Ensiling results of different forage types usually differ. For instance, legume silages usually contain more acetic acid, butyric acid and ammonia-N and are aerobically more stable than maize silages (Muck & O’Kiely, 1992; O’Kiely & Muck, 1992; Wilkinson & Davies, 2013). However, as to what extent these differences are related to chemical or microbial composition is yet unknown. In recent work by Mogodiniyai Kasmaei et al. (2015), a new ensiling methodology was introduced that enables sterilization of forages and inoculation with original microfloras. In the present experiment, we have further utilized this methodology to separate the confounding effects of microbial and chemical composition on silage fermentation and aerobic stability

Mogodiniyai Kasmaei, Kamyar

Spörndly, Rolf

Udén, Peter

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                                                                                                                                                                     Proceedings of the 6th Nordic Feed Science Conference, Uppsala, Sweden                                                      Institutionen för husdjurens Rapport 291 utfodring och vård Report  Swedish University of Agricultural Sciences Uppsala 2015 Department of Animal Nutrition and Management ISSN 0347-9838  ISRN SLU-HUV-R-291-SE  Published by: Organising committee of the 6th Nordic Feed Science Conference Department of Animal Nutrition and Management Swedish University of Agricultural Sciences (SLU) SE- 753 23 Uppsala, Sweden  Copyright © 2015 SLU  All rights reserved. Nothing from this publication may be reproduced, stored in computerised systems or published in any form or any manner, including electronic, mechanical, reprographic or photographic, without prior written permission from the publisher (SLU).  The individual contributions in this publication and any liabilities arising from them remain the responsibility of the authors.  Organising Committee: Peter Udén Rolf Spörndly Torsten Eriksson Marie Liljeholm  Editor in chief: Peter Udén  Editorial board: Torsten Eriksson Rolf Spörndly Ingemar Olsson Thomas Pauly Bengt-Ove Rustas Kamyar Mogodiniyai Kasmaei  Margareta Emanuelson Cecilia Kronqvist  Printed by: SLU Repro SE-75007 Uppsala, Sweden  Distributed by: Department of Animal Nutrition and Management, Box 7024, SE-75323 Uppsala, Sweden  www.slu.se/husdjur-utfodring-vard   QR code reader for accessing conference posters can be downloaded at: http://app.scanlife.com/appdownload/dl  Proceedings from the Nordic Feed Science conferences are indexed and archived in CAB Abstracts since 2010    Feed conservation and processing Separating the effects of pre-ensiled chemical and microbial composition on silage fermentation and aerobic stability K. Mogodiniyai Kasmaei, R. Spörndly & P. Udén                                                            Department of Animal Nutrition and Management, Swedish University of Agricultural Sciences, Box 7024, 750 07 Uppsala, Sweden Correspondence: Kamyar.mogodiniyai.kasmaei@slu.se  Introduction Ensiling results of different forage types usually differ. For instance, legume silages usually contain more acetic acid, butyric acid and ammonia-N and are aerobically more stable than maize silages (Muck & O’Kiely, 1992; O’Kiely & Muck, 1992; Wilkinson & Davies, 2013). However, as to what extent these differences are related to chemical or microbial composition is yet unknown. In recent work by Mogodiniyai Kasmaei et al. (2015), a new ensiling methodology was introduced that enables sterilization of forages and inoculation with original microfloras. In the present experiment, we have further utilized this methodology to separate the confounding effects of microbial and chemical composition on silage fermentation and aerobic stability.  Materials and Methods  Samples used were one second-cut of a mixed timothy (Phleum pratense)-meadow fescue (Festuca pratensis) sward harvested at early maturity (grass), one second-cut red clover (Trifolium pratense) (clover) at late flowering and one whole-crop maize (Zea mays) (maize) at early dent. Perennial forage samples were collected from fields around Uppsala, Sweden (59°51′N, 17°37′E) and maize sample was collected from southern Sweden (55°41′N, 14°10′E). Samples were collected in the autumn of 2013. The perennial forage samples were chopped to a length of 3-5 cm by a stationary chopper and the maize sample was chopped by a precision chopper before being frozen at -20°C.   Sampling procedure An amount of 3 kg of frozen samples was divided into two replicates which were thawed at room temperature before sampling of 150 g for microbial isolation and 500 g for drying at 60°C in a forced draught oven for 18 h. Sterilization The dried samples were milled on a hammer mill to pass a 1-mm screen before weighing 130 g in glass beakers. Samples covered loosely with aluminium foil were sterilized by the heating procedure described by Mogodiniyai Kasmaei et al. (2015), i.e. heating at 60°C for 3 h followed by heating at 103°C for 15 h in a forced draught oven. Thereafter, samples were tightly sealed with aluminium foil and were transferred into a desiccator to reach the room temperature.  Microbial isolation procedure A volume of 900 mL of 0.25-strength Ringer solution fortified with Tween®80 (Merck KGaA, Darmstadt, Germany) at 0.5 mL/L (O’Brien et al., 2007) was added to 150-g samples. The suspensions were kept on a bench for 30 min before being pummelled for 2 min in a laboratory stomacher (Seward 3500, Seward Ltd, Worthing, UK). Thereafter, an amount of 750 mL of the microbial solution, accounting for 75% of the total volume of Ringer solution and sample water, was centrifuged at 15,500 g for 90 min. The supernatant was discarded and Proceedings of the 6th Nordic Feed Science Conference                                                      55  Feed conservation and processing the pellet was re-suspended in 15 mL sterile 0.25-strength Ringer solution and stored at 4°C overnight.  Reconstitution and ensiling To estimate the amount of forage fresh matter corresponding to the isolated microbes two assumptions were made: i) microbial population was completely removed from each 150-g forage sample and evenly distributed into the Ringer solution after stomaching, ii) based on our preliminary observations (data not shown), recovery proportion of microbial population after centrifugation was considered to be 90%. Further, half the hypothetical ratio of microbes:fresh matter in original forage samples was targeted at reconstitution and all samples were reconstituted to a DM content of 40%.  The microbial isolates (n=6) were each divided into 3 equal volumes. The 5-mL inocula were added to the calculated amounts of sterilized DM so that each forage type received each of the three kinds of inoculum. Final reconstitution was carried out with addition of sterile distilled water. An amount of ~65 g was then put in sterile glass tubes (20 and 3 cm in length and inner diameter, respectively) and sealed with water locks. Silos were kept at 20±2°C for 71 day. Aerobic stability test An amount of 30 g of silage was placed in glass filter crucibles. The crucibles were insulated in foam polyethylene insulation pipe (15 mm wall thickness). Pipes were covered with aluminium foil and the samples were kept at 20°C with sample temperature being recorded at 2 h intervals for 8 days. Chemical analyses The sterilized forage samples were subjected to N determination by the Kjeldahl method, with Cu as a catalyst. Extracted juice was analyzed for short chain organic acids and alcohols by HPLC and ammonia-N by flow injection analysis. The pH of silage and aerated silage samples was measured also on the extracted juice by a laboratory pH-meter (Metrohm 654, Metrohm AG, Herisau, Switzerland). The DM content of the silage samples were estimated after drying at 103°C and corrected for volatiles as described by Mogodiniyai Kasmaei et al. (2015). Statistical analyses The effect of inoculum type (n=3), forage type (n=3) and their interactions on fermentation quality and aerobic stability variables was tested by the General Linear Model procedure. Total number of observations was 18.  The significant levels for the main and interaction effects were declared at P < 0.05 and P < 0.10, respectively. If significant, pairwise comparisons were made by the Tukey method.  Results and Discussion The effects of forage type, inoculum type and their interaction on silage variables are depicted in Table 1. Butyric acid concentrations were below 1 g/kg DM and are therefore not shown.  Formation of all the end-products was affected by the forage type. Clover silages had the highest contents of lactic and acetic acid. Silages made from maize had higher amounts of propionic, 2,3-butanediol and ammonia-N than grass  silages. They also formed more ethanol than clover silages.  56                                                       Proceedings of the 6th Nordic Feed Science Conference    Feed conservation and processing Inoculum type only affected the formation of ethanol where, the grass inoculum gave the highest ethanol concentration. This is surprising considering that counts and species of epiphytic lactic acid bacteria (LAB) differ among forage types (Andrieu & Gouet, 1991; Li et al., 1992). It may suggest that differences in fermentation quality of silage crops are mostly attributed to their chemical composition. It should be interesting to find out whether this also can be seen within the same forage type. An elaborate chemical analyses (e.g. water activity, amino acid and sugar composition) is then needed as chemical variables commonly measured (e.g. dry matter, crude protein, water soluble carbohydrates) poorly explained variations in fermentation results (Mogodiniyai Kasmaei et al., 2013). Clover samples inoculated with grass and maize inocula had the highest concentration of acetic acid, with inoculation having no negative effects on lactic acid formation.   Table 1 The effect of forage, inoculum and their interaction on silage variables Treatment pH Lactic acid Acetic acid Propionic acid Ethanol  2,3-butanediol  Ammonia-N   g/kg DM  g/kg N Forage (F)         Grass 4.12b 40.3b 12.4b 2.6b 4.3ab 0.9b  13.6b Clover 4.55a 57.0a 26.7a 3.0a 3.4b 2.0ab  15.5ab Maize 3.94c 27.5c 10.7b 3.0a 5.0a 3.0a  20.1a Inoculum (I)         Grass 4.28a 40.8 17.1 2.9 6.6a 1.5  15.4 Clover 4.25a 39.5 15.9 2.9 3.3b 2.2  17.7 Maize 4.08b 44.6 16.8 2.8 2.8b 2.2  16.1 SEM1 0.02 1.52 0.51 0.06 0.33 0.35  1.57 Interaction         Grass×Grass 4.20 36.1def 12.1c 2.6 7.0ab 0.2  12.7 Grass×Clover 4.18 39.5cde 12.4c 2.7 3.0c 1.0  16.3 Grass×Maize 3.99 45.4bcd 12.8c 2.6 2.8c 1.4  11.7 Clover×Grass 4.59 62.5a 28.5a 3.1 3.9bc 1.2  14.6 Clover×Clover 4.59 51.0abc 23.2b 2.9 3.8bc 2.5  13.7 Clover×Maize 4.47 57.6ab 28.4a 3.0 2.6c 2.4  18.2 Maize×Grass 4.05 23.8f 10.7c 2.8 8.9a 3.0  18.9 Maize×Clover 3.99 28.1ef 12.1c 3.2 3.0c 3.0  23.0 Maize×Maize 3.78 30.7def 9.2c 2.8 3.0c 3.8  18.3 SEM1 0.03 2.64 0.88 0.11 0.57 0.61  2.71 P value         F <0.01 <0.01 <0.01 0.01 0.03 0.01  0.04 I <0.01 0.10 0.25 0.48 <0.01 0.31  0.59 F×I 0.22 0.06 0.01 0.16 0.01 0.75  0.47 1Standard error of mean; abcdefvalues with different superscribed letters within ‘F’, ‘I’ or ‘F×I’ and analyte differ (P<0.05 for ‘F’, ‘I’ and P<0.1 for ‘F×I’). Proceedings of the 6th Nordic Feed Science Conference                                                      57  Feed conservation and processing Data from the aerobic stability test is shown in Table 2.  One of the replicates of grass with maize inoculum was discarded as the thermometer had been misplaced. The highest temperature and the greatest pH increase were observed in maize silages. At the same time, the maize inoculum had the best scores for the aerobic stability variables (Table 2).  Table 2 The effect of forage, inoculum and their interaction on temperature and pH rise (pH after – pH before) of silages aerated for 8 d (mean ambient temperature=20.2°C)  Treatment Maximum temperature (°C) pH increase  Forage (F)  Grass 20.9b 0.73b Clover 20.6b 0.03c Maize 21.7a 1.78a Inoculum (I)  Grass 21.4a 1.58a Clover 21.3a 0.93b Maize 20.7b 0.03c SEM1 0.1 0.07 Interaction  Grass×Grass 21.5abc 2.00b Grass×Clover 20.9bc 0.20c Grass×Maize 20.4c,A 0.00c Clover×Grass 20.5c 0.03c Clover×Clover 20.6c 0.01c Clover×Maize 20.7c 0.04c Maize×Grass 22.0ab 2.72a Maize×Clover 22.3a 2.58ab Maize×Maize 20.8bc 0.05c SEM1 0.2 0.12 P value  F <0.01 <0.01 I 0.01 <0.01 F×I 0.02 <0.01 1Standard error of mean; abcvalues with different superscribed letters within ‘F’, ‘I’ or ‘F×I’ and analyte differ (P<0.05 for ‘F’, ‘I’ and P<0.1 for ‘F×I’); ASEM=0.3. The results found here could be of a great potential in inoculant research. For instance, inoculating the difficult-to-ensile legume crops with strains of LAB obtained from grass or maize forages could be tested. Considering the effects of maize inoculum on silage pH (Table 1) and aerobic stability variables (Table 2), further characterization of maize LAB is warranted. It should be kept in mind the results presented were obtained from one forage sample of the forage types investigated and hence, caution needs to be undertaken when extrapolating. More studies of this kind are therefore needed before drawing firm conclusions.   58                                                       Proceedings of the 6th Nordic Feed Science Conference    Feed conservation and processing Conclusions Fermentation quality was affected by forage type but only to a very limited degree by inoculum type. Inoculation of clover sample with grass or maize inocula increased formation of acetic acid, but had no negative effect on lactic acid production. The maize inoculum improved aerobic stability. The study showed that the possibility to separate confounding effects of chemical and microbial composition can result in new insights as well as new research questions.   References Andrieu, J. P.& Gouet, J., 1991. Variation and modification of epiphytic microflora on standing crops and after their harvest for silage. In: Pahlow P. & Honig H. (eds.), Forage Conservation Towards 2000, Proc. Conf. Europ. Grassl. Fed. Institute of Grassland and Forage Research, Braunschweig, Germany, pp. 287-289. Lin, C.J., Bolsen, K.K., Brent, B.E. & Fung, D.Y.C., 1992. Epiphytic lactic acid bacteria succession during the pre-ensiling and ensiling periods of alfalfa and maize. J. Appl. Bacteriol. 73, 375-387. Moodiniyai Kasmaei, K., Rustas, B.-O., Spörndly, R. & Udén, P., 2013. Prediction models of silage fermentation products on crop composition under strict anearobic conditions: A meta-analysis. J. Dairy Sci. 96, 6644-6649. Moodiniyai Kasmaei, K., Passoth, V., Spörndly, R. & Udén, P., 2015. A new sterilization and inoculation method in silage research. Grass Forage Sci. doi: 10.1111/gfs.12153. Muck, R.E. & O'Kiely, P., 1992. Aerobic deterioration of lucerne (Medicago sativa) and maize (Zea mais) silages-Effects of fermentation products. J. Sci. Food Agric. 59, 145-149. O'Brien, M., O'Kiely, P., Forristal, P. D. & Fuller, H. T., 2007. Quantification and identification of fungal propagules in well-managed baled grass silage and in normal on-farm produced bales. Anim. Feed Sci. Technol. 132, 283-297. O'Kiely, P. & Muck, R.E., 1992. Aerobic deterioration of lucerne (Medicago sativa) and maize (Zea mais) silages-Effects of yeasts. J. Sci. Food Agric. 59, 139-144. Wilkinson, J.M. & Davies, D.R., 2013. The aerobic stability of silage: key findings and recent developments. Grass Forage Sci. 68, 1-19.      Proceedings of the 6th Nordic Feed Science Conference                                                      59                                          DISTRIBUTION: Sveriges Lantbruksuniversitet Institutionen för husdjurens utfodring och vård Box 7024 750 07 UPPSALA Tel.018/672817 Margareta.Norinder@slu.se 

Separating the effects of pre-ensiled chemical and microbial composition on silage fermentation and aerobic stability

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Separating the effects of pre-ensiled chemical and microbial composition on silage fermentation and aerobic stability

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