Supplementary effects of Noug Seed (Guizotia abyssinica) Cake with Sesbania (Sesbania sesban) Leaves on Feed Intake, Digestibility and Enteric Methane Emission in Arsi-Bale Sheep

Study Site

The experiment was conducted in the College of Agricultural and Environmental Science, Arsi University, located 3 km south of Asella town in the Arsi Administrative Zone of Oromia National Regional State. Assela is located at 175km southeast of Addis Ababa at 7057”N latitude and 3908” E longitude and has an altitude of 2400 m above sea level. The site has a bimodal rainfall pattern with a mean annual precipitation of about 725 mm. The main rainy season extends from June to September with a maximum rainfall in August, while the short rainy season is between Februarys to April. The mean minimum and maximum temperatures of the experimental site were 8.28°C and 23.3°C, respectively.

Experimental Diets, Treatments, and Design

The natural grass hay was harvested at about 50% flowering stage manually and sun-dried. The dried hay was piled and stored as loose hay under shade. The hay was chopped to a size of approximately 5-6 cm to facilitate intake. The S. sesban leaves meal was collected and prepared from the trees grown in the university area. Green leaves of the plant were harvested by cutting with a sickle manually from the branches. The harvested leaves were subjected to air-drying under shed separately for three to four days and turned up four times a day to ensure uniform drying and maintain green color. Noug seed (Guizotia abyssinica) cake (NSC) is purchased from Adama oil processing factory. The experimental diets were formulated according to the nutritional requirements and recommendations of the National Research Council NRC [7] to satisfy the maintenance requirements of adult sheep of 26.7 (±0. 14) kg body weight. The proportion of the dried S. sesban leaves’ meal and concentrates are calculated based on the CP contents to make supplements iso-nitrogenous in all treatments. The experimental diets were mixed in a uniform way to avoid the selection of feed intake of the experimental animals. The three treatments were Treatment 1: Hay + S. sesban leaves alone (T1) Treatment 2: mixed ration (27.6% hay +27.6% S. sesban leaves + 44.8% NSC) (T2) and Treatment 3: mixed ration (38.6% hay + 61.4% NSC) (T3)

Nine sheep were fed individually during the experimental period. The experimental feeds were offered twice a day in two equal portions at 08:00 and 16:00 hours. There was an adaptation period of 15 days to the experimental feeds before the commencement of data collection. Water was given ad libitum. Feed offered and refused was measured daily using a 5 kg sensitive balance with one gram precision, and the difference between the daily total feed offered and the daily refused was considered as daily feed intake on DM basis. Experimental lambs were housed in individual pens (0.70 m × 1.70 m) with concrete floors, a feeding trough, and a watering bucket. The pens were disinfected before moving animals in and then cleaned daily. The composition and nutrition levels of the three diets based on the 7 were shown above. The three diets were fed according to the 3 × 3 cross-over design over 90 days in three periods, each 30 days, including 14 days of pre-feeding and 15 days for the collection period.

Digestibility Trial

To determine the digestibility of the experimental diets, the sheep were fitted with fecal collection bags(harness) for at least five days of the adaptation period followed by a 7 days feces collection period; during which time (April-June/2022) daily feed intake of each animal was recorded. Samples of feed offered, feed refused and feces were collected every day in the morning. Total feces voided in the harness were weighed daily during the collection period. After the collected feces from each animal were mixed thoroughly, 20% representative samples were taken daily and kept in deep freezer at -200C. At the end of the collection period, each sample from each animal was thoroughly mixed and enough samples were taken and dried at 600C in a forced air oven for 72 hours to a constant weight. The apparent digestibility of DM, OM, CP, NDF, and ADF was determined using the following formula.

$$\text{Apparent DM digestibility} = \frac{DMI - Faecal DM output}{DMI} \times 100$$

$$\text{Apparent digestibility of nutrient (%)} = \frac{\text{Nutrient intake} - \text{Faecal nutrient excreted}}{\text{Nutrient intake}} \times 100$$

Laboratory Analysis of Feeds and Feces

Sample of feeds offered and refusals as well as the partially dried feces were ground to pass through a 1mm sieve. The DM, OM, CP, and ash contents were determined according to AOAC [8]. Neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid detergent lignin (ADL) were analyzed according to the procedure of Van Soest PJ [9]. The metabolizable energy (ME) concentration (MJ/kg DM) of the rations was estimated using the equation of Mcdonald P [10] based on digestible organic matter (OM) in DM (DOMD) as:

$$\text{DOMD} = \frac{(\text{OM intake} \times \text{kg/d}) - (\text{OM in feces} \times \text{kg/d})}{\text{DM intake} \times \text{kg/d}}$$

$$\text{ME} \left( \text{MJ/kg DM} \right) = 0.016 \times \text{DOMD} \left( \text{g/kg DM} \right)$$

Estimating Enteric Methane Emission Factor (EF)

A precise estimate of enteric methane emission (EME) would be necessary for accurate preparation of national GHG inventory and assessment of costs and benefits of GHG mitigation from sheep. Development of EME prediction models could precisely estimate methane emissions from sheep.

When GE intake (MJ/day) was not reported in the published papers, it was estimated from DM intake, and GE concentration (MJ/kg DM) were calculated from chemical composition of diets Jentsch W, et al. [11], as follows:

$$\text{GE intake} \left( \text{MJ/day} \right) = \text{DM intake} \left( \text{kg/day} \right) \times \left[ \frac{[23.6 \times \text{CP} \left( \text{g/kg} \right) + 39.8 \times \text{EE} \left( \text{g/kg} \right) + (17.3 \times \text{NFC} \left( \text{g/kg} \right) + 18.9 \times \text{NDF} \left( \text{g/kg} \right) ]}{1000} \right]$$

Emission factors for enteric fermentation for each animal sub-category were calculated based on gross energy intake and the estimated methane conversion factor [12, 13]. The sub-category enteric methane emission factors were then calculated using the following equation:

$$\text{EF} = \left[ \frac{\text{GE} \times \left( \frac{\text{Ym}}{100} \right) \times 365}{55.65} \right]$$

Where:

• EF = Emission factor, kg CH4 head -1 year -1
• GE = gross energy intake, MJ head -1 day -1
• Ym = methane conversion factor which is the percentage of gross energy in feed converted to methane, %
• 55.65 = the energy content of methane, MJ kg -1 CH4

Statistical Analysis

Data on feed intake, digestibility, BW change, gross energy intake, methane emission factor, and methane yield were subjected to ANOVA using Statistical Package for Social Sciences (IBM SPSS) software for window, Version 22.0 14. Whereas quantitative variables were analyzed using analysis of variance procedures and when the F-test showed significant differences, Tukey HSD at 5% significance level was used for comparison of means [14].

Chemical Composition of the Treatment Feeds

The chemical composition of the experimental diets is presented in Table 1. The DM content was low in S. sesban but high in natural grass hay and noug seed cake. Organic matter content was high in S. sesban and low in natural grass hay and noug seed cake. The CP content was high in S. sesban and noug seed cake but low in natural grass hay. The NDF, ADF, and cellulose content high in S. sesban leaves and natural grass hay but low in noug seed cake. In contrary, ADL and ash content high in noug seed cake and low in S. sesban leaves and natural grass hay. Semi- cellulose was higher than in natural grass hay noug seed cake and S. sesban leaves.

Natural grass hay has been used as vital feed for livestock in the tropics. In this study, the CP content of the natural grass hay was slightly below the maintenance requirement (7%) for ruminants that required for microbial protein synthesis in the rumen which is in line with the result of Daniel T, et al. [15], Melesse A, et al. [16] and higher than the CP content reported by Melesse A, et al. [16]. Getahun K. et al. [17]. In generally, the nutrient compositions of the experimental feeds were within the range of Ethiopian feeds. The CP content of S. sesban leaves recorded in this study was lower than the reported by Solomon M, et al. [18] for the S. sesban 15019 accessions, Mekoya A, et al. [19], Wondwosen B, et al. [20] Solomon G, et al. [21]. The OM, NDF, ADF, and ADL obtained in this study were higher than the values reported for S. sesban leaves [18, 19, 20]. The OM and content of S. sesban leave in this study were similar reported by Solomon G, et al. [21] but the NDF and ADF content of S.sesban lower than reported by the same authors.

The CP content of NSC in the present study was lower than the report by Abebaw N, et al. [22], Fentie B, et al. [23], Zinash, et al. [24] but higher than the report by Jemberu D, et al. [25]. NDF, ADF, ADL and cellulose content of NSC in the present study were lower than the report by Abebaw N, et al. [22], Jemberu D, et al. [25] but higher for the content of OM and hemicellulose and lower for the content of DM by the same authors.

DM=dry matter; OM= organic matter; CP=crude protein; NDF= neutral detergent fiber; ADF = acid detergent fiber; ADL= acid detergent lignin; HC=Hemicellulose; SSL=S. sesban leaves, NSC=Noug seed cake Table 1: Chemical composition of ingredients and experimental feeds.

DM and Nutrient Intake

Total daily DM intake was high significant (P<0.001) difference between T1 and T2 and also T3 (Table 2). Similarly, there is high significant (P<0.001) difference between T2 and T3. This is may be because of Anti-nutritional compounds found in the S. sesban leaves. The intake of DM as percent of BW was significantly (P<0.001) difference between T2 and T3 sheep compared to T1. The intake of OM, NDF, ADF, and ME were significantly (P<0.001) difference between T2 and T3 compared to T1 sheep. And also, the intake of CP was significantly (P<0.05) difference between T2 and T3 as compared to T1.

Total daily DM intake were significantly (p<0.001) different between sheep in mixed treatments than for the sole S.sesban leaves (Table 2). The low intake in sole S. sesban leaves may be due to the fact that S. sesban contains a substantial amount of anti-nutritional factors such as condensed tannins might have limited complete consumption of the same feed at a higher level of inclusion. Similarly, with the current finding, Solomon M, et al. [26], Solomon M, et al. [27], Solomon M, et al. [28] stated that variable quantities of refusals from feeding S. sesban to ewes and concluded that anti-nutritional factors in the same feed limit palatability and intake at a higher level of offer. It has been documented by Frutos P, et al. [29], that most browse species including S. sesban contain phenolic compounds that reduce voluntary feed intake and tannins were considered as anti-nutritive and/or toxic compounds when presented in feeds due to their decreasing the intake [30]. 10stated that tannins reduce the palatability of the browses and made them less preferred by animals. Other studies Bitende SN, et al. [31], Tibebu M, et al. [32] also showed that supplementation of Sesbania to grass or straw-based diet fed to different ruminant animal species increased the DM, OM and CP intakes. According to Ranjhan

SK, et al. [33] the ME requirement for sheep weighing 20-30 kg ranged between 5.86 to 8.37 MJ and in the current study, all the treatments satisfied the ME requirements.

Means followed by different superscript letters within a row for each treatment feed are significantly different at P < 0.05; C= Concentrate; R= Roughage; SEM = standard error of means; SSL; S. sesban leave; Noug seed cake; DM =Dry matter; OM=Organic matter; CP = Crude Protein; NDF= Neutral Detergent Fiber; ADF =Acid Detergent Fiber; ME= Metabolizable energy. Table 2: Daily feed and nutrient intakes of Arsi-Bale sheep fed dried S. sesban leaves, and mixed different percentage of hay, S. sesban leaves and noug seed cake.

Dry Matter and Nutrient Digestibility

Nutrient digestibility values were given in Table 3. The DM and OM digestibility was significantly (P<0.01) differences between T2 and T3 compared to T1 (Table 3). The digestibility of CP and NDF were significantly (P<.001) difference between the treatments. The lower DM and nutrient digestibility in the sole of S. sesban leaves indicated that sole S. sesban leaves might not be used solely as a supplement of sheep in such type of basal diet. Multipurpose trees (MPT) are seldom fed to ruminants as a sole source of feed and, therefore, their important attributes are their ability to improve the digestibility and utilization of fibrous feeds when used as a supplement [34]. The different effects of S. sesban supplementation on the total DMI may be attributed to its palatability, anti-nutritional content, stage of maturity, means of supplementation, and the amounts added [21]. Furthermore, it was apparent that nutrient digestibility showed a decreasing trend as the level of inclusion of S. sesban in the supplement increased. Khalili H [35] stated that dietary crude protein digestibility decreases with increasing S. sesban supplementation compared to concentrate supplementation. This could be attributed to anti-nutritional factors contained in S. sesban. The deleterious effect of secondary plant metabolites such as tannin on nutrient digestibility has been well documented [27, 28]. Tannins were considered as anti-nutritive and/or toxic compounds when present in feeds due to their decreasing the digestion of MPT 30. Similarly, tannin reacts with protein and form tannin–protein complex, which reduces rumen fermentation and eventually depress nutrient digestibility and voluntary feed intake [29]. In addition, Woldemeskel M, et al. [36] reported that the adverse effect on rumen metabolism and diminished digestibility due to tannins and diarrhoea induced by saponnins may reduce absorption and availability of ingredients in high level S. sesban (400 g) resulting in less adverse effects than low level (200 g).

Means followed by different superscript letters within a row for each feed type are significantly different at P < 0.05; SEM = standard error of means; DM =Dry matter; OM=Organic matter; CP = Crude Protein; NDF= Neutral Detergent Fiber; ADF =Acid Detergent Fiber SSL= S. sesban leave; NSC= Noug seed cake; T1 = S. sesban leave; T2 = 27.6% (hay: SSL): 44.8%NSC; T3 = 38.6 %hay: 61.4%NSC. Table 3: Dry matter and nutrient digestibility of Arsi-Bale sheep fed dried S. sesban leave, and mixed different percentage of hay, S. sesban leave and noug seed cake.

Prediction of Enteric Methane Emissions

Table 4 showed enteric CH4 emission factors and daily methane production of Arsi-Bale sheep fed different treatments of feed in the study area. Enteric fermentation emissions have been estimated using the IPCC Tier 2 model. The GE, EF, and DMP were significant (P<0.01) difference between the treatments. The quantification of methane emissions from livestock on a global scale relies on prediction models because measurements require specific equipment and may be expensive [37]. The average EF in the present study are similar for sheep Nandi and Bomet sheep (4.6 and 4.8 kg CH4per head per year) in Kenya [38]. Methane production from ruminants in small ruminant varies with the diet, which could be affected by the factors such as DM intake, diet composition, and digestibility. Because enteric CH4 emissions are strongly related to feed intake, all models include a measurement of intake, such as DMI, gross energy (GE) intake (GEI), ME intake, or NDFI. May be the reduction of CH4 production in S. sesban leaves in the current study could be associated with the DMI, GEI, NDFI Hristov AN [39] and fractions of tannin phenols and condensed tannins, which have the potential to modify rumen fermentation to reduce CH4 production [40]. An increased fibrous concentration, particularly NDF and ADF in treatment two and three resulted higher CH4 production. This might be explained by fact that high fiber in the feed could favor acetate production, thereby increasing hydrogen for methanogenesis [41]. In addition, it is a well-established nutrition principle that increasing the level of concentrate in the diet reduces the dietary energy converted to CH4 [42]. This occurs because feed rations rich in starch favor propionate production thereby reducing CH4 production per unit of fermentable organic matter in the rumen. Conversely, a roughage-based diet will favour acetate production and increase CH4 production per unit of fermentable organic matter in ruminants [43]. The reduction in CH4 output by shifting H2 flow towards alternative electron acceptors such as propionate significantly enhances the utilization of feeds and animal performance.

A significant portion of agricultural emissions in most developing countries come from ruminant livestock, which include cattle, sheep and goats [44]. The bulk of the emissions from ruminant livestock are in the form of methane produced through enteric fermentation.

The livestock feeding systems in Ethiopia are mainly dependent on natural grass, crop residues and high fiber diets that are deficient in nitrogen and digestible energy, limiting the animal performance. These high fiber diets rich in structural carbohydrates increase ruminal acetate-to-propionate ratio, thus produce more enteric CH4 but may limit manure CH4 production due to the resistance of excreted cell wall to microbial fermentation (Improving dietary quality can both improve animal performance and reduce CH4 production). It can also improve efficiency by reducing CH4 emissions per unit of animal product. In contrast, CH4 production in ruminants tends to increase with the age of the forage fed. The composition of both structural (i.e. fiber) and soluble carbohydrates is important in developing low CH4 emission rations. For example, CH4 production per unit of cellulose digested is reportedly three times that of hemicellulose [45]. The proportions of cellulose, hemicellulose and lignin in cell walls change as plants mature, leading to declines in both digestibility and CH4 emission per unit fed. Since structural carbohydrates produce more CH4 per unit of substrate digested than non-structural carbohydrates, adding grain to a forage diet increases starch intake and reduces fiber intake which, in turn, reduces rumen pH and favors the production of propionate rather than acetate in the rumen [46].