Effect of Walnut and Green Tea Ethanolic Extract to Mitigate Methane Emission and VFA Concentration in Ewes

Document Type : Research Paper

Authors

1 Department of Animal Science, Faculty of Animal Science, Urmia University, Urmia, Iran

2 Department of Internal Medicine and Clinical Pathology, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran

Abstract

This study was conducted to evaluate the effects of ethanol extracts of walnut leaves and green tea leaves on ruminal fermentation characteristics, methane and total gas production under in vitro conditions, and microbial population in lactating ewes using microbial-based methods. The experiment was arranged in a completely randomized design with four treatments: a control group (no extract) and three experimental groups receiving 100 mg extract per kg of live weight from walnut leaves, green tea leaves, or an 1:1 ratio. Extracts were administered orally twice daily for two months. At the end of the period, rumen fluid was collected from each treatment and subjected to in vitro gas production tests, including total gas production, methane production, and protozoal counts, conducted in three independent runs with four replicates per treatment. The results indicated that the control group had the highest potential gas production (A) at 373.60 mL, while the mixture of extracts showed the lowest (265.60 mL). The molar proportion of acetate decreased, and that of propionate increased significantly, with the highest propionate values observed in the mixture (34.39%) and walnut leaf (32.21%) treatments, and the lowest in the control (29.80%) and green tea leaf (29.66%) treatments. The combination of extracts led to a reduction in protozoa and methanogen populations, while fungal populations increased significantly (P < 0.02). Overall, the findings suggest that dietary inclusion of these extracts in lactating ewes may beneficially modify rumen fermentation patterns in terms of volatile fatty acid profiles, gas production, and microbial populations.

Keywords

Main Subjects

Extended Abstract

Introduction

     Improving feed efficiency in ruminants is achievable by reducing energy loss. This can be done through enhanced digestibility and better rumen fermentation management, particularly by increasing propionate production and consequently reducing methane production. Over the course of evolution, plants have developed a wide range of secondary metabolites that exert significant biological effects when consumed by herbivores. Among these, polyphenols—especially flavonoids—are valued for their antioxidant, anti-inflammatory, and immune-boosting properties, making them promising candidates for improving animal health and productivity. The use of plant extracts rich in these compounds in ruminant nutrition has attracted interest due to their low production cost, environmental compatibility, and ability to mitigate metabolic disturbances caused by oxidative stress. Walnut leaves and green tea are rich sources of flavonoids and antioxidant compounds capable of scavenging free radicals and modulating redox-sensitive biological pathways. Additionally, plant secondary metabolites such as tannins and saponins can help reduce methane production in the rumen and improve fermentation patterns by influencing the microbial population. However, there is limited information about the effects of ethanolic extracts of walnut leaves and green tea on rumen fermentation processes. This study aims to investigate the in vitro effects of these extracts on fermentation patterns, total gas production, methane emissions, and volatile fatty acid profiles, exploring their potential as a natural and sustainable approach to reducing methane production and the risk of ruminal acidosis.

 

Methods

The protocol for this study was approved by the Institutional Animal Care and Use Committee (IACUC). The experiment was conducted during autumn 2017 (Mehr to Azar 1396) on 20 Makui ewes allocated to four treatments: control (no extract), hydroalcoholic walnut leaf extract, green tea leaf extract, and a combination of both extracts. Walnut leaves were collected from Urmia University orchards, while green tea leaves were purchased from the National Tea Organization. Samples were collected in multiple rounds, dried, and ground. Extraction was performed in two stages using 96% and then 75% ethanol. The filtered extracts were concentrated at 50 °C using a rotary evaporator and sent to Turkey for GC-MS analysis. Ewes received the extracts at 100 mg per kg live weight twice daily for two months. Diets were formulated using SRNS software based on NRC (2007) requirements. For in vitro testing, rumen fluid was collected from all ewes in each treatment before morning feeding, filtered, pooled, and used as inoculum. The gas production test followed Menke & Steingass (1988), with 12 replicates and measurements at intervals up to 96 hours. Methane production was measured after injecting 10 M NaOH. Volatile fatty acids (VFAs) were analyzed via gas chromatography (GC), protozoa were counted using Dehority’s (2017) method, and ammonia nitrogen was determined using the colorimetric method of Broderick and Kang (1980). Apparent and true dry matter digestibility was assessed using the Blümmel et al. (1997) method, with samples incubated, dried, and weighed. Total DNA extraction was performed following Tajima and Yang protocols, purified with a ZYMO kit, and evaluated for purity with a NanoDrop spectrophotometer. Quantitative analysis of microbial populations was conducted using qPCR with SYBR Green dye.

 

Results

Results showed that different treatments had a significant effect on gas and methane production; the highest gas and methane production were observed in the control group, and the lowest in the group supplemented with a mixture of walnut and green tea extracts. The fermentation rate did not change, but the lag phase was longest in the combined extract treatment. In the volatile fatty acid profile, acetate decreased and propionate increased significantly in the combined extract treatment. The acetate to propionate ratio and ammonia nitrogen concentration were lower in this treatment. Protozoa population decreased in all extract treatments, with the lowest count in the combined treatment. Dry matter and organic matter digestibility improved in the walnut leaf extract and combined extract treatments, while the green tea extract alone had less effect. Microbial population analysis showed that the combined extract treatment caused the greatest reduction in protozoa and methanogens and the highest increase in fungal population. Individual extracts had lesser effects, and their increase in fungal population was not significant.

Conclusions

The combination of walnut leaf and green tea extracts effectively improved rumen fermentation, nutrient digestibility, and reduced methane production, highlighting its potential as a natural additive for enhancing ruminant performance and sustainability.

 

 

 

Author Contributions

Conceptualization, Investigation, Formal analysis, Software and Writing-original draft, M.S.A.; Supervision, Project administration, Conceptualization, Validation, Writing- review and editing, H.K.B.; Supervision, Validation, R.P.; Writing, review and editing, E.A.

 

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

 

Acknowledgements

The authors would like to appreciate Dr. Ramin Mazaheri-Khameneh, assistant professor of the department of radiology, Urmia University of Medical Sciences, Iran, for his technical assistance in this study.

 

Ethical considerations

The research protocol was approved by the university's Animal Care and Use Committee (IACUC Protocol #IR2018011), following the guidelines of the Iranian Council of Animal Care (1995).

Conflict of interest

The author declares no conflict of interest.

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Iranian Journal of animal Science
Volume 57, Issue 1
April 2026
Pages 79-99

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History

  • Receive Date: 21 May 2025
  • Revise Date: 11 August 2025
  • Accept Date: 18 August 2025

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