In vitro degradability of diets containing varying levels of Gliricidia sepium and cassava leaf meals

O O Adelusi, O J Idowu¹, K O Adebayo, M T Balogun and A O Oni

Department of Animal Nutrition, Federal University of Agriculture, P M B 2240, Abeokuta, Nigeria
¹ Department of Pasture and Range Management, Federal University of Agriculture, Abeokuta, Nigeria
adelusioo@funaab.edu.ng

Abstract

The present study evaluated the in vitro degradability of diets containing varying levels of Gliricidia sepium (G) and cassava (C) leaf meals. The treatments levels of the leaf meal inclusions were GC0, GC20, GC35 and GC40. The experiment was set up in a completely randomized design with four (4) treatments and six (6) replicates. Data was collected on chemical composition of diets, gas production recorded at 3, 6, 9, 12, 15, 18 and 24 hours, and methane gas was estimated at the end of the 24th hour of incubation. Volatile fatty acid (VFA) profile of the incubation fluid was done and dry matter digestibility of the substrate residue was carried out. Data obtained were analysed using one way analysis of variance. The results showed that crude protein content of the diets increased (p<0.05) with increase in the inclusion level of leaf meals with the highest value obtained at GC40 (14.7%). Total gas production, methane and dry matter digestibility decreased (p<0.05) with increase in the level of leaf meal inclusion. However, total VFA increased with the inclusion of leaf meals, and the highest was obtained at GC35 with 4.64 mmol/dl. From this study, it can be concluded that inclusion of Gliricidia sepium and cassava leaf meals at 20% or more in the diet has the potential to modify rumen by reducing total gas and methane production during fermentation which will result in higher post-ruminal nutrient utilization.

Keywords: rumen, methane, gas production, volatile fatty acids, chemical composition

Introduction

Ruminant livestock raised in tropics tend to exhibit cyclical variation in the quantity and quality of the available forages (Bamikole and Babayemi 2006). This variation is a continuous effect of climate change which is having a damaging impact on smallholder livestock production. Particularly during the dry season, ruminant feeding systems depend on poor quality tropical foliage, resulting in poor nutrition, low production performance, slow growth rate, loss of body condition and increased susceptibility to diseases and parasites. These poor-quality forages are usually augmented with crop residues or agro-industrial by-products (Oni et al 2012) and a number of browse plants worldwide serve as alternative feedstuffs for livestock (Rinehart 2008; Fayemi et al 2011). As a result, animals under semi-intensive and free-range systems have been observed feeding on them (Isah et al 2007).

Proteinaceous legumes like Gliricidia sepium are abundantly found in the tropics and considered as high-quality leguminous forage due to its protein content and moderate fibre fractions (Heuzé and Tran, 2015) when compared with grass foliage. Also, its content of plant secondary metabolites makes it fitting into dietary strategies for mitigating enteric methane targets modifying rumen fermentation (Bhatta 2015). Cassava leaf has an average crude protein content of 24.9% (Wanapat et al 1997), most of which can be regarded as true protein due to the presence of tannins in the leaves which forms tannin-protein complex that by-pass the rumen fermentation making the nutrient available for post-ruminal digestion and absorption. Cassava foliage has been fed to goats (Ukanwoko and Ibeawuchi 2014) and also used with brewers dried grain for cattle fattening (Phanthavong et al 2016). The in vitro method of feed evaluation for ruminants has gained wider acceptance due to its ease of adoption, repeatability, minimized use of animals and the decrease in funding requirement (Getachew et al 2005). The objective of this study was to evaluate the chemical composition, in vitro degradability and methane production of concentrate diets containing Gliricidia sepium and cassava leaf meals.

Materials and methods

Location

The experiment was carried out at the Animal Nutrition Laboratory, Federal University of Agriculture, Abeokuta, Ogun State. The site is located in the rain forest vegetation zone of South-Western Nigeria on Latitude 7 ^o 13ʹ 56.89ʺ N, longitude 3^o 26ʹ 13.47ʺ E and altitude 76m above the sea level (Google Earth, 2021).

Preparation of test materials

The leaves of Gliricidia sepium were collected from botanical gardens within the University campus environment through random and manual harvest from different parts of the tree branches to obtain both young and mature leaves. Cassava leaves were harvested from mature plantation that is ready for harvest. The leaves were air-dried for 7 days, ground in a mill to pass through 1mm sieve, and then used for formulation of feed with other ingredients.

Experimental diets and design

The experimental design was a completely randomized design (CRD) with four concentrate diets formulated to contain Gliricidia sepium and cassava leaf meals at 0% (GC0), 10% each (GC20), 20 and 15% (GC35) and 25 and 15% (GC40) respectively. The other ingredients in formulation are cassava peel, palm kernel cake, rice bran, wheat offal, salt, oyster shell and sulphur (see Table 1).

Incubation procedure

Rumen fluid was collected using the method of collection described by Babayemi et al (2006). Rumen liquor was collected from cattle into warm insulated flasks and used as the source of rumen fluid. The in vitro gas production technique employed was that described by Menke and Steingass (1988). Rumen liquor was collected from cattle into warm insulated flasks, filtered through layers of cheesecloth and used as the source of inoculum. The inoculum was then mixed with sodium and ammonium bicarbonate buffer (35g NaHCO₃ plus 4g NH₄ HCO₃ per litre) at a ratio of 1:2 (v/v) to prevent lowering the pH of the rumen fluid which could result in decreased microbial activity. A 200mg sample of diet substrate weighed into a fibre bag was replicated six times (n=6) for each treatment and were placed into 60ml calibrated syringes. Twenty millilitre of the buffered inoculum was then added to each syringe containing the ground samples and were then positioned in an incubator kept at 39°C. Blank syringes containing 20ml of the buffered inoculums only was included as control. Gas volume was recorded following; 0, 4, 8, 12, 16, 20, 24 and 48 hours of incubation. The syringes were shaken at regular intervals during the gas reading to simulate rumen motility.

Measurement of total and methane gas production, and dry matter degradation

Gas production was recorded for 24 hours of incubation at 3-hour intervals. The volume of methane gas from each sample was determined by dispensing 4mls of 0.4 sodium hydroxide (NaOH) in to the sample at the end of 24 hours of incubation (Fievez et al 2005). Substrate degraded (mg/200 mg) was determined by subtracting the un-degraded residue for each sample from the initial quantity incubated. The residue was recovered by filtering and drying at 105℃ for 12 h.

Analysis of volatile fatty acid concentrations and chemical composition of feed

Total volatile fatty acids concentration of incubation fluid was determined by titration of sample with 0.1N NaOH solution and expressed as volatile fatty acid content. The procedure was a modified protocol that replaces the conventional titration with the potentiometric titration system. The concentration of NaOH solution was matched with volatile fatty acid content in samples for all the samples (Siedlecka et al, 2008).

Chemical composition of feed was carried out using Near Infra-Red Spectroscopy (NIRS). Samples were analysed (in DM %) for ash content, crude protein, 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, 2000; Van Soest et al 1991). The NIRS instrument to be used is FOSS Forage Analyzer 2500 with software package WinISI II.

Statistical analysis

Data obtained from this experiment were analysed using one-way analysis of variance option of the SPSS (IBM SPSS Statistics 23) software (IBM, 2015). Treatment means were statistically compared using Duncan’s Multiple Range Test to identify differences between means and significant differences were declared if p<0.05.

Results

Table 2 shows the effect of inclusion at various levels of the leaf meals on chemical composition of formulated diets. Crude protein (CP) content increased with highest(P<0.05) value obtained with GC40 (14.7%) with the lowest at GC0 (11.3%). Higher (P<0.05) dry matter (DM) and neutral detergent fibre (NDF) values were obtained at G0C0 (91.2% and 49.9%). The highest (P<0.05) acid detergent fibre (ADF) was recorded for GC20 (35.5%)

The inclusion of the leaf meals affected the in vitro gas production values as seen in Table 3. Higher (p=0.04) gas volume was recorded for treatment containing GC0 (20.7ml) after 24 hours of incubation. The regression result in Figure 1 where R² = 0.94 showed that 94% of the gas production trend can be said to have resulted from the treatment variations applied.

Figure 1. Relationship between gas production and levels of leaf meals inclusion in diets

Methane production, % methane in total gas and dry matter digestibility (DMD) are presented in Table 4. Methane production reduced (p =0.01) with increase in the inclusion level of leaf meals with the lowest methane (4.00 ml) and % methane (24.8%) observed for diet containing GC35. Similarly, DMD was higher in the control treatment with 60.6%, while diets containing leaf meals were not different. In Figure 2, the negative value of the coefficient (-0.145) in the regression analysis shows that the increasing inclusion of leaf meals is expected to decrease the percentage of methane in the total gas produced.

Figure 2. Relationship between methane and levels of leaf meal inclusion in diets

The volatile fatty acid (VFA) values presented in Table 5 showed that inclusion of leaf meals increased (P<0.05) total rumen VFA and acetate. The highest (P<0.05) total VFA (4.52 mmol/dl) was obtained with feeding GC40 while the highest (P<0.05) acetate was obtained with feeding GC35 (3.18 mmol/dl). Propionate and butyrate values were not affected. Additionally, Figure 3 showed an increasing linear regression graph with R²=0.72 with a positive correlation value of +0.022.

Figure 3. Relationship between volatile fatty acid and levels of leaf meal inclusion in diets

Discussion

Dry matter (DM) values obtained in this study are similar to the range of 85.70 to 95.86 % reported by Asaolu et al (2012) for fodder, with the lowest obtained with the highest inclusion level of the leaf meals. The implication of high DM is that more nutrients will be available for rumen fermentation when the diets are consumed by ruminant animals. The CP content of the diets ranged between 10.9-14.71%, similar to the observation of Zain et al (2020) which reported an increase CP as the inclusion of Gliricidia sepium and cassava leaves increased. The CP level is above the minimum level for maintenance of 7.70 % for goats (NRC, 1981), with the highest obtained at GC40 (14.71%), and in agreement with the minimum of 8% CP observed by Norton (1994) to provide the ammonia levels required by rumen microbes for optimum activity. This result of this study is in agreement with report given by Gillespie (1998) that NDF is inversely related to the plants’ digestibility. The higher the NDF the lower the plant’s digestibility and vice versa. The values obtained for the various levels of leaf meal inclusion may imply a moderately high cell wall content.

The inclusion of Gliricidia and cassava leaf meals at various levels affected total gas and methane production. The observed lowered gas production could be adduced to rumen escape of nutrients facilitated by tannin presence in the diet. This is supported by the hypothesis of Preston et al (2021) which noted that the tannins present in the leaves of cassava combine with protein (and other soluble nutrients) making them resistant to fermentation in the rumen. The observed changes in methane production can be attributed to the tannin and saponin contents inGliricidia sepium, similar to the report of Jayanegara et al (2011) and Zain et al. (2020). The tannin level of Gliricidia leaves have been previously reported as high in various studies (Ahn et al 1997 – 19.9 g/kg; Heuzé and Tran, 2015 - 28.3 g/kg; Molina-Botero et al 2019 – 45.9 mg/g) along with high condensed tannin in cassava leaves (20.2 g/kg), reported by Heuzé and Tran (2016). The decrease in in vitro CH₄ production of plants containing condensed tannins which are often browsed by ruminants was similarly reported by Sebata et al (2011). Lower NDF values as obtained with diets containing leaf meals have also been reported to result in lower methane output. Gaviria-Uribe et al (2022) noted that the presence of soluble carbohydrates such as free sugars and starch result in lower methane production than highly structural carbohydrates because they are fermented more slowly.

Reductions in dry matter digestibility as the level of leaf meals inclusion increased could be attributed to the concomitant increase in lignin content of the diets. Ojo et al (2017) reported that although ruminants can digest the cellulose and hemicellulose components of fibre, however the lignin impedes the rate and degree of digestion especially when the proportion of lignin in fibre begins to increase. Dry matter degradation is related to fibre breakdown in the rumen, and the observation of Aderinboye et al (2020) noted that plants containing secondary metabolites with capacity to reduce methane emission has been attributed with inhibition of fibre degradation

In agreement with current findings, studies done on different tropical browses have showed substantial effects of plants containing phenolic compounds on the fermentation rate and level of diges­tion (Bhatta et al 2009; Sebata et al 2011). The effect of tannins on fermentation and digestion could be related to the formation of tannin-carbohydrate and tannin-protein complexes that are less degradable or to its toxicity to rumen microbes (Adelusi et al 2016). More­over, it acted as a toxicant to methanogens, reduced ac­etate and butyrate production (i. e. reduced fibre degrada­tion) or cause a decline in organic matter (OM) digestion (Patra and Saxena, 2011)

Conclusion and Recommendations

From the results of this study, it can be concluded that the chemical composition of diets was improved by inclusion of Gliricidia sepium and cassava leaf meals. It was also noted that gas production reduced as the inclusion increased. It is recommended that Gliricidia sepium and cassava leaf meals can be included in concentrate diets for ruminants at levels above 20% and up 40% of the total feed. Further studies are therefore recommended to investigate the response of animals in a feeding trial to these diets.

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