{'fraction_non_alphanumeric': 0.04236625142925919, 'fraction_numerical': 0.03454293795510622, 'mean_word_length': 4.5597189695550355, 'pattern_counts': {'":': 0, '<': 17, '<?xml version=': 0, '>': 2, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Effect of Fodder Tree Species with Condensed Tannin Contents on In vitro Methane Production

The objective was to evaluate the effect of fodder tree species (FTS) with condensed tannin contents: Cordia elaeagnoides, Platymiscium lasiocarpum, Vitex mollis, and Haematoxylon brasiletto, on in vitro methane (CH4) production at 24 h post incubation. The analysis was performed using the in vitro gas production technique, with three levels of inclusion/species: 600, 800, and 1,000 mg and with 4 replicates/species/level of inclusion. The substrate was incubated at 39°C, and the gas and CH4 production were recorded at 4, 8, 12, and 24 h post incubation. The data collected was analyzed through Pearson correlation, polinomial regression and fixed effects models. There were negative correlations between FTS-total gas volume (r = −0.40; p<0.001); FTS-volume of CH4 produced (r = −0.40; p<0.001) and between the inclusion level-volume of CH4 produced (r = −0.20; p<0.001). As well as a positive correlation between hours post incubation-total gas volume (r = 0.42; p<0.001) and between hours post incubation-volume of CH4 produced (r = 0.48; p<0.001). The FTS: C. elaeagnoides, V. mollis, and H. brasiletto have potential, in the three inclusion levels analyzed, to reduce CH4 emission on in vitro trials (>32.7%), taking into account the total CH4 production at 24 h of the forage used as reference (Avena sativa). It’s suggested that C. elaeagnoides-according to its crude protein, neutral detergent fiber, and condensed tannins content- is the best alternative within the FTS analyzed, for feeding ruminants and for the control of CH4 emissions during the dry season.


INTRODUCTION
Currently, global warming is one of the most important crises in the world. Livestock, especially ruminants, are major contributors to this crisis and to the deterioration of the ozone layer by releasing large amounts of greenhouse gases into the atmosphere, including carbon dioxide (CO 2 ) and methane (Leng, 2010). So CH 4 mitigation strategies in ruminants have focused on obtaining economic and environmental benefits (Thornton, 2010). The nutritional changes are more likely to be adopted by farmers because they increase the efficiency of feed utilization (Patra, 2012). Also, it has been shown that the use of different plants, such as trees and shrubs, have the potential to reduce CH 4 emissions in ruminants (Patra et al., 2006).
In the region of Tierra Caliente Michoacán, Mexico, a great variety of tree species have been referred by ranchers and identified as fodder tree species (FTS) which livestock (ruminants) consume mainly during the dry season. The FTS with condensed tannin (CT) contents, such as Cueramo (Cordia elaeagnoides), Atuto (Vitex mollis), Granadillo (Platymiscium lasiocarpum), and Brasil (Haematoxylon brasiletto), are identified in the region of Tierra Caliente, Michoacán, México. These trees are considered promising species in the region, since they have 4 or more of the following attributes: i) produce fruit, with possible energy supply for livestock, ii) provide comfort to the animal since they remain green (fully or partially), iii) contain less than 50% of neutral detergent fiber (NDF) and may be highly digestible, iv) have less than 5% of tannins, in order not to affect protein digestibility and v) have a concentration equal or greater than 0.2% of phosphorus. Therefore, the objective of this study was to evaluate the effect of the FTS as a source to inhibit in vitro CH 4 production.

Sampling of fodder tree species
Samples of Cueramo, Atuto, Granadillo, and Brasil were collected in December 2012 from Zacapungamio, municipality of Carácuaro (N 18° 57′ 46″ W 101° 03′ 39″) located in the region of Tierra Caliente, Michoacán, México. The municipality has elevations ranging from 300 to 1900 MASL. The area receives an annual rainfall of 800 to 1,000 mm, an average annual temperature greater than 22°C and winter temperature ranging around 18°C (INEGI, 2000). The leaves of the FTS were harvested (approximately 4 kg of each tree was collected, consisting of a pool of several trees from the same species) and transported for laboratory analysis to the Institute of Agriculture and Forestry Research, located in the municipality of Tarímbaro Michoacán, México. The samples were air dried indoors followed by oven drying for 48 h at 70°C and ground to pass a 1 mm sieve prior to chemical analysis and in vitro incubations.

In vitro gas production
Rumen liquor donor: Rumen fluid was obtained from a Creole bull (400 kg BW), which was fed oat hay (ad libitum), supplemented with minerals and had access to clear water (to simulate the feeding conditions during the dry season). The ruminal inoculum was obtained before the morning feeding (07:00 am) using a rumen fluid extraction equipment designed by Geishauser (1993). The collected rumen fluid was placed in warm insulated thermos (39°C) under anaerobic conditions and transported immediately to the laboratory. In the laboratory it was strained through four layers of cheesecloth and kept at 39°C under a N 2 atmosphere until utilization.

Culture medium and in vitro incubations:
The culture medium and the in vitro gas production test were carried out according to Menke and Steingass (1988), modified by using 70 mL serum bottles instead of calibrated glass syringes. The culture media consist of distilled water, macro and micromineral solution, buffer solution, resazurin and a reducing solution. It was prepared and inoculated using aseptic and anaerobic techniques. For the fermentation, 600, 800 and 1,000 mg of each species were incubated (with 4 replicates/species/level of inclusion) along with 10 mL of rumen fluid and 20 mL of the culture media. Additionally, serum bottles containing oat hay (without CT contents) were incubated in quadruplicate, serving as control for the incubation, no blanks were included. The bottles were sealed with rubber stoppers (Suba-Seal rubber septa network # 33, Sigma Aldrich, Milwaukee, WI, USA), the biogas in the headspace was flushed with N 2 and the bottles were incubated without agitation at 39°C for a period of 24 h. After each reading (4, 8, 12, 24 h post incubation) the bottles were agitated and returned to the incubator.
Determination of gas and methane production: To quantify the CH 4 production a gas chromatograph (GC) was used (Varian CP-3800, Walnut Creek, CA, USA), equipped with a flame ionization detector and a packed column Silica Gel 60/80 dimensions 18′×1/8″ stainless steel, the operating temperatures of the injector, the detector and the column were 170°C, 170°C, and 90°C respectively. With N 2 as the carrier gas at a flow rate of 30 mL/min. For analysis of CH 4 production, 30 µL were obtained from the headspace of each bottle with a gas-tight syringe (Hamilton Company, Reno, NV, USA; chromatography syringes) and injected in the GC at 4, 8, 12, and 24 h post incubation. Gas production was measured using a biogas measurement system (Beuvink et al., 1992).

Statistical analysis
With the gathered data, a database was developed for statistical analysis using Pearson correlation for continuous variables, the omega squared statistics of Hays (ω 2 ) to determine the degree of association between nominal and continuous variables, polynomial regression, and fixed effects models. The differences between treatments were tested using a least square means test (LSMEANS). The statistical package used was Scientific Applied Statistics (SAS, 2000). The model used to estimate polynomial methane gas production/FTS was: Where, Ŷ i = CH 4 production from: oat hay, Cueramo, Granadillo, Atuto, and Brasil, N = FTS inclusion level, t = hours post-incubation.
While for the fixed effects analysis the following model was used: Where, Y ij = in vitro CH 4 production at 24 h, μ = overall mean, T i = fixed effect of the ith treatment with i = oat hay at 600, 800, and 1,000 mg inclusion; Cueramo at 600, 800, and 1,000 mg inclusion; ... Brasil at 600, 800, and 1,000 mg inclusion, E ij = error (NID ~ 0, δ2).

Chemical composition
In order to evaluate the nutritional quality of the FTS during the dry season, the chemical composition analysis was done. Ash content in two FTS was >100 g/kg DM (Table 1). CP from the four FTS was higher than the reference feed (oat hay), and varied from 88 g/kg DM in Granadillo to 167 g/kg DM in Cueramo. The EE was very similar in all FTS but was much higher (32 to 35 g/kg DM) compared to the reference (6 g/kg DM). NDF in the FTS was very similar, ranging from 409 g/kg DM in Cueramo to 498 g/kg DM in Atuto. ADF varied from 220 g/kg DM in Cueramo to 339 g/kg DM in Atuto. Both NDF and ADF in FTS were lower than the reference oat hay with 633 and 392 g/kg DM, respectively.
CT and TP varied among the FTS (Table 1). Brasil showed the highest concentration of both CT and TP with 119 and 196 g/kg DM, respectively; in comparison with Cueramo, which contained the lowest concentrations of CT and TP with 0.2 and 22 g/kg DM, respectively.

Effect of fodder tree species on in vitro CH4 production
The degree of association observed between FTS and total volume of gas in vitro (ω 2 = 0.57; p<0.05) and between FTS and volume of CH 4 produced (ω 2 = 0.53, p<0, 05) was moderate (<70%) ( Table 2). Implying that 57% and 53% of the variance in the volume of gas and CH 4 produced in vitro, respectively, can be attributed to the presence of the FTS analyzed ( Figure 1). The total average of CH 4 production at 24 h was 44.7±28 mL/g DM. Average affected by treatment (FTS and inclusion level; p<0.001), hours post-incubation (p<0.001) and the interaction between treatment and hours post incubation (p<0.001).
Regarding the effect of the treatment on total CH 4 production (at 24 h post-incubation), it was found that CH 4 production was lower (p<0.05) with the species Cueramo and Brasil at the three inclusion levels analyzed (Table 3, Figure 2). The percent in CH 4 reduction from the FTS compared to oat hay (reference) showed a trend among the three levels of inclusion (Table 4). Granadillo was the species with the least CH 4 inhibited with a maximum of 18.6%.

DISCUSSION
The use of plant secondary metabolites has been previously studied for the inhibition of CH 4 production in ruminants. Hess et al. (2006) established that the strategic use of tannins is a means to limit CH 4 emission 13%. This is consistent with other researchers (Min et al., 2006), who determined that CT decreases (in a dose-dependent response) the rate of in vitro gas production. Dietary CP and NDF concentrations influence the amount of substrate organic matter fermented and the short chain fatty acids that are produced (Njidda and Nasiru, 2010). Crude protein concentrations below the threshold of 70 g CP/kg DM, restrict microbial activity due to a lack of nitrogen (Hariadi and Santoso, 2010). However, protein levels above this threshold, as seen in this study, are considered to enhance microbial multiplication in the rumen, thus improving fermentation (Njidda and Nasiru, 2010).    Nevertheless, Jayanegara et al. (2009) suggest that CT, in synergy with fiber digestion, decrease CH 4 production. The FTS in the region of Tierra Caliente, Michoacán, contain concentrations of CT, CP concentrations above the threshold and lower NDF concentrations than the rough forages used as feed during the dry season; thus, the four species used in our study were selected in order to test their capacity to decrease CH 4 production in vitro.

Effect of fodder tree species on in vitro CH4 production
Total CH 4 production decreased (mL/g DM) as the CT content increased, which occurred with the species Granadillo, Atuto, and Brasil. This was consistent with Bhatta et al. (2009) andJayanegara et al. (2011); they reported a negative correlation between tannin content and CH 4 production. Similarly, other researches O´kiely et al., 2011) concluded that a greater quantity of secondary metabolites present in FTS will lead to a lower in vitro gas and CH 4 production. The degree of association between level of inclusion and volume of CH 4 produced with the FTS was weak (ω 2 = 0.12; p<0.001), thus 12% of the variance in CH 4 production can be explained by the level of inclusion of the FTS analyzed. However, in other studies (Bhatta et al., 2009;Huang et al., 2010) it was reported that the greater the inclusion level of CT, the lower the CH 4 produced. This is consistent with the results found in this experiment (Table  4). As for the association between hours post-incubation and volume of gas produced (ω 2 = 0.30; p<0.001 and r = 0.42; p<0.001) and between hours post-incubation and volume of CH 4 produced (ω 2 = 0.24; p<0.001 and r = 0.48, p<0.001), these determined that as the hours of incubation elapse, gas volume and CH 4 production increase. Similarly, Abdalla et al. (2012) determined that the profile of cumulative gas production at 24 h is in function of the hours post-incubation, and depends on the CT concentration.
The reduction in CH 4 production by the species Brasil is attributed to its high CT content. Tannins have been shown to reduce CH 4 production by reducing fiber digestion (Tiemann et al., 2008); binding with proteins thus reducing degradation of the plant protein in the rumen and through the direct inhibition of the growth of methanogens . It has been established that a greater concentration of CT will lead to a lower total CH 4 production (Huang et al., 2010). Carulla et al. (2005) attributed the inhibition of CH 4 production from the species A. mearnsii to the direct effect of the CT.
The FTS Cueramo showed the least concentration of CT in comparison to the other species analyzed, but had a greater concentration of CP (167 g/kg DM) and a lower concentration of NDF (409 g/kg DM) compared to the rest of the FTS and oat hay ( Table 1). The CP and the NDF content in Cueramo may be the responsible for the inhibition of CH 4 production. Kume (2002) suggest that CH 4 reduction is attributed to a low content of NDF and a high content of CP. This is consistent with Meagher et al. (2005), they analyzed the species M. sativa, without CT, with 348 g of NDF/kg DM and 301 g of CP/kg DM, which led to a reduction in the volume of CH 4 produced. Jayanegara et al. (2009) determined that high concentrations of NDF in forages causes an increase of CH 4 production, due to a shift in the proportion of short chain volatile fatty acids towards acetate, which produces more H 2 .
It is generally accepted that feedstuffs which have higher gas production tend to have higher CH 4 production per gram DM incubated (Njidda and Nasiru, 2010;Jayanegara et al., 2011). This tendency was evident in the forages used in this study. This relationship is suggested to be a reflection of feed digestibility (Holtshausen et al., 2009). Increasing cell wall content (i.e. NDF and ADF) is considered to suppress microbial activity through a reduction in the availability of rapidly fermented carbohydrates (Wilson and Hatfield, 1997). This in turn, is negatively correlated with gas production (Njidda and Nasiru, 2010). This is consistent with the results obtained from the forage used as reference which had high concentrations of NDF (632 g/kg DM).
However, Getachew et al. (1998) indicate that the concentration level of the substrate reduces ruminal gas production per gram of DM, due to the low proportion of microorganisms in relation to the substrate or due to the buffer depletion. This could be associated with the decrease in CH 4 production by Cueramo and Brasil analyzed at a concentration of 1,000 mg.

CONCLUSIONS
The species with the greatest potential in CH 4 reduction were Brasil and Cueramo. The FTS with the most CH 4 produced at 24 h of incubation was Granadillo in the three inclusion levels (600, 800, and 1,000 mg). According to its CP, NDF, and CT content, Cueramo is the best alternative for ruminant nutrition during the dry season in the region of Tierra Caliente Michoacán and showed the best CH 4 reduction potential during the in vitro trials. However, there is a great variety of FTS which are used as a source of feed during the dry season in the region and with a CT content that will have no negative effects in ruminant nutrition. Further research is needed toward finding better alternatives in ruminant feeding and to determine how the FTS in the region of Tierra Caliente Michoacán can inhibit CH 4 production.

CONFLICT OF INTEREST
We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.