Research Article Received: 8 December 2014

Revised: 12 March 2015

Accepted article published: 21 May 2015

Published online in Wiley Online Library: 19 June 2015

(wileyonlinelibrary.com) DOI 10.1002/jsfa.7271

Effects of applying molasses, lactic acid bacteria and propionic acid on fermentation quality, aerobic stability and in vitro gas production of total mixed ration silage prepared with oat–common vetch intercrop on the Tibetan Plateau Lei Chen,a Gang Guo,b Xianjun Yuan,a Jie Zhang,a Junfeng Lia and Tao Shaoa* Abstract BACKGROUND: The objective of this study was to investigate the effect of molasses, lactic acid bacteria and propionic acid on the fermentation quality, aerobic stability and in vitro gas production of total mixed ration (TMR) silage prepared with oat–common vetch intercrop on the Tibetan plateau. TMR (436 g kg−1 dry matter (DM)) was ensiled with six experimental treatments: (1) no additives (control); (2) molasses (M); (3) an inoculant (Lactobacillus plantarum) (L); (4) propionic acid (P); (5) molasses + propionic acid (MP); (6) inoculant + propionic acid (LP). RESULT: All silages were well preserved with low pH (< 4.19) and NH3 -N contents, and high lactic acid contents after ensiling for 45 days. L and PL silages underwent a more efficient fermentation than silages without L. P and MP silages inhibited lactic acid production. Under aerobic conditions, M and L silage reduced aerobic stability for 15 and 74 h, respectively. All silages that had propionic acid in their treatments markedly (P < 0.05) improved the aerobic stability. After 72 h incubation, all additives treatments increased (P < 0.05) the 72 h cumulative gas production and in vitro DM digestibility (IVDMD) as compared with the control. L treatment decreased (P < 0.05) in vitro neutral detergent fibre degradability. CONCLUSIONS: Our findings show that TMR prepared with oat–common vetch intercrop can be well preserved. Although propionic acid is compatible with lactic acid bacteria, and when used together, they had minor effects on fermentation, aerobic stability and in vitro digestibility of TMR silage prepared with oat–common vetch intercrop. © 2015 Society of Chemical Industry Keywords: additives; aerobic stability; fermentation quality; in vitro gas production; oat–common vetch intercrop; total mixed ration silage

INTRODUCTION

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The Tibetan Plateau is one of the five most important livestock production areas in China with 82.1 million ha of grassland representing about 21% of the total natural grassland in the country.1 Dairy cows have long been fed as domestic animals and played a crucial role in alpine meadow of the Tibetan Plateau ecosystem and pastoral industry. They provide > 90% of milk and milk products for consumption by the local people. Also, their dung is one of the main sources of fertiliser and fuel in these cold highlands. However, the development of dairy cows industry in Tibet is relatively backward. During the past couple of years, the local government has encouraged herders to use total mixed ration (TMR) technology to feed dairy cows. Although the herders receive benefits from using this feeding technology, they found that normal TMR production was unsuitable for small and medium-size dairy cows’ feedlots as they needed to pay much more for buying the machines and machine maintenance as well raw material J Sci Food Agric 2016; 96: 1678–1685

detection of TMR. In this case, some Chinese scientists proposed that uniformly producing TMR silage and then dispensing the silage to each dairy farm could be a good solution for feeding cost. In recent years, the TMR silage feeding system has been widely accepted and used by herders in Tibet, particular based on local grass as roughage. TMR silage has the advantages of stabilising rumen function and avoiding self-selection by animals,2 and



Correspondence to: Tao Shao, Institute of Ensiling and Processing of Grass, Nanjing Agricultural University, Nanjing 210095, China. E-mail: [email protected]

a Institute of Ensiling and Processing of Grass, Nanjing Agricultural University, Nanjing 210095, China b College of Animal Science and Veterinary Medicine, Shanxi Agricultural University, Taigu 030801, China

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Effects of molasses, lactic acid bacteria and propionic acid on silage fermentation TMR silage can resist aerobic deterioration that is advantageous to long-distance transportation.3 Moreover, the TMR silage feeding system can alleviate the intensive grazing burden posed by livestock on the fragile alpine rangeland ecosystems. Oat (Avena sativa) and common vetch (Vicia sativa) are typically cultivated forages in Tibet. Both have high nutritive value and strong resistance against the harsh environment in which they grow. Farmers have used the practice of intercropping to plant oat and common vetch in Tibet for many years. The use of pea–cereal mixtures is preferable over that of pea or cereal monocultures, as it will result in higher forage yields and reduce the need for industrial fertilisers due to the nitrogen fixation of the legumes.4 Molasses has been used extensively as a fermentation stimulant, since it could provide fermentable substrates for lactic acid bacteria (LAB). Inoculants containing homomentative LAB have prepared as silage additives to provide sufficient LAB to ensure rapid and intensive fermentation resulting in improving DM and nutrient retention of silage.5 Propionic acid has been used to inhibit yeasts that assimilate lactic acid or water soluble carbohydrate (WSC) when silages are exposed to air; it is an effective inhibitor of aerobic deterioration of silage.6 However, this additive was less efficient on fermentation. Thus, farmers are often faced with a decision to use one or the other type of additive, realising that each often has a shortcoming. Feedback from the field suggests that some producers have applied both propionic acid additives and molasses or microbial inoculants on the same TMR silage, but there is no published information to support this practice. Previous studies have compared a range of legume–cereal bi-crops in terms of their fermentation characteristics, in vitro digestibility and aerobic stability, and generally shown positive effects on fermentation patterns and resultant silage nutritive and aerobic stability.7 – 9 However, to the best of our knowledge, few studies have investigated the effects of currently available additives on fermentation quality, in vitro digestibility and aerobic stability of TMR silage prepared with oat–common vetch intercrop as roughage under the Tibetan Plateau conditions. The objectives of this study were to determine the effects of molasses, lactic acid bacteria and propionic acid on the fermentation, aerobic stability and in vitro digestibility of TMR silage based on grass as roughage.

MATERIAL AND METHODS

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Table 1. Ingredient, chemical composition and pre-ensiled characteristics of total mixed ration (TMR) Item

TMR

Ingredient composition (g kg−1 DM) Oat Common vetch Mixed concentrate Chemical composition (g kg−1 DM) DM CP EE NDF ADF Hemicellulose Ash NFC Pre-ensiled characteristics pH WSC (g kg−1 DM) Buffering capacity (mEq kg−1 DM) Lactic acid bacteria (log10 CFU g−1 FM) Yeast (log10 CFU g−1 FM)

380 180 440 436 178 62.5 385 153 232 77.7 297 5.54 110 285 5.52 2.96

DM, dry matter; CP, crude protein; EE, ether extract; NDF, neutral detergent fibre; ADF, acid detergent fibre; WSC, water soluble carbohydrates; FM, fresh matter. Mixed concentrate = 7.5% crack corn, 20% rape cake meal, 20% cotton seed, 27.5% DDGS, 20% wheat bran, 5% vitamin–mineral. NFC (non-fibrous carbohydrate) = 100 − CP − aNDFom − EE − ash.

20% wheat bran, 5% vitamin–mineral). TMR (436 g kg−1 DM) was ensiled with six different treatments: (1) no additives (control); (2) molasses (sugarcane; 705 g kg−1 DM) were from Zhenjie Sugar Co., Ltd. (Lianyungang, Jiangsu, China) addition at 4% (M); (3) lactic acid bacteria (Lactobacillus plantarum, Ecosyl MTD/1, Ecosyl Products Ltd., Stokesley, North Yorkshire, UK) addition at 106 colony-forming units (CFU) g−1 (L); (4) propionic acid addition at 0.3% (P); (5) 4% molasses + 0.3% propionic acid addition (MP); and (6) 106 CFU g−1 lactic acid bacteria + 0.3% propionic acid addition (LP) on a FM basis of TMR. Additives were diluted with deionised water to an equivalent of 10 mL kg−1 fresh weight and spray mixed into the TMR samples. An equal volume of deionised water was applied to the control TMR. Silages were prepared using a small-scale system of silage fermentation. The TMR was ensiled in triplicate for each treatment in 1 L laboratory polyethylene silos (10 cm in diameter and 20 cm in length), at approximately 760 g (fresh weight), followed by being sealed with a screw top and plastic tapes, and then kept at the ambient temperature. These silos were opened on day 45 after ensiling, and then subjected to an aerobic stability test for 12 days. Chemical and microbiological analyses Fresh forage and pre-ensiled and ensiled TMR were analysed for chemical and microbiological composition. The DM content of unensiled forage samples and silage samples were determined by drying the samples at 65∘ C for 48 h to a constant mass, and then ground to pass through a 2 mm screen for later analysis. Dry matter, crude protein (CP) and ether extracts (EE) were determined according to Association of Official Analytical Chemists guidelines.10 The WSCs were determined by colorimetric after reaction with

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Silage preparation Oats (Avena sativa L. cv. Qingyin No.1) and common vetch (Vicia sativa L. cv. Ximu No.324) were intercropped at respective seed rates of 91 and 46 kg ha−1 on 18 May 2012 in the experimental field of the Grassland Station of Rikaze (29.27∘ N, 88.88∘ E, Tibet, China) on a fertilised (30 000 kg ha−1 cow dung). The site has an average annual rainfall of 420 mm, an annual average temperature of 6.3∘ C and 207 frost days annually. The pH of the silty loam soil was 8.1, total N content of 0.54 g kg−1 , total potassium content of 5.75 g kg−1 , total P content of 0.46 g kg−1 . The intercrops were harvested by hand using a sickle 90 days after sowing. Oats was at the milk stage (311 g kg−1 DM) and common vetch was at podding stage (298 g kg−1 DM). Crops were cut at approximately 5 cm above the ground, and then chopped with a conventional forage harvester to a length of 2–3cm and sampled to determine the chemical composition. The proportion of oat and common vetch in the intercrops on a fresh matter (FM) basis was 2:1. As shown in Table 1, TMR was prepared with oat–common vetch intercrops and mixed concentrate (7.5% crack corn, 20% rape cake meal, 20% cotton seed, 27.5% distillers dried grains with soluble (DDGS),

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anthrone reagent.11 The methods of Van Soest et al.12 were used for analysis of neutral detergent fibre (NDF) and acid detergent fibre (ADF) and the analyses were sequential. Amylase and sodium sulfite were used in the NDF analysis and the results of NDF and ADF were expressed on a DM basis exclusive of residual ash. Hemicellulose was calculated as NDF minus ADF. To measure fermentation indices of pre-ensiled and ensiled TMR, the top and lower layers of each TMR or TMR silage in the silo were discarded, and sampled 35 g from the middle layer that was blended with 75 mL of deionised water extracted at 4∘ C for 24 h. Then, the extracts were filtered through two layers of cheesecloth and a filter paper (Xinhua Co., Hangzhou, China). The filtrates were used for determining pH, buffering capacity (BC), ammonia-N (NH3 -N), lactic acid (LA) and volatile fatty acids (VFAs) contents. The pH of the silage was measured with a glass electrode pH meter (HANNA pH 211; Hanna Instruments Italia Srl, Villafranca Padovana, Italy). Buffering capacity was determined by the hydrochloric acid–sodium hydroxide method of Playne and McDonald.13 Ammonia-N, lactic acid and volatile fatty acids were determined using the method of Shao et al.14 To assess the quality of the silage, we calculated the V-score from the NH3 –N/Total N and VFAs concentrations.15 The mixed samples (10 g) were blended with 90 mL of sterilised water, and serially diluted in sterilised water. Enumeration of yeasts and LAB was done from the fresh forage and silages using the method of Guo et al.16 All the microbiological data were log10 transformed.

and filter bag using the methods described previously. Rate and extent of gas production was determined for each feed by fitting gas production data to the non-linear equation Y = b (1 − e−ct ),19 where Y is the volume of gas produced at time t, b is the potential gas production (mL), and c is the gas production rate constant. Parameters b and c were estimated by an iterative least square method using a non-linear regression procedure of the statistical analysis systems.20 Metabolisable energy (ME, in MJ kg−1 DM) was estimated using 24 h net gas production of the feeds as described Menke et al.21 ME = 2.20 + 0.136GP + 0.0574CP + 0.002859EE2 , where GP is 24 h net gas production (mL 200 mg−1 DM), CP is crude protein (% DM), and EE is ether extract (% DM).

Aerobic stability test After a silo was once thoroughly emptied, half (by weight) of the content from the middle layer in the silo was put into a polythene bottle (500 mL capacity) without compaction. The top was left uncovered and a thermocouple wire was placed in the centre of the silage mass, and double layers of cheesecloth was placed over each container to prevent drying and contamination, but allow penetration of air. The thermocouple wires were connected to data loggers (MDL-1048A; SMOWO Co., Ltd, Shanghai, China) that recorded the temperature every 30 min for 12 days. The bottles were kept in a room maintained at 25∘ C. Spoilage was considered to have occurred if the difference between silage and surrounding air reached 2∘ C.17 Triplicate silages of each treatment were sampled to determine pH values, lactic acid and WSC levels as well as yeast counts using the methods described previously at 0, 6, 9 and 12 days after aerobic exposure.

RESULTS

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In vitro gas production Samples (200 mg dry weight) were put into filter bags (F57; ANKOM Technology, Macedon, NY, USA) that were previously washed with acetone, dried at 55∘ C for 24 h and weighed. Rumen fluid was obtained from various locations within the rumen, composited and strained through two layers of cheesecloth under CO2 and then immediately transported to the laboratory, and kept at 39∘ C in a water bath while continually flushed with CO2 . Then the rumen fluid was mixed with a buffer solution as described by Menke and Steingass,18 which was maintained in a water bath at 39∘ C, and combined. All handling was under continuous flushing with CO2 . Readings of gas production were recorded before incubation (0 h) and 2, 4, 12, 24, 48 and 72 h after incubation. After the in vitro digestion process, samples were gently rinsed with cold tap water and dried at 65∘ C for 48 h to determine in vitro dry matter digestibility (IVDMD). The in vitro neutral detergent fibre (IVNDFD) was determined by analysing the digested sample

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Statistical analyses Data on fermentation quality and in vitro characteristic of ensiled TMR were analysed using the mixed model procedure (PROC MIXED) of SAS20 with treatments as fixed effect. In the aerobic stability test, silage pH, lactic acid, WSC and yeast data were analysed as repeated measures. Several covariance structures were tested. According to the Akaike’s information criteria (AIC), the covariance structure that gave the best fit to set was unstructured (UN) covariance structure. Statistical difference between means was determined by Tukey’s multiple comparison. Differences were considered significant when P < 0.05.

Chemical and microbial composition of materials and TMR before ensiling The ingredient, chemical composition and characteristics of TMR before ensiling are presented in Table 1. The DM content of TMR was 436 g kg−1 . The WSC content was 110 g kg−1 DM. The BC and CP contents of mixture were 285 mEq kg−1 DM and 178 g kg−1 DM, respectively. The numbers of epiphytic LAB on TMR were more than 1.0 × 105 CFU g−1 FM, and yeasts were less than 1.0 × 103 CFU g−1 FM. Fermentation quality of TMR silages after 45 days of ensiling The six TMR silages were well preserved as indicated by low pH values and NH3 -N/TN ratios, high lactic acid contents as well as V-scores (Table 2). There was a significantly (P < 0.05) positive effect of P, MP and LP treatments on DM recovery. The pH values of silages were not affected by M and P addition, while MP addition decreased (P < 0.05) the pH value as compared with the control silage. L and LP were more effective (P < 0.05) at reducing silage pH than MP. L and LP silages had higher (P < 0.05) lactic acid contents than the control silage. In contrast, silages treated with P and MP resulted in a marked decrease (P < 0.05) in the contents of lactic acid. L, MP and LP silages had lower (P < 0.05) acetic acid contents than the control silage, and LP silage had the lowest (P < 0.05) acetic acid content. Both of L and LP silages had higher (P < 0.05) lactic acid/acetic acid ratios than all the other silages. P application increased propionic acid (P < 0.05) content. The butyric acid content was higher (P < 0.05) in M silage than other silages, while that was not different among other treatments. The residual WSC content of the MP silage was higher (P < 0.05) than that of the control silage. NH3 -N/TN ratio was similar in the control and M silages, while silages treated with L, P and LP had lower (P < 0.05) NH3 -N/TN ratios than control silage. The counts of LAB in all silages were as high as 106 CFU g−1 FM, and L and PL silage had

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J Sci Food Agric 2016; 96: 1678–1685

Effects of molasses, lactic acid bacteria and propionic acid on silage fermentation

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Table 2. Fermentation characteristics of total mixed ration silages after 45 days of ensiling Treatments Item

Control

DM recovery (g kg−1 ) pH Lactic acid (g kg−1 DM) Acetic acid (g kg−1 DM) Lactic acid/acetic acid Propionic acid (g kg−1 DM) Butyric acid (g kg−1 DM) WSC (g kg−1 DM) NH3 -N/total-N (g kg−1 ) Lactic acid bacteria (log10 CFU g−1 FM) Yeast (log10 CFU g−1 FM) V-score

96.4b 4.19a 62.1c 10.4ab 5.99b 0.22b 0.04b 59.4bc 51.7a 6.72b 1.84a 93bc

M

L

97.4ab 4.17ab 61.0cd 10.7a 5.69b 0.26b 0.21a 64.0ab 52.6a 6.71b 1.75a 91cd

P

97.5ab 3.96c 76.6a 8.06d 9.54a 0.22b 0.02b 56.3c 40.0bc 7.29a 1.00c 95a

MP

97.6a 4.16ab 52.9e 9.48bc 5.58b 4.86a 0.03b 63.7ab 37.9c 6.07c 0.75c 90d

98.3a 4.12b 57.8d 9.19cd 6.29b 4.72a 0.02b 68.2a 47.6ab 6.17c 1.59ab 91cd

LP 98.1a 3.94c 70.3b 6.79e 10.4a 4.56a 0.01b 62.9b 37.8c 7.53a 1.43b 93b

SEM 1.73 0.02 1.92 0.34 0.48 0.55 0.02 1.03 1.64 0.14 0.10 0.43

P

Effects of applying molasses, lactic acid bacteria and propionic acid on fermentation quality, aerobic stability and in vitro gas production of total mixed ration silage prepared with oat-common vetch intercrop on the Tibetan Plateau.

The objective of this study was to investigate the effect of molasses, lactic acid bacteria and propionic acid on the fermentation quality, aerobic st...
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