Научная статья на тему 'Effects of NFC/NDF in diet on rumen fermentation parameters and microbial flora diversity in calves'

Effects of NFC/NDF in diet on rumen fermentation parameters and microbial flora diversity in calves Текст научной статьи по специальности «Животноводство и молочное дело»

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Ключевые слова
ТЕЛЯТА / СООТНОШЕНИЕ НЕСТРУКТУРНЫХ УГЛЕВОДОВ/НЕЙТРАЛЬНО РАСТВОРИМОГО ВОЛОКНА СЫРОЙ КЛЕТЧАТКИ / ПАРАМЕТРЫ ФЕРМЕНТАЦИИ РУБЦА / МИКРОБНЫЙ СОСТАВ РУБЦОВОЙ ЖИДКОСТИ / WEANED BEEF CALVES / NON-FIBER CARBOHYDRATE/NEUTRAL DETERGENT FIBER RATIO / RUMEN FERMENTATION PARAMETERS / RUMEN MICROBIAL COMMUNITY

Аннотация научной статьи по животноводству и молочному делу, автор научной работы — Цянь Чжао, Пэн Чжэн, Ягуан Тянь, Хе Хуан

This study assessed the effects of diets with different non-fiber carbohydrate (NFC)/neutral detergent fiber (NDF) levels on rumen fermentation parameters and bacterial community of male calves. Thirty 2-3 monthold healthy weaned male calves with 94.38±0.25 kg were randomly divided into 2 groups with 15calves each, and were fed 2 types of total mixed ratio diet with the same crude protein level and different NFC/NDF ratios (1.35 (A) and 0.80 (D)), respectively. The experiment lasted for 105 d, with 15 d of adaptation period and 90 d of formal period. Six calves in each group were selected to collect rumen fluid through the oral for 2 hours after morning feeding on the 120, 150 and 180 d. The pH, volatile fatty acids (VFA) and NH3-N concentration, and rumen microbial diversity of the rumen fluid samples were determined. The results showed that: 1) The dietary NFC/NDF levels had no significant effect on rumen fluid pH and total VFA concentration (P>0.05); 2) Acetate, NH3-N concentration and acetate/propionate ratio in group A were significantly lower than that in group D (P<0.05), propionate content in group A were significantly higher than that in group D (P<0.05); 3) The microbial diversity parameters of rumen fluid of calves in group A were significantly lower than that in group D (P<0.05); 4) At the phylum level, bacteriodetes proportion accounting for the total number of sequences of calves in group A was significantly higher than that in group D (P<0.05), Christensenellaceae R-7 and Prevotellaceae UCG-003 proportions accounting for the total number of sequences of calves in group D was significantly higher at the genus level than that in group A (P<0.05); 5) With the age increasing, the diversity indices except for Shannon index, were significantly different between the 2 groups (P<0.05), but there was no significant difference in rumen microbial composition between the 2 groups (P<0.05). In summary, the dietary NFC/NDF levels significantly affect the species and abundance of the rumen microorganisms, and the level of genus of microorganisms, finally affect the yield of acetate and propionate.

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ОЦЕНКА ВЛИЯНИЯ NFC/NDF НА ПАРАМЕТРЫ РУБЦОВОГО ПИЩЕВАРЕНИЯ И РАЗНООБРАЗИЕ МИКРОБИАЛЬНОЙ ФЛОРЫ ТЕЛЯТ

В этом исследовании представлены результаты влияния различных уровней неструктурных углеводов (NFC)/нейтрально растворимого волокна сырой клетчатки (NDF) на параметры ферментации и микробиальный статус в рубце телят. В качестве объектов исследования выступали 30 месячных здоровых телят с массой 94,38±0,25 кг, которые были случайным образом разделены на 2 группы по 15 голов в каждой. Каждая группа получала 2 варианта общего рациона с одинаковым уровнем сырого протеина и различными отношениями NFC/NDF (1,35(A) и 0,80(D) соответственно). Эксперимент продолжался в течение 105 дней, из которых 15 дней составлял период адаптации и 90 дней учётный период. У 6 голов телят из каждой группы был проведён отбор содержимого рубца через 2 часа после утреннего кормления на 120, 150 и 180 день. В качестве основных оцениваемых показателей были: pH, количество летучих жирных кислот (VFA), концентрация NH3-N и микробный состав рубца. Результаты показали, что: 1) внесение различных уровней NFC/NDF не способствовало существенному изменению рН среды рубца и общего количества летучих жирных кислот (VFA) (P>0,05); 2) количество ацетата и NH3-N, а также соотношение ацетат/пропионат в группе А было значительно ниже, чем в группе D (P<0,05), а содержание пропионата в группе А превышало аналогичные показатели в группе D; 3) параметры микробного разнообразия рубцовой жидкости телят в группе А были значительно ниже, чем в группе D (Р<0,05); 4) уровень микробного разнообразия рубцовой жидкости телят показал, что доля содержания бактероидов от общего числа сиквенсов в группе А была значительно выше, чем в группе D (P<0,05), а количество Christensenellaceae R-7 и Prevotellaceae UCG-003 в группе D значительно превышало показатели в группе A (P<0,05), 5) с возрастом показатели индекса разнообразия (индекс Шеннона) более разнились в опытных группах (P<0.05), однако различий в микробном составе рубца между 2 груп-пами не было (P<0.05). Таким образом, добавление различных уровней NFC/NDF оказывает значительное влияние на вид и количество микроорганизмов рубца, а уровень рода микроорганизмов оказывает влияние на выход ацетата и пропионата.

Текст научной работы на тему «Effects of NFC/NDF in diet on rumen fermentation parameters and microbial flora diversity in calves»

Инновационное направление науки

UDC 636.084.1

7

Effects of NFC/NDF in diet on rumen fermentation parameters and microbial flora diversity in calves

Qian Zhao, Peng Zheng, Yaguang Tian, He Huang College of Animal Science and Technology, Northeast Agricultural University, People's Republic of China, Harbin

Annotation. This study assessed the effects of diets with different non-fiber carbohydrate (NFC)/neutral detergent fiber (NDF) levels on rumen fermentation parameters and bacterial community of male calves. Thirty 2-3 month- old healthy weaned male calves with 94.38±0.25 kg were randomly divided into 2 groups with 15calves each, and were fed 2 types of total mixed ratio diet with the same crude protein level and different NFC/NDF ratios (1.35 (A) and 0.80 (D)), respectively. The experiment lasted for 105 d, with 15 d of adaptation period and 90 d of formal period. Six calves in each group were selected to collect rumen fluid through the oral for 2 hours after morning feeding on the 120, 150 and 180 d. The pH, volatile fatty acids (VFA) and NH3-N concentration, and rumen microbial diversity of the rumen fluid samples were determined. The results showed that: 1) The dietary NFC/NDF levels had no significant effect on rumen fluid pH and total VFA concentration (P>0.05); 2) Acetate, NH3-N concentration and ace-tate/propionate ratio in group A were significantly lower than that in group D (P<0.05), propionate content in group A were significantly higher than that in group D (P<0.05); 3) The microbial diversity parameters of rumen fluid of calves in group A were significantly lower than that in group D (P<0.05); 4) At the phylum level, bacteriodetes proportion accounting for the total number of sequences of calves in group A was significantly higher than that in group D (P<0.05), Christensenellaceae R-7 and Prevotellaceae UCG-003 proportions accounting for the total number of sequences of calves in group D was significantly higher at the genus level than that in group A (P<0.05); 5) With the age increasing, the diversity indices except for Shannon index, were significantly different between the 2 groups (P<0.05), but there was no significant difference in rumen microbial composition between the 2 groups (P<0.05). In summary, the dietary NFC/NDF levels significantly affect the species and abundance of the rumen microorganisms, and the level of genus of microorganisms, finally affect the yield of acetate and propionate.

Key words: weaned beef calves; non-fiber carbohydrate/neutral detergent fiber ratio; rumen fermentation parameters; rumen microbial community.

INTRODUCTION

Ruminants rumen contains a variety of microorganisms, these microorganisms interact with each other, so that the rumen internal environment is in a relatively stable state. Microorganisms are colonized in vivo within 24 hours of birth of the calf, but the rumen microflora of calves aged 3 to 6 months has not been fully established, so the diet that calf is fed on this stage has an important influence on the rumen microflora [1]. Some studies had found that animal species, age, diet structure, and season had an impact on rumen microorganisms [2]. Boots et al. using PCR technology found that the number of Neocallimasti-gales and assemblages significantly affected by the diet structure [3]. Shanks et al. used metagenomic sequencing to find that feeding patterns also affected rumen microflora [4]. The effect of age on rumen microflora was greater with the un-weaned calf lactobacillus and a certain species of proteolytic bacteria accounting for the major part. Jami et al. through metagenomic sequencing and real-time fluorescence quantitative PCR technology on beef cattle study found that with the increase of age, strict anaerobic bacteria in the rumen gradually replaced by aerobic and facultative anaerobes, and with the beef cattle with the increase of age, rumen microflora became more and more perfect [5]. Li et al. also used metagenome sequencing to conclude that day age affected rumen microflora [6]. In addition, rumen fermentation parameters are important indicators of rumen health. Studies had found that, with the increase of concentrate coarse material ratio, the rumen pH decreased significantly and the concentration of NH3-N significantly

8 Инновационное направление науки

increased, but the content of total volatile fatty acids (TVFA) was not affected by the concentrate coarse material ratio [7-8]. However, some experts believe that the ration of feedstuff does not affect rumen pH [9]. The rumen is the most important digestive organ of ruminants. The diets affect the rumen fermentation parameters and microflora. However, there are few studies on the calves aged 3-6 months, so the suitable diets can not be determined. In this study, the rumen fermentation parameters and microbial diversity of weaned calf aged 3-6 months old with different levels of NFC/NDF were studied to investigate the changes of rumen fermentation and microbial flora.

MATERIALS AND METHODS

Experiment materials

The experiment uses single factor experiment design. Thirty calves of early weaned calves aged 23 months were selected and randomly divided into 2 groups with 15 in each group. The average body weight was 94.38±0.25 kg. According to the calf s body weight and nutrition requirements, the standard of cattle feeding in China (2004), the CP content is about 11.7 %. NFC/NDF levels were 1.35 (group A) and 0.80 (group D). The nutrient levels were shown in Table 1 (dry matter basis). Experiment period was 105 d (pre-test period 15 d, treatment period 90 d).

Item Group Treatment A Group Treatment D

Corn 43.62 30.03

Wheat bran 15.00 0.00

Soybean meal 2.90 2.57

Dry distiller's grains and their soluble

DDGS 15.00 15.00

Limestone 0.20 0.21

Calcium hydrogen phosphate 1.78 0.69

Premix 1.00 1.00

NaCl 0.50 0.50

Alfafa 20.00 35.00

Chinese wild rye 0.00 15.00

Total 100.00 100.00

Dry matter 91.80 91.58

Crude protein 16.34 16.38

Crude fat 3.71 3.82

Ash 7.57 7.44

Neutral detergent fiber 34.43 45.33

Acid detergent fiber 15.34 25.44

Calcium 1.05 1.14

Phosphorus 0.45 0.47

Metabolism/(MJ kg-) 11.20 9.79

NFC/NDF 1.35 0.80

The premix is provided the following per kg of the concentrate: VA 15 000 IU, VD 5 000 IU, VE 50 mg, Fe 90 mg, Cu 12.5 mg, Mn 60 mg, Zn 100 mg, Se 0.3 mg, I 1.0 mg, Co 0.5 mg. Data of dietary nutrient levels were measured values, methane energy was measured using SF 6 tracer method [10], ME=GE-FE-UE-CH 4 E.

Инновационное направление науки 9

Feeding and Management

The calves were weighed in an empty stomach in the early morning after wearing the ear tag. The calves were treated with deworming and placed in a single bar in the calf island (4.5 m*1.5 m). Each calf was provided sinks, trough, cleaned and disinfected once a week. Dry matter feed intake of 3.3 % by weight of the full mixed diet, daily feed intake of cattle per day before harvest to calculate the amount of daily feed intake.

Sample Collection and Determination

Rumen Fluid

Six calves were selected from each group, and were taken by oral vacuum for extracting rumen fluid at 120, 150 and 180 days of age, respectively. One part of rumen fluid aliquoted in two 10 mL centrifuge tube, stored at -20 0 for the determination of VFA and NH3-N concentration after 4 layers of sterile gauze filter. Unfiltered rumen fluid immediately put into liquid nitrogen for the determination of microorganisms.

Rumen pH Measurement

The pH was measured in time using a portable pH meter (testo-206-pH2).

Rumen Fluid Ammonia Nitrogen and Volatile Fatty Acid Concentration Determination

The concentration of NH3-N was measured by phenol-sodium hypochlorite colorimetric method [11], and the concentration of VFA was determined by gas chromatography [12].

Determination of Microbial Diversity

The DNA in the rumen contents was first extracted, PCR amplified and purified, then the Miseq library was constructed and sequenced. Then the index and bar code sequences distinguished each sample data and determine the length of the distribution of high quality sequence. Finally, OTU classification was performed on all the sequences, and the RDP Classifier algorithm was used to analyze the OTU representative sequences to obtain the species information of each community (for details, refer to the E.Z.N.A.TMSoil DNA Kit kit manufactured by Omega manufacturer).

Data Analysis

The experiment data were analyzed by one-way ANOVA and significance test used the MIXED process in SAS 8.1 software, and LSD was used for multiple comparison test when the difference was significant. P<0.05 indicated significant difference, 0.05<P<0.10 indicated significant difference.

RESULTS

Effect of Diet NFC/NDF on Rumen Fermentation Parameters of Calves

As shown in Table 2, the dietary NFC/NDF levels had no significant effect on the rumen pH of calves (P>0.05); the concentrations of NH3-N, acetic acid, and acetic acid/propionic acid ratio in group A were significantly lower than those in group D (P<0.05), while the propionic acid concentration in group A was significantly higher than that in group D (P<0.05), and TVFA and butyrate concentrations had no significant difference (P>0.05). NH3-N concentration was significantly affected by age (P<0.05), others indexes were not significantly affected by age (P>0.05). The concentration of propionic acid was significantly affected by the interaction between day age and the treatment (P<0.05), while the other indexes were not significantly affected by the interaction between the age and the treatment (P>0.05).

Table 2. Effects of diets with different NFC/NDF levels on rumen fluid fermentation parameters in bull calves from 120 to 180 days old

Item Treatment P-value of fixed effects

A D SEM Treatment Day TreatmentxDay

pH 6.55 6.83 0.068 0.1016 0.0760 0.3273

NH3-N 14.69b 20.96a 1.271 0.0021 0.0003 0.0694

TVFA 100.01 96.25 1.349 0.2391 0.5449 0.5651

Acetate 56.35 b 60.76 a 0.919 0.0328 0.2737 0.0664

Propionate 24.89 a 19.86 b 0.771 0.0010 0.6601 0.0201

Butyrate 12.93 13.95 0.615 0.4933 0.9118 0.6435

Acetate/Propionate 2.32 b 3.23 a 0.160 0.0058 0.9080 0.0601

10 Инновационное направление науки

In the same row, values without letter or with the same letter superscripts mean no significant difference (P>0.05), while with different small letter superscripts mean significant difference (P<0.05). The same as below.

Rumen Microbial Otus Basic Analysis

Rumen Microbial Diversity

The analysis of diversity of microbial OTUs by QIME software included Chaol, Observed-species, PD-whole-tree and Shannon indices. The coverage rate of each group was above 99%, it showed that the sequencing quantity of each sample is reasonable and can well reflect the species and structure diversity of microbial communities in the rumen. As shown in Table 3, each index of group D was significantly higher than that of group A (P<0.05). Except for the Shannon index, the other indexes were significantly affected by the age (P<0.05). The indexes were not significantly affected by the interaction between treatment and day-old (P>0.05).

Table 3. Effects of diets with different NFC/NDF levels on rumen microbial Diversity indices in bull calves from 120 to 180 days old

Item Treatment P-value of fixed effects

A D SEM Treatment Day Treatment X Day

OTUs 335.61 b 578.28 a 33.531 <0.0001 <0.0001 0.3424

Chaol 381.77b 615.33a 1.271 0.0002 <0.0001 0.1366

Goods coverage 0.985 b 0.991 a 0.001 0.0004 <0.0001 0.1352

Observed species 295.72 b 517.02 a 28.634 0.0001 <0.0001 0.1720

PD whole tree 34.00 b 47.69 a 2.262 0.0001 <0.0001 0.1002

Shannon 5.14 b 6.40 a 0.155 0.0002 0.1855 0.2731

Venn Diagram

The rumen microflora of calves fed on different NFC/NDF diets was analyzed using the shared and unique bacterial abundance in the bacterial community as shown in Figure 2. At the 97 % similarity level, a total of 1713 OTUs were produced in the two treatment groups. The numbers of OTUs in group A and group D were 1281 and 1596, respectively, and 1164 OTUs were shared in group 2 and 67.95 % of total OTU in group A, and the number of D group were respectively accounted for 6.83 % and 25.22 % of the total number of bacterial OTU.

A D

Unique objects: All = 1 713; SI = 1 281; S2 = 1 596

The left circle represents the group A (S1), and the right cir cle represents the group D (S2) Fig. 2 - The microbial diversity Venn of rumen microorganismin bull calves

Rumen Microbial Taxonomy Analysis

As shown in Table 4, at the Phylum level, the dominant bacteria in both groups A and D were Firmicutes, Bacteroidetes and Proteobacteria. The proportion of the total number of sequences accounted for more than 1%. The content of Bacteroidetes in group A was significantly higher than that in group D (P<0.05). The contents of Ficus and Fusarium were not significantly affected by dietary NFC/NDF

Инновационное направление науки 11

(P>0.05). At genus level, Prevotella was dominant in both A and D groups, but there was no significant difference between the two groups (P>0.05). The content of Prevotellaceae_UCG-003 and Christensenel-laceae_R-7 in group A was significantly lower than that in group D (P<0.05). The contents of Eubacte-riumcroprostanoligenes and Lachnospiraceae ND 3007 decreased with the decrease of dietary NFC/NDF (P=0.0875, P=0.0789, respectively). The content of Rikenellaceae_RC 9 gut increased with the decrease of dietary NFC/NDF (P=0.052 6). All the other indexes were not significantly affected by the level of dietary NFC/NDF (P>0.05). The indexes were not significantly affected by the interaction between age and treatment (P>0.05).

Treatment P-value of fixed effects

Item A D SEM Treatment Day Treatment X Day

Firmicutes 47.25 49.78 2.177 0.6590 0.8651 0.7779

Bacteroidetes 51.22 a 39.35 b 2.005 0.0239 0.4594 0.5396

Proteobacteria 1.16 2.89 0.523 0.2427 0.8929 0.3172

Ruminococcaceae NK 4 A 214 5.01 5.98 0.513 0.4983 0.4098 0.0891

Ruminococcus 2 4.06 2.05 0.455 0.1501 0.2529 0.0709

Eubacterium coprostanoligenes 4.84 2.23 0.542 0.0875 0.9905 0.7881

Lachnospiraceae ND 3007 1.99 0.29 0.346 0.0789 0.4135 0.4114

Lachnospiraceae NK 3 A 20 3.69 2.67 0.465 0.4257 0.1755 0.6248

Prevotella 1 21.76 17.23 2.093 0.3459 0.7444 0.9162

Prevotellaceae UCG-003 0.60 b 3.28 a 0.321 0.0004 0.9252 0.8712

Rikenellaceae RC 9 _gut 3.83 6.08 0.595 0.0526 0.1023 0.7870

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Christensenellaceae R -7 4.23 b 9.02 a 0.682 0.0044 0.6753 0.7043

unidentified 17.97 16.27 1.249 0.4931 0.4119 0.2128

Comparison of Rumen Microbiological Samples Rumen Microbial Similarity Analysis

UPGMA method for calf rumen microbiological DGGE pattern similarity analysis shown in Figure 3. The same treatment of calf rumen microbes except A511 non-polymerization together, other calves treated the same high similarity together to form two major branches. This result illustrated the effect of different NFC/NDF diets on rumen microbes.

■D721

■ D7II

D522

-D512 ■D422 -D432 D4I2

■ D232 D222 D212 -D221 D421 ■D4II

D2II - A5II

■ D731 D431 D231 -A53I

■ AI3I

■ A231

■ A221 -A211

• A521

■ A7I2 -A832

an::

■ A812 " A732

■ A532 A522

• A5I2

■ AI2I

■ Alll

A111, D211, A121, D421, A231, D431, Fig.

A211, A511, A512, A712 and A812are rumen fluid samples of calves in group A at 120days old, D411, D711, D212, D412 and D512are rumen fluid samples of calves in group D at 120days old; A221, A521, A522 and 822 are rumen fluid samples of calves in group A at 150 days old, D221, D721, D222, D422 and D522 are rumen fluid samples of calves in group D at 150 days old; A131, A531, A532, A732 and A832 are rumen fluid samples of calves in group A at 180 days old, D231, D731, D232, D432 and D532 are rumen fluid samples of calves in group D at 180 days old 3 - The UPGMA dendrogram for illustrative similarity of rumen microorganism in bull calf

12 Инновационное направление науки

Rumen Microbial Principal Component Analysis

Using UniFracPCA to study the difference of bacterial communities in the rumen of calves fed different levels of NFC/NDF diet, the distance between samples was measured by phylogenetic distance, and PCA could display this difference in PCA map in two- or three-dimensional space on. As shown in Figure 4, the contribution of the first principal component and the second principal component based on the UniFrac weighted principal coordinate analysis were 13.82 % and 9.85 %, respectively. Calves fed diets with different NFC/NDF levels, the rumen fluid samples showed obvious differences in bacterial community structure and could be well separated from each other. The samples fed the same diet could be well clustered together and the community similarity was high.

0.25

g 0,00

'r.

ОС

IN

У

^ -0.25

-0.50

-0.6 -0.4 -0.2 0 0.2 0.4 PCI (13.82%!

The dots represent the samples in group A, and the trianglesrepresent the samples in group D Fig. 4 - The PCA of rumen microorganism in bull calf

NMDS Analysis of Rumen Microorganisms

It can be seen from Fig. 5 that most of the samples of groups A and D were in their respective circles after eating different diets with different levels of NFC/NDF, and there was no intersection with each other, indicating that the feed differences in rumen microbiota after different NFC/NDF diets were observed.

0.6 0.4 0.2

z

-0.2 -0.4 -0.6

-15 -1.0 -0.5 0.0 0.5 NMDSI

The triangles represent the samples in group A, and the dots represent the samples in group D. NMDS. Nonmetric multi -dimensional scaling

Fig. 5 - The NMDS of rumen microorganism in bull calf

Инновационное направление науки 13

DISCUSSION

Effect of Diet NFC/NDF on Rumen Fermentation Parameters in Calves

Rumen fermentation can be reflected by the rumen fluid pH, and the rumen fluid pH was affected by the structure of the diet, the nutrient content of the diet, the salivary secretion, the flow velocity of the chyme, and the absorption of VFA by the rumen epithelium. The rumen pH was significantly reduced with increasing NFC levels [13-14]. Fermented carbohydrates contained in high-NFC diets were rapidly fermented by rumen microorganisms and produced large amounts of VFA and organic acids that decreased rumen pH. However, studies had shown that dietary NFC levels did not affect the rumen pH level [9]. In this experiment, the rumen of animals had not yet developed completely, the nutrients can not be completely digested and utilized, the total amount of volatile acids produced was limited, but also due to the absorption of the rumen epithelium and saliva buffer effect, rumen pH had no significant difference between groups. In addition, this experiment through the oral collection of rumen fluid, may be mixed with a small amount of saliva, that also affected the determination of pH results.

The nitrogenous material in the diet was the main source of ruminal NH3-N and its concentration was influenced by the rumen's degradation rate and absorption rate of nitrogenous substances [15], which can reflect the utilization of nitrogen by microorganisms. It had been found that the optimum concentration of NH3-N for growth of microorganisms was 6.3-27.5 mg • dL-1 [16]. The concentration of NH3-N in this experiment was within its range. Increasing the level of dietary NFC can increase the activity of microorganisms, and then improved the utilization and deamination of protein in diets, and finally increase dthe nitrogen metabolism. Previous research results showed that the nitrogen metabolism of calves in high NFC/NDF group was higher than that of other groups [17]. Liu et al. found that NH3-N concentration increased with the increase of dietary NFC/NDF levels [18]. The higher NFC content of diets provided more energy to microorganisms after decomposition, increased microbial activity and promoted microbial degradation of dietary protein. The study found that feeding high-NFC diets reduced rumen pH. Released ammonia was immobilized in the form of NH+4, and NH+4 was not easily absorbed through the rumen wall. Therefore, ammonia immobilized in the form of NH+4 increased and NH3-N concentration increased [19-20]. However, the results of this test were the opposite, probably because the concentration of NH3-N was limited by the requirements of the microorganisms on the fermentation substrate [20]. In addition, the difference between the animal species and the diet was also one of the reasons. The main product of rumen fermentation was VFA, which was produced mainly by fermenting carbohydrates in the diet. Studies had found that VFA provided 70% to 80% of the body's energy, acetic acid, propionic acid, and butyric acid accounted for about 95% of total volatile fatty acids, so acetic acid, propionic acid, and butyric acid concentration or its proportion can reflect the rumen fermentation [21]. The type of rumen fermentation was divided into acetic acid type, propionic acid type, and butyric acid type according to the ratio of acetic acid, propionic acid, and butyric acid. The acetic acid type and propionic acid type were generally determined by the ratio of acetic acid/propionic acid [22]. Studies had found that TV-FA increased both by reducing the fiber content in the diet or by increasing the concentrate level [22-23], since high-concentrate diets provided more fermentation substrate for microbial fermentation [22]. Studies had also found that dietary concentrate coarse material ratio had no significant effect on TVFA, but feed concentrate levels increased, the proportion of propionic acid increased, crude material levels increased, the proportion of acetic acid increased [24-25]. Increasing the level of NFC in the diet resulted in a predominance of amylo-lytic bacteria in the rumen, while fibrolytic bacteria predominance increased the level of crude material [26]. In this experiment, with the decrease of dietary NFC/NDF level, the proportion of acetic acid in TVFA increased while the proportion of propionate decreased, which led to a significant increase of acetic acid/propionic acid ratio, which indicated that the decrease of dietary NFC content could promote the type of rumen fermentation from the propionic acid to acetic acid type transition. Feeding more concentrate, increasing the proportion of soluble carbohydrates, with the same feed intake, can promote calf weight gain [27]. The rumen development of calves aged 3-6 months still needed to be improved. The digestibility of fibrous material was weaker than that of adult cattle, and the ability of supplying energy by fiber digesting products was lower. It was more suitable for feeding ration with higher proportion of concentrate.

14 Инновационное направление науки

Otus Basic Analysis Results

Although the diversity index did not directly reflect which microbial communities the samples consist of, it can reflect the diversity of microbial community types and structures, that was the abundance of microorganisms. The Venn diagram can intuitively display the uniqueness of each sample at the similar level of 97%. The number of OTUs shared can be very intuitive to see whether the samples were different.

Taxonomy Analysis Results

Qin et al. and Ley et al. showed that the predominant microflora in rumen micro-organisms of ruminants was Firmicutes and Bacteroidetes, and its dominant position did not change with changes in dietary fiber and starch levels [28-30]. This study got the same result. It showed that the degradation of feed had an important role. Bacteroidetes play an important role in the degradation of non-fibrous materials, while Fibrous bacteria mainly decomposed fibrous substances [31]. The production of propionic acid increased with the fermentation of non-fibrous material by rumen microorganisms. The type of rumen fermentation was mainly propionic acid. However, the concentration of acetic acid in the fiber increased after fermentation, and the type of rumen fermentation was acetic acid. In this study, with the reduction of dietary NFC level, the content of Firmicutes decreased but the difference was not significant. Our previous study found that NDF apparented digestibility of 1.35 group was higher than 0.80 group [27], probably because other bacteria digested it. Studies had found that about half of the rumen fibrinolytic enzymes came from protozoa [32]. At the same time, the content of Bacteroidetes in 1.35 group was significantly higher than that of 0.80 group, with high apparent digestibility of non-fiber material and apparent digestibility of organic matter, which eventually promoted calf growth [27]. Kim et al. and Petri et al. found Compared with beef cattle fed high grain ration, the content of Bacteroidetes in beef manure and rumen decreased significantly with high coarse feed [33-34]. Ellison et al. and Han were attributed to differences in animal species and sequencing regions [35-37]. Tajima et al. found that the content of Prevotella decreased with the increase of dietary NDF content, indicating that it played an important role in the decomposition of the concentrate [38]. Jami et al. found that the content of Prevotella in the rumen was unaffected by the diet structure and had always been in a dominant position, basically consistent with the results of this experiment [30]. Description Prevotella was a rumen predominant microflora in calves. However, the content of Prevotel-laceae_UCG-003 in group 0.80 was significantly higher than that in group 1.35, probably due to the poor palatability of diet in group 0.80 and the pick-up of calf. Studies had found that when the body fed high-concentrate diets, preferred choice of coarse feed; and when feeding high-coarse feed, preferred feed concentrate [39]. Prevotella mainly degraded the starch in the diet [40], so propionic acid content increased after feeding 1.35 group of diets in this experiment. Huo et al. found that the increase of Prevotella in the rumen after the goats fed hay may be due to differences in animal species and diets [41]. Christensenellaceae _R - 7 was mainly related to the production of acetic acid, so the acetic acid content of this experiment was higher than that of 0.80 group. Rumen pH affected the activity of microorganisms. Too low pH was not conducive to the survival of acid-fast bacteria such as fibrolytic bacteria, but the pH was not conducive to the survival of acid-fast bacteria such as starch-degrading bacteria. However, the rumen pH in this experiment was not significantly different between the groups, not the impacted on the activity of the bacteria, so our previous study found that 1.35 groups of calves fed diets of nutrients of the highest digestibility of nutrients, and the fastest growing [27]. CONCLUSION

Feeding high NFC diet decreased the variety and abundance of rumen microorganisms in calves, and also affected the content of some bacteria in the genus, which ultimately affected the proportion of volatile fatty acids in the rumen fluid. In rumen fluid of 120-180 day-old calves, the dominant bacteria at Phylum level were both Firmicutes and Bacteroidetes, and the dominant bacteria at Genus level was Prevotella.

ACKNOWLEDGEMENT

This research was financially supported by the Science Foundation (CARS-35-04), National Natural Science Foundation of China (31472131) and Scientific And Technological Innovation Talent Project Of Harbin Science And Technology Bureau (2016RAQXJ083).

Инновационное направление науки 15

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Инновационное направление науки 17

AUTHOR INFORMATION

Qian Zhao, Doctor, Associate Professor, Northeast Agriculture University, 150030, Harbin, People's Republic of China, +86 0451-55190264, [email protected]

Peng Zheng, Doctor, Lecturer, Northeast Agriculture University, Animal Science and Technology College, +86 0451-55190364, [email protected]

Yaguang Tian, Master, Expert Experimenter, Northeast Agriculture University, Animal Science and Technology College, 150030, Harbin, People's Republic of China, +86 0451-55190464, [email protected]

He Huang, Doctor, Associate Professor, Northeast Agriculture University, Animal Science and Technology College, 150030, Harbin, People's Republic of China, +86 0451-55191705, [email protected]

Поступила в редакцию 14 марта 2018 года

UDC 636.084.1

Цянь Чжао, Пэн Чжэн, Ягуан Тянь, Хе Хуан

Колледж зоотехнии и технологии, Северо-Восточный сельскохозяйственный университет, Китайская Народная Республика, Харбин, e-mail: [email protected] Оценка влияния NFC/NDF на параметры рубцового пищеварения и разнообразие микробиальной флоры телят

Аннотация. В этом исследовании представлены результаты влияния различных уровней неструктурных углеводов (NFC)/нейтрально растворимого волокна сырой клетчатки (NDF) на параметры ферментации и микробиальный статус в рубце телят. В качестве объектов исследования выступали 30 месячных здоровых телят с массой 94,38±0,25 кг, которые были случайным образом разделены на 2 группы по 15 голов в каждой. Каждая группа получала 2 варианта общего рациона с одинаковым уровнем сырого протеина и различными отношениями NFC/NDF (1,35(A) и 0,80(D) соответственно). Эксперимент продолжался в течение 105 дней, из которых 15 дней составлял период адаптации и 90 дней - учётный период. У 6 голов телят из каждой группы был проведён отбор содержимого рубца через 2 часа после утреннего кормления на 120, 150 и 180 день. В качестве основных оцениваемых показателей были: pH, количество летучих жирных кислот (VFA), концентрация NH3-N и микробный состав рубца. Результаты показали, что: 1) внесение различных уровней NFC/NDF не способствовало существенному изменению рН среды рубца и общего количества летучих жирных кислот (VFA) (P>0,05); 2) количество ацетата и NH3-N, а также соотношение аце-тат/пропионат в группе А было значительно ниже, чем в группе D (P<0,05), а содержание пропио-ната в группе А превышало аналогичные показатели в группе D; 3) параметры микробного разнообразия рубцовой жидкости телят в группе А были значительно ниже, чем в группе D (Р<0,05); 4) уровень микробного разнообразия рубцовой жидкости телят показал, что доля содержания бактероидов от общего числа сиквенсов в группе А была значительно выше, чем в группе D (P<0,05), а количество Christensenellaceae R-7 и Prevotellaceae UCG-003 в группе D значительно превышало показатели в группе A (P<0,05), 5) с возрастом показатели индекса разнообразия (индекс Шеннона) более разнились в опытных группах (P<0.05), однако различий в микробном составе рубца между 2 группами не было (P<0.05). Таким образом, добавление различных уровней NFC/NDF оказывает значительное влияние на вид и количество микроорганизмов рубца, а уровень рода микроорганизмов оказывает влияние на выход ацетата и пропионата.

Ключевые слова: телята, соотношение неструктурных углеводов/нейтрально растворимого волокна сырой клетчатки, параметры ферментации рубца, микробный состав рубцовой жидкости.

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