Effect of ensiling and silage additives on fatty acid ...

Effect of ensiling and silage additives on fatty acid composition of ryegrass and corn experimental silages S. P. Alves, A. R. J. Cabrita, E. Jerónimo, R. J. B. Bessa and A. J. M. Fonseca J ANIM SCI published online March 11, 2011

The online version of this article, along with updated information and services, is located on the World Wide Web at: http://www.journalofanimalscience.org/content/early/2011/03/11/jas.2010-3128

www.asas.org

Downloaded from www.journalofanimalscience.org by guest on March 16, 2013

Running head title: Fatty acid composition of silages

Effect of ensiling and silage additives on fatty acid composition of ryegrass and corn experimental silages1

S. P. Alves* †, A. R. J. Cabrita‡, E. Jerónimo*§, R. J. B. Bessa* § 2 and A. J. M. Fonseca†

*

INRB - Instituto Nacional dos Recursos Biológicos, Unidade de Produção Animal, Fonte-Boa,

2005-048 Vale de Santarém, Portugal. †

REQUIMTE, ICBAS, Instituto de Ciências Biomédicas de Abel Salazar, Universidade do

Porto, Rua Padre Armando Quintas, 4485-661 Vairão VC, Portugal. ‡

REQUIMTE, Departamento de Geociências, Ambiente e Ordenamento do Território, Faculdade

de Ciências, Universidade do Porto, Rua Padre Armando Quintas, 4485-661 Vairão VC, Portugal. §

Faculdade de Medicina Veterinária, Universidade Técnica de Lisboa, CIISA, Pólo Universitário

do Alto da Ajuda, Av. da Universidade Técnica, 1300-477 Lisboa, Portugal.

1

The authors gratefully acknowledge the assistance of technicians of the Unidade de Produção Animal

(INRB) and Eng. Isabel Carvalhais in ensiling and chemical analyses. We would like to thank Sr. José Félix for the supply of ryegrass and also Eng. Susana Silva and Eng. José França for providing the corn. Individual PhD grant (SFRH/BD/37793/2007) to S.P. Alves from Fundação para a Ciência e a Tecnologia (FCT), Ministério da Ciência e Ensino Superior, Portugal, is greatly acknowledged. 2

Corresponding author: Rui J.B. Bessa; Tel. (+351) 213652871; Fax. (+351) 213652884; E-mail:

[email protected].

1 Downloaded www.journalofanimalscience.org guest on March 16, 2013 Published Onlinefrom First on March 11, 2011 asbydoi:10.2527/jas.2010-3128

ABSTRACT Two experiments were conducted using laboratory mini-silos to study the effect of ensiling and silage additives on fatty acid (FA) composition, including minor or unusual FA, of ryegrass and corn silages. Ryegrass was ensiled for 12 wk with no additives, with the addition of a bacterial inoculant or formic acid. Corn was ensiled for 9 wk without additives, with the addition of a bacterial inoculant or calcium formate. Ensiling affected both total FA content and FA composition of ryegrass silages. Total FA concentration increased (P < 0.001) during ryegrass ensiling. The proportions (g/100g of total FA) of the major unsaturated FA, 18:3n-3 and 18:2n-6, were not affected (P > 0.05) by ensiling. However, their concentration (mg/g DM) in silages was greater (P = 0.017 and P = 0.001, respectively) than in fresh ryegrass. Two 18:2 FA (trans11,cis-15 and cis-9,cis-15) that were not originally present in the fresh ryegrass were detected in silages. Silage additives affected the FA composition of ryegrass silages, mostly by increasing the proportions of saturated FA, but not on total FA concentration. Ensiling did not affect (P = 0.83) total FA content of corn silages, however, FA composition was affected, mostly by decreasing the proportions of 18:2n-6 and 18:3n-3. Silage additives had no effect on corn silages FA composition. Exposing corn silages to air resulted in no oxidation of FA or reduction in total FA content or composition.

Keywords: additives, corn, ensiling, experimental silage, fatty acids, ryegrass.

2 Downloaded from www.journalofanimalscience.org by guest on March 16, 2013

INTRODUCTION Corn and ryegrass silages constitute the base diet in many ruminant production systems as in the coastal dairy region of Portugal. The influence of ensiling process on forage preservation and its major nutrients is well studied. However, forages are also an important dietary source of αlinolenic acid (18:3n-3) and linoleic acid (18:2n-6) that are biohydrogenated in the rumen originating a complex pattern of C18 FA (Jenkins et al., 2008). Effect of ensiling on total FA content of forages is contradictory, with some studies reporting a decrease (e.g., legume-grass, Ward and Allen, 1957; perennial ryegrass, Elgersma et al., 2003) and others an increase (e.g., timothy, Boufaied et al., 2003). However, as far as we know, there are no studies on this subject using corn silages. Additives became an important part of silage management, studies have reported slight effects on the FA composition of grass silages by the use of additives like formalin, formic acid or enzymes (Dewhurst and King, 1998; Shingfield et al., 2005; Arvidsson et al., 2009). The occurrence of trans FA isomers in silages were found by few authors. Lough and Anderson (1973) reported the occurrence of trans 18:1 and 18:2 isomers on mixed grasses ensiled without additives. Vanhatalo et al. (2007) reported the presence of trans 18:1, but not trans 18:2, in silages comprising timothy – meadow fescue, and red clover swards, using a formic acid based additive. These results suggest that isomerization and hydrogenation may occur during ensiling. Indeed, lactic acid bacteria commonly found in forages have the ability to biohydrogenate 18:2n6 and 18:3n-3 (Ogawa et al., 2005). We hypothesized that the metabolism of the 18:2n-6 and 18:3n-3 might be affected by the ensiling process and type of additive used (inhibitor or stimulant). Hence, this study aimed to evaluate the effect of ensiling and silage additives on FA composition of corn and ryegrass silages, including the occurrence of minor and unusual FA.


MATERIALS AND METHODS

Grass and Corn Samples Fresh samples of ryegrass (Lolium multiflorum, var. Major, DLF-Trifolium, Denmark) used in Experiment 1 were sown in the field in November 2007 at Santarém region (39º13’N/8º43’W), Portugal. Ryegrass received one fertilizer application before the first cut at February 2008 (7 kg N/ha, 21 kg P2O5/ha, 7 kg K2O/ha). On the morning of 06 May of 2008, ryegrass was harvested (second cut) at 5 cm height from soil and immediately taken to the laboratory. Before ensiling, grass was manually chopped with a length of approximately 2 cm and directly ensiled without wilting. Corn (Zea mays L., var. Klimt, KWS-SAAT-AG, Portugal) used in Experiment 2 were sown in the field in June of 2006 at Benavente region (38º57’N/8º53’W), Portugal. On the morning of 16 October of 2008, whole-plant was harvested using a Claas harvester (Claas KGaA, Harsewinkel, Germany) with a length of approximately 1 cm. Immediately after collection, samples were taken to the laboratory and ensiled in laboratory mini-silos.

Silage Preparation Immediately after cutting two experiments were made in the laboratory, using 3.5 L jars of PVC (polyvinyl chloride). Both grass and corn were compacted by hand as much as possible and jars were sealed with a lid and joints filled with paraffin to prevent entry of air. Both jars and lids did not allow the entering of light. Cement weights of approximately 2 kg were placed on top of each silo. Each jar contained a small tap in the bottom to discharge effluents. In Experiment 1, about 2 kg of fresh ryegrass were ensiled using no additives; a commercial inoculant (Sil-ALL 4x4,


Alltech, Sintra, Portugal, with 1x10E11 CFU/g of silage; 2 mL of 5 g/L of inoculant solution in water per kg of fresh material) containing the bacteria Lactobacillus plantarum, Enterococcus faecium, Pediococcus acidilactici, and Lactobacillus salivarius, and also the enzymes cellulase, hemicellulase, pentosanase and amylase; or formic acid (Sigma-Aldrich; diluted 1:5 in water and added 20 mL/kg of fresh material). Silos were stored in a laboratory that had temperatures ranging from 17 to 26 ºC, and were opened after 12 wk. Taps on the bottom of each silo were opened weekly to examine the appearance of effluents. After 5 wk of ensiling, effluents started to emerge from silos with formic acid, after 6 wk from silos with inoculant and after 7 wk effluents emerged from all silos. All effluents were discharged. Every silage treatments were made in three replicates. In experiment 2, about 2 kg of fresh corn were ensiled using no additives; a commercial inoculant (Sil-ALL 4x4, Alltech, Sintra, Portugal, with 1x10E11 CFU/g of silage; 2 mL of 5 g/L of inoculant solution in water per kg of fresh material); or a commercial source of formic acid (calcium formate 70%, Silenergi from Zoopan, Portugal; 0.3 g/kg of fresh material). Silos were stored at laboratory temperatures (7 to 12ºC), and were opened after 9 wk. Taps on the bottom of each silo were opened weekly to examine the appearance of effluents, however, no effluent was observed in any silo during the ensiling period. Every silage treatments were made in three replicates. At the end of the ensiling period, the effect of exposing corn silages to air for 48 hours on the FA composition and content was also evaluated.

Chemical Analyses Chemical analyses were performed in duplicate and presented on DM basis. The DM content of fresh ryegrass and fresh corn was determined by oven drying the fresh materials at 65ºC for 24 h


and then to constant weight. The DM content of silages was determined by the method of Bidwell and Sterling (1925). A fraction of all samples were freeze-dried (72 h) for acid ether extraction (AEE) and FA analysis. Acid ether extraction of freeze-dried ryegrass and ryegrass silages was determined by hydrolysis with HCl 3M followed by Soxhlet extraction for 6 h with petroleum ether (Portuguese Standard NP876, 2001). Ash of the oven dried materials was obtained after 3 h at 550ºC. Neutral detergent fiber (NDF), acid detergent fiber (ADF) and acid detergent lignin (ADL) of the oven dried materials were determined by the detergent procedures (Robertson and Van Soest, 1981; Van Soest et al., 1991); during NDF extraction, amylase and sodium sulphite were not added; NDF and ADF values were expressed inclusive of residual ash. Water soluble carbohydrates (WSC) and starch of the oven dried materials were determined by a colorimetric method (DuBois et al., 1956). Total N concentration was determined by using the macro-Kjeldahl technique (Portuguese Standard NP2030, 1996). A saline solution of silages was used to measure the pH and ammonia-N was measured by the micro-diffusion method (Portuguese Standard NP4038, 1990). Gross energy was determined by calorimetric method (ISO 9831, 1998) and digestibility was measured in vitro by the method of Tilley et al. (1969). A water extract of silages were prepared by adding deionised water (50 mL) to 10 g of silage that was used to measure fermentation acids (lactic acid, VFA) and ethanol. Briefly, 1 mL of phosphoric acid (25%, vol/vol) was added to 1 mL of extract, after homogenization at vortex and centrifugation, the liquid extract was quickly analyzed by gas-liquid chromatography using an Agilent HP6890 (Agilent Techn. Inc., Palo Alto, CA, USA) equipped with a flame ionization detector and a Permabond-FFAP semi-capillary column (50 m, 0.25 mm I.D., 0.25 m film thickness, Macherey-Nagel). The chromatographic conditions were as follows: injector temperature, 230ºC; detector temperature, 220ºC; helium was used as carrier gas at constant flow


of 1.0 mL/min and the split ratio was 1:50. The oven temperature program was: 45ºC (maintained for 2 min), followed by a 10ºC/min ramp to 220ºC (maintained for 20 min). Volatile fatty acids and lactic acid were identified by comparison with retention times of known standards and quantified by external standard calibration. Ethanol was quantified using internal standard according to Porter and Murray (2001).

Fatty Acid Analyses Fresh and ensiled ryegrass and corn samples were frozen at -80ºC, and then freeze-dried, ground and stored under vacuum at -80ºC. Fatty acid methyl esters (FAME) from all silages and fresh materials (~250 mg) were prepared by using HCl/methanol as catalyst of the direct transesterification method, as described by Alves et al. (2009). Additionally, samples of ryegrass and ryegrass silages were submitted to solid-phase extraction before FAME analysis by gasliquid chromatography to remove contaminants, according to Alves et al. (2008). The nonadecanoic acid was used as internal standard, as recommended by Alves et al. (2009). Fatty acid methyl esters were determined using a gas chromatograph system from Agilent HP6890 (Agilent Techn. Inc., Palo Alto, CA, USA) equipped with a flame ionization detector and a CPSil 88 capillary column (100 m, 0.25 mm I.D., 0.20 m film thickness; Chrompack, Varian Inc., Walnut Creek, CA, USA). The chromatographic conditions were as follows: injector temperature, 250ºC; detector temperature, 280ºC; helium was used as carrier gas and the split ratio was 1:50. The oven temperature program was: 50ºC (maintained for 4 min), followed by a 13ºC/min ramp to 175ºC (maintained for 27 min), then increased at 4ºC/min to 215ºC (maintained for 60 min). Identification of FA and non-FA compounds was accomplished by using authentic standards and also by GC-MS analysis using a Varian Saturn 2200 system


(Varian Inc.,Walnut Creek, CA, USA). The ion trap parameters used in the present analyses are similar to those described in Alves and Bessa (2007). Additionally, the tandem mass spectrometry technique of covalent adduct chemical ionization (CACI-MS/MS) using acetonitrile as reagent of chemical ionization (CI) was used for localization of double bond position of the minor 18:2 isomers. Tandem MS/MS parameters were as follows: emission current, 10 mA; scan time, 0.32 s; mass isolation window, 3 m/z; and excitation storage level, 82.3 m/z. The resonant excitation amplitude used to collisionally dissociate the [M+54]+ ions was 1.0 eV.

Statistical Analysis The effect of silage additives on chemical composition of silages was evaluated by analysis of variance with silage additive as fixed effect. Comparison of total FA content and FA composition between fresh materials and silages was studied by analysis of variance considering the type of material as fixed effect. Additionally, orthogonal contrasts were constructed to evaluate: (1) the effect of ensiling; (2) the effect of using additives; and (3) the effect of using inoculant or acid as additives. All the computations were conducted using the GLM procedure of SAS (SAS Inst. Inc. 2002, Cary, NC).

RESULTS

Chemical Composition The chemical composition of fresh ryegrass and corn used are presented in Table 1. In Experiment 1 (Table 2), the use of formic acid as silage additive decreased pH (P = 0.038),


ammonia-N (P = 0.018), and the concentrations of butyric acid (P = 0.037) and ethanol (P = 0.041), and increased the concentration of the WSC (P = 0.002) compared with silages with no additives. In Experiment 2, the use of calcium formate as additive (Table 3) only decreased the concentration of WSC (P = 0.009) and increased the concentration of ethanol (P = 0.001).

Fatty Acid Composition In Experiment 1, the concentration of AEE and total FA after ensiling increased 30% and 29%, respectively (Table 4). Comparing to fresh ryegrass, silages had greater proportions of 12:0, cis11 18:1, cis-9,cis-15 18:2, and 23:0, and lower proportions (F contrast, P < 0.05) of 14:0, trans-3 16:1, cis-9 16:1, 17:0, 18:3 and 26:0. Proportions of major FA (18:3n-3, 16:0, 18:2n-6) in fresh ryegrass remained unchanged after ensiling, but their concentration increased (P = 0.001, P = 0.002, and P < 0.001, respectively). The use of silage additives (inoculant and formic acid) did not affect total FA content, but had some effect on the FA composition of ryegrass silages, mostly on the saturated 12:0, 14:0, 16:0, 18:0, 20:0, and 22:0, whose proportions were lower in silages produced with additives than in those produced without. The use of formic acid, comparing to inoculants, lowered proportions of saturated FA, 16:0, 20:0, 22:0, 23:0, and 24:0, and of the monounsaturated cis-11 18:1. Two FA that were not originally present in fresh ryegrass were produced during ensiling, the trans-11,cis-15 18:2 and cis-9,cis-15 18:2. The proportion of the trans-11,cis-15 18:2 did not differ between silages. Conversely, the proportion of cis-9,cis-15 18:2 was greater in silages produced without additives than in ryegrass silages produced with inoculant or formic acid. The CACI-MS/MS technique was used for structural elucidation of the unusual trans-11,cis-15 18:2 and cis-9,cis-15 18:2 peaks in ryegrass silages. Figure 1 shows acetonitrile CACI-MS/MS spectra of both 18:2 isomers present in ryegrass


silages, as well as a synthesized standard of cis-9,cis-15 18:2. The synthesis of this isomer was already described by our group (Alves and Bessa, 2009), however, its CACI-MS/MS spectra had never been published in the literature. In Experiment 2 (Table 5), ensiling and the use of additives did not affect total FA content of corn silages. However, ensiling had some effect on the FA composition of silages. Comparing to fresh corn, the proportion of cis-11 18:1 in silages was greater, and the proportions of 10 FA were lower, including the major FA, 18:2n-6, cis-9 18:1, 16:0, and 18:3n-3. However, when expressed in mg/g DM, no differences (P = 0.63, P = 0.74, and P = 0.66, respectively) were observed for the 18:2n-6, cis-9 18:1, and 18:3n-3 in silages compared with fresh corn. Silage additives did not affect FA composition of corn silages, with the exception of 24:0. The effect of air exposure on FA composition of corn silages was also studied. However, after 48 hours of exposure to air, neither FA composition nor total FA content of silages differed from that at the time of opening the silos. During that time, we observed temperature increases in silages of about 2ºC and the laboratory temperature was around 9.5ºC, which probably prevented temperature increases and ensured conservation of silages.

DISCUSSION

Silage Quality As expected, the use of acid as silage additive, in Experiment 1, reduced pH and ammonia-N. The rapid reduction in pH is known to reduce the proteolysis by inhibiting plant proteases enzymes, preventing degradation of amino acids and a consequent reduction of volatile nitrogen (McDonald and Whittenbury, 1973; Charmley, 2001). The greatest concentration of butyric acid


in ryegrass silages produced without additives and with inoculant may indicate the presence of a higher saccharolytic clostridial activity compared to silages produced with formic acid (McDonald and Whittenbury, 1973). Conversely to grass silages, corn silages were all well preserved. The pH was lower than 3.7 and seems that both clostridial and proteolytic activity were inhibited because both butyric and ammonia-N concentrations were quite low (McDonald and Whittenbury, 1973; Harrison et al., 1994).

Effect of Ensiling on Fatty Acid Composition of Silages Some studies report a decrease on the total FA content of ryegrass silages compared to those of fresh products (Dewhurst and King, 1998; Elgersma et al., 2003). However, in these studies the losses of FA were probably generated during field manipulations, like shading or wilting, that promote FA oxidation. In our study, total FA content was increased in ryegrass silages (Experiment 1) and maintained in corn silages (Experiment 2). The maintenance of total FA content in corn silages may be explained by the direct ensilage of corn without wilting or shading which would prevent losses from oxidative degradation. The increase of total FA content in ryegrass silages is not completely understood. However, it may be explained by the concentration of lipid matter due to losses of DM content. In silo DM losses can result from either respiration (aerobic), fermentation (anaerobic), and effluent losses, and may total as much as 10% to 50% of the DM ensiled (McDonald and Whittenbury, 1973). Losses of DM due to respiration of organic compounds (including lipids/FA) by plant enzymes and aerobic microorganism are oxygen dependent, and will continue in the silo until anaerobic conditions are achieved. However, in well compacted and sealed silos anaerobic conditions are established very


quickly, so these losses are expected to be small (Dewhurst and King, 1998). Losses due to fermentative activity in unwilted grass pasture were reported to reach 12% of DM (Jarrige et al., 1982) and were due to catabolization of non-lipid organic compounds (mostly WSC and proteins), whereas the anaerobic degradation of FA is improbable (Mackie et al., 1991) and of the cell wall carbohydrates is rather slow. This would lead to the increased concentration of FA, NDF and ash in silages. Indeed, along with the increase in FA content (29% of DM), ryegrass silages DM contains more 19.5% of ash and 4.1% of NDF than fresh ryegrass. Effluent DM losses can also contribute to FA enrichment of silages. Indeed, in our experiment, effluent losses were visible and started at 5 wk of ensiling. Effluents usually contain sugars, soluble nitrogenous compounds, fermentation acids and minerals (McDonald and Whittenbury, 1973; Randby, 1997; Haigh, 1999). Ash content can reach up to 16% of effluent DM (Savoie et al., 2002), while FA are not described to occur in effluents. Ryegrass silages showed a DM recovery among silages of about 83.7%, therefore the expected FA concentration should be 28.4 mg/g DM, which is not very distant from the actual values obtained for ryegrass silages. Recently, other studies also found greater concentrations of lipid in silages compared to fresh materials (Boufaied et al., 2003; Arvidsson et al., 2009; Van Ranst et al., 2009b). Acid ether extract was determined to confirm the results that indicate greater FA content in ryegrass silages than in fresh ryegrass. The acid ether extracts include significant amounts of nonsaponifiable lipids (like waxes, chlorophyll, cutin), so greater values were expected for the AEE than for the total FA content. However, the same trend to increase fat content in silages compared to fresh ryegrass was also observed. So, our results corroborate the hypothesis that DM losses due to anaerobic fermentation and effluent during ensiling are a major determinant of


total FA concentration in silages. In ryegrass silages of high quality, with lower DM losses, the FA concentration is expected to be substantially lower. Most of the studies on the FA composition of silages are concerned with losses on the levels of 18:2n-6 and 18:3n-3 during ensiling, given that high levels of both 18:2 and 18:3 in animal diets might increase their transfer to milk or meat of ruminants and, consequently, increase their concentration in the human diet. In the present study, ensiling increased the concentration of either 18:2n-6 and 18:3n-3 in ryegrass silages, which might be explained by the increase of total FA content due to DM losses in silages as previously discussed. In our experiment, lower proportion of 18:2n-6 and 18:3n-3 were observed in silages compared with fresh corn. Losses of FA are probably due to oxidation by lipoxygenases during the silage making and as long as the conditions are not anaerobic, which in well compacted and sealed silos, are established quite fast. Lipoxygenases, which were detected in the seed and seedlings of corn (Gardner, 1970; Gardner and Weislede, 1970), are recognized to convert non-esterified 18:3n-3 and 18:2n-6 into hydroperoxy PUFA that are further catabolised to yield a range of volatile compounds. During ensiling, the microbial population may increase the concentration of FA that are recognized to be of microbial origin, like odd- and branched chain FA (Vlaeminck et al., 2006). However, we did not detect any branched chain FA in either ryegrass or corn silages despite of the method used is effective in the detection of these FA in other matrices. Despite some significant effect on the proportions of 17:0 in ryegrass silages (Experiment 1), and on 15:0 and 17:0 in corn silages (Experiment 2), we observed a decrease in proportions of both FA compared to the fresh materials. However, when results are expressed in mg/g DM (data not shown) only


the concentration of 17:0 decreased (P < 0.001) in both ryegrass and corn silages compared with fresh materials. The occurrence of minor mono-unsaturated 16:1 isomers was observed in both fresh ryegrass and ryegrass silages of Experiment 1. In fresh corn and corn silages of Experiment 2, the cis-9 16:1 was the only hexadecenoic isomer identified, although it was not affected by ensiling. However, in ryegrass materials the major hexadecenoic isomer was the trans-3 16:1 which presence was previously discussed by our group (Alves et al., 2008). Apart from 18:2n-6, two minor 18:2 isomers were detected in ryegrass silages. In our chromatographic conditions, the first 18:2 isomer elutes just before 18:2n-6 and the second immediately after. Comparing their retention times with other matrixes such as milk or meat, we believe that these isomers might be the trans-11,cis-15 and cis-9,cis-15 18:2 isomers. Despite their presence in milk, meat and abomasal digesta (Bessa et al., 2007; Rego et al., 2009), as far as we know, there are no reports of their occurrence in silages. Thus, for an accurate characterization of both isomers, we used the CACI-MS/MS technique with acetonitrile as reagent of chemical ionization (Figure 1). The presence of trans-11,cis-15 18:2 and cis-9,cis-15 18:2 in ryegrass silages, may be indicative of biohydrogenation activities in silages. Indeed, some lactic acid bacteria were described to isomerize 18:2n-6 into conjugated 18:2 FA and proceed to reductive step producing trans-10 18:1. Moreover, these bacteria also isomerizes 18:3n-3 into cis-9,trans-11,cis-15 18:3, trans-9,trans-11,cis-15 18:3, and trans-10,cis-15 18:2 (Ogawa et al., 2005; Kishino et al., 2009). Both trans-11,cis-15 and cis-9,cis-15 18:2 isomers found in ryegrass silages were not described to be produced by lactic acid bacteria. On the other hand, they have been described as intermediates in the biohydrogenation of 18:3n-3 by rumen bacteria. Therefore, the occurrence of both 18:2 isomers in ryegrass silages suggests


biohydrogenation pathways of 18:3n-3 similar to those that occur by microbial action in the rumen. Rumen biohydrogenation has been extensively studied, however its role on ruminal microbial ecology is not completely understood (Jenkins et al., 2008). Moreover, only few hydrogenating rumen bacteria are known, so identification of the responsible bacteria for biohydrogenation in silages could be useful to study biohydrogenation in general. Nevertheless, the presence of 18:2 with trans double bonds in silages was previously reported by Lough and Anderson (1973), although they did not identify those isomers. Other authors reported the presence of trans 18:1 FA in grass and red clover silages (Vanhatalo et al., 2007). We detected the presence of only trace amounts of trans 18:1 FA, however their concentrations were well below the limit of quantification of our GC-FID system. A minor 18:3 isomer (confirmed by GC-MS) was also detected in fresh ryegrass and in ryegrass silages. Comparing the GC-FID retention time of this 18:3 peak with a authentic standard containing all 18:3n-3 geometric isomers, we suspect that it may be a cis-9,cis-12,trans-15 18:3 or trans-9,trans-12,cis-15 18:3. Due to its low concentration in samples, an unequivocally characterization by CACI-MS/MS was not possible. Indeed, this 18:3 isomer was already detected in others grasses and legumes (Alves et al., 2008).

Effect of Additives on Fatty Acid Composition of Silages A few studies showed that the use of additives had small effects on the FA composition of silages prepared from ryegrass (Dewhurst and King, 1998; Van Ranst et al., 2009a). In fact, in ryegrass silages (Experiment 1), additives had small, but significant effect in some saturated FA, especially when formic acid was used as silage additive. Indeed, the use of acids as additive are recognized to inhibit the fermentation process, thus lower microbial FA synthesis are expected in


silages with the addition of formic acid. Dewhurst and King (1998) evaluated the use of formic acid as additive and found minor, but significant differences, in the proportions of 18:3 and 16:0, in perennial ryegrass. In corn silages (Experiment 2), only 24:0 was affected by the type of additives used. Nevertheless, the minor effect in FA composition on both silages could not only be due to the additives used but also to different DM at time of ensiling. Regarding the two FA produced during ensiling of ryegrass, only the cis-9,cis-15 18:2 was affected by the use of silage additives. Its proportion was greater in silages produced without additives than in silages produced with additives, although with a large variation among silo replicates, as shown by the high standard error of mean. Although independent of additives, the concentration of trans-11,cis-15 18:2 also showed a great variance among silos.

CONCLUSIONS The results on total FA content of ryegrass silages suggest that DM losses, which are due to anaerobic fermentation and effluent during ensiling, may have a significant influence on the concentration of lipid material in silages, resulting in an increase of fat content and consequentially an increase on concentration of major FA, 18:3n-3 and 18:2n-6. The use of silage additives had small effects on the FA composition of ryegrass and corn silages. Furthermore, FA composition and total FA content of corn silages was not affected by exposure of silages to air up to 48 h. Two 18:2 FA that were not originally present in the fresh material were detected in ryegrass silages. Both FA were probably produced by biohydrogenation pathways similar to those that occur by microbial action in the rumen.


LITERATURE CITED Alves, S. P. and R. J. B. Bessa. 2007. Identification of cis-12,cis-15 octadecadienoic acid and other minor polyenoic fatty acids in ruminant fat. Eur. J. Lipid Sci. Technol. 109:879883. Alves, S. P., A. R. J. Cabrita, A. J. M. Fonseca, and R. J. B. Bessa. 2008. Improved method for fatty acid analysis in herbage based on direct transesterification followed by solid-phase extraction. J. Chromatogr. A 1209:212-219. Alves, S. P. and R. J. B. Bessa. 2009. Comparison of two gas-liquid chromatograph columns for the analysis of fatty acids in ruminant meat. J. Chromatogr. A 1216:5130-5139. Alves, S. P., A. R. J. Cabrita, A. J. M. Fonseca, and R. J. B. Bessa. 2009. Effect of a Purification Step and the Type of Internal Standard Used on Fatty Acid Determination of Grass and Maize Silages. J. Agric. Food Chem. 57:10793-10797. Arvidsson, K., A. M. Gustavsson, and K. Martinsson. 2009. Effects of conservation method on fatty acid composition of silage. Anim. Feed Sci. Technol. 148:241-252. Bessa, R. J. B., S. P. Alves, E. Jeronimo, C. M. Alfaia, J. A. M. Prates, and J. Santos-Silva. 2007. Effect of lipid supplements on ruminal biohydrogenation intermediates and muscle fatty acids in lambs. Eur. J. Lipid Sci. Technol. 109:868-878. Bidwell, G. L. and W. F. Sterling. 1925. Preliminary notes on the direct determination of moisture. Ind. Eng. Chem. 17:147-149. Boufaied, H., P. Y. Chouinard, G. F. Tremblay, H. V. Petit, R. Michaud, and G. Belanger. 2003. Fatty acids in forages. I. Factors affecting concentrations. Can. J. Anim. Sci. 83:501-511. Charmley, E. 2001. Towards improved silage quality - A review. Can. J. Anim. Sci. 81:157-168. Dewhurst, R. J. and P. J. King. 1998. Effects of extended wilting, shading and chemical additives on the fatty acids in laboratory grass silages. Grass Forage Sci. 53:219-224. DuBois, M., K. A. Gilles, J. K. Hamilton, P. A. Rebers, and F. Smith. 1956. Colorimetric Method for Determination of Sugars and Related Substances. Anal. Chem. 28:350-356. Elgersma, A., G. Ellen, H. van der Horst, B. G. Muuse, H. Boer, and S. Tamminga. 2003. Comparison of the fatty acid composition of fresh and ensiled perennial ryegrass (Lolium perenne L.), affected by cultivar and regrowth interval. Anim. Feed Sci. Technol. 108:191-205.


Gardner, H. W. 1970. Sequential Enzymes of Linoleic Acid Oxidation in Corn Germ Lipoxygenase and Linoleate Hydroperoxide Isomerase. J. Lipid Res. 11:311-321. Gardner, H. W. and D. Weislede. 1970. Lipoxygenase from Zea Mays - 9-D-HydroperoxyTrans-10, Cis-12-Octadecadienoic Acid from Linoleic Acid. Lipids 5:678-683. Haigh. 1999. Effluent production from grass silages treated with additives and made in largescale bunker silos. Grass Forage Sci. 54:208-218. Harrison, J. H., R. Blauwiekel, and M. R. Stokes. 1994. Fermentation and Utilization of GrassSilage. J. Dairy Sci. 77:3209-3235. Jarrige, R., C. Demarquilly, and J. P. Dulphy. 1982. Forage Conservation. In: J. B. Hacker (Ed.) Nutritional limits to animal production from pastures. Commonwealth Agricultural Bureaux, Farnham Royal, UK. Jenkins, T. C., R. J. Wallace, P. J. Moate, and E. E. Mosley. 2008. BOARD-INVITED REVIEW: Recent advances in biohydrogenation of unsaturated fatty acids within the rumen microbial ecosystem. J. Anim Sci. 86:397-412. Kishino, S., J. Ogawa, K. Yokozeki, and S. Shimizu. 2009. Metabolic diversity in biohydrogenation of polyunsaturated fatty acids by lactic acid bacteria involving conjugated fatty acid production. Appl. Microbiol. Biotechnol. 84:87-97. Lough, A. K. and L. J. Anderson. 1973. Effect of Ensilage on Lipids of Pasture Grasses. Proc. Nutr. Soc. 32:A61-A62. Mackie, R. I., B. A. White, and M. P. Bryant. 1991. Lipid Metabolism in Anaerobic Ecosystems. Crit. Rev. Microbiol. 17:449-479. McDonald, P. and R. Whittenbury. 1973. The Ensilage Process. In: G. W. butler and R. W. Bailey (Eds.) Chemistry and Biochemistry of Herbage. pp. 33. Academic Press, London. Ogawa, J., S. Kishino, A. Ando, S. Sugimoto, K. Mihara, and S. Shimizu. 2005. Production of conjugated fatty acids by lactic acid bacteria. J. Biosci. Bioeng. 100:355-364. Porter, M. G. and R. S. Murray. 2001. The volatility of components of grass silage on oven drying and the inter-relationship between dry-matter content estimated by different analytical methods. Grass Forage Sci. 56:405-411. Randby, A. T. 1997. Quality of silage and silage effluent as influenced by storage of effluent in tower silos. Acta Agric. Scand. , Sect. A - Anim. Sci. 47:9-19. Rego, O. A., S. P. Alves, L. M. S. Antunes, H. J. D. Rosa, C. F. M. Alfaia, J. A. M. Prates, A. R. J. Cabrita, A. J. M. Fonseca, and R. J. B. Bessa. 2009. Rumen biohydrogenation-derived fatty acids in milk fat from grazing dairy cows supplemented with rapeseed, sunflower, or linseed oils. J. Dairy Sci. 92:4530-4540.


Robertson, J. B. and P. J. Van Soest. 1981. The detergent system of analysis. In: W. P. T. James and O. Theander (Eds.) The Analysis of Dietary Fibre in Food. pp. 123-158. Marcel Dekker, NY. Savoie, P., A. Amyot, and R. Thériault. 2002. Effect of moisture content, chopping, and processing on silage effluent. Trans. ASAE 45:907-914. Shingfield, K. J., P. Salo-Väänänen, E. Pahkala, V. Toivonen, S. Jaakkola, V. Piironen, and P. Huhtanen. 2005. Effect of forage conservation method, concentrate level and propylene glycol on the fatty acid composition and vitamin content of cows' milk. J. Dairy Res. 72:349-361. Tilley, J. M. A., R. A. Terry, R. E. Deriaz, and G. E. Outen. 1969. Digestibility of Structural Carbohydrates of Grasses by Rumen Micro-Organisms in Vitro. J. Brit. Grassl. Soc. 24:238-243. Van Ranst, G., V. Fievez, J. De Riek, and E. Van Bockstaele. 2009a. Influence of ensiling forages at different dry matters and silage additives on lipid metabolism and fatty acid composition. Anim. Feed Sci. Technol. 150:62-74. Van Ranst, G., V. Fievez, M. Vandewalle, J. De Riek, and E. Van Bockstaele. 2009b. Influence of herbage species, cultivar and cutting date on fatty acid composition of herbage and lipid metabolism during ensiling. Grass Forage Sci. 64:196-207. Van Soest, P. J., J. B. Robertson, and B. A. Lewis. 1991. Methods for Dietary Fiber, Neutral Detergent Fiber, and Nonstarch Polysaccharides in Relation to Animal Nutrition. J. Dairy Sci. 74:3583-3597. Vanhatalo, A., K. Kuoppala, V. Toivonen, and K. J. Shingfield. 2007. Effects of forage species and stage of maturity on bovine milk fatty acid composition. Eur. J. Lipid Sci. Technol. 109:856-867. Vlaeminck, B., V. Fievez, A. R. J. Cabrita, A. J. M. Fonseca, and R. J. Dewhurst. 2006. Factors affecting odd- and branched-chain fatty acids in milk: A review. Anim. Feed Sci. Technol. 131:389-417. Ward, R. M. and R. S. Allen. 1957. Silage Evaluation, Polyunsaturated Fatty Acids in LegumeGrass Silage. J. Agric. Food Chem. 5:765-767.


Table 1 – Chemical composition (g/kg of DM, unless otherwise stated) of fresh ryegrass and corn used in Experiments 1 and 2, respectively

Dry Matter, g/kg Ash NDF ADF ADL WSC Starch Total Nitrogen Ammonia-N, g/kg of total N In vitro DM Digestibility In vitro OM Digestibility, g/kg OM

Ryegrass

Corn

216 104 523 299 31 167 n.d. 25 15 650 600

270 49 477 277 26 96 211 12 n.d. 640 630

n.d. = not determined.


Table 2 - Chemical composition (g/kg of DM unless otherwise stated) of experimental ryegrass silages after 12 wk of ensiling without additives and with a commercial inoculant or formic acid as silage additives (Experiment 1)

Silages

Dry Matter, g/kg pH Ash NDF ADF ADL WSC Gross Energy, MJ/kg DM Ammonia-N, g/kg total N Total Nitrogen In vitro DM Digestibility In vitro OM Digestibility, g/kg OM Ethanol Fermentation acids Lactic Acetic Propionic Iso-Butyric Butyric a,b

SEM

P

167 4.32ab 129b 561 369 37.1 10.2a 18.9 128ab 26.7 566 508

Formic Acid 182 3.95a 115a 529 334 30.6 56.9b 19.1 99.0a 21.3 568 519

13.2 0.146 2.4 7.2 8.8 2.07 5.85 0.16 8.7 2.55 14.9 17.5

0.66 0.038 0.007 0.05 0.08 0.15 0.002 0.56 0.018 0.35 0.22 0.17

8.1b

9.0b

3.8a

1.16

0.041

23.7 7.7b 1.5 0.44 15.2b

25.5 5.6a 1.4 0.55 15.1b

11.9 6.5ab 0.46 0.69 3.8a

7.48 0.36 0.303 0.126 2.68

0.43 0.016 0.10 0.43 0.037

No additives 167 4.66b 129b 544 353 32.0 8.5a 18.9 149b 25.5 531 467

Inoculant

Within a row, means without a common superscript letter differ (P < 0.05).

SEM = standard error of the mean.


able 3 - Chemical composition (g/kg of DM unless otherwise stated) of experimental corn silages after 9 wk of ensiling without additives and with a commercial inoculant or calcium formate as silage additives (Experiment 2) Silage

Dry Matter, g/kg pH Ash NDF ADF ADL WSC Starch Gross Energy, MJ/kg DM Ammonia-N, g/kg of total N Total Nitrogen In vitro DM Digestibility In vitro OM Digestibility, g/kg OM Ethanol Fermentation acids Lactic Acetic Propionic Butyric a,b

SEM

P

282 3.67 54.3 455 264 26.0 11.5a 233 18.3 18.6 11.0 610 592

Calcium Formate 283 3.69 54.3 461 291 31.3 9.0b 231 18.5 16.9 11.5 614 593

4.1 0.060 0.72 11.1 7.3 3.34 0.44 5.7 0.32 1.06 0.58 5.5 5.2

0.08 0.41 0.93 0.55 0.10 0.22 0.009 0.37 0.87 0.14 0.49 0.73 0.68

5.9ª

16.5b

15.1b

1.17

0.001

13.3 2.3 0.13 0.07

12.9 2.2 0.14 0.05

12.8 2.1 0.17 0.05

3.34 0.14 0.009 0.021

0.99 0.52 0.06 0.62

No additives 268 3.58 54.0 444 274 35.3 11.6a 243 18.4 20.5 12.0 617 598

Inoculant

Within a row, means without a common superscript letter differ (P < 0.05).

SEM = standard error of the mean.


Table 4 – Effect of ensiling and use of additives in acid ether extract (AEE, mg/g DM), total FA content (mg/g DM) and FA composition (g/100 g of total FA) of fresh ryegrass and experimental ryegrass silages after 12 wk of ensiling without additives and with a commercial inoculant or formic acid as silage additives (Experiment 1) Silages FA

Fresh No Inoculant Ryegrass additives

Contrast P-value Formic acid

SEM

F2

AEE 32.13 44.50 42.65 38.16 1.916 0.002 Total FA 23.79 30.44 29.85 31.81 0.705