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In vitro fermentation of corn silage using rumen fluid buffered or not and different sample amounts/Fermentacao in vitro da silagem de milho utilizando liquido ruminal tamponado ou nao e tres quantidades de amostras.


The in vitro gas production technique is used to estimate methane (C[H.sub.4]) production, rate and degree of digestibility of feed given to ruminants. In this technique, it is indicated to use rumen fluid diluted with buffer solutions, and the values obtained for C[H.sub.4] production have good correlation with respirometric methods (BLUMEL et al., 1997; RYMER et al., 1999, 2005; BHATTA et al., 2006, 2008; STORM et al., 2012).

According to the review on gas production technique conducted by FONDEVILLA & BARROS (2001), several dilutions of rumen fluid (10, 25, or 33% rumen fluid) and sample amount (0.66, 0.83, 1.0, and 1.25g of DM 100[mL.sup.-1] rumen fluid) have been used, but there is no study using pure rumen fluid (PRF). MOULD et al. (2005) stated that the use of buffer solution had little influence on gas production and only minimized pH variations. However, RYMER et al. (2005) found that the increase in the ruminal fluid/ buffer solution ratio reduced the time of colonization and pH of the medium, and increased gas production.

In this study, it was evaluated the effect of using ruminal fluid buffered or not and three sample amounts in the in vitro fermentation of corn silage on final pH, total gas volume, C[H.sub.4] production and DM degradation.

The study evaluated the effect of three sample amounts (0.6, 1.3 and 2.6g of DM 100[mL.sup.-1] of rumen fluid) incubated in rumen fluid buffered (BRF-buffered rumen fluid) or not (PRF-pure rumen fluid) on total gas volume (TV), C[H.sub.4] production, dry matter degradation (DM Deg) and final pH after 24 hours in vitro incubation at 39[degrees]C. The incubated sample consisted of corn silage dried at 55[degrees]C for 72h and ground to 1mm. Rumen fluid was collected before feeding from three cattle cannulated in the rumen and adapted to corn silage. A portion of fluid was used without adding buffer solution, and another portion of fluid was buffered at a ratio of 1:2 (rumen fluid: buffer ANKON[R]).

Incubation was performed in 250-mL Erlenmeyer flasks closed with silicone stoppers, and gases produced were transported by capillarity to the storage bottle. There in, it was measured the gas column height to estimate TV. After determining the TV, an aliquot of 0.5mL gas was collected with a syringe and injected into a gas chromatograph to determine C[H.sub.4] concentration.

Final pH was measured after 24 hours incubation using a digital potentiometer. DM Deg was calculated after centrifugation for 3 minutes at 3000rpm, separation and drying the residue in an oven, subtracting the blank value (CHAUDHRY & KHAN, 2012). Data were analyzed as a randomized complete block design in a 3 x 2 factorial arrangement (3 sample amounts x 2 types of ruminal fluid) with 3 replications and 3 blank for each type of fluid. Data were subjected to analysis of variance and means were compared by Tukey's test. Statistical significance was set at P[less than or equal to] 0.05.

There was no interaction effect between sample amount x type of ruminal fluid (P>0.05). Regardless of fluid, the greatest amount of sample incubated (2.6g DM 100[mL.sup.-1]) resulted in lower TV and DM Deg (P<0.05), while the amounts of 0.6 and 1.3g DM 100[mL.sup.-1] were not significantly different from each other (P>0.05; Figure 1).

Methane production per g of incubated DM (mL [g.sup.-1] of DMI) was higher using the PRF (P<0.05; Table 1). Sample amount had no effect on C[H.sub.4] production using BRF. However, with PRF, the greatest sample amount (2.6g of DM 100 [mL.sup.-1] of rumen fluid) resulted in lower (P<0.05) C[H.sub.4] production (mL [g.sup.-1] of DMI), but the amounts of 0.6 and 1.3 (g of DM 100[mL.sup.-1] of rumen fluid) were not significantly different from each other for C[H.sub.4] production (P>0.05).

The greater the sample amount was, the lower the final pH of incubation, regardless of ruminal fluid types (P<0.05). PRF showed a higher final pH of incubation with sample amounts of 0.6 and 1.3g of DM 100[mL.sup.-1] compared with BRF (P<0.05). However, for the greatest sample amount (2.6g of DM 100[mL.sup.-1] rumen fluid), there was no difference in final pH between the types of fluids used (P>0.05). The final pH was lower than 5.4 using 2.6g of DM 100[mL.sup.-1] rumen fluid.

In the present study, the reduction in pH with greater amount of sample was probably due to the saturation of fluid, which had higher concentration of volatile fatty acids produced by fermentation, which can inhibit the activity of rumen microorganisms. Consequently, it may reduce DM Deg and C[H.sub.4] production per g of DM incubated (RAMIN & HUHTANEN, 2012). Higher C[H.sub.4] production using PRF, regardless of the sample amount, indicated no negative effect in the absence of buffer in the ruminal fluid. Thus, it is possible to conduct in vitro incubation of corn silage without buffer solution.

The final pH at 5.8 indicated the excess of sample, which is possibly the decisive factor in reducing DM Deg (Figure 1), since the microbiota fermenting fiber carbohydrates is inhibited by pH reduction. RYMER et al. (2005) incubated hay or wheat grain using different ratios of rumen fluid/buffer solution and reported reduction in final pH of in vitro incubation related to the decreased amount of buffer solution, differently from the findings of this study.

The lowest amount of sample incubated (0.6g of DM 100[mL.sup.-1] rumen fluid) associated with PRF improved the DM Deg, resulting in higher TV, which may be related to lower production rate of fermentation end products. Unlike, in in vivo conditions, end products leave the rumen by absorption or passage, but in in vitro system, they remain in the rumen, indicating that the use of buffer is important when greater amounts of corn silage are incubated.

The results suggested the possibility of incubation with the lowest sample amount, because it provides higher final pH and higher DM Deg. The use of buffered rumen fluid reduces C[H.sub.4] production from corn silage. Further studies should be carried out to verify the possibility of in vitro ruminal incubation with pure rumen fluid using other feedstuffs.


Authors declaration

We, authors of the article entitled In vitro fermentation of corn silage using buffered rumen fluid or not and three amounts of sample, declare that the data from this study were not submitted for evaluation to the Ethics and Biosafety Committee of the Faculdade de Ciencias Agrarias e Veterinarias (FCAV), Universidade Estadual Paulista (UNESP) in Jaboticabal, Sao Paulo State, but we are aware of the rules, and therefore, the experiment was conducted in accordance with the laws and regulations which controlls experiments that use live animals in Brazil-Conselho Nacional de Controle de Experimentacao Animal (CONCEA) <>, < content/view/310553.html>. Thus, the authors assume full responsibility for the data and are available for possible questions.


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Antonio Carlos Homem Junior (I) Jane Maria Bertocco Ezequiel (II) Henrique Leal Perez (III) Marco Tulio Costa Almeida (III) Josimari Regina Paschoaloto (III) Vanessa Barbosa de Carvalho (III) Leonardo Fernandes Cremasco (II) Macella Borsonello da Costa (II)

(I) Centro Estadual de Educacao Tecnologica Paula Souza, Faculdade de Tecnologia (FATEC), R. Francisco Henrique Lemos, 51, 15900-000,Taquaritinga, SP, Brasil. E-mail: Corresponding author.

(II) Departamento de Zootecnia, Faculdade de Ciencias Agrarias e Veterinarias (FCAV), Universidade Estadual Paulista (UNESP), Jaboticabal, SP, Brasil.

(III) Programa de Pos-graduacao em Zootecnia, FCAV, UNESP, Jaboticabal, SP, Brasil.

Received 06.13.14

Approved 04.07.15

Returned by the author 07.30.15


Table 1--Methane production and final pH of in vitro
incubation of corn silage using PRF or BRF with three
sample amounts.

Item               Sample         Rumen fluid
                 amount (2)
                                 PRF          BRF         Mean

C[H.sub.4] (1)      0.6       22.38 (Aa)   10.00 (Ab)   16.19 (A)
                    1.3       19.44 (Aa)   12.11 (Ab)   15.77 (A)
                    2.6       13.53 (Ba)   8.82 (Ab)    11.17 (b)
                    Mean      18.45 (A)    10.31 (b)
Final pH            0.6       6.65 (Aa)    6.49 (Ab)    6.57 (A)
                    1.3       6.13 (Ba)    5.85 (Bb)    5.99 (B)
                    2.6       5.38 (Ca)    5.32 (Ca)    5.35 (c)
                    Mean      6.05 (a)     5.89 (b)

Item                            Effect

                 RMSE   Amount   Fluid   Interaction

C[H.sub.4] (1)   1.27    ***      ***        ***
Final pH         0.03    ***      ***        ***

Mean values in the same row followed by different lowercase
(a,b) letters and mean values in the same column followed by
different uppercase (A,B,C) letters are significantly
different by Tukey's test (P>0.05); (1) mL [g.sup.-1] DM
incubated; (2) Sample amount g 100[mL.sup.-1] rumen fluid;
PRF--Pure rumen fluid, BRF--Buffered rumen fluid;
RMSE--Root mean square error; *** P<0.0001.
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Title Annotation:produccion animal; texto en ingles
Author:Homem, Antonio Carlos, Jr.; Ezequiel, Jane Maria Bertocco; Perez, Henrique Leal; Almeida, Marco Tuli
Publication:Ciencia Rural
Date:Dec 1, 2015
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