Effects of carob (Ceratonia siliqua L.) pod supplementation of lamb diets on in vitro methane production, digestion, and microbial yield

1. Introduction

Methane (CH₄) emissions from ruminant animals have emerged as a significant concern owing to their contribution to global greenhouse gases and their detrimental effects on global warming and climate change (FAO, 2023). Enteric fermentation (the digestive process in ruminants) is the main source of CH₄ emissions (Zhao et al., 2020). Enteric fermentation not only results in global warming but also loss of energy (Johnson and Johnson, 1995). Various strategies could be utilized to mitigate CH₄ emissions from ruminant animals, including improved management practices, superior genetics, nutritional interventions, and innovative technology (Cottle et al., 2011; Hristov et al., 2013; Kumar et al., 2014; Patra and Yu, 2013; Beauchemin et al., 2022). One of the most important dietary interventions is the use of feedstuffs with high water-soluble carbohydrates (WSC) and condensed tannins (CT). The WSC and CT in diets can mitigate CH₄ production through a variety of ways, including promoting the production of propionate, which acts as a hydrogen sink, reducing the hydrogen available for methanogens, thus decreasing CH₄ production, altering the rumen microbial community to favor propionate-producing bacteria over CH₄-producing archaea, shifting fermentation patterns from fiber to more efficient carbohydrate fermentation, which generates less CH₄ (Johnson and Johnson, 1995; Newbold et al., 2005; Patra and Yu, 2013; Ramin and Huntanen, 2013).

The carob tree (Ceratonia siliqua L.) is a legume native to or widely grown in Mediterranean nations. Its abundant pods are used by humans and animals equally. (Karabulut et al., 2006; Kotrotsios et al., 2012; Bulca, 2016; Saratsis et al., 2016; Al-Nawass and Al-Saady, 2019; Richane et al., 2022; Basharat et al., 2023; Kurt, 2023). It is an evergreen legume tree indigenous to Southern Europe, widely cultivated for its edible pods known as carob pods. Carob pods have gained importance as a potential source of animal feed, especially for ruminants, due to their unique nutritional properties (Ikram et al., 2023). The distribution of carob pods the Mediterranean region is considerable, as the tree is well adapted to the climate of the region and has been a staple food for centuries (Winer, 1980). Carob pods have been traditionally used in the past as a sweetener and to treat various diseases, and more recently, they have been explored as an inexpensive food source for animals and humans (Naghmouchi et al., 2012). Ruminants find carob pods appealing as a food source because of their high sugar content (Naghmouchi et al., 2012). The availability of high-quality fodder is minimal throughout the summer months, constraining the performance of ruminants in most regions of Türkiye due to insufficient calorie and protein intake. Small ruminants depend on the leaves and pods of trees and bushes for sustenance during this critical summer phase. Carob pods could improve the productivity of sheep and goats and increase farmers’ profits in Southern Europe during periods of traditional feed shortages (Aloueedat et al., 2019). Ruminant nutrition and performance issues are prevalent in Türkiye throughout the summer due to a lack of high-quality feed. Proper planning and use of different feed sources are necessary to guarantee sufficient livestock nourishment throughout the year.

Results from both in vitro and in vivo investigations show that increasing WSC may decrease CH₄ production by decreasing the acetate-to-propionate ratio (Rivero et al., 2020; Lovett et al., 2006). Tannins may limit methanogens by reducing the population of protozoa that symbiotically harbor archaea and by directly suppressing some archaea like WSC (Aboagye and Beauchemin, 2019). The use of tannins in animal diets may effectively reduce CH₄ emissions by directly targeting protozoal populations and methanogenic archaea. This combined action not only mitigates greenhouse gas emissions but also underscores the promise of tannin-rich forages as a sustainable method for enhancing ruminant nutrition and environmental outcomes. Recently, Kurt (2023) has shown that carob pods from different growing regions are rich in WSC and moderate in CT.

While there is some literature regarding the impact of supplementing sheep diets with carob pods on feed intake, milk yield, and some blood parameters is available, there is currently no evidence concerning the effects of carob pod supplementation in lamb diets on CH₄ emissions, digestibility, and MPY. Therefore, it was hypothesized that supplementation lamb diets with carob pods, which contain WSC and CT, would reduce CH₄ emission and promote microbial protein production by altering fermentation patterns without compromising digestibility. The present study aimed to determine the impact of including carob pods in lamb diets on digestibility, PF, MPY, EMPY, gas production (GP), and CH₄ emissions.

2. Materials and Methods

Carob pod sample used in the current study was acquired from Mersin (36° 48’ 43.5708’’ N, 34° 38’ 29.3244’’ E) provinces, Türkiye. The diet consisted of barley grain, carob pods, wheat bran, sunflower seed meal, and alfalfa hay, which are commercially available and widely used in sheep diets. The dietary components were pulverized to pass through a one-mm filter and kept in nylon bags for further in vitro gas generation and chemical analysis.

3. Result

3.1 Effect of supplementation of lamb diet with carob pod on gas, methane, digestibility, and microbial yield

The addition of carob pods to the lamb diet significantly affected GP (mL) and CH4 production (mL), while not impacting the percentage of CH₄ in the gas produced (Table 3). The GP and CH₄ production ranged from 107.75 - 120.75 mL and 15.88 - 18.58 mL, respectively. Gas and CH₄ production were significantly lower in diets III and IV than I and II. The relationship between the ratio of carob pod supplementation and GP in the diet is shown in Fig. 1. Lambs that consumed a higher percentage of carob pods exhibited reduced gas emissions. GP decreased by 0.0485 units for each milligram of carob pod inclusion

Table 3. Effect of supplementation of lamb diet with carob pod on gas, methane, digestibility and microbial yield.

Parameters	Diets				SEM	p
	I	II	III	IV
Gas (mL)	120.00a	120.75a	109.00b	107.75b	2.561	<0.001
CH4 (mL)	18.54a	18.58a	16.13b	15.88b	0.522	<0.001
CH4 (%)	15.45	15.39	14.83	14.74	0.468	0.333
Truly degraded substrate (mg)	344.59	345.65	345.00	346.11	3.916	0.980
Partitioning factor	2.87b	2.86b	3.17a	3.21a	0.073	<0.001
MPY (mg)	80.59b	80.00b	105.20a	109.06a	5.940	<0.001
EMPY (%)	23.37b	23.14b	30.49a	31.50a	1.629	<0.001
True digestibility (%)	74.03	74.21	74.22	74.23	0.789	0.993
CH4 (mL/ per gram digested substrate)	53.83a	53.76a	46.79b	45.92b	1.588	<0.001

^ab Row means with common superscripts do not differ at p<0.05. SEM: Standard error mean, MPY: Microbial protein yield, EMPY: Efficiency of microbial protein yield.

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Fig. 1.

Relationship between gas production and supplementation ratio.

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Fig. 2 demonstrates the correlation between the supplementation ratio of carob pods in the diet and CH₄ produced. CH₄ production reduced as the ratio of carob pods in the lambs’ diet increased. The mean decrease in CH₄ production (mL) per mg of carob pod supplement was 0.0104 units.

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Fig. 2.

Relationship between methane production and supplementation ratio.

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On the other hand, the supplementation of carob pods had no effect on TDS and true digestibility, while the PF increased with the supplementation of carob pods. The PF of the diets was between 2.87 and 3.21, with the highest values determined for diets III and IV. The relationship between the PF and the supplementation ratio of carob pods in the diet is shown in Fig. 3. The PF improved with increasing levels of carob pod in the lamb diets.

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Fig. 3.

Relationship between partitioning factor and supplementation ratio.

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The supplementation of carob pods increased MPY and EMPY. The MPY and EMPY ranged from 80.00 - 109.06 mg and 23.14 - 31.50%, respectively (Table 3). The highest values were recorded for diets III and IV. The relationship between MPY and carob pod supplementation ratio is shown in Fig. 4. The MPY improved with an increasing ratio of carob pods in lamb diets. The mean increase in MPY per mg of carob pod supplement was 0.111 units.

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Fig. 4.

Relationship between microbial protein yield and supplementation ratio.

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Fig. 5 shows the correlation between the production of gases and the MPY ratio. A negative association exists between GP and microbial yield.

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Fig. 5.

The relationship between gas production and microbial yield.

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The relationship between CH₄ (mL/digested DM) and the supplementation ratio is shown in Fig. 6. The CH₄ (mL/digested DM) decreased as the degree of carob pods in the lamb diets increased. The mean decrease in CH₄ (mL/digested DM) per mg of carob pods supplement was 0.0307 units.

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Fig. 6.

The relationship between methane (mL/DDM) and supplementation ratio. DDM: digested dry matter.

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4. Discussion

The essential process of carbohydrate fermentation in the digestive tracts of ruminants significantly influences their energy metabolism and overall health. The fermentation of carbohydrates in vitro using buffered rumen fluid yields many products, including microbial biomass, short-chain fatty acids (SCFAs), and gases, primarily carbon dioxide (CO₂) and CH₄ (Beuvink and Spoelstra, 1992; Blummel and Orskov, 1993). Therefore, it is essential to understand the dynamics of carbohydrate fermentation in rumen fluid to enhance ruminant nutrition and reduce CH₄ emissions. Producers may attain energy efficiency and reduce greenhouse gas emissions by careful regulation of the types and amounts of fermentable substrates.

The supplementation of diet with carob pod decreased the gas and CH₄ production without compromising the digestibility of diets (Table 3). The mean decreases in gas (mL) and CH₄ (mL) per mg of carob pods supplement were 0.0485 and 0.0104 units, respectively. Therefore, the decrease in gas and CH₄ production might be associated with a shift in the composition of volatile fatty acids (VFAs) produced during fermentation. It would be more informative to obtain sufficient details about VFAs composition to adequately interpret these results. This is one of the limitations of the current experiment. However, Lee et al. (2003) showed that a linear increase in the proportion of propionic acids was obtained at the expense of acetate when WSC contents increased. Such a shift in the VFA profile of rumen will decrease the amount of H₂ and thus, gas and CH₄ production. This is also in agreement with the findings of Mills et al. (2001). On the other hand, the mean decrease in CH₄ production per mg carob pod supplementation was 0.0307 units when the CH₄ production was expressed as CH₄ mL/ per gram digested substrate. This result agrees with the findings of Purcell et al. (2014), who showed that there was a linear decrease in CH₄ production (mL) per gram of DMD with increasing WSC content of the incubated substrate.

Ruminant nutrition and metabolism are significantly influenced by VFAs, which are generated during the fermentation of carbohydrates in the rumen. The kind of fermentation substrate used significantly influences the molar ratios of these VFAs. Enhancing ruminant diets and reducing greenhouse gas emissions necessitates comprehension of the effects of different substrates on VFA profiles and gas emissions. Unlike substrates that promote elevated levels of acetate and butyrate, those abundant in water-soluble carbohydrates facilitate the production of propionate, reducing gas and CH₄ emissions. (Newbold et al., 2005; Ramin et al., 2013; Patra and Yu, 2013) This is because the fermentation of substrate to acetate and butyrate may yield more hydrogen, depending on the metabolic pathways and micro-organisms involved. By promoting propionate formation, the hydrogen that would have been used by methanogens to form CH₄ is instead used for propionate production, thereby reducing CH₄ production.

The supplementation of diets with carob pod increased the portioning factor. The mean increase in partitioning factor per mg carob pod supplementation was 0.0013 units. The partitioning factor is one of the important fermentation parameters and ranges from 2.87 to 3.21. The partitioning factors obtained in the current study fell into the theoretical range (2.75 to 4.41) indicated by Blümmel et al. (1997b). Blümmel et al. (1997a) suggested that roughages with a high partitioning factor had a higher intake. Therefore, the increase in partitioning factors due to carob pod supplementation might improve feed intake, thereby milk production, or growth performance of lambs.

As the quantity of carob pod in the lamb meal enhanced, the MPY correspondingly increased. The MPY improved by an average of 0.111 units per mg of carob pod supplementation. An inverse relationship exists between the two variables to some extent since the partitioning factor of fermented matter between microbial cells and SCFAs (gas) is not uniform (Blümmel et al., 1997b).

The dietary WSC and CT content enhanced with the increase of carob pod supplementation (Table 2). The presence of WSC and tannin may positively influence nutrient partitioning, favoring enhanced microbial output over the synthesis of SCFAs (gas) (Baba et al., 2002). The inclusion of tannins in the diet is predicted to positively influence the reduction of CH₄ emissions by directly suppressing some archaea and indirectly decreasing protozoa populations (Aboagye and Beauchemin, 2019). The previously proposed hypothesis was supported by the fact that supplementation lamb diet with carob pods containing WSC and tannin reduced CH₄ emissions and improved microbial protein production without affecting digestibility.

The decrease in gas and CH₄ production without compromising digestibility is desirable because the fermentable substrate has been diverted into microbial protein production rather than VFA production. The reason for shifting the fermented substrate into MPY instead of VFA production (GP) could be related to the WSC and tannin content of the carob pods.

Microbial protein plays an important role in providing a rich source of high-quality and digestible protein to ruminant animals for growth, milk production, reproduction and health. As can be seen from Table 3, the supplementation of lamb diets with carob pod increased the microbial protein. This result might have some practical implication since microbial protein plays an important role in providing a rich source of high-quality and digestible protein for ruminant animals for growth, milk production, reproduction, and health.

The comparison of in vitro and in vivo assessments of energy metabolism, specifically the EMPY, shows significant variations in study approaches. Although it is often expected that these estimations may vary owing to the absence of product flow in in vitro systems, research has shown substantial correlations between the two methodologies. Blümmel et al. (1999) indicated a correlation between in vitro GP estimates and in vivo EMPY estimations derived from renal allantoin excretion in steers. It was proposed that assessing gas volume and substrate degradability at substrate-specific incubation intervals rather than a standard 24 hours might enhance the correlation.

The current in vitro experiment has some limitations in terms of correlating with in vivo experiments due to differences in rumen dynamics. Although the current experiment is very important in providing possible insights on the effects of supplementation of diets with carobs, the long-term effects of carob pod supplementation on production and health remain unexplained.

Therefore, before large implications this should be tested in vivo animal experiments to evaluate the long-term effect of the carob pod supplementation on animal performance and health.

5. Conclusions

The carob pod has the potential to improve microbial protein production and reduce CH₄ emissions from lambs. It was found that carob pods can be used ∼30% in the diet for lambs to increase microbial protein production by ∼35.32 mg and reduce CH₄ emissions by 15% without compromising the digestibility of the diet. Carob pods deserve further investigation to explore the impact on CH₄ emissions per unit of animal product.