Effect of Long-Term Supplementation With Silkworm Pupae Oil on the Methane Yield, Ruminal Protozoa, and Archaea Community in Sheep

Abstract

Supplementation with lipids and oils is one of the most efficient strategies for reducing enteric methane emission. However, high costs and adverse impacts on fiber degradation restrict the use of conventional oils. Silkworm pupae, a non-conventional oil source rarely used for human consumption in India, could be one of the cheaper alternatives for methane mitigation. The objective of this study was to investigate the effect on sheep of long-term supplementation (180 days) of silkworm pupae oil (SWPO) with two distinct supplementation regimes (daily and biweekly) on daily enteric methane emission, methane yield, nutrient digestibility, rumen fermentation, ruminal archaea community composition, and protozoal population. The effect of the discontinuation of oil supplementation on enteric methane emission was also investigated. Eighteen adult male sheep, randomly divided into three groups (n = 6), were provisioned with a mixed diet consisting of 10.1% crude protein (CP) and 11.7 MJ/kg metabolizable energy formulated using finger millet straw and concentrate in a 55:45 ratio. SWPO was supplemented at 2% of dry matter intake (DMI) in test groups either daily (CON) or biweekly (INT), while no oil was supplemented in the control group (CTR). DMI (p = 0.15) and CP (p = 0.16) in the CON and INT groups were similar to that of the CTR group; however, the energy intake (MJ/kg) in the supplemented groups (CON and INT) was higher (p < 0.001) than in CTR. In the CON group, body weight gain (kg, p = 0.02) and average daily gain (g, p = 0.02) were both higher than in the CTR. The daily methane emission in the CON (17.5 g/day) and INT (18.0 g/day) groups was lower (p = 0.01) than the CTR group (23.6 g/day), indicating a reduction of 23–25% due to SWPO supplementation. Similarly, compared with the CTR group, methane yields (g/kg DMI) in test groups were also significantly lower (p < 0.01). The transient nature of the anti-methanogenic effect of SWPO was demonstrated in the oil discontinuation study, where daily methane emission reverted to pre-supplementation levels after a short period. The recorded methanogens were affiliated to the families Methanobacteriaceae, Methanomassilliicoccaceae, and Methanosarcinaceae. The long-term supplementation of oil did not induce any significant change in the rumen archaeal community, whereas minor species such as Group3b exhibited differing abundance among the groups. Methanobrevibacter, irrespective of treatment, was the largest genus, while Methanobrevibacter gottschalkii was the dominant species. Oil supplementation in CON and INT compared with CTR decreased (p < 0.01) the numbers of total protozoa (× 107 cells/ml), Entodiniomorphs (× 107 cells/ml), and Holotrichs (× 106 cells/ml). SWPO continuous supplementation (CON group) resulted in the largest reduction in enteric methane emission and relatively higher body weight gain (p = 0.02) in sheep.

Effect of an anti-methanogenic supplement on enteric methane emission, fermentation, and whole rumen metagenome in sheep

“…The samples were then analyzed using a gas chromatograph. For the analysis of methane and SF 6 , a gas chromatograph (GC 2010 plus, Shimadzu, Japan) equipped with flame ionization and electron capture detectors was used under the same circumstances as previous studies ( Malik et al, 2021 ; Thirumalaisamy et al, 2022b ).…”

“…Methane (ppm) and SF 6 (ppt) concentrations were calculated using the canister pressures at the beginning, end, and after N 2 dilution ( Lassey et al, 2014 ). The local elevation and atmospheric pressure at the study site ( Thirumalaisamy et al, 2022b ) were also taken into consideration while calculating the gas concentration.…”

“…Rumen fluid samples were collected at the completion of the methane measurement study (38th day of feeding). The samples were collected 3 h post-feeding from the individual sheep with the help of 1 m perforated nylon stomach tube connected to an airtight collection vessel and handheld vacuum pump ( Malik et al, 2015 ; Thirumalaisamy et al, 2022a , b ). To avoid saliva contamination, the first 30 ml of ruminal fluid was discarded, and then 45 ml of rumen fluid was collected and strained through a single layer of muslin cloth for the separation of liquid and solid digesta fractions.…”

“…The samples were thawed at room temperature and the VFA concentration was estimated in accordance with Filípek and Dvořák (2009) using a gas chromatograph (Agilent 7890B, Santa Clara, United States). During the analysis, the following GC conditions were upheld ( Malik et al, 2021 ; Thirumalaisamy et al, 2022b ): temperature program: 59°C–250°C (20°C/min, 10 min), injector temperature: 230°C, and detector temperature: 280°C.…”

“…Tannins, which are polyphenolic metabolites, can modulate rumen fermentation and mitigate enteric methane emissions ( Cieslak et al, 2012 ; Hassanat and Benchaar, 2013 ). Based on previous research by our group ( Bhatta et al, 2001 ; Malik et al, 2017a , b ; Baruah et al, 2019 ; Poornachandra et al, 2019 ; Thirumalaisamy et al, 2022a , b ), we have formulated a patent-applied (Indian patent application 201,941,004,992) tanniferous anti-methanogenic supplement “Harit Dhara.” The product was formulated primarily using phyto-sources, furnishing the condensed and hydrolysable tannins in a 3:1 ratio in the final product. We hypothesized that the supplementation of anti-methanogenic product in the straw and concentrate-based diet when included at the appropriate level would mitigate the enteric methane emissions and rumen microbial composition without any detrimental implications on the rumen fermentation.…”