Ruminal Methane Emission, Microbial Population and Fermentation Characteristics in Sheep as Affected by Malva sylvestris Leaf Extract: in vitro Study

Document Type: Research Articles

Authors

1 Department of Animal Science, Faculty of Agriculture, Razi University, Kermanshah, Iran

2 Department of Animal Science, Faculty of Agriculture, University of Kurdistan, Sanandaj, Iran

Abstract

The objective of this study was to investigate in vitro effect of Malva sylvestris leaf extract (at 0, 25, 50 and 100 µL/30 mL of medium) on sheep ruminal cellulolytic and total viable bacteria growth, protozoa populations, methane production, neutral detergent fiber degradability (NDFD) and fermentation efficiency of oat hay. The addition of Malva sylvestris leaf extract at 25, 50 and 100 µL led to a linear increase (P<0.01) in vitro truly degraded dry matter (INTDDM), NDFD and partitioning factor (PF) of oat hay and decrease (P<0.01) methane emission after 24 hours of incubation. The addition of Malva sylvestris leaf extract resulted in a decrease (P<0.01) in potential of gas production at 25 and 50 µL and increase in lag time (0.95, 1.01 and 1.13 h relative to 0.61 h), constant rate of gas production (b)and gas produced at half-life (c)at 25, 50 and 100 µL. The addition of this extract decreased a number of total protozoa and Entodinium, Isotrichae, Diplodinium and Ophryoscolex species (P<0.01). The number of total and cellulolytic bacteria was not influenced by the addition of Malva sylvestris leaf extract. The result of this study demonstrated that Malva sylvestris extract had some potential for improving the rumen fermentation.

Keywords


INTRODUCTION

With increase in the probability of transmissible antibiotic resistance to human, the use of growth-promoter antibiotics in animal production systems has been banned in most developed countries (European Union, 2003(. The removal of antibiotics has led the scientists’ interest on investigation for the alternatives (Busquet et al. 2005; Iason, 2005; Durmic et al. 2014). Medicinal plants and their extracts as natural alternative to antibiotics showed the potential of manipulating the rumen fermentation to improve the utilization of nutrients (Busquet et al. 2005; Kim et al. 2013; Kim et al. 2015(. The bioactive constituents of these compounds are secondary metabolites that are produced by plants to protect themselves against plant diseases and insects (Wallace, 2004). Malva sylvestris usually known as common Mallow is native to Asia, Europe, and north Africa and have been used as a medicinal plant since a long time ago. In the Mediterranean region, Malva sylvestris has a long history of use as food and potent drug in traditional and ethno veterinary medicines because of its anti-inflammatory, antioxidant, anti-complementary, anticancer and skin tissue integrity characteristics (Gasparetto et al. 2011). The whole aerial parts of Malva sylvestris or its leaves have been administrated to ruminant to treat colic, blocked rumen, mastitis, young calves’ diarrhea, respiration problem and reproductive disorder (Gonzalez et al. 2010; Idolo et al. 2010). Enteric methane arising due to fermentation of feeds in the rumen not only is a great source of energy loss but contributes substantially to the greenhouse gas emissions. In the recent years with increase horse farm in Kermanshah province the request for oat grain increased. Therefore, increase in oat cultivation resulted to enhance in oat hay and straw production that mostly have been used in the sheep nutrition. Thus, like an evaluation of chemical composition and nutritive values of feeds, methane production potential of each feed should be determined (Patra et al. 2015). The effect of Malva sylvestris on ruminant colic and improve the blocked rumen, may be because of its effect on rumen microbial activity. Therefore, the objective of the present experiment was to investigate the effect of Malva sylvestris leaf extract on rumen microbial populations, methane production and microbial fermentation of oat hay in sheep rumen liquor.

 

MATERIALS AND METHODS

Malva sylvestris plant was collected from Kermanshah province, Iran. For juice extraction, the fresh leaves of Malva sylvestris (pre-flowering) were finely grounded and blended in a commercial blender, squeezed through 2 layers of cheesecloth (Davys et al. 1969). The extract centrifuged at 454 × g for 15 minutes and the supernatant collected. The supernatant was saved and kept frozen at -80 ˚C until use. The effects of four doses [0.0 (as control), 25, 50 and 100 µL] of Malva sylvestris leaf extract were examined in vitro using mixed ruminal microbiota from sheep rumen liquor. The substrate used for batch cultures was oaten hay (crude protein (CP)=15.5, neutral detergent fiber (NDF)=54.2 and acid detergent fiber (ADF)=29.4% of dry matter (DM)) wich was grounded to pass from 1 mm screen. Rumen content was obtained from three fistulated sheep (39±4.5 kg body weight) before morning feeding. Animals were fed 0.6 kg alfalfa hay and 0.6 kg concentrate (16.5% CP, 25% NDF and 45% nonfiber carbohydrates (NFC)). For eliminating of large feed particles, immediately rumen content was filtered through two layers of cheesecloth and transferred to the laboratory in a prewarmed thermos (38 ˚C). In the laboratory, under anaerobic conditions, 30 mL of buffered rumen fluid [ratio of rumen fluid to buffer was 1:2, buffer prepared as proposed by McDougall (1948)], using pipettor pump was added into 125 mL bottles containing 0.2 g of oaten hay (12 replicates for each treatment in two runs). Then, bottles were sealed by a rubber stopper and aluminum cap and placed in shaking water bath for 96 h at 38.6 ˚C. Accumulation of gas produced in the bottles, head space was measured by a pressure transducer at 2, 4, 8, 12, 18, 24, 36, 48, 72 and 96 h after the incubation and the gas released (Theodorou et al. 1995). After 24 h of incubation 8 tubes from each treatment were withdrawn to determine in vitro truly degraded DM (INTDDM), partitioning factor (PF) and NDF degradability (NDFD) and enumeration of total viable and cellulolytic bacteria and rumen protozoa. Then each bottle content was filtered (42 μm pore size) and solid residues were used for determining IVDMD, PF and NDFD. A 10 mL sample of each filtrate bottle was taken and transfer into the separate flask (50 mL, 4 replicate/treatment). The flasks were closed and allowed to stand for 1 h at 38.6 ˚C. Rumen fluid was collected by suction from the middle of each flask. The ruminal fluid contained bacteria were serially diluted (10-fold increments) in the liquid version of mediums in the Hungate tubes (3 replicate). For enumeration of total viable and cellulolytic bacteria, the anaerobic technique proposed by Bryant (1972) was used for preparation medium. For cellulolytic bacteria ball-milled cellulose used as a single source of energy. The tubes were incubated at 38.6 ˚C for 72 h and 14 d (for cellulolytic bacteria) and in the end of incubation, growth was scored (+ or -) by the increase in optical density (650 nm). The cellulolytic and total viable bacteria population size was estimated using most probable number (MPN) procedure from replicate (3 tubes/dilution) dilutions. For protozoa enumeration, 5 mL of filtered rumen fluid was preserved using 5 mL of 50% Formalin (18.5% concentration of formaldehyde) as described by Dehority et al. (1984). Two drops of Brilliant green dye was added to 1 mL of rumen fluid (n=2) and stored overnight at the laboratory temperature. Then, 9 mL of 30% glycerol solution was added. Protozoa were enumerated microscopically in a Sedgwick-Rafter counting chamber. Protozoa species were identified from photographs and descriptions given by Ogimoto and Imai (1981).

 

Calculation and statistical analysis

Gas pressure was converted into volume using an experimentally calibrated curve. To estimate kinetic parameters of gas production, gas production data were fitted using France et al. (1993) equation as:

Y= A × ((1-exp)–(b(t-L))–(c(t½-L½ )))

Where:

A: volume of gas produced from quickly and slowly degradable fraction.

b and c: constants of the fractional rate (%/h).

t: incubation time (h).

L: lag time (h).

Y: volume of gas produced at time t.

The half-life (t1/2, h) of the fermentable fraction of each substrate was calculated as the time taken for gas accumulation to reach 50% of its asymptotic value. Methane production was measured according to the method proposed by Fievez et al. (2005). True substrate degradability was measured following the procedure of Blummel et al. (1997). Briefly, the contents of the bottles were transferred into the Berzelius beaker by repeated washings with 50 mL of neutral detergent solution (double strength), refluxed for 1 h, filtered through silica crucibles (Grade 1) and then for determining organic matter (OM) concentration, residues were burnt in a furnace at 600 ˚C for 3 h. Then INTDDM, INTDOM and NDFD were calculated as the ratio of residues DM or OM after 24 h of incubation/ incubated DM or OM. PF was calculated as the ratio of milligram organic matter truly degraded/mL gas produced after 24 h of incubation (Blummel et al. 1997). The population size of total viable and cellulolytic bacteria were calculated using most-probable-number tables (Alexander, 1982) with values derived from the number of tubes that showed positive growth. Data were analyzed using the SAS (SAS, 2002) and significance between individual means was identified using Duncan multiple range test. For enumeration total and cellulolytic bacteria and protozoa, least significant difference (LSD) was used to compare the means.

 

RESULTS AND DISCUSSION

In vitro rumen fermentation

Effect of addition of Malva sylvestris leaf extract on INTDDM, NDFD and PF of oat hay are summarized in Table 1. Relative to the control, the addition of Malva sylvestris leaf extract at 25, 50 and 100 µL/30 mL of medium resulted in a linear increase (P<0.01) in INTDDM and NDFD. Relative to the control, the PF was increased (P<0.05) with addition Malva sylvestris leaf extract at 25, 50 and 100 µL (2.74 vs. 2.81, 2.82 and 2.94). As it is shown in Table 1, the addition of Malva sylvestris leaf extract at 25, 50 and 100 µL, after 24 h of incubation decreased (P<0.01) the methane production (except at 50 µL doses) relative to the control. Gas production not affected with Malva sylvestris leaf extract supplementation, except at 50 µL (41.22 vs. 40.16 in control, P-value=0.001). According to results shown in Table 2, the addition of Malva sylvestris leaf extract resulted in a decrease in half-life (h) of gas production at 25 and 50 µL and increase at 100 µL. In addition, supplementation of Malva sylvestris at 25, 50 and 100 µL increased the lag time (h)and constant rate of gas production at half-life (c) (P<0.01). Half-life was decreased as a result of increased constant rate of gas production (c) during the half of incubation time (P<0.01).

 

Total viable and cellulolytic bacteria and protozoa population counting

Effect of Malva sylvestris leaf extract on genus diversity and total population of protozoa, total viable and cellulolytic bacteria population are shown in Tables 3 and 4. Results of the present study showed that the addition of Malva sylvestris leaf extract resulted in a decrease (P<0.01) in total protozoa (highest at 100 µL and lowest at 25 µL) and Entodinium, Isotrichae, Diplodinium and Ophryoscolex genus. Relative to the control, the addition of Malva sylvestris leaf extract at 25 and 50 µL increased and at 100 µL decreased total viable and cellulolytic bacteria (P>0.05). Partitioning factor (PF) is reported to be valuable in the accuracy of voluntary dry matter intake (DMI) prediction and microbial biomass synthesis efficiency of temperate-tropical crop residues and Mediterranean hays and forages with high PF result in high dry matter intake (DMI) (Blummel et al. 1997). The PF is known as the index of microbial biomass synthesis efficiency (Blummel et al. 1997). Therefore, results of the present study showed that Malva sylvestris leaf extract had a potential to increase the efficiency of ruminal microbial protein synthesis. The increase in PF by supplementation of Malva sylvestris leaf extract demonstrated that proportionally more of the truly degraded substrate was incorporated in microbial mass which can increase the efficiency of microbial protein synthesis. Blummel et al. (2005) reported 48.8% and 2.81 mg/mL for INTDDM and PF (at 24 h incubation) in oat hay, respectively. The inhibitory effect of Malva sylvestris leaf extract on methane production confirmed their anti-methanogens potent on ruminal microbes involved in methanogenesis. In ruminants, methane emission represents 8 to 12% loss of intake energy (Johnson and Johnson, 1995). On the other hands, methane is a greenhouse gas that has a global warming potential 21 times that of CO2 (Crutzen et al. 1995). Therefore, supplementation of additives that decrease ruminal methane emission can increase the efficiency of energy utilization in ruminant and decrease environment contamination (Kim et al. 2013; Kim et al. 2015). In addition, utilization of low methane producing feeds that are available at livestock farms could be strategically considered to feed ruminants decreasing environmental impacts (Patra et al. 2015). Increase in gas production with the addition of 50 µL of Malva sylvestris leaf extract and decrease in gas production at 100 µL may be due to raise in the concentration of antimicrobial secondary compounds of Malva sylvestris that added to the medium at this level. Our findings agree with previous in vitro batch culture reports where high doses of essential oils or extract have been tested (Jahani-Azizabadi et al. 2011; Busquet et al. 2005).

 

Table 1 Effect of Malva sylvestris leaf extract on in vitro fermentation characteristics of oat hay

 

IVTDMD: in vitro truly degraded dry matter; NDFD: nuteral detergent fiber degradability; PF: partitioning factor and DM: dry matter.

The means within the same column with at least one common letter, do not have significant difference (P>0.05).

SEM: standard error of the means.

 

Table 2 Effect of Malva sylvestris leaf extract on in vitro gas production parameters

 

A: potential gas production; b,c: constant rate; L: lag time and DM: dry matter.

The means within the same column with at least one common letter, do not have significant difference (P>0.05).

SEM: standard error of the means.

 

Table 3 Effect of Malva sylvestris leaf extract supplementation on in vitro protozoa population (×105/mL)

 

ND: not detected.

The means within the same row with at least one common letter, do not have significant difference (P>0.05).

SEM: standard error of the means.

 

Table 4 Effect of Malva sylvestris leaf extract on in vitro total and cellulolytic bacteria (log10/mL of medium)

 

The means within the same column with at least one common letter, do not have significant difference (P>0.05).

NS: non significant.

SEM: standard error of the means.

 

Linearly increasing in lag time withe increase in the concentration of Malva sylvestris leaf extract could be due to the increase in time required for bacterial colonization on feed particles. Therefore, results of the present study demonstrated that high doses of Malva sylvestris leaf extract in high-forage diets can decrease in dry matter intake due to increase in colonization time and rumen filling. Results of the present study demonstrated that Entodinium species accounted for around 90% of total counted protozoa. This finding was in agreement with values observed by other researchers (Santra et al. 1998; Hindrichsen et al. 2002). In addition, results of the present study suggested that Entodinium, Diplodinum and Ophryoscolex species were more sensitive to the addition of Malva sylvestris leaf extract. Relative to the control, abundance of Entodinium, Diplodinum and Ophryoscolex species were decreased by 59.8%, near to 100% and 82.3%, respectively. Therefore, in the present study decrease in methane production with addition of Malva sylvestris leaf extract was accompanied by a significant decrease in protozoa count. Around 10-20% of ruminal methanogenic bacteria are attached to the surface of protozoa, especially Entodinium species (Stumm et al. 1982). The methanogens take up hydrogen, which appears too toxic for Entodinium species activity. Previous studies have demonstrated that rumen protozoa defaunation reduced methane production ranging from 13 to 35% (Morgavi et al. 2008; Morgavi et al. 2012) and 9 to 25% in vitro (Newbold et al. 1995). Neutral detergent fiber degradability (NDFD) is an important index of forage quality. Increased NDFD may result to greater voluntary feed intake by reducing physical fill in the rumen (Dado and Allen, 1995). In vitro or in situ one unit increase in forage NDFD was associated with 0.17 kg increase in dry matter intake (Oba and Allen, 1999).On the other hand, results of the present study showed that addition of Malva sylvestris leaf extract resulted in a significant linear (P<0.01) increase in NDF disappearance. No significant changes in rumen cellulolytic bacteria and increase in NDF disappearance of oaten hay suggest that probably, the addition of Malva sylvestris leaf extract could result in an increase in the relative abundance of obligate cellulolytic bacteria with higher cellulolytic activity and persistence to main secondary compounds of Malva sylvestris leaf extract. However, unfortunately, there is not any information about the effect of Malva sylvestris leaf extract on rumen microbial fermentation.

 

CONCLUSION

In conclusions, Malva sylvestris leaf extract used in the present study resulted in the valuable effect on rumen microbial fermentation. For determine the mechanisms that Malva sylvestris leaf extract affects ruminal degradation of NDF and methane emission, more studies on the main rumen cellulolytic and methanogens bacteria are needed. As regards that this is first report about effect of Malva sylvestris extract on rumen microbial fermentation, future research is required to determine optimal doses and the effect of Malva sylvestris organic extract and essential oil on in vitro and in vivo rumen microbial fermentation patterns and animal performance.

 

ACKNOWLEDGEMENT

The authors thank the University of Razi (Kermanshah, Iran) for the financial support.

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