Introduction

The uncontrolled emission of greenhouse gases (GHG) into the ecosystem is worriment for society. Over time, the variations in the concentrations and proportions of detrimental GHG produced in the atmosphere have caused an unprecedented change in the ecosystem (Lackner et al. 2022; Elghandour et al. 2023). It is estimated that the temperature of the globe might increase by 4 °C in the following decades due to GHG emissions (IPCC 2014). In the current scenario, the agriculture industry and deforestation contribute about 25% of total GHG released into the atmosphere (Ahmed et al. 2020). Livestock industries are considered a source of GHG emissions, contributing approximately 15% of total anthropogenic production (Khusro et al. 2022a). The socio-economic and environmental impact of GHG emissions from animals is expected to increase worldwide in the coming years; thus, its mitigation are an urgently needed.

Dietary manipulation of animals is one of the paramount strategies implemented to minimize the emission of GHG from ruminants. Strategies such as the addition to diets of herbal extracts, plants’ metabolites (saponins, tannins, essential oils, organosulfides, etc.), probiotics, yeasts, exogenous enzymes, organic acids, ionophores, algae, and metallic nanoparticles into the fodder had shown to reduce the GHG emission from ruminants (Palangi and Lackner 2022). Among the plants, the inclusion of Moringa oleifera (Moringaceae) in diets has been studied due to its ample nutritional properties (proteins, minerals, vitamins, amino acids, etc.) and low anti-nutrient contents (tannin, lignin, phytate, etc.) (Su and Chen 2020; Pedraza-Hernández et al. 2021; Magalhães et al. 2021). M. oleifera is a rapidly growing perennial softwood plant (5–12 m in height) primarily distributed in tropical and subtropical regions, M. oleifera leaves contain high amounts of crude protein, vitamins, minerals, fatty acids, and different phytochemicals, while seeds contain odorless oil which is resistant to autoxidation process (Ebeid et al. 2020). Therefore M. oleifera had been included as an additive in animal’s diet to improve the productivity and feed utilization (Pedraza-Hernández et al. 2021; Alvarado-Ramírez et al. 2023).

Probiotics are non-pathogenic direct-fed microorganisms extensively used in animal nutrition as additives (Gado et al. 2017) to stimulate the growth of ruminal bacteria and enhance the total bacterial count by providing them certain with nutritional constituents. Although sometimes probiotic bacteria reduce the methanogenesis process by directly inhibiting of the growth of methanogens (Doyle et al. 2019), some probiotics also might inhibit specific bacteria of the rumen that produce secondary metabolites, which reduce the methanogenesis process. Previous reports have critically analyzed the role of probiotic bacteria, mainly lactic acid bacteria, in mitigating of GHG production of ruminants (Doyle et al. 2019).

Because of the role of dietary feed supplements in livestock industries, the present study aimed to assess the potentialities of M. oleifera seeds and probiotic bacteria (Pediococcus acidilactici BX-B122 and Bacillus coagulans BX-B118) as feed additives in the mitigation of biogas [methane (CH4), carbon monoxide (CO), and hydrogen sulfide (H2S)] production from steers and sheep but also explore its fermentation profile under in vitro conditions.

Materials and methods

Experimental treatments

The treatments consisted of ruminant diets with the inclusion of 0, 6, 12, and 18% of M. oleifera seeds meal and a commercial probiotic product (INSILATO AL@, BIORGANIX MEXICANA S.A. DE C.V, Coahuila, Mexico), which contained probiotic bacteria [ P. acidilactici BX-B122 (1 × 1011 cfu m/L) and B. coagulans BX-B118 (1 × 1011 cfu m/L)] at a dose of 0.2 ml/g of diet. The ingredients of the diet were purchased from a feed store, while M. oleifera seeds were obtained from wild trees in the municipality of Iguala de la Independencia Guerrero, Mexico, with an approximate age of 4 years and under the criterion that the pods had to be mature (brown color and open valves). The seeds were subjected to the dehydration process at room temperature (area free from solar radiation and humidity), and subsequently, it was powdered using a forage grinder to generate flour. Mixing of the ingredients, including M. oleifera seed flour, was done manually.

Chemical composition of diets

Three representative samples of each diet were obtained were dehydrated at 60 °C for 72 h, and ground in a hammer mill (Thomas Wiley® Laboratory Mill model 4, Thomas Scientific™, Swedesboro, NJ, USA) with a 1 mm sieve. Ash (method ID 942.05) and nitrogen content (N; method ID 954.01) were quantified (g/kg DM) according to the standard methods of the Association of Official Analytical Chemists (AOAC 1997). From the obtained values, the organic matter (OM) and the crude protein (CP) were calculated as follows:

$$\text{OM}=100 - \text{Ash}$$
$$\text{CP}= N \times 6.25$$

The content of neutral detergent fiber (NDF) and acid detergent fiber (ADF) was estimated using the ANKOM200 Fiber Analyzer (ANKOM Technology Corp., Macedonia, NY, USA) following the methodology of Van Soest et al. (1991). In addition, sodium sulfite and thermostable α-amylase were used in the NDF analysis, and the NDF and ADF values were expressed without residual ash. The chemical composition of the diets is presented in Table 1.

Table 1 Ingredients and chemical composition of diets for ruminants with the inclusion of different concentrations of M. Oleifera seeds
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In vitro incubations

The ruminal content was obtained from 4 steers (430 ± 20 kg BW) and 4 sheep (40 ± 5 kg BW) that were slaughtered in a local slaughterhouse, regulated by the Official Mexican Standard NOM-033-SAG/ZOO-2014, which establishes methods to kill domestic and wild animals. The ruminal contents were transported to the laboratory in air-tight thermoses pre-heated to 39 °C, where it was filtered with four layers of cheesecloth to obtain only ruminal fluid, which was subsequently used as inoculum for fermentation. The nutrient medium was prepared following the methodology described by Goering and Van Soest (1970) and contained buffer solution, macrominerals, microminerals, reducing agent, resazurin, and distilled water. Fermentation was carried out in glass vials (120 ml) containing 500 mg of diet, probiotic doses (only if applicable), 10 ml of ruminal inoculum, and 40 ml of nutrient medium in each vial. Rubber stoppers and aluminum seals were used to seal the vials hermetically. Further, vials were shaken lightly and placed in a water bath at 39 °C for 48 h. In total, three fermentation cycles were carried out, and in each one, there were 51 vials, including the white ones (containing only ruminal inoculum and nutrient medium).

Biogas estimation

Total biogas production was quantified up to 48 h, following the methodology proposed by Theodorou et al. (1994) and using a digital manometer with a precision of ± 2% (Manometer model 407,910, Extech® Instruments, Nashua, NH, USA). The biogases (CH4, CO, and H2S) were quantified following the methodology of Acosta et al. (2022) using a portable gas detector (Dräger X-am®, model 2500, Dräger, Lübeck, SH, Germany) connected to an external pump (Dräger X-am®, Dräger, Lübeck, SH, Germany). Furthermore, at the end of each measurement, the accumulated biogas was released to avoid over-estimation.

Rumen pH and dry matter degradability (DMD)

The contents of the vials were filtered at the end of the fermentation using filter bags with a porosity of 25 μm (Filter bags F57, ANKOM Technology Corp., Macedonia, NY, USA) to obtain the residues of the diets and collect the liquid part in beakers (Alvarado-Ramírez et al. 2023). pH was measured in the collected liquid using a potentiometer with a glass electrode (pH wireless electrode HALO® model HI11102, Hanna® Instruments, Woonsocket, RI, USA), while DMD (%) was estimated after dehydrating and weighing the residue of the diets by measuring the difference between the initial and final weights (Elghandour et al. 2014).

Calculous

The production (ml/g DM incubated) of total biogas, CH4, CO, and H2S was used to estimate the asymptotic gas production, the production rate, and the time of the lag phase before the production of each gas, using the NLIN protocol of the Statistical Analysis System (SAS 2002) and the model proposed by France et al. (2000) as mentioned below:

$$y=b \times [1-e^{-c(t-Lag)}]$$

where y is the production (ml/g MS) of total biogas, CH4, CO, and H2S at time t (h); b is the asymptotic production (ml/g MS) of total biogas, CH4, CO, and H2S; c is the production rate (ml/h) of total biogas, CH4, CO, and H2S; and Lag is the lag phase (h) before the production of total biogas, CH4, CO, and H2S.

The metabolizable energy (ME; MJ/kg DM) and short-chain fatty acids (SCFA; mmol per 200 mg of DM) were calculated with the equations proposed by Menke et al. (2009) and Getachew et al. (2002), respectively. The CH4 conversion efficiency was estimated based on CH4 production per unit of SCFA, ME, and MO in mmol/mmol (CH4:SCFA), g/MJ (CH4:ME), and ml/g (CH4:OM), respectively.

Statistical analyses

The variance analysis (ANOVA) model considered the experimental design (completely randomized) with a factorial arrangement (2 × 4 × 2), where factor 1 was the source of ruminal inoculum (steer and sheep), factor 2 was the inclusion of M. oleifera seeds (0, 6, 12, and 18%), and factor 3 was the addition of probiotic (without and with) in triplicates. The triplicate data of each treatment in each run were calculated as a mean, and the average values obtained were used as the experimental unit of each treatment. The data were analyzed using the GLM procedure of SAS (2002). The last minimum significance (LSD) was used for the comparison of means; it was calculated from the standard error (SE) by Proc Mixed (SAS 2002), considering the error degrees of freedom (DF) from variance analysis (ANOVA) and a P = 0.05.

Results

Total biogas production and fermentation kinetics

Table 2 shows the total biogas production from steers and sheep by supplementing M. oleifera seeds and probiotic bacteria (P. acidilactici BX-B122 and B. coagulans BX-B118). In steers, the asymptotic biogas production was increased by supplementing different concentrations (6–18%) of M. oleifera seeds in the presence of probiotics. M. oleifera seeds (P = 0.0381) and probiotics depicted (P = 0.0021) increment in asymptotic biogas production (259.7 to 344.7 ml/g DM). However, there were not interaction M. oleifera seeds × probiotics (P = 0.8774) effect on asymptotic biogas production. Similarly, the rate of biogas production was increased (0.039 to 0.048 ml/h) due to the supplementation of varied concentrations of M. oleifera seeds in the presence of probiotic bacteria, while the effect was not significant (P = 0.8062) for M. oleifera seeds and significant (P < 0.0001) for probiotics inclusion. M. oleifera seeds × probiotics was not significant (P = 0.5973) influence on the biogas production rate. The lag period was reduced (6.34 to 4.86 h) at higher concentrations of M. oleifera seeds in the presence of probiotics. However, the effect of M. oleifera seeds × probiotics interaction on the lag period was not significant (P = 0.7525). In a like manner, total biogas production (ml total biogas/g DM incubated) was increased (P < 0.05) from 2 to 48 h at distinct concentrations of M. oleifera seeds in the presence of probiotics. However, there were not interaction M. oleifera seeds × probiotics (P = 0.0318) at 2 h of incubation.

Table 2 Parameters and total biogas production from steers and sheep as a source of inoculum using different concentrations of M. Oleifera seeds in the presence or absence of probiotic (P. acidilactici BX-B122 and B. coagulans BX-B118)
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The asymptotic biogas production was increased (150.44 to 390.23 ml/g DM) in sheep, due to the supplementation of varied concentrations (6–18%) of M. oleifera seeds in the presence of probiotics (Table 2). M. oleifera seeds, probiotics, and there was interaction M. oleifera seeds × probiotics (P < 0.0001) for asymptotic biogas production. Similarly, the rate of biogas production was increased (0.0076 to 0.0339 ml/h; P < 0.05) due to the inclusion of varied concentrations of M. oleifera seeds and probiotics. However, M. oleifera seeds × probiotics interaction did not affect (P = 0.3095) the rate of biogas production. The addition of varied concentrations of M. oleifera seeds and probiotics also showed no effect (P > 0.05) on the lag period. M. oleifera seeds and probiotics exhibited a significant increment in total biogas production up to 48 h. Maximum biogas production of 333.6 ml total biogas/g DM was estimated using 6% of M. oleifera seeds in the presence of probiotics. Overall, M. oleifera seeds × probiotics interaction depicted an influence (P < 0.05) on asymptotic biogas production and total biogas production up to 48 h, while the rate of biogas production (P = 0.1751) and the duration or onset of the lag period (P = 0.2871) was not significantly affected.

Figure 1 illustrates total biogas production kinetics from steers and sheep using M. oleifera seeds at different concentrations in the presence and absence of probiotics. The results showed higher total biogas production using 6% of M. oleifera seeds in the presence of probiotics.

Fig. 1
figure 1

Kinetics of ruminal total gas (TG) production from steers and sheep as a source of inoculum using different concentrations of M. oleifera seeds in the presence or absence of probiotic (P. acidilactici BX-B122 and B. coagulans BX-B118)

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In vitro CH4 production

Methane production is due to the inclusion of M. oleifera seeds and probiotic bacteria, using steers as a source of ruminal inoculum is shown in Table 3. The addition of various concentrations of M. oleifera seeds, probiotics, and M. oleifera seeds × probiotics interaction revealed an effect (P < 0.05) on the asymptotic CH4 emission from steers. The rate of CH4 emission was reduced (P < 0.05, 0.1694 to 0.0447 ml/h) using 6 and 18% of M. oleifera seeds, while the effect was not significant (P = 0.7246) due to the addition of probiotics. The supplementation of M. oleifera seeds at higher concentrations caused a reduced lag period (21.02 to 8.48 h), however, the effect was not significant (P = 0.1285), while the presence of probiotics (P = 0.0262) and M. oleifera seeds × probiotics interaction (P = 0.0347) showed a significant effect on lag period reduction. On the other hand, CH4 production was increased up to 48 h using various concentrations of M. oleifera seeds in the presence of probiotics. However, the production due to M. oleifera seeds and probiotics supplementation was estimated to be lower than that of the control.

Table 3 Parameters and CH4 production from steers and sheep as a source of inoculum using different concentrations of M. Oleifera seeds in the presence or absence of probiotic (P. acidilactici BX-B122 and B. coagulans BX-B118)
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On the contrary, using sheep as a source of ruminal inoculum, the addition of various concentrations of M. oleifera seeds, probiotics, and M. oleifera seeds × probiotics interaction exhibited an increment (P < 0.05) in CH4 production up to 48 h to the control. Overall, the interaction of M. oleifera seeds × probiotics affected the CH4 production (P < 0.05). Figure 2 illustrates CH4 production from steers and sheep using M. oleifera seeds at different concentrations in the presence and absence of probiotics. The results revealed a low CH4 production using 18% of M. oleifera seeds in the presence of probiotics.

Fig. 2
figure 2

Kinetics of ruminal CH4 production from steers and sheep as a source of inoculum using different concentrations of M. oleifera seeds in the presence or absence of probiotic (P. acidilactici BX-B122 and B. coagulans BX-B118)

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In vitro CO production

Carbon monoxide production from ruminants due to the addition of M. oleifera seeds and probiotic bacteria, using steers and sheep as a source of ruminal inoculum is shown in Table 4. Findings showed that various concentrations of M. oleifera seeds, probiotics, and M. oleifera seeds × probiotics interaction depicted an increment (P < 0.05) in asymptotic CO production (0.007 to 1.026 ml/g DM), lag period (0.0135 to 0.1710 h), and CO production (up to 48 h; 0.0076 to 0.9367 ml CO/g DM incubated) from steers. In contrast, the rate of CO production was decreased (P < 0.05, 0.0008 to 0.0003 ml/h) due to the inclusion of M. oleifera seeds in the presence of probiotics. Likewise, in sheep, asymptotic CO production (0.1626 to 1.4293 ml/g DM) as well as CO production (0.1538 to 1.4085 ml/g DM incubated) were estimated to be increased (P < 0.05) by supplementing varied concentrations of M. oleifera seeds in the presence of probiotics.

Table 4 Parameters and CO production from steers and sheep as a source of inoculum using different concentrations of M. Oleifera seeds in the presence or absence of probiotic (P. acidilactici BX-B122 and B. coagulans BX-B118)
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Figure 3 shows CO production (ml/g DM) from steers and sheep using M. oleifera seeds at different concentrations in the presence and absence of probiotics. Results showed low CO production using 12% of M. oleifera seeds in the presence of probiotics.

Fig. 3
figure 3

Kinetics of ruminal CO production from steers and sheep as a source of inoculum using different concentrations of M. oleifera seeds in the presence or absence of probiotic (P. acidilactici BX-B122 and B. coagulans BX-B118)

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In vitro H2S production

Hydrogen sulfide production from ruminants due to the supplementation of M. oleifera seeds and probiotic bacteria, using steers and sheep as a source of ruminal inoculum is shown in Table 5. In steers, asymptotic H2S production was decreased from 0.0672 to 0.0114 ml/g DM, but M. oleifera seeds × probiotics interaction was not significant (P = 0.3390). Similarly, M. oleifera seeds × probiotics interaction depicted no significant effect on the biogas production rate (P = 0.2977) and lag period (P = 0.2952). Adding 12% of M. oleifera seeds along with probiotics revealed a reduction in H2S production from 0.06745 to 0.01116 ml H2S/g DM incubated at 48 h, however there were not interaction M. oleifera seeds × probiotics (P = 0.4490).

Table 5 Parameters and H2S production from steers and sheep as a source of inoculum using different concentrations of M. Oleifera seeds in the presence or absence of probiotic (P. acidilactici BX-B122 and B. coagulans BX-B118)
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In sheep, the inclusion of M. oleifera seeds with probiotics exhibited a reduction in asymptotic H2S production from 0.0363 to 0.0029 ml/g DM. However, the influence of M. oleifera seeds × probiotics interaction was not significant (P = 0.8631). The interaction showed no significant (P = 0.1557) effect on the biogas production rate, but the lag period was reduced (P = 0.0044) from 0.1534 to 0.0026 h. On the other hand, the inclusion of M. oleifera seeds along with probiotics in the diet caused mitigation in H2S emission from 0.03636 to 0.00293 ml/g DM up to 48 h, but the interaction was not significant (P = 0.7744).

Figure 4 estimates H2S production from steers and sheep using M. oleifera seeds at different concentrations in the presence and absence of probiotics. Results showed low H2S production using 18% of M. oleifera seeds in the presence of probiotics.

Fig. 4
figure 4

Kinetics of ruminal H2S production from steers and sheep as a source of inoculum using different concentrations of M. oleifera seeds in the presence or absence of probiotic (P. acidilactici BX-B122 and B. coagulans BX-B118)

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Fermentation profile and CH4 conversion efficiency

In steers, the supplementation of varied concentrations of M. oleifera seeds in the presence of probiotics resulted in a reduction (P < 0.0001) in pH from 7.11 to 6.42. The DMD was reduced from 85.82 to 60.68% using 12% M. oleifera seeds in the presence of probiotics, but the interaction was not significant (P = 0.9706). The supplementation of M. oleifera seeds along with the probiotics increased SCFA (3.70 to 6.35 mmol/g DM) and ME (5.82 to 7.18 MJ/kg DM). M. oleifera seeds × probiotics did not significant affected (P = 0.3368) the CH4:SCFA, but it affected (P = 0.0004) the CH4:ME and CH4:OM (Table 6).

Table 6 Rumen fermentation profile and CH4 conversion efficiency from steers and sheep as a source of inoculum using different concentrations of M. Oleifera seeds in the presence or absence of probiotic (P. acidilactici BX-B122 and B. coagulans BX-B118)
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In sheep, including of M. oleifera seeds and probiotics yielded reduced the pH (P = 0.0032) and DMD (P = 0.0208) from 6.95 to 6.18 and 72.94 to 61.40%, respectively. The SCFA and ME were increased from 1.33 to 6.11 mmol/g DM and 4.60 to 7.05 MJ/kg DM, respectively, using 18% M. oleifera seeds along with probiotics. M. oleifera seeds × probiotics interaction exerted a significant effect (P = 0.0022) on CH4:SCFA, but the effect was not significant for CH4:ME (P = 0.2044) and CH4:OM (P = 0.1994) (Table 6).

Discussion

The anthropogenic GHG emissions have become a pivotal topic globally because of their detrimental impact on climate change and global warming. In the coming years, the release of GHG will exhibit significant ecological and socio-economic effects worldwide due to the significant rise in temperature. Since livestock is one of the prime contributors towards increments in GHG release, followed by a change in the earth’s climate (Mangar et al. 2022), it is imperative to minimize GHG emissions from livestock by developing alternative feed resources.

The volume of biogas produced from livestock depends on the nature of feed digestion and the fermentation process. Some feed additives affect animal biogas emissions (Santillán et al. 2023). A plethora of dietary supplements, such as the inclusion of plants and probiotics, have been tested to investigate their roles in the rate of biogas production from ruminants and non-ruminants (Khusro et al. 2022a).

In the present study, supplementing different concentrations (6–18%) of M. oleifera seeds along with probiotics (P. acidilactici BX-B122 and B. coagulans BX-B118) in diets increased the total biogas production of steers and sheep. The increase in biogas production from steers and sheep shows the availability and digestibility of diets. Similarly, Pedraza-Hernandez et al. (2019) found that including different doses of M. oleifera extract increased the rate of in vitro total biogas production from goats. On the contrary, Elghandour et al. (2017) and Mangar et al. (2022) observed a reduction in total biogas production from dairy calves and cows, respectively, after the supplementation of M. oleifera as a feed additive. The potential effects of M. oleifera inclusion on biogas production might depend on factors such as genetic differences, soil fertility, nutritional content of the plant, and type of livestock used (Fritsche et al. 2017). In another study, Abdelbagi et al. (2021) estimated increased biogas production from steers by supplementing probiotics as additives. Likewise, Elghandour et al. (2018) observed higher horse biogas production by adding probiotics to an oat straw-containing diet. The increment in biogas production is mainly associated with better microbial fermentation, followed by a nutrient digestibility enhancing.

The mitigation of CH4 emissions from ruminants and non-ruminants is the main target of veterinarians because livestock causes approximately 35–40% of the CH4 emissions (Vohra et al. 2016; Khusro et al. 2022b). In ruminants, 90% of CH4 emissions are derived from enteric fermentation (Doyle et al. 2019). Among the different types of GHG produced, the CH4 ranks second after CO2 and absorbs more energy than CO2, with a global warming potential of ~ 28 (Króliczewska et al. 2023). Thus, an innovative approach is required to reduce CH4 production from ruminants to ensure a cleaner ecosystem. In this regard, in the present context, adding different doses of M. oleifera seeds, probiotics, and M. oleifera seeds × probiotics interaction revealed a significant (P < 0.05) effect on asymptotic CH4 emission from steers. The rate of CH4 emission was significantly (P < 0.05) reduced using 6 and 18% of M. oleifera seeds, while the effect was not significant (P = 0.7246) due to the addition of probiotics. Including M. oleifera seeds, probiotics, and M. oleifera seeds × probiotics interaction exhibited a significant increment in CH4 production up to 48 h in sheep. Similar observations were estimated by Pedraza-Hernández et al. (2019), Dong et al. (2019), and Mangar et al. (2022), who suggested the utilization of M. oleifera as feed additives to mitigate CH4 emission from goats and cows. The reduction in CH4 production might be because of the cell plant wall content that might decrease the microbial action, thereby causing reduced emissions of CH4 (Elghandour et al. 2017). However, Elghandour et al. (2018) depicted increased CH4 production in horses after adding probiotic to the feeding diet. Overall, the source, concentrations, and strains of probiotics are factors that could affect the emission of CH4 from animals (Vohra et al. 2016).

Carbon monoxide is an indirect GHG because it has the potential to react with other molecules (i.e. the hydroxyl radical, OH*) present in the air and create another GHG, mainly CO2. It generally causes a lower absorption of energy in the infrared region. However, it enhances global warming by reacting with certain chemical species in the atmosphere, thereby increasing the amount of primary GHG and modulating CH4 and ozone production (Sobieraj et al. 2022). Including different levels of M. oleifera seeds, probiotics, and M. oleifera seeds × probiotics promoted increments of CO production from steers. However, the present study´s findings differ from the reports of Santillán et al. (2023), who demonstrated that CO emissions could be reduced in horses fed diets with plant leaf extract.

In livestock, H2S is known as a toxic signalling molecule after NO (nitric oxide) and CO (Shah et al. 2020). The anaerobic digestion of organic materials through the action of sulphate-reducing bacteria releases H2S in the ecosystem. The gut bacteria cause the metabolism of dietary SO42- (sulfate) and produce H2S in animals, which is rapidly absorbed through the intestinal wall and exhibits toxicological effects (Pal et al. 2018). Since the accumulation of H2S gas causes poliomyelitis in ruminants (Binversie et al. 2016), it is imperative to regulate the synthesis of H2S in the rumen. In the rumen, H2S (sulphide) production depends on the amount of SO42- in the diet. Ruminal microbes use sulphur or SO42-, which is present in the diet, to synthesize H2S. A competitive relationship is observed among methanogens and sulphide-reducing bacteria to require H+ for the metabolic process. Correspondingly, sulphide-reducing bacteria reduce SO42- to H2S, and methanogens reduce CO2 to CH4 in the rumen (Shah et al. 2020). Depicted that the inclusion of sulphur in the diet of steers enhanced H2S production (Drewnoski et al. 2012), the addition of S-containing amino acid and SO42− in the diet of swine might mitigate the H2S production (Sutton et al. 1999). Santillán et al. (2023) found a reduction of H2S emission from equines by supplementing M. oleifera plant extract in the diet.

Saksrithai and King (2018) summarized extensively the potential role of different additives in reducing the emission of H2S from poultry and animals. In the line with prior reports, the present investigation revealed reduction in H2S production from steers and sheep due to the inclusion of M. oleifera seeds along with probiotics in the diet.

Plants are sources of saponins, tannins, flavonoids, and other metabolites, which directly or indirectly could mitigate ruminants’ digestion-associated biogas emission, mainly CH4 (Króliczewska et al. 2023). Saponins are known to inhibit the growth of ciliate protozoa present in the rumen (Hartinger et al. 2018) and reduce the production of CH4 indirectly through the defaunation process (removal of protozoa from the rumen), which is known to disrupt the protozoan cell membrane in the rumen.

A hydrophilic sugar moiety and a hydrophobic steroid or triterpenoid aglycone are saponins’ components that allow to the formation of complexes with sterols of cell membranes, leading to cell death (Patra and Saxena 2009). Additionally, saponins affect CH4 emission by reducing the viability of methanogens and deactivating methanogenesis-associated genes, slowing down the methanogenesis process. Saponin also affects specific microbes in the rumen and alters biochemical mechanisms in the rumen (Ramos-Morales et al. 2017).

Tannins are another secondary polyphenolic plant metabolites that affect the rumen ecosystem (Broucek 2018). Tannins cause indirect inhibition of hydrogen-producing microbes and direct inhibition of methanogenic microbes in rumen (Kumar et al. 2014). Anti-methanogenic properties of tannins may be bactericidal or bacteriostatic and may depend on the type of bacterial species present in the rumen (Vasta et al. 2019). Overall, the anti-methanogenic traits of tannins rely on the binding of tannins to protein through the interaction of phenolic hydroxyl groups with amino acid residues by hydrogen bonds and hydrophobic interactions (Vasta et al. 2019); similarly, flavonoids decrease the viability of protozoa and methanogens, and thus, inhibit the methanogenesis process in the rumen by absorbing H2 after the breakdown of their carbon ring structures (Oskoueian et al. 2013).

Probiotics (lactobacilli, bacilli, pediococci, lactococci, bifidobacteria, and propionibacteria) are known to affect the ruminal fermentation process and improve animals’ health by controlling the gastro-intestinal microflora (Tavendale et al. 2005). Probiotics present in the rumen increase feed efficiency, which may decrease the production of GHG, particularly CH4 emissions (Islam and Lee 2019). Since the increase in propionate production and reduction in CH4 emission are co-related (Haque 2018), probiotics can help promote fermentation mechanisms to release hydrogen-based propionate. However, probiotic bacteria affect methanogenesis in ruminants by other possible mechanisms such as (1) Shifting of the ruminal fermentation process so that there is a prominent reduction in CH4 emission, (2) Directly inhibiting the methanogens present in the rumen, and (3) Inhibiting H2 or methyl-containing compounds producing specific bacterial species present in the rumen that are responsible for the methanogenesis process (Doyle et al. 2019).

In the present study, steers and sheep showed a reduction in pH and DMD because of the addition of 6 and 18% of M. oleifera seeds. In addition, the supplementation of M. oleifera seeds along with probiotics increased SCFA and ME and reduced the rate of CH4 emission.