Effect of extraction solvents and encapsulation on the efficacy of certain medicinal plant extracts to inhibit enteric methane emission

A series of in vitro and in vivo experiments were conducted to evaluate the antimethanogenic properties of four medicinal plants (Aloe vera, Jatropha curcas, Moringa oleifera, and Piper betle) extracts as alternative rumen modifiers to antibiotics to modulate rumen fermentation and inhibit methane production. In the first study, two solvents (methanol and water) were used in three different combinations (70, 85, and 100%) to extract bioactive compounds from the four studied medicinal plants as some of their metabolites have been reported to possess rumen modulation properties and improve nutrient utilization in ruminants, thereby reducing enteric methane emission per unit of animal product. The extracts were evaluated at 50 mg kg-1 DM feed as an additive to Eragrostis curvula hay substrate in vitro. Results showed an increase in extract yields with increasing water content in the extraction solvents. The effect of extraction solvents was also observed in the concentration of the bioactive compounds in each plant extract when analysed with UPLC-MS, these plant bioactive compounds showed different solubility values. Subsequently, promising plant extracts were selected based on yield and methane inhibition potentials for subsequent encapsulation with alginate as wall material. The encapsulated products were scanned using electron microscope for morphological characterisation and later their methane inhibition attributes were investigated using both in vitro and in vivo studies. The particle characterization of the encapsulated extracts was carried out using a scanning electron microscope (SEM) and images were generated for a morphological study. Gas measurements were taken at regular intervals of 3, 6, 12, 24, and 48 h during the incubation period. Methane emission was determined for each gas sample with the use of gas chromatography. During the first phase of the in vitro study, the plant extract treatments were incubated with feed samples to test their methane inhibition potentials, their effect on total gas production (TGP), and their organic matter digestibility (IVOMD). In the second phase of the in vitro study, Aloe vera and Moringa oleifera extracts were encapsulated with alginate and/or alginate-chitosan wall materials and the final product is incubated with feed samples to evaluate their methane inhibition potentials, their effect on TGP, and IVOMD. The results from the first in vitro study indicated that the four plant extracts from three aqueous-methanol extractions (70%, 85%, and 100%) generally reduced methane emission in different proportions at 50 mg kg-1 DM without adversely affecting TGP and IVOMD of E. curvula hay. However, the methane inhibition potentials of the tested plant extracts were not influenced by the extraction solvents. For the second in vitro study, alginate encapsulated and alginate-chitosan encapsulated plant extracts were also tested together with non-encapsulated plant extracts on E. curvula hay and also reduced methane production. Higher methane inhibitions were noted for the alginate encapsulated and alginate-chitosan encapsulated extracts as against the non-encapsulated extracts, without negatively affecting the TGP and IVOMD. Finally, encapsulated M. oleifera plant extract was selected for in vivo evaluation to determine its effect on enteric methane production, rumen fermentation parameters, growth performance, feed intake, apparent digestibility, and nutrient utilization of South African Mutton Merino (SAMM) lamb. A total of thirty (30) SAMM lambs were first ranked according to their body weight and divided into three groups with approximately equal body weight measurements. The three treatment groups were randomly allotted to one of the three treatments (two plant extract treatments and control). The plant extract treatments included non-encapsulated M. oleifera extract (NME) and encapsulated M. oleifera extract (EME) additives. These additives were provided to the lambs at 50 mg kg-1 feed DM intake by incorporating them into a ball-like molasses mix and presented at 7:30 am daily to the experimental lambs. All treatments were placed on the same total mixed ration formulated with a 42% roughage component. The growth performance study lasted for 14 weeks, including adaptation, and the feed digestibility study lasted for 14 days. The lambs were moved into the open circuit respiratory chambers for methane emission measurements in six batches. The methane emission measurements and digestibility study were done concurrently with the growth performance study. For the in vivo study, both the NME and EME reduced (p < 0.05) enteric methane emission by approximately 22.61% and 20.06%, respectively; reduced rumen ammonia nitrogen (NH3-N), urinary nitrogen excretion and feed conversion ratio but increased nitrogen retained as a percentage of intake without adverse effect on nutrient intake, apparent digestibility, and performance of SAMM lambs. Supplementation of SAMM lambs with NME and EME as dietary additives did not affect the rumen fermentation parameters. The alginate encapsulation of MO plant extract is safe and did not reduce the efficacy of MO plant extracts compared to non-encapsulated plant extracts, and is therefore recommended for practical use as antimethanogenic dietary additives in the feeding system of ruminant animals. Further study needs to be conducted to determine the effect of alginate encapsulated MO plant extracts on the rumen microbial populations and meat quality of the lambs. Also, other wall materials that can act as good carriers of active compounds in rumen medium should be evaluated for methane inhibition in livestock production.