| Livestock Research for Rural Development 38 (3) 2026 | LRRD Search | LRRD Misssion | Guide for preparation of papers | LRRD Newsletter | Citation of this paper |
Phytogenic additives have been known to promote animal production performance by inhibiting the production of methane gas, hence the use of Parquetina nigrescens leaf meal as an additive. This study evaluated the potentials of Parquetina nigrescens leaf meal (PNLM) as phytogenic additive in cassava residue-based diet for West African Dwarf (WAD) sheep. The experiment was conducted to investigate the in vitro total gas, methane production and rumen fermentation parameters of diets containing PNLM. The experimental substrate for the experiment included cassava peels, cassava leaves, Megathyrsus maximus and a formulated concentrate diet which contained 0, 1, 2, 3, 4 and 5% DM PNLM to form six different treatments. Substrates were incubated for 48 hours and in vitro total gas, methane gas production and rumen fermentation parameters were evaluated. Data obtained were analyzed using one-way analysis of variance procedure (SPSS). Results showed that inclusion of 2 to 5% PNLM in the feed caused a reduction (p<0.05) in in vitro total gas (16.80 - 10.80 ml), methane (6.21 - 3.53%), ammonia nitrogen (20.14 - 15.15 mg/100ml) and in vitro dry matter degradation (66.37 - 54.14%). Increase in total volatile fatty acids (36.70 - 54.90 mM/ 100 ml) was observed from 3 - 5% PNLM inclusion. The result showed no significant (p>0.05) effect on the growth performance parameters. The study concluded that 3 - 5% of PNLM could be included as phytogenic additive in the diet of West African Dwarf sheep to reduce in vitro methane and ammonia production.
Keywords: ammonia-nitrogen, degradation, innoculum, Megathyrsus maximum, metabolite
In vitro fermentation techniques simulate microbial digestion, allowing researchers to assess feed degradation and nutrient availability (Mengwei et al 2025). Methane production in ruminants results in an energy loss of 2–12% of dietary intake (Yanza et al 2018). Various herbs and spices contain secondary metabolites that can influence rumen fermentation, leading to reduced methane emissions (Chaudhry and Khan, 2012; Pawar et al 2014). Plants contain secondary metabolites such as saponins, tannins, essential oils, organosulfur compounds, and flavonoids, which have significant biological activities (Palazon and Alcalde, 2025). These plant-based alternatives are considered natural and safe replacements for synthetic feed additives (Tsiplakou et al 2021). The use of phytogenic feed additives has consistently demonstrated methane reduction potential, albeit often accompanied by decreased feed degradation (Joch et al 2019). Analysis of P. nigrescens leaves has revealed the presence of alkaloids, saponins, tannins, oxalates, glycosides, and flavonoids (Oloruntola et al 2018). Various phytogenic substances and their combinations have demonstrated potential in reducing methane emissions from ruminants (Flachowsky and Lebzien, 2012). Tannins have demonstrated potential in decreasing methane emissions by suppressing methanogenic microbes (Bhatta and Behera, 2023). Total volatile fatty acid (TVFA) levels serve as key indicators of rumen fermentation efficiency and play a crucial role in energy metabolism in ruminants. Studies have reported that saponins lower ruminal ammonia nitrogen concentrations in both in vitro (Jayanegara et al 2020) and in vivo (Gunun et al 2019; Liu et al 2019) conditions. This study therefore aimed to evaluate the in vitro gas production and rumen fermentation of West African dwarf sheep fed varying inclusion levels of Parquetina nigrescensleaf meal as phytogenic feed additive.
The study was conducted at the analytical laboratory of Animal nutrition department in the College of Animal Science and Livestock Production at Federal University of Agriculture, Abeokuta, Ogun State, Nigeria. The experimental site is located in the South-Western area of Nigeria and it falls within Latitude 7.24°N and Longitude 3.34°E (Google Earth, 2026).
The experimental feed substrate used for in vitro studies consisted of 50% cassava peels, 20% cassava leaves, 20% Megathyrsus maximus and 10% concentrate on dry matter basis as shown in Table 2. Parquetina nigrescens leaf meal was included in the substrate at varying levels of 0, 1, 2, 3, 4 and 5% shown in Table 1. Concentrate diet was compounded using 50% wheat offal, 27% corn bran, 20% soya bean meal, 2% bonemeal and 1% salt, which was sourced from a nearby feed mill.
The in vitro gas production technique of Menke and Steingass (1988) was followed for the measurement of total gas production. Rumen liquor was collected from the rumen of cattle immediately after slaughter into a warm, insulated flask and taken to the laboratory. The rumen fluid collected was strained through four-layered muslin cloth. A medium containing a micro element solution, buffer solution, macro element solution, resazurin and reduction solution was prepared. The prepared medium and rumen fluid was mixed in ratio of 2:1 (v/v), respectively to obtain the inoculum for digestion. The mixture was handled under continuous flow of carbon-dioxide. Approximately 200 mg (n = 8 per treatment) of the substrate of each of the six treatments were weighed into well-labelled dacron bags of known weights and inserted into 100 ml calibrated glass syringes. Each of the syringes were filled with 30 ml of the prepared medium. In order to expel air bubbles completely from the syringes, each syringe was tapped with the finger to move air bubbles to the top, pushed upward by the piston, and expelled. Silicon tube attached to the tip of the syringes were tightened by a metal clip so as to prevent escape of fermentation gas. Incubation was carried out at 39oC and the volume of gas produced was measured and recorded at three hours (h) interval from 0 to 48 hours. Three blanks containing 30 ml of innoculum only was included in the run. To correct for gas production not arising from substrate fermentation, the average of the volume of gas produced from the blanks were deducted from the volume of gas produced per sample.
The volume of methane gas produced from fermentation of substrate from each treatment group (n = 4 per treatment) was determined after the incubation of syringes at 48 hours and after the final gas volume had been recorded. This was done by dispensing 4ml of 10 M sodium hydroxide into the syringes through the fitted silicon tube. The sodium hydroxide was introduced into the 100 ml syringe with the aid of a 5 ml syringe and needle, avoiding the escape of gas. The content of the 100 ml syringe was mixed with sodium hydroxide to allow the absorption of carbon-dioxide. The gas volume remaining in the syringe was then read and considered as methane volume according to Fievez et al (2005).
After 48 hours of incubation, inoculum from syringes were recovered. About 10 ml sample of the inoculum was taken for the determination of ammonia nitrogen using the micro-kjeldahl method (A. O. A. C. 2002).
Samples of rumen fluid after 48 h in vitro gas production trial was collected per treatment (n=4) centrifuged at 10,000 × g for 15 minutes and supernatant collected. The total volatile fatty acid (TVFA) in rumen fluid was determined by the method of Barnett and Reid (1957) with slight modification as described by Roy et al (2014). The volume of total volatile fatty acid in sample was then calculated using standard titration equation.
Where N is the normality of the sodium hydroxide, V is the volume of sodium hydroxide used in titration and W is the volume of rumen fluid used.
The in vitro dry matter degradability (IVDMD) was then calculated using the equation below:
The substrates were analyzed (in DM %) for ash content, crude protein, ether extract, crude fibre and fibre components (NDF and ADF using near infra-red spectroscopy (NIRS) equipped with globally calibrated equations developed by International Livestock Research Institute (ILRI) from conventional analysis of proximate chemical fractions (AOAC, 1990 and Van Soest et al1991). The cyanide content in cassava peels and leaves were determined using the picrate method as described by Nwokoro et al (2009). The tannin content in Parquetina leaf meal was determined using the Folin Ciocalteu method according to Makkar (2003). Total phenol was determined using the Folin Ciocalteu reagent method following Do (2014). Flavonoid was determined using aluminum chloride colourimetric method as described by Nasseri et al(2019). Oxalate was determined by the permanganate titration method as described by Mishra (2017). Saponin was determined as described by Mir et al(2016) while alkaloid was determined by a gravimetric method as described by Adeniyi (2009).
During a feeding trial of 90 days, experimental animals were fed at 5% body weight on dry matter basis with clean water offered ad-libitum. Animals in each dietary treatment group were fed in the morning at 08:00 h. Quantities of feed offered and feed refused for each animal was recorded daily to compute feed intake. Initial body weight of the animals were taken at the commencement of the trial and thereafter on a weekly basis to compute weekly weight changes.
The feed conversion ratio (FCR) of animals were estimated by dividing the quantity of feed dry matter intake (g/day) by weight gained by the animal (kg).
Data obtained were subjected to a one-way analysis of variance procedure (SPSS, 2007). The level of significance was tested at 5% probability. Where significant differences occur between means, Duncan's multiple range test (SPSS, 2007) was used to separate the means.
The statistical model is shown below.
Yij= µ+Ti+Ɛij
Where,Yij is the observation, μ is the population mean, Ti is the effect of treatments, Ɛij is the residual error.
The chemical composition of basal diet used in this study is presented in Table 1. The diet contained 12.19% crude protein, 21.80% crude fibre and 8.96Mj/kg DM metabolizable energy This composition indicates a feed that can support maintenance and growth in sheep according to NRC, (2007).
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Table 1. Chemical composition of basal diet used for the experiment |
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Parameters (%) |
Concentration |
|
|
Crude protein (%) |
12.19 |
|
|
Crude fibre (%) |
21.80 |
|
|
Metabolizable energy (MJ/kg DM) |
8.96 |
|
The result of the phytochemical composition of Parquetina nigrescens leaf meal is shown in Table 2. The result showed that PNLM contained tannin (226.23 mg/100g), total phenol (169.38 mg/100g), flavonoids (339.06 mg/100g), phytate (21.45 mg/100g), oxalate (46.24 mg/100g), alkaloids (4.1%) and saponin (3.11%). The Parquetina nigrescens leaf meal used in this study was found to contain alkaloids, saponins, tannins, oxalates, phytates, total phenols, and flavonoids, which corroborates the report by Oloruntola et al (2018). The result of this study aligned with the study of Patra and Saxena (2011) who showed that alkaloids like quinolizidine and pyrrolizidine may inhibit specific microbial groups that affects fiber degradation and volatile fatty acid production.
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Table 2. Phytochemical composition of air-dried Parquetina nigrescens leaf meal |
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Phytochemical content |
Concentration (mg/100 g) |
|
|
Tannin |
226.23 |
|
|
Total phenol |
169.38 |
|
|
Flavonoids |
339.06 |
|
|
Phytate |
21.45 |
|
|
Oxalate |
46.24 |
|
|
Alkaloids |
4.10% |
|
|
Saponin |
3.11% |
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The study recorded a reduction in methane production with decreased feed degradation at varying inclusion of Parquetina nigrescens agreed with a reduction in methane production with decreased feed degradation with the use of phytogenic feed additives reported by Joch et al (2019). The reduction in methane recorded in this study maybe due to the presence of tannins which have demonstrated potential in decreasing methane emissions by suppressing methanogenic microbes (Bhatta and Behera, 2023). Tannin suppressing the methanogenic microbes will thereby alter the microbial population in the rumen and will enhance TVFAs concentration corroborating the research conducted by Patra and Saxena (2009) which revealed that phytogenic additives enhance TVFAs concentrations by altering the microbial population in the rumen. For instance, cumin and coriander, known for their high terpenoid and phenolic content, have been found to elevate propionate production, consequently increasing TVFAs levels (Elghandour et al 2018). The presence of saponin in P. nigrescensleaf meal is consistent with the observations of Wang et al (2019), who reported that dietary saponins elevate TVFAs by improving microbial cellulolytic activity. Fadiyimu et al (2017) reported changes in ammonia nitrogen concentration and volatile fatty acid production following the inclusion of Parquetina nigrescens leaf meal, further supporting these observations. The reduction in methane production and ammonia nitrogen concentration with increasing levels of P. nigrescens supplementation, aligns with the findings of Adebayo et al(2023) when Morinda lucidaBenth leaf powder was used at varying inclusion levels (0, 2, 4, 6, 8, and 10 mg/g DM) that showed a decrease in these parameters at higher inclusion rates (8 and 10 mg/g DM).
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Table 3. Effect of diets containing varying inclusion levels of Parquetina nigrescens on in vitro gas and rumen fermentation parameters |
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Parameters |
Dietary inclusion level of Parquetina leaf meal |
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|
0% |
1% |
2% |
3% |
4% |
5% |
SEM | p value | ||||
|
48th hours incubation |
16.80a |
15.80a |
13.00ab |
13.00ab |
11.20b |
10.80b |
0.991 |
0.021 |
|||
|
Methane (ml/200 mg DM) |
6.21a |
5.88a |
4.67b |
4.51bc |
4.10c |
3.53d |
0.236 |
0.000 |
|||
|
Ammonia nitrogen concentration (mg/100 ml) |
20.14a |
18.61ab |
16.73b |
16.08bc |
15.27c |
15.15c |
1.050 |
0.017 |
|||
|
Total volatile fatty acids concentration (mM/100 ml) |
36.70c |
34.50c |
47.8bc |
51.6ab |
53.0a |
54.9a |
4.030 |
0.003 |
|||
|
IVDMD (%) |
66.37a |
65.18a |
64.39a |
60.22b |
55.26c |
54.14c |
0.943 |
0.000 |
|||
|
a, b, c Means along the same row with different superscripts are significantly different (p < 0.05) SEM: Standard error of means, OMD: Organic matter digestibility, ME: Metabolizable energy, NE: Net energy, IVDMD: In vitro dry matter degradation. |
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 |
| Figure 1. Methane gas production of West African dwarf sheep fed varying levels of Parquetina nigrescens leaf meal |
Dry matter intake (DMI) and overall nutrient intake remained unchanged across all treatments. In contrast, studies on other leafy plants like Leucaena leucocephala and Moringa oleifera have reported improved nutrient digestibility and intake, leading to better growth performance in sheep (Salem et al 2018. This suggests that P. nigrescens may contain lower levels of essential nutrients or bioactive compounds required for such improvements. Regarding feed conversion ratio (FCR), no statistical differences were observed among treatments, contrasting with the findings of Greathead (2003), who reported improved FCR in sheep and goats fed phytogenic additives such as cumin and coriander. This discrepancy may indicate that P. nigrescens lacks the necessary bioactive properties or is present in insufficient quantities to significantly enhance feed conversion.
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Table 4. Growth performance of West African dwarf sheep fed diets containing varying levels of Parquetina nigrescens leaf meal |
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Parameters |
Dietary inclusion level of Parquetina leaf meal |
SEM |
p - value |
||||||||
|
0% |
1% |
2% |
3% |
4% |
5% |
||||||
|
Average initial weight (kg) |
12.99 |
12.74 |
12.76 |
13.11 |
12.78 |
13.20 |
1.47 |
0.63 |
|||
|
Average final weight (kg) |
15.70 |
15.54 |
15.51 |
16.06 |
15.68 |
16.23 |
1.62 |
0.25 |
|||
|
Total weight gain (kg) |
2.71 |
2.80 |
2.75 |
2.95 |
2.90 |
3.03 |
0.14 |
0.49 |
|||
|
Average daily weight gain (g) |
30.14 |
31.25 |
30.50 |
32.80 |
32.18 |
33.63 |
0.46 |
0.09 |
|||
|
Total dry matter consumed (kg) |
63.14 |
62.87 |
63.21 |
64.36 |
62.71 |
64.09 |
0.65 |
0.19 |
|||
|
Average daily dry matter intake (g/day) |
701.61 |
698.55 |
702.36 |
715.13 |
696.76 |
712.14 |
2.17 |
0.31 |
|||
|
Feed conversion ratio |
23.28 |
22.35 |
23.03 |
21.80 |
21.65 |
21.03 |
0.38 |
0.172 |
|||
|
a, b, c means along the same row with different superscripts are significantly different (p< 0.05), SEM: Standard error of mean |
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The phytochemical profile of Parquetina nigrescens leaf meal showed that it contained most of the phytochemicals that can influence rumen fermentation. The result of this study showed that the varying inclusions of P. nigrescens leaf meal reduced in vitro gas produced which suggested an inhibitory effect on rumen fermentation. The result of the in vitro study showed that at 4% and 5% inclusion of P. nigrescens leaf meal, in vitro dry matter digestibility reduced which was a reflection of antimicrobial activity. Based on the result of this study, inclusion of P. nigrescensleaf meal from 3% to 5% could be used in the diet of WAD sheep for effective reduction of methane and ammonia degradation in the rumen.
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