The Efficacy of Mootral Supplementation on Methane Production and Rumen Fermentation Characteristics in Ruminants Fed Different Styles

DOI: https://doi.org/10.21203/rs.3.rs-148722/v1

Abstract

Background: Using natural feed supplements to mitigate methane emissions from ruminants is a promising strategy. Many antimethanogenic compounds have been used to alter rumen fermentation, yet their potential to reduce methane production effectively is not consistent across different kinds of feeding styles (forage:concentrate ratios). Therefore, this study was conducted to investigate the impacts of Mootral (MT), a natural combination of garlic powder and bitter orange extract, on methane production, rumen fermentation, and digestibility in different feeding models commonly used for ruminants. The dietary treatments were 1000 g grass/kg ration (10 GRS), 8 GRS + 200 g concentrate/kg ration (2CON), 6GRS + 4CON, 4GRS + 6CON, and 2GRS + 8CON. MT was supplemented at 200 g/kg of the feed. Each group consisted of 6 replicates. The experiment was performed as a batch culture for 24 h at 39 °C. This procedure was repeated in 3 consecutive runs.

Results: The results of this experiment showed that supplementation with MT strongly reduced methane production in all kinds of feeding models (P<0.001). Its efficacy in reducing methane/digestible dry matter was 44% in the 10GRS diet, and this reductive power increased with the inclusion of CON up to a 69.5% reduction with the 2GRS + 8CON diet. MT application significantly increased gas and carbon dioxide production and the concentration of ammonia-nitrogen, but decreased the pH (P<0.001). In contrast, it did not interfere with organic matter and fiber digestibility. Supplementation with MT was effective in altering rumen fermentation toward less acetate and more propionate and butyrate. Additionally, it improved the production of total volatile fatty acids in all feeding models (P<0.001).

Conclusions: The MT combination showed effective methane reduction by improving rumen fermentation characteristics without exhibiting adverse effects on fiber digestibility. Thus, MT could be used with all kinds of feeding models to effectively mitigate methane emissions from ruminants.

Introduction

Due to the continuous expansion of the world population, human demand for meat and milk is expected to increase by 73% and 58%, respectively, by 2050 compared with 2010 levels [1]. Therefore, to meet future needs, animal production must be increased. Although the livestock sector, especially ruminants, plays an essential role in food security, it is considered a significant source of greenhouse gases (GHGs), such as methane (CH4) and carbon dioxide (CO2), representing approximately 14–18% of global anthropogenic GHG emissions depending on the accounting approaches by various sources (IPCC, FAO, EPA or others) [2, 3]. These GHGs are directly related to global warming and climate change, which threaten the well-being of current and future generations [4]. Ruminants emit CH4 as a byproduct of their normal digestive process due to fermentation of feed. The CH4 released from enteric fermentation through eructation represents a loss of up to 15% of their gross energy intake, thus being one of the most important inefficiencies in ruminant production systems in addition to its environmental impact, since CH4 is 28 times more powerful than CO2 at trapping the sun’s heat [3, 5].

Currently, studies are being performed of abatement strategies to reduce CH4 emissions and to improve the performance of ruminants. Through these studies, it has been proven that manipulation of the rumen microbiome with dietary supplements is one of the best mitigation strategies and it has a two-sided benefit for both the environment and efficient livestock production [6, 7]. Several research groups worldwide are investigating different kinds of feed additives/supplements with antimicrobial activity; however, the results reported in the literature are variable and show inconsistent efficacy [8, 9]. One of the major reasons for the inconsistent effectiveness is related to the type of diet: forage- or concentrate-based diets [9, 10]. It is often assumed that diet composition has a role in CH4 formation [11, 12]. Therefore, there is an urgent need for a feed supplement that could achieve effective reduction of emissions with different kinds of ruminant diets without impairing rumen fermentation; meanwhile, it should be natural, as there is global interest in using plants and their secondary metabolites as alternatives to chemical compounds in animal feed, and they are acceptable to consumers [13]. These compounds, which are called phytochemicals, show the ability to alter rumen fermentation and have direct toxic effects on methanogens [14].

Mootral (MT) (Mootral SA, Rolle, Switzerland), a natural mixture of organosulfur compounds extracted from garlic (Allium sativum) and flavonoids extracted from bitter orange (Citrus aurantium), showed the ability to reduce CH4 production without impairing rumen fermentation characteristics in two in vitro studies [15, 16]. However, there are still limitations to proving the efficacy of this new combination with different feeding models (grass:concentrate ratios). Therefore, this study was conducted to investigate the potential of MT to be used as an antimethanogenic feed supplement with different feeding styles of ruminants, considering its impact on rumen fermentation and nutrient digestibility.

Materials And Methods

Rumen fluid collection

At 3 h after the morning feeding, approximately 3 L of rumen fluid was collected from two ruminally fistulated, nonlactating Holstein cows (880 kg average body weight). The cows were maintained on a daily diet of Orchard grass (Dactylis glomerata) hay (organic matter (OM), 980 g/kg; crude protein (CP), 132 g/kg; neutral detergent fiber (NDF), 701 g/kg; acid detergent fiber (ADF), 354 g/kg; acid detergent lignin (ADL), 40 g/kg; on dry matter (DM) basis) with free access to clean drinking water and mineral blocks (KOEN® E250 TZ, Nippon Zenyaku Kogyo Co., Fukushima, Japan). The rumen fluid from each cow was collected from four different locations in the rumen. The collected rumen fluid was strained through four layers of surgical gauze into a thermos flask that was prewarmed to 39 °C and then immediately transferred to the laboratory within 15 minutes. The animal management and sampling procedures were approved by the Obihiro University of Agriculture and Veterinary Medicine, Animal Care and Use Committee (Approval number, 20–119).

Experimental treatments and in vitro incubation technique

Prior to the in vitro incubation, ten experimental groups, with six replicates each, were prepared with approximately 500 mg of ground substrate comprised of Kleingrass (Panicum coloratum) hay and commercial concentrate mixture at different ratios with and without MT inclusion. The MT mixture is composed of 90% garlic granules and 10% citrus extract powder. The garlic powder used for MT preparation was sourced from cultivated and carefully processed and dried non-GMO garlic of Chinese origin. The dried garlic granules were standardized to contain 1% (w/w) Allicin potential (S-Prop-2-en-1-yl prop-2-ene-1-sulfinothioate). Allicin concentration was determined by High Performance Liquid Chromatography (HPLC) as described in details by Eger et al. [15]. The citrus components for the MT mixture (Naringin, Naringenin, Neohesperidin, Rhoifolin, and Neoeriocitrin) were developed from commercially available citrus extracts (Khush Ingredients, Oxford, United Kingdom) mainly extracted from bitter oranges (Citrus aurantium). The total polyphenol content of the citrus extract was standardized to 45% (w/w) by the Folin-Ciocalteau method [17]. Flavonoid concentrations were analyzed by HPLC using standards from (Sigma-Aldrich Ltd., Dorset, United Kingdom). Further information on the MT preparation was described in details by Eger et al. [15]. This mixture was provided by a Swiss company (Mootral SA, Rolle, Switzerland).

The experimental diets were as follows: 1- 1000 g grass/kg ration (10GRS), 2- 10GRS + 200 g MT/kg of substrate (2MT), 3- 8GRS + 200 g concentrate/kg ration (2CON), 4- 8GRS + 2CON + 2MT, 5- 6GRS + 4CON, 6- 6GRS + 4CON + 2MT, 7- 4GRS + 6CON, 8- 4GRS + 6CON + 2MT, 9- 2GRS + 8CON, and 10- 2GRS + 8CON + 2MT. Five hundred milligrams of each of the experimental substrates (GRS and CON) was added to preweighed ANKOM filter bags (F57, ANKOM Technology, Macedon, NY, USA), which were heat-sealed and placed in 120 mL glass bottles, while the MT feed supplement was added directly to the bottles one day before incubation. The MT dose used in the current study was based on the most effective dose in our previous study [16]. The chemical composition of the substrates and the MT are described in Table 1.

Table 1

Chemical composition of ration and Mootral (g/kg of dry matter) used in 24 h in vitro incubation.

(g/kg dry matter)

Kleingrass hay

Concentrate

Mootral

Dry matter (g/kg fresh matter)

844.9

843.0

871.5

Organic matter

904.7

934.2

955.5

Crude ash

95.3

65.8

44.5

Crude protein

134.8

223.1

210.7

Ether extract

38.4

38.0

17.1

Neutral detergent fiber

662.5

232.6

35.5

Acid detergent fiber

362.6

109.1

33.7

Acid detergent lignin

52.2

30.1

1.8

The procedure of in vitro batch culture was performed as described by Menke and Steingass [18]. In the laboratory, the collected rumen fluids from the two cows were mixed together in one beaker under a constant stream of CO2. Forty milliliters of fresh buffer solution at a pH of 6.8 prepared according to McDougall [19] with twenty mL of rumen fluid was added to each 120 mL bottle under continuous CO2 flushing to maintain anaerobic conditions. Thereafter, the fermentation bottles were flushed with CO2 before sealing with butyl rubber stoppers and aluminum caps (Maruemu Co., Ltd, Osaka, Japan). All bottles were incubated for 24 h at 39 °C. This batch culture procedure was repeated in three consecutive runs on three different days. In each run, two blanks without substrate (empty filter bag plus 60 mL of buffered rumen fluid) were included to be used for digestibility and gas production correction. In total, 180 bottles plus 6 blank bottles were examined in this study.

Sample collection

After 24 h of incubation, the total gas production was measured, and gas samples (3 mL) were collected from the headspace of the glass bottles into vacutainer tubes (BD Vacutainer®, Becton Drive, USA). The tubes were stored at room temperature until CH4 and CO2 determination. Thereafter, the bottles’ caps were removed, and the pH of each tube was recorded using a pH meter (LAQUA F-72, HORIBA Scientific, Kyoto, Japan). Then, aliquots of the culture fluid were transferred into 1.5 ml Eppendorf tubes and centrifuged at 16,000×g and 4 °C for 5 minutes. The supernatant was collected and transferred into a new Eppendorf tube® (Eppendorf AG, Hamburg, Germany), which was stored at − 20 °C until use for volatile fatty acid (VFA) and ammonia nitrogen (NH3-N) analysis. The bags were removed from the bottles, washed under running tap water until the draining fluid became clear, and then dried at 60 °C for 48 h to determine the in vitro dry matter digestibility (IVDMD). After IVDMD determination, the bags were used for the determination of in vitro organic matter digestibility (IVOMD), in vitro neutral detergent fiber digestibility (IVNDFD), and in vitro acid detergent fiber digestibility (IVADFD). The residues in the fermentation bottles were discarded.

Chemical analysis

The chemical composition of the GRS, CON, MT and remaining substrate in the bags was determined following the standard procedure of AOAC [20]. DM content was measured by drying the samples in an air-forced oven at 135 °C for 2 h (930.15). OM and ash were measured by placing the samples into a muffle furnace at 500 °C for 3 h (942.05). Nitrogen (N) was measured according to the method of Kjeldahl (984.13) using an electrical heating digester (DK 20, VELP Scientifica, Usmate (MB), Italy) and an automatic distillation apparatus (UDK 129 VELP Scientifica, Usmate (MB), Italy), and then CP was estimated as N × 6.25. aNDF, ADF, and ADL were measured and expressed as inclusive residual ash using an ANKOM200 Fiber Analyzer (Ankom Technology Methods 6, 5 and 8, respectively; ANKOM Technology Corp., Macedon, NY, USA). aNDF was measured using sodium sulfite with heat-stable α-amylase.

Gas composition analysis

The concentrations of CH4 and CO2 in the gas samples were determined by injection of 1 mL using a Hamilton gastight syringe (Hamilton Company, Reno, Nevada, USA) into a gas chromatograph (GC-8A, Shimadzu Corp., Kyoto, Japan). The carrier gas was helium. The temperatures of the infuser port, column, and detector were 70 °C, 150 °C, and 150 °C, respectively. The identification of CH4 and CO2 was based on the retention time.

Volatile fatty acids and NH3-N analysis

The concentration of VFA was determined using high-performance liquid chromatography (Shimadzu Corp., Kyoto, Japan) after diluting the supernatant 3 times with distilled water. Briefly, the analytical specifications were as follows: column, Shim-pak SCR-102H (7 mm, i.d. 8.0 mm×300 mm, Shimadzu Corp., Kyoto, Japan); eluent flow rate and mobile phase for organic acid analysis (Shimadzu Corp., Kyoto, Japan) at 0.8 mL/min; column temperature, 40 °C; reaction reagent and flow rate, pH buffer for organic acid analysis (Shimadzu Corp., Kyoto, Japan) at 0.8 mL/min; conductivity detector (CDD-10AVP, Shimadzu Corp., Kyoto, Japan). Quantification of the VFA concentration was performed using an external standard quantitation method [16].

The NH3-N concentration was measured by diluting samples 50 times with 0.1 M phosphate buffer (pH 5.5) and then they were analyzed following the procedure of the modified Fujii-Okuda method [21] using an NH3 kit (FUJIFILM Wako Pure Chemical Corp, Osaka, Japan). The plate was read by a microplate reader (SH-1000 Lab, Corona Electric Co., Ltd., Japan) at an optical density of 630 nm.

Statistical analysis

All variables were analyzed using PROC MIXED by SAS version 9.4 (SAS Institute Inc., Cary, NC, USA). The model included the treatment (diet) effect, MT effect, and their interaction as fixed effects, while the experimental runs were considered random effects. Least square means and standard error (SEM) were calculated, and the differences of means were estimated by pairwise t-tests (PDIFF option of PROC MIXED). Significance was declared at P < 0.05, and a tendency toward significance was declared when the P value was between 0.05 and 0.10.

Results

Effect of Mootral supplementation on in vitro pH, gas production, and gas composition

Supplementation of MT to all feeding models reduced the pH (P < 0.001) when compared with its corresponding group without MT supplementation in the same feeding model (Table 2). Moreover, the inclusion of MT increased the absolute total gas production when correlated with DM and digestible DM in all feeding styles (P < 0.001, Table 2).

Table 2

Effect of Mootral supplementation on gas production and pH in different feeding styles after 24 h in vitro incubation (n = 18).

Treatments1

Parameter

10GRS

8GRS+2CON

6GRS+4CON

4GRS+6CON

2GRS+8CON

 

SEM

 

P-value

0

2

0

2

0

2

0

2

0

2

 

Trt

MT

Trt×MT

Gas production (ml)

30.06

45.53***

40.18

51.19***

43.50

53.08***

44.17

57.14***

45.94

54.47***

0.74

< 0.001

< 0.001

0.07

Gas/DM2 (ml/g)

71.17

107.67***

94.90

120.93***

102.89

125.44***

104.31

135.09***

108.68

128.87***

1.76

< 0.001

< 0.001

0.07

Gas/Digestible DM (ml/g)

53.83

79.77***

62.34

84.88***

64.23

82.59***

62.54

82.54***

62.38

75.67***

0.95

< 0.001

< 0.001

0.001

pH

6.58

6.50***

6.55

6.47***

6.53

6.44***

6.51

6.41***

6.50

6.40***

0.01

< 0.001

< 0.001

0.36
1 GRS: grass; CON: concentrate; 0: 0 g Mootral/kg; 2: 200 g Mootral/kg of substrate. 2 DM: dry matter. Asterisks in 2 mean significant difference between 0 and 200 g Mootral/kg in the same feeding model, * (P < 0.05), ** (P < 0.01), *** (P < 0.001). Trt: treatment; MT: Mootral; Trt×MT: interaction between treatment and Mootral. SEM: standard error of the mean.

Adding MT to all feeding styles decreased the proportion of CH4 but increased the proportion of CO2 in the produced gas (P < 0.001, Fig. 1A, 1C). The proportion of produced CH4/g digestible DM decreased (P < 0.001) by 61.9%, 61.4%, 67.1%, 72.3%, and 73.6% due to MT inclusion in all feeding styles (10GRS, 8GRS + 2CON, 6GRS + 4CON, 4GRS + 6CON, and 2GRS + 8CON, respectively, Fig. 1B). Furthermore, the CH4/CO2 ratio in the produced gas (ml/ml) decreased in all feeding models due to MT’s effect (P < 0.001, Fig. 1D).

MT inclusion was effective with all diets in reducing the production of CH4/DM (ml/g) (P < 0.001, Fig. 2A); moreover, it reduced the production of CH4/digestible DM (ml/g) by 44.2%, 49.1%, 60.4%, 63.9%, and 69.5% in 10GRS, 8GRS + 2CON, 6GRS + 4CON, 4GRS + 6CON, and 2GRS + 8CON, respectively (P < 0.001, Fig. 2C). In contrast, the production of CO2/DM and CO2/digestible DM (ml/g) increased (P < 0.001) due to the effect of MT in all feeding styles (Fig. 2B, 2D, respectively).

Effect of Mootral supplementation on in vitro nutrient digestibility and ammonia-nitrogen concentration

MT supplementation did not affect the IVDMD in the different experimental diets except in the 8GRS + 2CON and 6GRS + 4CON diets, where adding MT to these styles reduced the IVDMD (P < 0.01, Table 3). However, IVOMD, IVNDFD, and IVADFD did not show any differences when MT was added as compared with their corresponding groups without MT supplementation (P > 0.05, Table 3). MT inclusion increased the NH3-N concentration (P < 0.01) in 10GRS and 6GRS + 4CON and it tended to increase in 4GRS + 6CON (P = 0.088), while there was a non-significant numerical increase in 8GRS + 2CON (P = 0.106) and 2GRS + 8CON (P = 0.32) (Table 3).

Table 3

Effect of Mootral supplementation on digestibility and ammonia-nitrogen in different feeding styles after 24 h in vitro incubation (n = 18).

Treatments1

Parameter

10GRS

8GRS + 2CON

6GRS + 4CON

4GRS + 6CON

2GRS + 8CON

SEM

P-value

0

2

0

2

0

2

0

2

0

2

  

Trt

MT

Trt×MT

IVDMD2

0.55

0.57

0.64

0.60*

0.68

0.64**

0.71

0.69

0.73

0.72

0.01

< 0.001

0.002

0.04

IVOMD3

0.47

0.44

0.55

0.52

0.60

0.55

0.62

0.62

0.66

0.64

0.02

< 0.001

0.03

0.80

IVNDFD4

0.36

0.36

0.40

0.38

0.38

0.32

0.36

0.34

0.32

0.35

0.01

0.61

0.56

0.76

IVADFD5

0.21

0.24

0.25

0.21

0.21

0.23

0.20

0.23

0.22

0.23

0.01

0.97

0.62

0.64

NH3-N6

(mg/dL)

4.46

6.43***

6.30

7.09

6.14

7.51**

6.01

6.84

7.35

7.83

0.18

< 0.001

< 0.001

0.17
1 GRS: grass; CON: concentrate; 0: 0 g Mootral/kg; 2: 200 g Mootral/kg of substrate. 2 IVDMD: In vitro dry matter digestibility. 3 IVOMD: In vitro organic matter digestibility. 4 IVNDFD: In vitro neutral detergent fiber digestibility. 5 IVADFD: In vitro acid detergent fiber digestibility. 6 NH3-N: ammonia-nitrogen. Asterisks in 2 mean significant difference between 0 and 200 g Mootral/kg in the same feeding model, * (P < 0.05), ** (P < 0.01), *** (P < 0.001). Trt: treatment; MT: Mootral; Trt×MT: interaction between treatment and Mootral. SEM: standard error of the mean.

Effect of Mootral supplementation on in vitro volatile fatty acids

MT supplementation did not show any effect on the acetate concentration in all feeding models; however, the interaction between MT and treatment showed a difference for the MT supplemented groups to be increased in 10GRS (P < 0.001) and to be decreased in 2GRS + 8CON (P < 0.05) compared with its corresponding treatment without MT inclusion (Table 4). In contrast, the acetate ratio decreased in all feeding models due to MT supplementation (P < 0.01), while the interaction between MT and treatment did not have a significant effect (P > 0.05, Table 4). The concentration and the ratio of propionate and butyrate increased (P < 0.001) by adding MT in all feeding models. Additionally, the concentration of total volatile fatty acids (TVFA) showed the same finding (Table 4). The acetate/propionate (A/P) ratio decreased (P < 0.001) with MT supplementation in all feeding styles (Table 4).

Table 4

Effect of Mootral supplementation on volatile fatty acids in different feeding styles after 24 h in vitro incubation (n = 18).

Treatments1

Parameter

10GRS

8GRS + 2CON

6GRS + 4CON

4GRS + 6CON

2GRS + 8CON

SEM

P-value

0

2

0

2

0

2

0

2

0

2

  

Trt

MT

Trt×MT

Acetate (mmol/L)

62.54

66.02**

64.81

66.12

64.81

65.66

65.61

65.91

66.80

64.15*

0.54

0.45

0.21

0.01

Propionate (mmol/L)

15.24

20.19***

18.57

22.95***

19.38

24.34***

20.61

26.82***

22.21

26.78***

0.29

< 0.001

< 0.001

0.20

Butyrate (mmol/L)

7.14

10.86***

8.53

11.38***

9.01

12.36***

9.17

12.71***

9.84

12.89***

0.16

< 0.001

< 0.001

0.21

TVFA2 (mmol/L)

84.91

97.07***

91.92

100.45***

93.20

102.36***

95.40

105.44***

98.85

103.81*

0.78

< 0.001

< 0.001

0.07

Acetate

(mol/100 mol)

73.64

67.93***

70.53

65.67***

69.47

64.00***

68.72

62.39***

67.54

61.59***

0.30

< 0.001

< 0.001

0.07

Propionate

(mol/100 mol)

17.95

20.80***

20.20

22.92***

20.86

23.86***

21.66

25.49***

22.46

25.92***

0.20

< 0.001

< 0.001

0.04

Butyrate

(mol/100 mol)

8.41

11.27***

9.27

11.41***

9.67

12.14***

9.62

12.12***

10.00

12.50***

0.13

< 0.001

< 0.001

0.50

A/P ratio3

4.11

3.29***

3.49

2.88***

3.34

2.69***

3.19

2.46***

3.02

2.40***

0.04

< 0.001

< 0.001

0.03
1 GRS: grass; CON: concentrate; 0: 0 g Mootral/kg; 2: 200 g Mootral/kg of substrate. 2 TVFA: total volatile fatty acids. 3 A/P: Acetate/Propionate. Asterisks in 2 mean significant difference between 0 and 200 g Mootral/kg in the same feeding model, * (P < 0.05), ** (P < 0.01), *** (P < 0.001). Trt: treatment; MT: Mootral; Trt×MT: interaction between treatment and Mootral. SEM: standard error of the mean.

Discussion

CH4 emissions from ruminants are not only a serious environmental issue but also a significant source of energy loss to the animals. Different kinds of antimethanogenic compounds have already been studied to investigate their potential to reduce CH4 production; however, there are limitations to their use due to their negative impacts on rumen fermentation characteristics [22], and they exhibited inconsistent efficiency with different feeding styles [23]. Therefore, sustainable and immediate CH4 mitigation strategies for the livestock industry are in high demand. MT, a natural plant-based combination of garlic and citrus extracts, showed promising results when used as a feed supplement for methane mitigation from ruminants [15, 24]. Therefore, this study was performed to evaluate the efficacy of MT with different kinds of feeding styles in ruminants.

Similar to the findings of the current study, MT increased gas production when used as a feed supplement with rumen fluid collected from sheep, which may reflect a stimulating effect of MT on rumen microbes [16]. This finding has been reported previously from a 48 h in vitro gas production study conducted by Hansen and Nielsen [25]. Furthermore, MT increased the concentration of ruminal NH3-N, which might be due to the role of MT in enhancing the proteolysis process. This nitrogen source can be used by rumen microorganisms to build their own protein, which in turn would be used as a protein source for the animal [26]. A similar effect has been reported when MT was used as a feed supplement with a 70 forage:30 concentrate diet in the RUSITEC system [27]. The same finding has also been observed with garlic oil with a 50 forage:50 concentrate diet for 24 h incubation by Busquet et al. [28].

MT supplementation did not interfere with fiber degradability in all feeding models, which was similar to the findings of García-González et al. [29], who reported that inclusion of garlic bulbs in the substrate in an in vitro trial did not affect IVNDFD, and Wanapat et al. [30], who declared that adding garlic powder to concentrates did not change the NDF and ADF digestibility through an in vivo trial using steers. Rumen microbiome analysis in upcoming studies would provide a better understanding of MT’s effect on nutrient digestibility and proteolytic bacteria.

The synergism between the organosulfur compounds and flavonoids in the MT mixture was effective in decreasing CH4 production in all feeding models. The reduction in CH4 may be due to the direct inhibitory effect of MT on methanogenic archaea. Eger et al. [15] and Ahmed et al. [16] reported a lower abundance of the family Methanobacteriaceae, which is the major CH4 producer in the rumen, in MT supplemented treatment. This was attributed to the toxicity of organosulfur compounds of garlic, such as diallyl sulfide and allicin, to inhibit certain sulfhydryl-containing enzymes essential for the metabolic activities of methanogenic archaea [31, 32]. It has been established that ruminal ciliated protozoa could enhance methanogenesis, as they are major H2 producers in the rumen and are in symbiotic relationships with methanogens [33]. Although the impact of MT on protozoa has not yet been investigated, allicin and flavonoids have shown toxic effects on protozoa [34, 35]. Any effect of MT on protozoa has to be confirmed in additional studies.

It is well established that CH4 formation has been positively associated with more acetate production and negatively associated with increased propionate production [36]. MT was able to shift rumen fermentation toward less acetate and more propionate and butyrate. This increase in propionate may be due to the role of MT in increasing the abundance of the Prevotellaceae and Veillonellaceae families, which was confirmed by Ahmed et al. [16]. Prevotellaceae is one of the dominant families in rumen fluid, and it is well known to produce propionate by utilizing hydrogen (H2) produced during the fermentation of carbohydrates [37]. This pathway is the main pathway for H2 consumption and it represents a competitive and alternative pathway to methanogenesis [38, 39]. Moreover, the family Veillonellaceae showed high relative abundance due to the effect of flavonoids extracted from citrus [34], and it was associated with propionate production [40]. Supplementation of steers with garlic powder reduced the A/P ratio [30]. Similarly, the current study showed the same finding. An increase in butyrate was also associated with a reduction in CH4 production when the basal diet of ewes was supplemented with garlic extract [41].

Reports about the effects of garlic and flavonoid components on TVFAs are inconsistent. Some studies reported that they had no effect on TVFA [8, 29, 42, 43], while others reported an adverse effect [28, 34, 44] using an in vitro batch culture system. In contrast, in the current study, the MT formulation improved the production of TVFA. This may be attributed to the role of MT in stimulating the metabolic activity of some rumen microbes, which may be proven by increasing the production of total gas and CO2. This finding has also been observed previously in studies using in vitro batch culture [16] and the RUSITEC system [15].

Conclusion

In the present study, we investigated the efficiency of the MT mixture on CH4 production, rumen fermentation and digestibility in different feeding styles commonly applied in ruminant farms. This study has confirmed the potential of MT to effectively reduce CH4 production with all feeding styles. MT showed a high reducing power up to 70% when the amount of CON comprised up to 800 g/kg of the ration. Moreover, MT supplementation improved the production of TVFA by shifting the fermentation profile toward less acetate and more propionate and butyrate. Additionally, MT did not impair fiber digestibility. Therefore, MT could be used as a feed supplement with all feeding styles to efficiently reduce CH4 production by ruminants.

Abbreviations

GRS: grass, CON: concentrate, MT: Mootral, CH4: methane, CO2: carbon dioxide, GHGs: greenhouse gases, H2: hydrogen, DM: dry matter, OM: organic matter, CP: crude protein, NDF: neutral detergent fiber, ADF: acid detergent fiber, ADL: acid detergent lignin, IVDMD: in vitro dry matter digestibility, IVOMD: in vitro organic matter digestibility, IVNDFD: in vitro neutral detergent fiber digestibility, IVADFD: In vitro acid detergent fiber digestibility, TVFA: total volatile fatty acids, A/P: acetate/propionate, NH3-N: ammonia nitrogen.

Declarations

Authors’ contributions

Eslam Ahmed: Conceptualization, Methodology, Formal analysis, Investigation, Writing - original draft. Naoki Fukumaand Takehiro Nishida: Project administration, Resources, Supervision, Writing - review & editing. Masaaki Hanada: Supervision, Writing - Review & Editing.

Acknowledgements

The authors would like to thank Mootral company to provide the feed supplement used in the experiment.

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Ethics approval

All animals used in the present study were humanely treated and the experimental protocol and procedures used were approved by the Animal Ethics and Care Committee of Obihiro University of Agriculture and Veterinary Medicine, Japan.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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