Effect of Alfalfa Hay Substitution by Raw Garlic Leaves on In vitro Gas Methane Production and Ruminal Fermentation

In the recent years there is an increasing global demand for garlic consumption which leads to the inescapable production of agricultural waste consisting primarily of husk, straw and leaves (Kallel and Ellouz, 2017). In fact, garlic leaves generated abundantly during the harvesting period, are usually made into waste which is incinerated (Han et al., 2013). Nevertheless, agricultural waste generated in the garlic production may be used as forage, since the fiber and protein contents in leaves are higher than the ones contained in common forages sources. Moreover, raw garlic leaves contains several compounds, including sulfur compounds as thiosulfates and allicin, as well as enzymes, free amino acid, sterols, glycosides, flavonoids and phenols (Lawson, 1996). Some of these compounds have shown activity on decreasing methane production on in vitro assays (Kamra, et al., 2012). Moreover, Busquet et al., (2005), using the same in vitro system, showed that garlic components reduced the proportions of acetate and branched-chain volatile fatty acids (VFA) and increased the proportion of propionate and butyrate and small peptides. In this study, we hypostatized that under in vitro conditions the alfalfa hay substitution by raw garlic leaves can improve the ruminal fermentation and reduce the methane production in ruminants diets. Therefore, the aim of present research was to evaluate under in vitro conditions, the effect of alfalfa hay substitution by raw garlic leaves on ruminal fermentation patterns and methane production in diets ruminants.

Study location
 
The experiment was conducted at the animal metabolic unit and the laboratory of animal nutrition of the Faculty of Veterinary and Husbandry of Juarez University at the Durango (México).
 
Raw garlic leaves sampling
 
The samples (leaves with 25 cm of length) of raw garlic (Allium sativum) used in this study, were collected from North region of Mexico. To ensure representative sampling, the samples were collected eight times between June 2019 and January 2020.
 
Experimental treatments
 
Four treatments were evaluated: (T1) alfalfa hay (50%, DM) + raw garlic leaves (0%, DM); (T2) alfalfa hay (33%, DM) + raw garlic leaves (17%, DM); T3 alfalfa hay (17%, DM) + raw garlic leaves (33%, DM); T4 alfalfa hay (0%, DM) + raw garlic leaves (50%, DM). The other 50% of the DM was supplied for a concentrate composed of ground corn, cottonseed and minerals. Nutritional composition of experimental treatments is shown in Table 1.


 
Chemical composition
 
The samples of each experimental treatment were analyzed in triplicate for dry matter (DM), organic matter (OM) crude protein (CP) and ether extract (EE) AOAC, (2000). Acid detergent fiber (ADF), neutral detergent fiber (NDF) and acid detergent lignin (ADL) analyses were determined using the filter bag technique with a fiber analyzer (ANKOM Technology, Fairport, NY, USA). Total carbohydrates (TCHO) contents were calculated according to the equation proposed by Sniffen et al., (1992): %TCH = 100 - (%CP+%EE+% ash); whereas, the nonfibrous carbohydrate (NFC) content was calculated using the difference between %TCH and %NDF.
 
True degradability of dry matter
 
The true degradability of dry matter (TDMD) at 48 h was carried out in polyethylene bags (ANKOM®), to which they were weighed 0.5 g of each diet. The bags were introduced into glass bottles of ANKOM gas production system. Immediately, 125 mL of ruminal fluid and a buffer solution were added to each glass bottle. Ruminal fluid was obtained from of two rumen cannulated steers fed with a diet containing fed 60% oat hay and 40% concentrate. The glass bottles were introduced in Daisy incubator (ANKOM Technology, Fairport, NY, USA) with controlled temperature (39°C).
 
In vitro gas parameters and methane production
 
In vitro gas production was measured using the ANKOM gas production system. Ruminal fluid was collected approximately 3 h after morning feeding from two rumen fistulated steers. Approximately 1 g of dried and ground samples was weighed and placed into glass bottles. The gas production was recorded after 2, 4, 6, 8, 12, 16, 24, 36, 48, 72 and 96 h of incubation. The cumulative gas production kinetic was fitted to model proposed by France et al., (2002) as: GP = A * [1-e-kd(t-L)]. Where: GP is the volume of gas produced at time t, “A” is potential gas production (ml/g DM) from the fermentable fraction of forage, “kd” is the fractional rate of gas production (h-1) and “L” is the discrete lag time prior to gas production. The average GP rate (AGPR) at half of A was calculated according to the equation of Garcia-Martinez et al., (2005). For methane production measure once the incubation period is over at 24 h, pressure release valve was opened during 2 secs in every glass module and the released gas in each module was passed through a tube and connected to a portable gas analyzer (GEMTM5000, LANDTEC, USA).
 
Ruminal fermentation patterns
 
After termination of the incubation at 24 h, two samples (5 mL) of the glass bottle fluid were collected. The first subsample was acidified with 0.3 mL of 50% H2SO4 and the second subsample with 2.5 mL of 25% metaphosporic acid. Both subsamples frozen immediately at -40°C and later analyzed for ammonia nitrogen (NH3-N) and total volatile fatty acids (TVFA), respectively (Uden et al., 1980).
 
Calculation
 
The microbial biomass synthesis (MBS) yield was calculated using the TDMD (mg) and gas volume (24 h) and stoichiometric factor as follows: MBS (mg-1g DM) = TDMD (GP24 × 2.25) (Blummel et al., 1997). Additionally, the efficiency of microbial biomass synthesis (EMBS) was estimated as the ratio of MBS to TDMD (Blummel et al., 1997). The partitioning factor (PF) was calculated as the ratio between TDMD (mg) and the gas produced at 24 hours of incubation of substrate truly degraded (Blummel et al., 1997).
 
Statistical analysis
 
Analysis of variance for completely random design was carried out to compare the in vitro gas production, methane and ruminal fermentation patterns using the procedure GLM of SAS (2002).

Chemical composition
 
In this study, the forage to concentrate ratios were 50%:50%, respectively. As would be expected, the four experimental treatments provided the same amount of protein and energy. Nevertheless, the highest content of NDF was recorded in the treatment containing a higher proportion of alfalfa (T1). Alfalfa provides a high fiber content (NDF, 18.68% DM) (Van Soest, 1994). Cell wall content of forages is related to their dry matter digestibility. In fact, in this study NDF content seems to have reduced the TDMD of T1.
 
In vitro gas parameters and methane production
 
The in vitro gas parameters and methane production of experimental treatments are summarized in Table 2. The “A” fraction was affected significantly by treatments (P<0.05). The higher “A” value was found in T4 and the lower in T1 (P<0.05). The higher values fractional rate of gas production “kd” and average gas production rate when half of “A” occurred “AGPR” were found in T4 and the lower in T1 (P<0.05); though “kd” and AGPR values was unaffected by T2 and T3 (P>0.05). The average “A” value in the present study, was of 116.5 mL 200 DM mg-1. These values agree with values reported by Sahli et al., (2018) when incubated in vitro, garlic powder a doses of 32 mg in a diet composed of 50% ryegrass hay and 50% commercial concentrate. The “kd” value registered with T4 (9.0 mL h-1), was higher to that found by Anassori et al., (2012) (3.5 mL h-1), who evaluated the in vitro gas production kinetics of raw garlic bulb. Likewise, the AGPR value registered in T4 suggests that the inclusion raw garlic leaves accelerated microbial fermentation of digestible components in the diet. There were differences in the methane production among treatments (P<0.05). The low methane concentration was obtained in T4 (high in garlic leaves) may be compared with results reported by Kongmun et al., (2010) who report a decrease in the methane production when evaluated garlic powder in ruminants diets. These results suggest two possible reduction pathways for methane: a) an inhibitory action in growth and expression of methanogens and, b) the acétate pathway was affected by affecting microorganisms which contribute to produce methane from it (Murillo et al., 2018).


 
True degradability of dry matter, microbial biomass synthesis and efficiency microbial biomass synthesis
 
The true dry matter degradability, microbial biomass synthesis and efficiency microbial biomass synthesis are presented in Table 3. The TDDM and GP24 were different between treatments (P<0.05). The highest TDDM values and GP24 were recorded in T4 and the lowest in T1 (P<0.05). In the same way, the MBS values were affected significantly by treatments (P<0.05). The highest MBS value was recorded in T4 and the lowest in T1. However, the highest EMBS value was recorded in T3 and the lowest in T2 and significant differences were observed between both treatments (P<0.05). The highest PF was recorded in T1 and was different to the others treatments (P<0.05). The TDDM values obtained in study current, partially agreement with reported by Zafarian and Manafi (2013) who evaluated under in vitro conditions garlic powder in wheat straw based diets. The result obtained in T1 for MBS values is not agreement with reported by Arbabi et al., (2017) who evaluated under in vitro conditions alfalfa hay more concentrate in a range 50:50; though the MBS obtained with T4, could be explained from by CP and NFC contents which promoting a better balance between protein and energy provided by the diet (Van Soest, 1994). The average PF value in all treatments, was of 5.9 mg of TDMD/mL was higher to the suggested range of 2.74 to 4.41 mg TDMD/mL gas produced for stimulate the microbial protein production (Makkar, 2004).


 
Fermentation ruminal patterns
 
The fermentation ruminal patterns and methane production are presented in Table 4. The highest NH3-N concentration was recorded in T4 and the lowest in T1 (P<0.05). There was no difference between T2, T3 and T4 in TVFA (P>0.05); but both treatments were different to T1 (P<0.05). The highest acetate concentration was recorded in T1 and the lowest in T4 (P<0.05); whereas, the highest propionate concentration was recorded in T4 and the lowest in T1 (P<0.05). Ruminal NH3-N concentrations recorded in all experimental treatments evaluated, were maintained within range of 15 to 30 mg/100 mL suggested for optimal microbial growth (Wanapat and Pimpa, 1999). In contrast, with the results obtained in this study, several studies in vitro and in vivo report that garlic oil or garlic powder reduced or have no effect on ruminal fluid NH3-N concentration (Cardozo et al., 2014). The TVFA concentrations obtained with T2, T3 and T4 are consistent with other in vitro studies where did not differ with addition of garlic oils (Klevenhusen et al., 2011). The propionate concentration observed in T4 was higher than in the other treatments; this is evidenced with decreased ratio of acetate:propionate. A lower acetate:propionate ratio improves ruminal fermentation efficiency as well as energy is available for rumen microbes activities.

These results indicated that the substitution of alfalfa hay by raw garlic leaves improved the in vitro gas production parameters, ruminal ferrmentation patterns, microbial protein synthesis and decreased the methane production when measured in vitro.