5.2. Experimental design, procedures and sampling In vitro fermentation was used to test the effects of the unsaturity of long chain fatty acids on the ruminal fermentation. Six fatty acids with different numbers of double bond as shown in Table 5, stearic acid (C18:0, group A, control group), oleic acid (C18:1, n-9, group B), linoleic acid (C18:2, n-6, group C), α-linolenic acid (C18:3, n-3, group D), arachidonic acid (C20:4, n-6, group E), and eicosapentaenoic acid (C20:5, n-3, group F) were tested and each fatty acid was included as 3% (w/w) of total substrate. Stearic acid contains nil double bond and was used as control in this experiment. Chalupa et al. (1984) supported that stearic acid did not affect ruminal fermentation. Zhang et al. (2008) also found that stearic acid slightly increased rumen microbes in vitro. All fatty acids were purchased and added as the solid form and the characteristics of them were shown in Table 5. In addition to the fatty acids, the culture substrate consisted of 25.50% starch, 4.25% xylan, 4.25% araban, 4.25% glucan, 4.25% mannan, 34.00% cellulose, 4.25% pectin, 4.25% lignin, 3.60% urea and 8.40% casein. Rumen fluid was collected from 3 goats as inoculum.
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Around 300 mL of rumen fluid was collected from each goat before morning feeding using a vacuum pump through the rumen fistula. The fluid from three goats was pooled, filtered through four layer gauze into an aseptic saline bottle. The bottle was flushed with CO2 and sealed. The fermentation fluid was prepared by mixing a portion of the rumen fluid with two portions of the artificial saliva prepared according to Menke and Steingass (1988). The artificial saliva was added to neutralize fatty acids produced during fermentation in order to maintain pH in a normal range. The fermentation was carried out in glass culture bottle, into which 1.50 g of the substrate and 150 mL of the fermentation fluid were added, flushed with CO2 and then sealed. The bottles were placed in water-bath with constantly shaking at 50 r min–1 at 39°C for 24 h. Samples of the fermentation fluid, 5 mL each, were drawn from the bottles using syringe respectively at 0, 3, 6, 9, 12, 18 and 24 h for the measurements as listed below. Each treatment (i.e., fatty acid) group had 3 replicates.
5.3. Analysis Activities of glutamic oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) were determined using commercial kits (nanjing Jiancheng Bioengineering Institute, nanjing, China). The sample preparation was performed according the procedures as described in the kits, and the enzyme activity was quantitated using an MD-SpectraMax M5 plate reader (Molecular Devices Corporation, USA). The total dehydrogenase activity in the fermentation fluid was determined using the triphenyltetrazolium chloride (TTC) method as described by Dror et al. (1969) with some modifications. The 2 mL fermentation fluid was transferred into a 10-mL glass tube, then 0.2 mL of 1.5% TTC was added. The tube was incubated in water bath at 38°C for 10 min. The reaction was stopped by an addition of 5 mL isopropanol into the tube. After vortexing, the tube was centrifuged at 3 220×g for 10 min. Then an aliquot of the supernatant was taken, and diluted 15 times with water,
Table 5 The characteristics of the long-chain fatty acids used in this experiment Group
name
A
Stearic acid
B C D E F
Oleic acid Linoleic acid α-Linolenic acid Arachidonic acid Eicosapentaenoic acid
Length of carbon chain 18
number of double bonds 0
18 18 18 20 20
1 2 3 4 5
Position of Chemical first double Abstracts Source bond Service (CAS) – 21-12-8 Beijing Beilingwei Scientific and Technical Co. Ltd., China 9 112-80-1 Beijing Beilingwei Scientific and Technical Co. Ltd. 6 60-33-3 Beijing Beilingwei Scientific and Technical Co. Ltd. 3 463-40-1 Beijing Beilingwei Scientific and Technical Co. Ltd. 6 506-32-1 Beijing Beilingwei Scientific and Technical Co. Ltd. 3 10 417-94-4 Shanghai Kewei Chemical Co. Ltd., China
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and its absorbency intensity at 485 nm was registered using a UV-7502C spectrophotometer (molecular devices, Shanghai Precision Instruments Co., Ltd., China). The unit of the enzyme activity was defined as the rate absorbency intensity per 0.1 mL per min. Protein contents of bacteria and protozoa in the fermentation fluid were determined using the method described by Wang et al. (2008). To isolate protozoa, the sample was diluted in equal volume saline, incubated in water bath constantly shaking at 125 r min–1 at 39°C for 1 h, then vortexed thoroughly and squeezed through four layer gauze. The mixture was then centrifuged at 150×g for 15 min. Sediment, regarded as the protozoal fraction, was collected, rinsed twice using saline and then re-suspended in saline and stored at –20°C until analysis. For bacteria, the suspension was collected from the above centrifugation, and then further centrifuged at 22 000×g for 15 min. Sediment, regarded as the bacterial fraction, was collected, rinsed twice using saline and then re-suspended in saline and stored at –20°C until analysis. Microbial true protein was estimated as microbial production using the method of trichloroacetic acid precipitation, and then calculated using the equation as followings: Protein (mg mL–1)=(1.45×OD280–0.740×OD260)×Dilution
5.4. Statistical analysis Analysis of variance (AnOVA) for repeated measures was used to examine the effects of the treatments (i.e., fatty acids), time (i.e., hours of the incubation) and potential interaction between treatment and time. The measurement at 0 h was considered as a co-variate in the model. The procedure of residual maximum likelihood (REML) for repeated measures in GenStat (ver. 16, VSn International Ltd.) was used to analyze the data, and treatment (i.e., fatty acid), time (hours) and their interactions were included as fixed effect in the model. To focus on the differences among the fatty acid groups, the multiple comparisons were performed for the means of the fatty acids across all the six sampling times, as well as for the means of the sampling times (hours) across all the fatty acids, and the corresponding standard error of means (SEM) are presented in the Tables respectively for fatty acid and time. P values less than 0.05 (P<0.05) were declared to be significant.
Acknowledgements The authors would like to give special thanks to MSc Yu Pi (Yangzhou University, China) for the help in this experiment. This work was financially supported by the Innovation Foundation for Undergraduate of Yangzhou University, China (201311117034), the Domestic Cooperative Innovation of
Industry-University-Research (XT20140012), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), China.
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