1 Introduction

Rumen fermentation is important for the digestion and absorption of nutrients ingested by ruminants. During this process, several by-products including hydrogen sulfide (H2S) and carbon monoxide (CO) are absorbed through the intestinal wall. The quantity of sulfide produced by rumen microbes is influenced by the diet sulfate content [1]. CO and H2S can also serve as therapeutic purposes and could be alternative means of hydrogen sinking [2, 3].

Meanwhile, increasing greenhouse gas emissions, such as methane (CH4) continues to be a great concern due to their potential adverse effects on global warming [4]. This gas is produced in both the foregut and hindgut during anaerobic fermentation by methanogens. The reduction of enteric methane production enhances nutrient utilization and productivity and reduces environmental pollution. While there have been efforts to mitigate CH4 production both in vivo and in vitro [5], additional research on plants with the capacity to reduce methane synthesis in needed. This would ensure that each country could adopt feeding practices to mitigate CH4 production regardless of the livestock reared.

C. coriaria, a tanniniferous tropical tree, has the potential to mitigate the enteric CH4 production for optimum ruminant output. Pods of this tree are rich in tannin, phenols, and flavonoids. Previous studies have shown that C. coriaria extract reduces methane production in vitro [6, 7] apparently by creating alternative sink for H2 and by competing and metabolizing H2 for other uses, thereby preventing methanogens from using them for methane production [8], which improves performance and rumen fermentation in goats [9].

Despite all the above benefits, additional research on methane inhibitor additives is needed. Administration of unconventional feed additives to livestock often results in contrasting growth performance, gut manipulation, health, and greenhouse gases production. Thus, this study aims to evaluate the long-term effect of oral administration of C. coriaria fruit extract to lambs on reduction of ruminal CH4, CO, and H2S production.

2 Materials and methods

2.1 Cascalote fruit collection

For the preparation of the extract, fruits of C. coriaria (Jacq) wild were randomly and manually harvested from 10 trees in different zones of the State of Guerrero, Mexico, during March and April. Leaf samples were also collected and stored at room temperature (i.e., 30–35 °C) in the dark for subsequent chemical components determination, secondary metabolites, and preparation of extract for the in vitro gas production trials.

2.2 Preparation of the aqueous extract

Fruits were ground to a particle size of 1 mm using a blender and extracted at 1 g fruits/8 mL of plain water. The ground fruits were immersed in water at room temperature for 72 h in closed 5-L jars. After incubation, jars were filtered through 4–5 layers of gauze, and the extract was collected. Extracts were prepared weekly (stock volume of 8 L each); this mixture was stored at 4 °C before daily oral administration to lambs. The extract chemical composition is shown in Table 1.

Table 1 Ingredients and chemical composition of experimental diets
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2.3 Ruminal liquor

The rumen liquor (RL) was collected from lambs at the end of the experiment (800 g from two lambs per group) of three groups of lambs, each composed of 8 mixed-breed (crosses of Pelibuey, Black belly, and Criollo) male lambs (22 to 28 kg live weight). Lambs were offered a balanced diet (Table 2) twice a day, and feed intake was registered daily. The control group was fed the balanced diet; a second group received the balanced diet plus 30 mL aqueous extract of the C. coriaria fruit; group three was offered the balanced diet plus 60 mL of the aqueous extract of the C. coriaria fruit. Lambs received the aqueous extract for 60 consecutive days.

Table 2 In vitro ruminal fluid gas production kinetics and total production of the incubated and degraded diets at 48 h of incubation
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2.4 In vitro incubations

Two lambs from each animal group (i.e., 0-, 30-, or 60- mL) were slaughtered at the end of the experimental period (60 days), and immediately the rumen contents were used as inoculum for the in vitro incubation with the same diet fed to lambs during the experiment. Rumen contents of each group were mixed with the Goering and Van Soest [10] buffer solution without trypticase at 1:4 vol/vol ratio.

The newly mixed RL was managed according to the method of Salem et al. [11] with different doses (0-, 0.6-, 1.2-, and 1.8- mL) of the C. coriaria extract, considering the bottles without the extracts as blanks. After filling all bottles with the substrate, extract, Goering, and Van Soest buffer solution, they were flushed with CO2, rubber stoppered, shaken, and placed in an incubator at 39 °C.

Total gas production (psi) was recorded at different hours after incubation, starting at 2 h until 48 h following the technique of Theodorou et al. [12]. In addition, CH4, CO, and H2S production was measured at the same hours of incubation using a diffusion-based gas detector (MONITOR de Dräger Safety X-am 20,500, Lübeck, Germany) using a 5 mL sample. After each recording, the gas was dispersed using a syringe needle to avoid gas accumulation.

2.5 Degraded substrate dry matter

At the end of the incubation period (48 h), the pH was measured according to Rodriguez et al. [13], and the residual of each bottle was filtered and rinsed. Fermentation residues were dried at 45 °C for 72 h to estimate DM degradability [10], according to [11].

2.6 Diet chemical analyses

Proximate analysis of diet samples (3 subsamples) was performed according to AOAC [14]. The fiber fractions were determined out using an ANKOM200 Fiber Analyzer Unit (ANKOM Technology Corp., Macedon, NY) according to AOAC [14], with the acid detergent fiber (ADF) and neutral detergent fiber (NDF) determined according to Rodriquez et al. [13].

2.7 Secondary metabolites of the Cascalote fruit extract

2.7.1 Determination of the total phenolic content and total condensed tannins

Total phenolic content of the extracts was determined by a colorimetric method utilizing Folin-Ciocalteu reagent [15], and the absorbance was measured at 765 nm against a reagent blank. The total phenolic content was expressed as mg of gallic acid equivalent per g. The tannin content was determined using tannic acid as a reference compound, following the method of Ayalew and Emire [16].

2.7.2 Determination of total flavonoid content

Modified AlCl3 colorimetric method was used according to the technique of Sembiring et al. [17], and the absorbance was measured against methanol blank at 510 nm. The flavonoid was expressed as µg of quercetin equivalent per 1 g of dry extract.

2.8 Calculations

To estimate the kinetic variables of gas production (GP), CH4, CO, and H2S (mL/g DM) were fitted using the NLIN option of SAS [21] using the following model [18]:

$$A=b\times \left(1-{e}^{-c\left(t-lag\right)}\right)$$

where A is the volume of GP, CH4, CO, and H2S at time t; b the asymptotic GP, CH4, CO, and H2S (mL/g DM); c is the rate of GP, CH4, CO, and H2S (/h); and lag (h) is the discrete lag time before GP, CH4, CO, and H2S.

Metabolizable energy (ME, MJ/kg DM) and in vitro organic matter digestibility (IVOMD, g/kg OM) were estimated according to Menke et al. [19] as:

$$ME=2.20+0.136 GP+0.0057 CP \left({~}^{g}\!\left/ \!{~}_{kg}\right. DM\right)$$

SCFA was calculated according to [20] as:

$$SCFA\left({~}^{mmol}\!\left/ \!{~}_{200 mg DM}\right.\right)=0.0222 GP-0.00425$$

where GP is the 24-h net gas production (mL/200 mg DM).

3 Statistical analyses

Data of in vitro ruminal gas production variables were analyzed as a 3 × 4 factorial experiment (i.e., C. coriaria 3 ruminal fluids (fixed effect) and 4 extract doses (random effect)), according to a randomized block design using the PROC MIXED procedure of SAS [21] using the following statistical model:

$${Y}_{ijk}=\mu +{S}_{i}+{R}_{j}+{S}_{i}*{R}_{j}+{\varepsilon }_{ijk}$$

where Yijk represents every observation of the dose when incubated in the jth rumen type, Si = the dose effect (0, 0.6, 1.2, and 1.8 mL), Rj (j = 0-, 30-, or 60-mL aqueous extract fed to lambs) is the rumen liquor type effect, Si*Rj is the interaction rumen liquor type and C. coriaria extract dose, and εijk is the experimental error.

4 Results

4.1 Total gas production

GP (mL gas/g DM incubated and degraded) linearly increased (P = 0.002; P = 0.04) with increasing RL type at 24 h of incubation (Table 2, Fig. 1). In addition, doses of C. coriaria extract used during incubation linearly improved (P = 0.031) asymptotic gas production kinetics and gas production rate (/h) (P < 0.001).

Fig. 1
figure 1

Rumen total gas production (mL/g dry matter (DM)) at different hours of incubation as affected by the dietary inclusion with the aqueous extract of Caesalpinia coriaria (Jacq.) wild fruit

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4.2 Methane production

No trend was observed for CH4 production during the incubation period. RL30 produced the highest CH4, while RL0 lambs produced the least. In contrast, 0 and 1.8 mL/ g DM extract of C. coriaria had the highest CH4 production, while 0.6 and 1.2 ml/g DM extract had the lowest CH4 output (Fig. 2).

 Lambs of RL30 had the shortest delay of CH4, while RL0 had the most extended delay (Table 3). In Table 4, RL type had a linear effect on g CH4/kg DM at 24 h (P = 0.013), whereases RL30 had the lowest, while control lambs (i.e., RL0) had the highest at 24 h. Furthermore, there was a dose-dependent linear (P < 0.05) decrease in CH4 production (mL CH4/100 mL gas; mg CH4/mL gas) at 24 and 48 h of incubation (Table 4). The RL x C. coriaria extract dose showed that when measured as CH4 (g CH4/kg DM), RL0, 1.8 mL/g DM had the lowest CH4 production at 24 and 48 h; RL30, 0 mL/g DM had the lowest (P = 0.0002) CH4 production at 24 h, while 1.8 mL/g DM had the lowest (P = 0.031) at 48 h of incubation (Table 4, Fig. 2).

Table 3 In vitro ruminal fluid methane production kinetics and total production of the incubated and degraded diet at 48 h of incubation
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Table 4 In vitro ruminal fluid methane proportions1 of incubated and degraded diet at 48 h of incubation
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Fig. 2
figure 2

Rumen methane (CH4) production (mL/g dry matter (DM)) at different hours of incubation as affected by the dietary inclusion with the aqueous extract of Caesalpinia coriaria (Jacq.) wild fruit

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4.3 Carbon monoxide (CO) and hydrogen sulfide (H2S)

Table 5 and Fig. 3 showed that RL type and doses had no linear effect on CO production kinetics (mL/g DM incubated) and CO production (mL/g DM degraded) at 24 and 48 h of incubation. However, RL type × dosage affected CO production (mL/g DM incubated) at 24 (P = 0.028) and 48 h (P = 0.018) of incubation.

Table 5 In vitro ruminal fluid carbon monoxide (CO) production kinetics and total production of the incubated and degraded diet at 48 h of incubation
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Fig. 3
figure 3

Rumen carbon monoxide (CO) production (mL/g dry matter (DM)) at different hours of incubation as affected by the dietary inclusion with the aqueous extract of Caesalpinia coriaria (Jacq.) wild fruit

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Table 6 and Fig. 4 showed that extract dose (P = 0.004), rumen type × extract dose (P < 0.001) affected the H2S production rate. Dose 1.8 mL/g DM had the highest H2S production, while 0.6 mL/g DM produced the lowest. RL0 and RL30, in combination with 1.2 and 1.8 mL/g DM, produced the lowest H2S, while RL60, 0.6 mL/g/DM, had the slowest gas production rate. Lambs of RL0 produced the lowest H2S (mL/g degraded DM) at 24 h, and RL30 lambs produced the highest. Dose level of C. coriaria extract had a linear effect where H2S decreased with the increasing dose of the extract in 24 h (P = 0.039); the reverse occurred in 48 h (P < 0.0001). The ruminal fluid × dose of C. coriaria extract showed that at 48 h, 1.8 mL/g DM had the lowest (P = 0.0001) H2S production in RL0 and RL30, whereas, in RL60 lambs, the inverse occurred, and 0 mL/g DM produced the lowest.

Table 6 In vitro ruminal fluid hydrogen sulfide (H2S) production kinetics and total production of the incubated and degraded diet at 48 h of incubation
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Fig. 4
figure 4

Rumen hydrogen sulfide (H2S) production (mL/g dry matter (DM)) at different hours of incubation as affected by the dietary inclusion with the aqueous extract of Caesalpinia coriaria (Jacq.) wild fruit

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4.4 Fermentation profile

Table 7 shows the rumen fermentation profile and CH4 conversion efficiency of diet after in vitro digestibility. Diets fermented with RL0 produced the highest SCFA (P = 0.001) and ME (MJ/kg DM at 24 h) (P = 0.001), while diets incubated with RL30 produced the lowest. Furthermore, there was a linear (P < 0.001) and quadratic (P = 0.002) dose-dependent increase in SCFA and ME. The CH4:ME and CH4:OM showed that diets fermented with RL0 produced the highest CH4 level for every unit of ME, while RL30 produced the lowest. The CH4 to SCFA ratio showed that for every increase in C. coriaria extract, less (P < 0.001) CH4 was produced per SCFA.

Table 7 In vitro ruminal fluid fermentation profile1 and methane conversion efficiency of the incubated diet
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Rumen fluid type × dose of C. coriaria extract showed that with RL0, the decrease in CH4 to ME ratio, OM, and SCFA ratio, there was a dose-dependent decrease in CH4 with increasing C. coriaria extract. Moreover, RL60 and RL30 generated the lowest CH4 output per ME and OM, while 0.6 mL/g DM generated more CH4. Similarly, 1.8 mL/g DM produced the lowest (P = 0.0003) CH4 per SCFA, while 0.6 mL/g DM produced the highest.

5 Discussion

5.1 Gas production

Phytogenic additives manipulate rumen fermentation due to their secondary metabolites which may improve nutrient digestion and availability [22]. It has been reported that some phenolic compounds stimulate the growth of microbial communities [23]. C. coriaria extract increased GP kinetics and decreased the lag time. This outcome disagrees with Campos‑Perez et al. [6], who observed that higher condensed tannin concentrations from C. coriaria decreased biogases production. Despite the condensed tannin present in the extract used in the present study, the increased gas production in the current study may be associated with a high level of phenols and flavonoids of the C. coriaria fruit, which may have stimulated the growth of a particular microbial community. Besides, the short lag time suggests that the phenol and flavonoid helped microbes to easily adapt to the diet, and fermentation rapidly began. This view is further supported by the interaction of RL type and dose of C. coriaria extract, where the groups with the highest dose of C. coriaria extract had the shortest lag time, while those with no extract had the longest lag time, indicating that the absence of C. coriaria extract prolonged the microbe adaptation to the diet. This suggests that the inclusion of C. coriaria extract enhances ruminal fermentation, benefiting ruminants receiving this diet.

However, when gas production was measured per dry matter incubated and digested, lambs fed C. coriaria had the lowest gas production. This indicates that long-term in vivo use of C. coriaria extract altered the rumen fermentation pattern. Furthermore, Manuel-Pablo et al. [6] showed that C. coriaria fruit offered to goats reduced the population of some rumen microbes. This suggests that prolonged use of C. coriaria fruit at higher dose affected the rumen microbial population without affecting goat growth. It is, however, interesting that when C. coriaria fruit extract was added in vitro, the gas production improved compared with RL without C. coriaria fruit extract at 24 and 48 h of incubation. This implies that the phenolic and flavonoid compounds of the C. coriaria fruit extract exerts a “restorative/booster” activity on rumen microbes, increasing their fermentative activities.

5.2 Rumen methane production

Lambs with RL given C. coriaria had the highest CH4 production. However, when CH4 was measured per gram of DM incubated or digested, C. coriaria decreased CH4 output for every gram of incubated and digested feed. The decreased CH4 production can be attributed to the tannin content of this plant, which exhibited antimethanogenic activity. Campos‑Perez et al. [6] reported that C. coriaria fruit can reduce CH4 output by creating an alternative form of H2 sink/utilization preventing methanogens from using H2. In the present study, the alternative form of hydrogen sink could be the sulfide-reducing bacteria (SRB). This is because there is an interactive and competitive relationship between methanogens and sulfide-reducing bacteria. This bacaterial speceies can also competitively attach to hydrogen ions since the energy provided by the sulfates is greater. Besides, the incubation temperature and rumen temperature (37 °C) favor the sulfur-reducing bacteria, which dominate methanogen for hydrogen use [24]. Therefore, if sulfate levels exceed a particular concentration in the rumen, the sulfate-reducing bacteria proliferates, creating alterative hydrogen sink and decreasing CH4 production [25, 26].

Manuel-Pablo et al. [9] reported the reduction of rumen protozoa in goats fed C. coriaria fruit. This suggests that the C. coriaria antimethanogenic activity reduced CH4 through creation of an alternative sink for H2 or the decrease in protozoa which reduced the hydrogen exchange relationship with methanogens [6]. Despite the apparent tendency of C. coriaria to reduce CH4 production, care must be taken to use the right combination.

Lambs of RL30 had the lowest CH4 output, while a dose level of 1.8 mL/g DM had the lowest level with similar CH4 production between RL0 and RL60 lambs. This condition suggests that the action of phenols and flavonoids of C. coriaria could have neutralized the antimethanogenic activity of tannins at a higher level to the extent that it would be not different or even produce more CH4 than the unsupplemented groups. This indicates that a balance is needed to use the right combination of C. coriaria fruit to avoid an antimethanogenic neutralization effect. This earlier submission can be observed in Table 4, where RL60 with CC extract began to increase CH4 output per DM incubated even higher than the control. This is similar to what was observed in the in vitro report of Jack [27] for the percentage of CH4 per total gas volume, using RL from rams fed water washed neem, where RL with the highest water washed neem produced the highest CH4 production, while the lower water washed neem produced the lowest. Nonetheless, the advantage of reducing CH4 production per gram DM degraded is that, if fed to ruminants, it could reduce CH4 eructed.

5.3 CO and H2S production

An imbalance between oxidants and antioxidants causes oxidative stress. This stress could be induced by the constant contact with ingested materials and microbial pathogens. H2S and CO are endogenous gaseous mediators implicated in gut function [2]. However, CO and H2S protect the gut against inflammation and serve as antioxidant enzyme to adapt to stress. Endogenous CO can initiate a compensatory expression of antioxidant enzymes and other adaptations to oxidative stress [3]. The higher CO and H2S in the rumen of C. coriaria-fed lambs suggests that the gut is protected against inflammation or irritation due to ingestion of unconventional feed ingredient or additives. It also suggests that the prevention of gastrointestinal inflammation will aid the absorption of nutrients by the rumen and intestine and limit the compromise of tight junctions of the gut. In addition, CH4 formation is also driven by reactive oxygen species across all living organisms, and these respond to inducer of oxidative stress by enhanced CH4 formation [28]. This suggests that CO and H2S ability serve as antioxidant enzyme which could be a factor in reducing CH4 in this study. Thus, supplementation of C. coriaria extract has the potential to protect the gut from oxidative stress, hence offering a therapeutic function and improving the antioxidative status of the gastric mucosa [29], as well as indirectly reducing CH4 production.

5.4 Rumen fermentation profile

Adequate ruminal pH is required for rumen health and microbial proliferation. For optimal microbial stability, pH should range between 6.0 and 6.8 [30]. In the present study, pH after incubation was optimal for microbial function. The SCFA level indicates energy availability and can provide about 80% of livestock’s daily energy requirement [31, 32]. The lower SCFA in RL types of lambs given C. coriaria fruit indicates the effect of tannin present in the fruit, which, when given for a prolonged time, might have affected the rumen microbes compared with the control lambs. Nevertheless, the C. coriaria extract had a dose-dependent increase in SCFA concentration. This suggests that phenols had a stimulatory effect on the microbes to aid feed fermentation and SCFA production. The predominance of SCFA could be ascribed to increased proportion of volatile fatty acids [22] and could enhance milk production [33]. The increased ME indicates the availability of energy which will be useful for microbial protein production [34], and the ME also followed the pattern of SCFA production.

The CH4 conversion efficiency ratio showed that the RL type of lambs ingesting the higher C. coriaria fruit extract was more efficient in producing CH4 as they had the lowest value. This attests to the anti-methanogenic properties of C. coriaria fruit extract.

6 Conclusion

Rumen liquor of lambs orally administrated with 60 mL of C. coriaria fruit extract and 1.2–1.8 mL/g DM resulted in the best antimethanogenic activity and reduced CO and H2S, reduced total gas production, and had better ME and SCFA. However, without adding extra fruit extract, 30 mL/day/lamb oral supplementation is optimal for digestion, ME, and SCFA production. Therefore, oral supplementation with 60/mL/day/lamb of fruit extract is optimal for rumen fermentation and methane reduction. The results on antimethanogenic activity of C. coriaria secondary metabolites indicated their potential as feed additives for decreasing the enteric CH4 emission, which is important for the sustainable development of ruminant production.