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8 April 2026

13 Pages

In Vitro Ruminal Fermentation and Gas and Methane Production of Eragrostis curvula Supplemented with Searsia lancea Leaf or Silage Meal

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1
Agricultural Research Council-Animal Production: Animal Nutrition, Private Bag X2, Irene 0062, South Africa
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Department of Agriculture and Animal Health, College of Agriculture and Environmental Sciences, Science Campus, University of South Africa, Pretoria 1709, South Africa
3
School of Interdisciplinary Research and Graduate Studies, College of Graduate Studies Muckleneuk Campus, University of South Africa, Pretoria 0003, South Africa
4
Department of Agricultural Economics and Animal Production, School of Agriculture and Environmental Sciences, Turfloop Campus, University of Limpopo, Sovenga 0727, South Africa
Methane2026, 5(2), 12;https://doi.org/10.3390/methane5020012
This article belongs to the Special Issue Mitigating Methane Emissions Through Innovative Animal Nutrition Strategies
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Abstract

Livestock represent a key asset in the livelihood of smallholder farmers and play a critical role in the social dynamics and nutritional security of resource-poor communities. However, within these resource-poor communities, livestock productivity remains low. This is often due to seasonal changes in the quantity and quality of available feed from the natural veld, which in turn also contributes to methane production. This study aimed to evaluate the effects of supplementing Eragrostis curvula hay with Searsia lancea leaf or silage meal on in vitro fermentation efficiency and gas and methane production. Therefore, an in vitro study using a semi-automated pressure transducer technique was conducted on grass hay alone (control) and grass hay supplemented with 15% or 30% of either S. lancea leaf or silage meal. The dietary treatments were arranged in a complete randomized design, with each treatment replicated four times. Total gas and methane production was recorded at 3, 6, 12, 24 and 48 h using a pressure transducer attached to a data logger. After incubation, samples were collected to determine volatile fatty acids. Supplementing grass hay with 15% S. lancea leaf meal increased gas production by 76%, 52%, 32% and 12% in the first 24 h of incubation. Similarly, increasing the supplementation level to 30% increased gas production by 75%, 63%, 45% and 14%. However, supplementing grass hay with silage meal at 15% significantly reduced gas production by 37% during the first 3 h of incubation, whereas supplementation at 30% had no effect. Supplementing grass hay with S. lancea meals effectively reduced methane production at 24 and 48 h. Grass hay supplemented with 15% or 30% silage meal reduced methane by 46% and 39% at 24 h, while at 48 h, methane was reduced by 39% and 49%, respectively. Supplementing grass hay with S. lancea meals, however, did not affect volatile fatty acids. In conclusion, S. lancea can be strategically used as a supplementary feed source to modulate the rumen ecosystem by attenuating enteric methane production. Further studies are required to determine the effect of S. lancea on rumen microbial composition and its metabolic function.

1. Introduction

Livestock are vital to the livelihoods of smallholder farmers, providing income, enhancing food security, and contributing to cultural practices and social standing [1,2]. Moreover, livestock play a fundamental role in the social fabric and nutrition of resource-poor communities, sustaining billions of people worldwide [3]. With the global population predicted to reach 10.2 billion in 2100 [4], a concurrent increase in livestock-derived food will be needed [5]. Therefore, this necessitates an increase in livestock production to meet the increasing demand for livestock products. However, livestock, particularly ruminants, are blamed for emitting greenhouse gases, with enteric methane being a major concern as the primary contributor to global warming [6,7,8]. Due to enteric rumen fermentation, ruminant animals contribute about 9.2% of global greenhouse gas emissions [9] and up to 32% of global anthropogenic methane production [10]. Methane gas has a global warming potential over a 100-year time horizon that is approximately 28–34 times greater than that of carbon dioxide [11,12]. Besides the negative effects on the environment, enteric methane production represents a loss of energy, diverting up to 12% of dietary gross energy from microbial protein synthesis, thereby negatively affecting livestock production [13,14]. Thus, this necessitates modulation of rumen fermentation to reduce enteric methane production.
Mitigation of enteric methane production from ruminants and the reduction in its negative environmental and global warming impacts are priorities [3,15]. Therefore, research in livestock production, particularly nutrition, has focused on modulating the rumen ecosystem to reduce enteric methane production [16,17,18,19]. Hence, different strategies such as feed manipulation through feed formulation, additives, and direct-fed microbes have been evaluated to reduce ruminal enteric methane production [20,21]. The integration of fodder trees into livestock diets has recently become a research focus, as these trees are rich in phytogenic compounds that have the potential to modulate the rumen ecosystem [22]. Searsia lancea is an evergreen tree native to Southern Africa, commonly known as the karee tree [23,24]. The species contains a protein content ranging from 8.3% to 19% [23,25,26]. The wide range in protein content could be attributed to the harvesting stage and the area in which the plant was harvested. These protein levels are indicative that S. lancea fodder can be used as a protein source for livestock by resource-poor farmers. In addition, the plant contains phytogenic compounds such as saponins and tannins [27]. The presence of phytogenic compounds, particularly tannins, in tree fodder reduces ruminal protein degradability by binding protein [28,29,30,31]. Furthermore, it can reduce enteric methane production by modulating rumen microbiota [32,33,34]. However, the tanniferous fodder must be fed cautiously to avoid exceeding the livestock threshold intake limit of 5% [32].
In this study, S. lancea fodders (shade-dried and ensiled), hereafter referred to as leaf and silage meals, were evaluated for their effects as a supplement to poor-quality Eragrostis curvula grass hay. To test the effects of the S. lancea (leaf and silage) supplementation on poor-quality grass hay, an in vitro gas production technique was used as proposed by Menke and Steingass [35]. In vitro ruminal fermentation is an important technique for evaluating the nutritional value of feedstuffs, particularly tree fodder that contains phytogenic compounds [36]. Therefore, in vitro approaches (e.g., pressure transducer or rumen simulation techniques) provide an effective means of assessing the impacts of fodder tree supplementation of poor-quality grass hay without exposing animals to the potential adverse effects of phytogenic compounds. Moreover, the technique does not require bulky feed, unlike an in vivo study. According to Wang et al. [37], the in vitro technique eliminates variations due to individual animals, nutrient absorption, and the rumen feed retention period, as would occur in an in vivo study. However, the technique does not provide data on the animal’s performance, such as the feed intake, growth performance and meat quality. Therefore, the objective of this study was to evaluate the effects of supplementing poor-quality grass hay with S. lancea leaf meal or silage meal on in vitro ruminal fermentation and gas and methane production. Hence, it was hypothesized that supplementing poor-quality grass hay with S. lancea leaf or silage meal would affect the fermentation profile.

2. Results

2.1. In Vitro Gas and Methane Production

Table 1 shows in vitro gas production of grass hay influenced by S. lancea leaf or silage meal supplementation at different incubation times. Supplementing grass hay with S. lancea leaf improved fermentation significantly (p < 0.05) in the first 24 h of incubation. Supplementing grass hay with 15% S. lancea leaf meal increased gas production by 76%, 52%, 32% and 12% in the first 24 h of incubation, whereas increasing supplementation to 30% increased gas production of grass hay by 75%, 63%, 45% and 14%. However, grass hay supplemented with 15% S. lancea silage meal significantly (p < 0.05) decreased gas production by 37% compared with the control treatment during the first 3 h of incubation; thereafter, no further effects were recorded. Increasing the supplementation level to 30% also did not affect gas production, except at 48 h, when gas production was lowest.
Table 1. In vitro gas production of E. curvula grass hay supplemented by S. lancea leaf or silage meal over 48 h.
Table 2 shows methane production from grass hay supplemented by S. lancea leaf or silage meal. Methane gas production was very low in the first 12 h of incubation. At 24 h of incubation, methane gas production was significantly (p < 0.05) higher in grass hay alone (T1) and grass hay supplemented with 15% S. lancea leaf meal (T2). However, supplementing grass hay with 15% S. lancea leaf meal reduced methane production effectively by 30% at 48 h post-incubation. On the other hand, supplementing grass hay with 30% S. lancea leaf meal reduced methane production by 29% and 43% at 24 and 48 h post-incubation, respectively. Silage meal supplementation at 15% reduced methane production by 46% and 39% at 24 and 48 h post-incubation, respectively. However, increasing the supplementation level of silage meal to 30% reduced methane production by 39% and 49% at 24 and 48 h post-incubation, respectively.
Table 2. In vitro methane production of E. curvula grass hay supplemented by S. lancea leaf or silage meal over 48 h.

2.2. Volatile Fatty Acids and Fermentation Ability

The fermentability of grass hay supplemented with S. lancea leaf or silage meal is shown in Table 3. This study showed that supplementing grass hay with S. lancea leaf or silage meal did not affect total volatile fatty acids—acetic acid, propionic acid, iso-butyric acid or the ratio between acetic acid and propionic acid. However, butyric acid, isovaleric acid and valeric acid were significantly (p < 0.05) influenced by S. lancea supplementation. Butyric acid was reduced by 7.5% for grass hay that was supplemented with 15% S. lancea leaf meal, whereas increasing the supplementation level of S. lancea leaf meal to 30% led to a reduction in butyric acid of 11.8% as compared to the control. A similar pattern was observed when silage meal was used as a supplement to grass hay. Supplementing grass hay with S. lancea silage meal at 15% and 30% significantly (p < 0.05) reduced butyric acid by 4.1% and 5.5%, respectively, compared to the control. This study showed that supplementing grass hay with S. lancea silage meal at a 30% inclusion level significantly (p < 0.05) increased isovaleric and valeric acid by 13.9% and 12.7%, respectively. However, it did not affect the rumen non-glucogenic-to-glucogenic ratio, while it showed a tendency (p < 0.10) to reduce fermentation efficiency.
Table 3. Volatile fatty acids, non-glucogenic-to-glucogenic acid ratio and fermentation efficiency of E. curvula grass supplemented with S. lancea leaf or silage meal.

3. Discussion

The quality of E. curvula grass used in this study was clear, as it was of low quality, similar to what would be utilized for livestock in marginal times in South Africa. The protein content of the grass hay was below the threshold limit of 7% required for rumen microbe growth and normal functioning [38]. This means microbes cannot break down the cell wall components of grass, thereby prolonging the grass’s retention time in the rumen. The high fiber content (particularly NDF) in the grass hay is associated with methane production [39,40]. The NDF value of the grass hay was above the threshold limit of 60%, above which digestibility is reduced [41]. This is in line with the gas production recorded for grass hay alone (control), which was significantly lower. This means that rumen microbes would take time to break down the feed due to high NDF levels coupled with low protein content [42]. The pH of 4.2 recorded for S. lancea silage indicates that it fermented well, as this is the recommended pH value for correctly preserved silage [43].
The in vitro gas production of forages indicates the fermentability of the feed material in the rumen. Therefore, the low gas production from grass hay incubated alone is associated with the quality of the grass. However, dietary supplementation of S. lancea leaf meals to grass hay significantly increased gas production. This could be attributed to the fact that supplementation with 15% and/or 30% S. lancea leaf meal is expected to increase protein content in the mixture to about 6.9% and 8.5%, respectively. These values are adequate for the normal functioning of rumen microbes. However, a different pattern was observed when grass hay was supplemented with S. lancea silage meal. Gas production was not improved, although the protein content in the mixture was expected to increase to 7.5% and 9.6%. This may be attributed to fermentation during ensiling, which increases the protein content by 33.3% compared to leaf meals. Searsia lancea plants contain secondary metabolites, including tannins [27]. Therefore, the increase in protein content after ensiling is attributed to the dissociation of protein from tannins as ensiling reduces their levels [44]. However, the elevated NDF concentration in the silage meal may account for the lack of an effect on gas production when it was used as a supplement for grass, notwithstanding its higher protein content relative to the leaf meal. This indicates a need for further study to determine the effect of ensiling on the nutrient bioavailability of fodder trees.
In this study, it was also shown that in vitro methane production increased with increasing incubation time. Furthermore, it was shown that grass hay supplementation with S. lancea leaf meal significantly increased methane in the first 6 h of incubation compared to control and silage meal supplements. This same pattern was reported by Nel et al. [45] when E. curvula grass hay was supplemented with enzymes. However, as incubation time advanced, methane gas production was significantly reduced. On the other hand, supplementing grass hay with S. lancea silage meal had no effect on methane production during the first 12 h of incubation, but as the incubation period advanced, methane production was significantly reduced. These results showed that supplementing grass hay with S. lancea meal (leaf or silage) reduces the negative environmental impact of livestock by reducing enteric methane production, since methane production from ruminant animals is a major contributor to climate change [3,15]. In addition, methane production from ruminant livestock represents a gross energy loss, negatively impacting livestock production. Therefore, the reduction in methane production resulting from the inclusion of S. lancea in grass hay means that more energy will be retained in the livestock’s body and become available for production [14].
Furthermore, this study showed that supplementing grass hay with S. lancea leaf meal or silage meal did not interfere with total volatile fatty acid production. However, this must be discussed with caution as volatile fatty acids were determined at 48 h post-incubation, which may explain the lack of change observed. The longer the feed is exposed to rumen microbes, the greater the loss of fermentation efficiency. On the other hand, more VFAs were produced in a short incubation period, indicating effective substrate degradability, which could be attributed to the presence of fermentable material [46]. The reduction in butyric acid reported in this study due to the inclusion of S. lancea meals in grass is not consistent with the result reported by Mhlongo et al. [47], who reported that Acacia mearnsii inclusion in maize did not affect butyric acid production. However, the reduction in butyric acid production reported in this study due to supplementation of S. lancea leaf and silage meals is in line with the methane reduction reported in Table 2. A study conducted by Williams et al. [48] showed that butyric acid positively correlates with enteric methane production. Butyric acid production in the rumen is associated with the release of hydrogen (H), which methanogenic bacteria use to produce methane [49,50,51]. Therefore, the reduction in methane production reported in this study could be attributed to the low H availability in the rumen due to the reduction in butyric acid production as influenced by the supplementation of S. lancea leaf and silage meals. The increase in isovaleric and valeric acids observed in this study could indicate that grass hay supplemented with 30% S. lancea silage meal may have a greater proteolysis capability for rumen-degraded protein. This may influence ammonia nitrogen (NH3-N) production in the rumen; however, NH3-N was not recorded in this study. The observed decrease in fermentation efficiency suggests that the inclusion level of S. lancea should not exceed 30%, as higher levels may negatively impact ruminal fermentation.

4. Materials and Methods

4.1. Ethical Statement

The experimental protocol was approved by the Agricultural Research Council-Animal Production (ARC-AP), the Animal Ethics Committee (Ref APIEC 23/08) and the University of South Africa (UNISA), and the College of Agriculture and Environmental Sciences–Animal Research Ethics Committee (Ref 2022/CAES_AREC/056).

4.2. Harvesting and Preparation of S. lancea Leaf and Silage Meals

The S. lancea trees used in the trial were collected from the Agricultural Research Council-Animal Production (ARC-AP) Roodeplaat experimental farm (25°34′11.27″ S:28°22′05.36″ E) in Pretoria, South Africa, which receives annual precipitation of 700 mm with a minimum ambient temperature of 2–20 °C in winter and maximum of 20–29 °C in summer. At the Roodeplaat farm, S. lancea trees of similar size were identified and marked for harvesting. Prior to harvesting, all marked trees were pruned in order for subsequent harvesting to occur at a standardized time of regrowth. Eight weeks after pruning, the new leaves were manually harvested and used to produce either leaf meal or silage meal. After harvesting, the forage material was transported to the ARC-AP Irene experimental farm (25°53′53″ S and 28°11′24″ E) located in Irene, South Africa. Upon arrival at ARC-AP in Irene, the harvested plant material was passed through a hammer mill and chopped to approximately 2–3 cm in length. The chopped material was then separated into four portions (each with a fresh mass of 2.5 kg). Three portions were shade-dried separately until constant mass was achieved, while the other portion was ensiled for 60 days to produce silage meal. During the drying, the forage materials were turned over every four hours during the day to reduce mold development. After drying, the plant materials were milled to pass through a 1 mm sieve, and they were subsequently used to determine dry matter (DM), crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF) and tannins. Determination of DM and CP followed a standard procedure of AOAC [52], while NDF and ADF were determined according to Van Soest et al. [53], and tannin was determined according to the procedure of Makkar [54].
The remaining fresh S. lancea fodder was used to produce silage meals by ensiling for 60 days. Before ensiling, the material was thoroughly hand-mixed with 5% molasses and compacted into 500 mL bottles. Bottles were closed with a glass lid and a rubber seal, which helped to reduce air ingress while allowing for the release of fermentation gas to reduce internal pressure. A total of six bottles were used to have enough material at opening (60 days post-ensiling). In each bottle, about 350 g of chopped S. lancea was compacted; thus, a total of 2.1 kg of fresh S. lancea material was ensiled. The ensiled material was stored at room temperature (±24 °C) in a dark place till opening at day 60 post-ensiling. At opening, the top 2 cm per bottle was discarded to reduce contamination. The contents of the whole bottles were emptied into a 5 L clean bucket and thoroughly hand-mixed. Two samples of 20 g each were taken and each mixed with 180 mL of distilled water, shaken vigorously for 2 min and stored in a fridge at 5 °C overnight [55]. The mixture was filtered the following day using Whatman No. 1 filter paper, and the filtrate was used to measure pH using a pH meter (Thermo Orion Model 525, Thermo Fisher Scientific, Waltham, MA, USA). The remaining silage was oven-dried at 60 °C until constant weight. The oven-dried silage samples were milled to pass through a 1 mm sieve and used for chemical composition analysis as indicated above for shade-dried materials. The rest was kept as silage meal to be used for an in vitro gas transducer technique.

4.3. Treatment Setup

The two S. lancea meals (leaf and silage) were further used as a supplement to E. curvula. Eragrostis grass was purchased from a local supplier; a sample of about 4 kg was oven-dried till constant mass, milled to pass through a 1 mm sieve and used as a substrate for the in vitro study. Eragrostis curvula (hereafter referred to as grass hay) was analyzed for DM, CP, NDF, ADF, and tannins as described in Section 4.2 above. The chemical compositions and tannin content of the grass hay, leaf meal, and silage meal are presented in Table 4.
Table 4. Chemical composition and tannin content of the grass hay, leaf meal and silage meal.
Searsia lancea silage at day 60 post-ensiling had a pH value of 4.2 and a dry matter content of 27.7%. However, the aerobic stability test was not conducted after opening. Grass hay was supplemented with S. lancea meal at two levels of inclusion each, and grass hay alone was used as a control. The treatments are presented in Table 5. The in vitro study was arranged in a completely randomized design comprising five treatments with four replicates per treatment. The inclusion of S. lancea meals was calculated as a percentage of the weight of grass hay on a dry-mass basis.
Table 5. Treatment arrangement of grass hay supplemented with S. lancea leaf or silage meal.

4.4. Buffer Solution Preparation and Collection of Rumen Fluid

The preparation of buffer, macro and micro minerals, resazurin and reducing solutions followed the procedure of Goering and van Soest [56]. However, the macro solution procedure was modified according to that of Mould et al. [57], using magnesium chloride instead of magnesium sulphate to reduce sulphate in the solution. After preparing the solutions separately, they were mixed (i.e., 1000 mL macro solution, 1000 mL buffer solution, 100 mL micro mineral solution, 100 mL resazurin solution and 96 mL reducing solution) and purged with carbon dioxide (CO2) gas to reduce oxygen and promote anaerobic conditions. The anaerobic condition of the buffer solution was indicated by the change from a strong pink color to a pale pink color. Two Holstein dry cows, about four years old (average body weight of 650 ± 5 kg) with permanent rumen cannulas, were used as rumen fluid donors. These animals were fed grass hay (E. curvula) ad libitum as basal hay which was supplemented by concentrate at 0.5% body weight daily. The concentrated diet with a CP of 14% was composed of hominy chops (42%), wheat bran (34%), soybean meal (17%), feed lime (1.5%), salt (0.5%), molasses meal (5%) and premix (1 pack per ton of feed). The animals had free access to clean and fresh water. Rumen fluid was collected by a trained animal health technician from two cows between 06:30 and 06:50, which was before morning feeding. The rumen fluid was strained through four layers of cheesecloth into a pre-warmed flask and taken to the laboratory within 20 min of collection, where it was purged with CO2 before mixing with buffer solution. After purging with CO2, 15 mL of rumen fluid and 25 mL of buffer solution were mixed, and the mixture was purged continuously with CO2 gas.

4.5. In Vitro Gas and Methane Production

The in vitro study was conducted following the procedure of Menke and Steingass [35]. Five forage mixtures of grass hay and S. lancea meals (leaf or silage) were weighed into serum bottles of about 125 mL each. In each bottle, there was 200 mg of grass hay alone or grass hay plus 15% or 30% of each of the S. lancea meals. Therefore, the control treatment had 200 mg of grass hay alone, whereas T2 and T4 had 170 mg of grass hay mixed with 30 mg of leaf meal or silage meal for 15% inclusion, while T3 and T5 had 140 mg of grass hay mixed with 60 mg of leaf meal or silage meal for 30% inclusion. In each bottle, 40 mL of buffered rumen fluid (i.e., 15 mL rumen fluid plus 25 mL buffer solution) was added, the bottle was flushed with CO2 and it was immediately closed as described by Mpanza and Hassen [58]. In addition, four serum bottles containing only buffered rumen fluid served as a negative control to correct for gas and methane production by rumen microbes in the absence of a substrate. The serum bottles were incubated in a continuous-shaking incubator (120 revolutions per minute) and were kept at 39 °C to mimic an animal’s body temperature. Total gas production was measured by gas pressure at 3, 6, 12, 24 and 48 h post-incubation, and incubation was terminated after 48 h. A semi-automated pressure transducer (PX 4200-015GI, Omega Engineering Inc. Laval, QC, Canada), which was attached to a digital data logger (Tracker 220 series indicators; Omega Engineering Inc. Laval, QC, Canada), was used to record gas pressure. More details on recording gas pressure are described in Akanmu et al. [59]. Total gas produced at each time of reading was added to estimate cumulative gas production for the incubation period (48 h).
Data on methane gas production were recorded by taking gas samples at 3, 6, 12, 24 and 48 h post-incubation. The collected gas samples (about 2 mL) were injected manually (pull-and-push methods) into a gas chromatograph (GC) equipped with a flame ionizer detector. The gas chromatograph column temperature was at 60 °C, injector temperature was 200 °C, detector temperature was 240 °C, and helium was used as the carrier gas at a flow rate of 10 mL/min. The GC model used in this study is the 8610C, SRI Instruments GmbH, Bad Honnef, Germany. Prior to using the GC as a methane gas analyzer, it was calibrated by injecting methane gas standards at 200, 500, and 1000 ppm. The methane gas percentage reading from the GC for 2 mL samples was corrected using the total gas volume recorded at different times, as indicated above, to determine the total volume of methane gas produced.

4.6. Volatile Fatty Acid Measurement

At the end of the incubation period (48 h), the serum bottles were removed from the incubator and placed in an ice-filled container to immediately stop the fermentation. After that, three serum bottles per treatment were centrifuged at 5000× g for 10 min and about 5 mL of supernatant was taken and used to determine volatile fatty acids (VFAs), which include acetic, propionic, butyric, iso-butyric, valeric, and isovaleric acids, following the procedure of Wang et al. [60]. To estimate the total VFAs, the six acids were summed using the technique of Wei et al. [36]. The non-glucogenic-to-glucogenic acid ratio (NGR) and fermentation efficiency (FE) were calculated from VFAs using the procedure of Wang et al. [37]. The two following equations were used for NGR and FE calculations, respectively:
NGR = (AA + 2 × BA + VA)/(PA + VA)
FE = (0.622 × AA + 1.092 × PA + 1.56 × BA)/(AA + PA + BA)
where AA, PA, BA, and VA represent acetic acid, propionic acid, butyric acid and valeric acid, respectively.

4.7. Statistical Analysis

The data were tested for normality using the Shapiro–Wilk test. Thereafter, data on gas production, methane production, volatile fatty acids, NGR and FE were subjected to one-way analysis of variance using the GLM procedure of SAS version 9.0 (SAS Institute Inc., Cary, NC, USA). In the case of a significant difference between the means, a post hoc analysis was conducted using the PDIFF statement. Significance was declared at p ≤ 0.05, and a trend was declared at 0.05 < p ≤ 0.10.

5. Conclusions

This study showed that S. lancea has the potential to be strategically used as a supplementary feed source for poor-quality grass hay. Searsia lancea leaf meal was more effective in improving the fermentation of poor-quality grass hay, while S. lancea silage meal showed a different pattern, and this indicates a need for further investigation, particularly on the phytochemical changes due to ensiling and nutrient bioavailability in silage meal. This study showed that S. lancea meals were effective in reducing methane production by decreasing butyric acid, a precursor of methane in the rumen. This could mean that S. lancea supplementation to grass hay inhibited the rumen metabolic function that favors the butyrate pathway. However, this study did not include rumen bacterial profiles to evaluate the effects of S. lancea meals on rumen microbial composition and their metabolic functions. Furthermore, an in vivo study is required to determine the effects of S. lancea meals on animal performance, including intake, growth, health, digestibility, and meat and carcass quality.

Author Contributions

Conceptualization, T.D.E.M., S.T.J. and K.R.M.; methodology, S.T.J., T.D.E.M., K.R.M. and M.J.M.; software, T.D.E.M., S.T.J. and F.L.M.; validation, T.D.E.M., S.T.J., K.R.M. and F.L.M.; formal analysis, M.J.M. and T.D.E.M.; investigation, M.J.M., T.D.E.M. and F.L.M.; resources, T.D.E.M. and F.L.M.; data curation, T.D.E.M., F.L.M. and S.T.J.; writing—original draft preparation, T.D.E.M. and M.J.M.; writing—review and editing, F.L.M., K.R.M. and S.T.J.; visualization, F.L.M., K.R.M. and S.T.J.; supervision, S.T.J., K.R.M. and T.D.E.M.; project administration, T.D.E.M. and F.L.M.; funding acquisition, T.D.E.M. and F.L.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the DEPARTMENT OF LAND REFORM AND RURAL DEVELPOMENT (DLRRD), South Africa, grant number API012501000001 (Agricultural Research Council-Animal Production).

Institutional Review Board Statement

This in vitro study protocol was approved by the University of South Africa (UNISA), and the College of Agriculture and Environmental Sciences-Animal Research Ethics Committee (Ref 2022/CAES_AREC/056) on 15 March 2022 and Agricultural Research Council-Animal Production (ARC-AP), Animals Ethics Committee (Ref APIEC 23/08) on 10 January 2024.

Data Availability Statement

Data may be made available to any interested individuals upon reasonable request, and authors will provide data at their convenience.

Acknowledgments

The first author would like to acknowledge the financial support from AGRISETA, which covered the student leaving allowance, and the bursary from the University of South Africa, which covered student registration fees.

Conflicts of Interest

The authors declare no conflicts of interest associated with this manuscript.

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