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Agrociencia

versão On-line ISSN 2521-9766versão impressa ISSN 1405-3195

Agrociencia vol.52 no.8 México Nov./Dez. 2018

 

Animal Science

In vitro ruminal fermentation and emission of gases of diets with different inclusion of sunflower seed (Helianthus annuus)

Jerónimo Herrera-Pérez1 

María M. Crosby-Galván1  * 

José R. Bárcena-Gama1 

David Hernández-Sánchez1 

Omar Hernández-Mendo1 

Nicolás Torres-Salado2 

Rosy G. Cruz-Monterrosa3 

1Ganadería. Campus Montecillo. Colegio de Postgraduados. 56230. Montecillo. Estado de México.

2Universidad Autónoma de Guerrero, Unidad Académica de Medicina Veterinaria y Zootecnia No. 2 Carretera Acapulco-Pinotepa Nacional km 198, CP. 41940. Cuajinicuilapa Guerrero. México.

3Universidad Autónoma Metropolitana, Unidad Lerma, Avenida Hidalgo Pte. 46, Colonia La Estación, Lerma de Villada, CP. 52006, Estado de México

Abstract

Enteric methane (CH4) is produced during the process of energetic fermentation and represents an energy loss of 2 to 15 %. The seeds of oleaginous plants in the feed of ruminants are an alternative for reducing the production of CH4. Therefore, the objective of the present study was to determine in vitro the production of CH4, carbon dioxide (CO2) and the fermentative characteristics in diets for lambs with different levels of sunflower seed. Treatments were 0 (T1), 6 (T2), 12 (T3) and 18 % (T4) of inclusion of sunflower seed in the base diet. The diets were evaluated for production of CH4, CO2, production of volatile fatty acids (VFA), degradation of dry matter (DEGDM), neutral detergent fiber (DEGNDF) and acid detergent fiber (DEGADF), as well as the total bacteria count (TB) at 72 h of incubation. The experimental design was completely randomized, and an analysis of orthogonal polynomials was made to evaluate the linear and quadratic effects of the treatments. When the content of sunflower seed in the diet was increased, there was a reduction of DEGDM, DEGNDF and DEGADF (p≤0.05). The content of VFA after 72 h of fermentation showed a linear reduction (p≤0.05) when the content of sunflower seed was increased. The production of CH4, CO2 and TB count did not present differences among treatments (p>0.05). Therefore, increasing the amount of sunflower seed in the diet reduced the degradation capacity of its components.

Keywords: gas production; methane; in vitro; degradation of dry matter; sunflower seed

Introduction

The atmospheric concentrations of greenhouse gases (GEI) increased with anthropogenic activities (IPCC, 2007); therefore, they are considered precursors of climate change (Smith et al., 2007: Kumar, 2012: Hill et al., 2016). Livestock production contributes 18 % of the global emissions of GEI, and enteric methane produced by ruminants represents 37 % of the total anthropogenic production (Steinfeld et al., 2006; Key and Tallard, 2012; Kumar et al., 2014). CH4 is produced during ruminal fermentation of feed and represents 2 to 15 % of the energy consumed (Kumar et al., 2009: Eckhard et al., 2010). The efficiency of the energy consumed by the ruminant depends on the genetics of the animal, environmental conditions, along with quality and quantity of feed supplied (Shibata and Terada, 2010; Kumar et al., 2014).

The use of oilseeds in the feed of ruminants is due to the content of energy, protein and polyunsaturated fatty acids (Matthäus and Luciana, 2003). Oilseeds, such as those of sunflower (Helianthus annuus), reduce the emission of CH4 by 13 % (Beauchemin et al., 2009), and do not affect digestibility, ruminal fermentation (Soder et al., 2013; Vanegas et al., 2017), nor the content of fatty acids in ewes’ milk (Zhang et al., 2006). Chuntrakort et al. (2011) showed a reduction of 9 % of CH4 in cattle by giving a supplement with cottonseed, sunflower seed and coconut pulp.

Climate change and the loss of energy during ruminal fermentation of the feed stimulates the interest in finding different biotechnological alternatives that minimize the production of enteric CH4 (Eckard et al., 2010; Patra, 2012). Therefore, the objective of this study was to determine in vitro the production of CH4, CO2 and the fermentative characteristics in diets for lambs with different levels of sunflower seed.

Materials and methods

Location of the study area

The study was carried out in the laboratories of Animal Nutrition and Ruminal Microbiology and Microbial Genetics of the Graduate Program in Genetic Resources and Productivity - Livestock Production, Campus Montecillo of the Colegio de Postgraduados, State of Mexico.

Treatments

Treatments (Table 1) were elaborated according to the National Research Council (NRC, 2007) to satisfy the nutritional requirements of growing lambs. The ingredients of the diets were ground in a Thomas Wiley mill (Thomas Scientific®, Swedesboro, N.J., USA) with a 1 mm mesh prior to the elaboration of the feed.

Table 1 Composition and bromatological analysis of the treatments. 

Ingredientes T1 T2 T3 T4
Composición (g kg-1 MS)
Alfalfa 100 80 80 80
Maíz grano 500 500 500 520
Rastrojo de maíz 160 150 150 120
Pasta de soya 60 30 30 0
Semilla de girasol 0 60 120 180
Salvado de trigo 160 160 100 80
Premezcla mineral 20 20 20 20
Composición química
Materia seca, % 93.81 93.25 93.48 93.95
Proteína cruda, % 14.23 14.15 14.47 14.27
Extracto etéreo, % 1.96 1.98 2.06 2.16
Fibra detergente neutro, % 35.65 33.63 34.23 37.82
Fibra detergente ácida, % 18.59 19.59 21.57 22.29
Cenizas, % 7.09 6.71 6.40 6.27

T1 = 0 % sunflower seed; T2 = 6 % sunflower seed; T3 = 12 % sunflower seed; T4 = 18 % sunflower seed.

Bromatological analysis

Dry matter (DM), crude protein (CP), ash (Ce) and ether extract (EE) were determined according to the AOAC (2005) in the experimental diets. Neutral detergent fiber (NDF) and acid detergent fiber (ADF) were quantified using an ANKOM analyzer (200/220, USA) based on the method of Van Soest et al. (1991).

Culture medium

The culture medium contained 52.6 m mL of distilled water, 30 mL of clarified ruminal liquid [filtered with gauze, centrifuged at 20,817 xg for 15 min in a centrifuge (Eppendorf 5804, Germany) and was sterilized for 15 min at 121 °C and 15 psi in an autoclave (Tuttnauer 2540, Israel)], 5 mL of mineral solution I [6 g K2PO4 (Sigma) per 1000 mL distilled H2O], 5 mL of mineral solution II [6 g K2PO4; 6 g (NH4)2SO4 (Merck); 12 g NaCl (Sigma-Aldrich), 2.45 g MgSO4 (Sigma) and 1.6 g CaCl2H2O (Sigma) per 1000 mL of distilled H2O], 5 mL Na2CO3 (Merck) in 8 % solution [8 g Na2CO3 (Merck) in 100 mL distilled H2O], 2 mL solution of cysteine sulfite [2.5 g L -cysteine (Sigma) dissolved in 15 mL NaOH (2N) and 2.5 g Na2S-9H2O (Meyer) in 100 mL distilled H2O], 0.1 mL resazurin at 0.1 % [p/v; 0.1 g resazurin (Sigma-Aldrich) in 100 mL distilled H2O], 0.2 g soy peptone and 0.10 g yeast extract (Sánchez-Santillán and Cobos-Peralta, 2016; Ley-de Coss et al., 2016).

Traps for capture of biogas

The trap vials were prepared by placing a saturated saline solution [350 g NaCl (common salt) in 1 L distilled H2O and 5 mL methyl orange (Meyer) at 0.1 % in serological vials (120 mL). The pH of the saturated saline solution was adjusted with a potentiometer (Thermo Scientific® Orion 720A) to pH 2 with HCl (2N). The traps were maintained at room temperature until ready for use.

Biodigestors

From each treatment, 0.5 g of sample were placed in serological vials (120 mL) and sterilized for 15 min at 121 °C and 15 psi. Then, 45 mL of sterile culture medium were added to each vial according to the method of Cobos and Yokoyama (1995), under a flow of CO2, to verify sterility. The biodigestors were inoculated with 5 mL of fresh ruminal liquid (filtered with three layers of gauze and centrifuged for 3 min at 1257 xg) and were incubated 72 h at 39 °C.

The biodigestors were connected to a trap vial with a Taygon® hose (2.38 mm inside Ø and 45 cm length) with hypodermic needles (20 G x 32 mm) at the ends. A needle (20 G x 32 mm) was placed obliquely in the trap vial as escape valve and was placed inverted over a modified test tube of 50 mL.

Biogas production

Biogas production was measured as the displacement of the saturated saline solution at 24, 48 and 72 h of incubation. The proportion of CH4 and CO2 was determined of the biogas produced at 72 h contained in the trap vial by gas chromatography. This was performed in a chromatograph (PerkinElmer® Claurus 500, USA) equipped with a thermal conductivity detector and a Porapak® packed column. The conditions of analysis were as follows: temperature of oven, detector and column of 80, 130 and 170 ºC; helium as carrier gas (22.3 mL min-1) and injection volume of 300 mL. Retention times were 0.73 and 1.05 min for CH4 and CO2. The molar concentration of CH4 and CO2 was estimated by substituting the mL of gas produced in the general equation of ideal gases (Posadas and Noguera, 2005).

Fermentative characteristics

The in vitro degradation of DM (DEGDM) at 72 h of incubation was obtained by filtering the content of the biodigestors in ANKOM® (F57) bags. The bags with the nondegraded matter were dried 48 h at 60 °C in an oven (RIOSSA® HCF-41, Mexico). The DEGDM was calculated by difference of weight. The ANKOM® bags were sealed with heat and placed in a fiber analyzer based on the method of Van Soest et al. (1991) to estimate NDF and ADF. The percentage of degradation of the NDF (DEGNDF) was calculated with the formula DEGNDF = (initial NDF - residual NDF / initial NDF)* (100). In the degradation of ADF (DEGADF) a similar formula was used to that of DEGNDF (Hernández-Morales et al., 2018).

The total count of bacteria was obtained by placing 1 mL of the liquid part of the biodigestor with 72 h of incubation in test tubes (Pyrex®) 13 x 150 mm with 0.25 mL of formaldehyde (Sigma Aldrich) at 10 %. This was done with the direct count technique in a Petroff-Hausser® camera (Hausser #39000, Electron Microscopy Sciences, USA) and a microscope (Olympus® EX51, USA), at a magnification of 1000X. The total bacteria count was calculated with the following equation: total bacteria count = (average) (dilution factor; 2x107) (Sánchez-Santillán et al., 2016; Sánchez-Santillán and Cobos-Peralta, 2016).

An aliquot of 1 mL of the liquid part of the biodigestor was taken at 72 h of incubation and was placed in tubes for microcentrifuge (Eppendorf) with 0.25 mL of metaphosphoric acid (Meyer) at 25 % (ratio 4:1). The tubes were centrifuged at 20,817 xg in a centrifuge (Iletich zentrifuguen® EBA-21, Germany) and the supernatant was transferred to vials for chromatography (1.5 mL, Perkin Elmer®, USA). The analysis of volatile fatty acids (VFA) was performed in a chromatograph (PerkinElmer® Claurus 500, USA) equipped with a flame ionization detector (FID). The conditions of analysis were as follows: 1 µL of injection volume; column 15 m length x 0.32 mm inside diameter, film thickness of 0.25 µm, temperature limits of 40 to 250 °C (Elite FFAP PerkinElmer®, USA); temperature of 80, 250 AND 140 °C in oven, injector and column; nitrogen as carrier gas (flow 8 mL min-1); H2 and O2 as gases for generating flame (flow 45 and 450 mL min-1). Retention times were 1.26, 1.50 and 2.09 min for acetate, propionate and butyrate (Cobos et al., 2011).

Statistical analysis

The experimental design was completely randomized (eight independent replications). The data were analyzed with the GLM procedure of SAS® (2011). The mean values were compared with the Tukey test (p ≤ 0.05) and were analyzed using orthogonal polynomials for linear and quadratic effect.

Results and discussion

The concentrations of CH4 and CO2 (Table 2) did not present differences among treatments (p>0.05). Mao et al. (2010) mentioned that the production of enteric methane in diets that include sunflower seed increases rapidly after feeding and slowly decreases until the next feeding. The above justifies the results of our study because the CH4 was measured at 72 h of incubation, which caused a low quantification of CH4 and without differences among treatments (p>0.05). Furthermore, the synthesis of acetate and butyrate in the rumen increases the production of H2, and consequently the methanogenic arcs increase the production of CH4 by utilizing the H2 and the CO2 as energy source (Widiawati and Thalib, 2007; Kim et al., 2012; Chuntakort et al., 2014).

Table 2 Production of CH4 and CO2 in vitro (mM g-1) at 72 h in integral diets for lambs that includes four levels of sunflower seed. 

Tratamiento CH4 CO2
T1 0.82 7.32
T2 0.94 7.65
T3 0.78 6.87
T4 0.70 6.96
EEM 0.06 0.39
Lineal 0.07 0.30
Cuadrático 0.12 0.76

Average values with different letter in a column are statistically different (p≤0.05).

T1 = 0 % of sunflower seed; T2 = 6 % sunflower seed; T3 = 12 % sunflower seed and T4 = 18 % sunflower seed. SEM: Standard error of the mean; CH4: methane; CO2: carbon dioxide.

The DEGDM at 72 h of incubation (Table 3) presented differences among treatments (p≤0.05). T2, T3 and T4 decreased (p≤0.05) the DEGDM by 3, 5.4 and 8 % with respect to T1 by the linear effect among treatments (Table 3), which could be directly related to the fiber content of the seed and its fat content. These results are similar to those of Beauchemin et al. (2009), who reported a reduction in the digestibility of the DM and organic matter of 8 to 20 % by using ground rapeseed (9 %) and sunflower seed (10 %) or fat protected with calcium salts in diets for cows.

Table 3 Fermentative characteristics in integral diets for lambs that include four levels of sunflower seeds at 72 h of incubation. 

Tratamiento DEGMS, % DEGFDN, % DEGFDA, % [Bacterias]
T1 88.80a 79.24a 69.70a 7.5 x 109
T2 86.18b 73.07b 62.44b 6.8 x 109
T3 84.01c 68.45c 57.13c 6.5 x 109
T4 81.86d 63.96d 53.67d 7.0 x 109
EEM 0.31 0.56 0.80 0.27
Lineal 0.01 0.01 0.01 0.50
Cuadrático 0.46 0.15 0.29 0.36

a,b,c,d Average values with different letter in a column are statistically different (p≤0.05).

T1 = 0 % sunflower seed; T2 = 6 % sunflower seed; T3 = 12 % sunflower seed and T4 = 18 % sunflower seed. SEM = Standard error of the mean; DEGDM = Degradation of dry matter; DEGNDF = Degradation of neutral detergent fiber; DEGADF = Degradation of acid detergent fiber. [Bacteria] = Concentration of total bacteria mL-1.

The DEGNDF decreased between 8 and 19 % (p≤0.05) when increasing the sunflower seed content in the treatments (Table 3). The above may be due to the changes in the fat content of the treatments, which is related to a reduction of protozoa and bacteria concentration in the rumen (Yang et al., 2009). It should be pointed out that the fibrolytic bacteria are sensitive to the fat content in the diet (Patra and Yu, 2012).

The increase in the proportion of sunflower seed in the treatments reduced (p≤0.05; Table 3) the DEGNDF by 8 to 19 % and the DEGADF by 10 to 33 %, which is similar to the reduction of degradability of the DM (8.9 to 19.2 %), NDF (10.1 to 19.8 %) and ADF (3.7 to 28.8 %) by including in the diet coconut pulp, cottonseeds and sunflower seeds (Chuntrakort et al., 2014). The addition of 10 % of oilseeds (linseed, canola and sunflower) in a basal diet with orchardgrass (Dactylis glomerata L.) did not affect the digestibility of the DM and NDF (Soder et al., 2013).

The amounts of total bacteria at 72 h of incubation did not show differences among treatments (p>0.05); Table 3). This result is similar to that reported by Ley de Coss et al. (2013), but lower than that published by Dehority (2003). The former published a count of 109 bacteria mL-1; whereas the latter reported from 1010 to 1012 bacteria mL-1 in rumen. The results of our study are related to ecological principles, given that the microbial population of the rumen is integrated by a variety of species that constantly change based on the medium that surrounds it. Therefore, the elimination or suppression of any microbial group causes the adaptation of another group to fill its space in the ruminal ecosystem (Hungate, 1966; Czerkawski, 1986; Weimer, 1998).

The concentration of total VFA showed a linear decrease (p≤0.05) with respect to the control (T1; Table 4). The concentration of acetic, propionic and butyric showed a behavior similar to that of total VFA, which may be related to the decrease in the DEGDM. The decrease in the concentration of VFA in the rumen was related to a lower concentration of H2 (Dohome et al., 1999), because it is the principal byproduct after the synthesis of acetic acid and butyric acid in the rumen. Furthermore, the H2 generated and the presence of methanogenic archaea increment the production of CH4 by utilizing the H2 and CO2 as energy source (Kim et al., 2012). The treatments presented a linear effect (p ≤ 0.05) in the reduction of VFA as the sunflower seed content increased, which is congruent with that reported by Jordan et al. (2006), who mentioned that the total concentration of VFA decreased by utilizing coconut oil in diets for beef cattle, because the digestibility of the DM and of the components of the NDF and ADF was reduced.

Table 4 Concentration of volatile fatty acids (mM) at 72 h in integral diets for lambs that include four levels of sunflower seed. 

Tratamiento Acético Propiónico Butírico A:P AGV total
T1 73.93a 66.05a 14.80a 1.12 154.78a
T2 72.72a 63.34ab 14.41a 1.15 150.47ab
T3 69.64ab 62.03b 14.00ab 1.12 145.62bc
T4 65.85b 59.70b 13.31b 1.10 138.86C
EEM 1.17 0.91 0.26 0.17 2.01
Lineal 0.01 0.01 0.01 0.34 0.01
Cuadrático 0.29 0.84 0.63 0.16 0.55

a,b,c,d Average values with different letter in a column are statistically different (p≤0.05).

T1 = 0 % sunflower seed; T2 = 6% sunflower seed; T3 = 12 % sunflower seed and T4 = 18% sunflower seed. SEM: Standard Error of the Mean; A:P ratio acetic propionic; VFA: volatile fatty acids.

Conclusions

The addition of up to 18 % of sunflower seed in diets for sheep does not affect the production of greenhouse gases. The increment of sunflower seed reduces the degradation of the nutrients of the diet and the fermentation of the volatile fatty acids.

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Received: November 2017; Accepted: February 2018

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