Effect of Plantain ( Musa paradisiaca ) Leaves Ash Extract and the Source of Rumen Fluid on in vitro Digestibility of Rice Straw Complemented with Calliandra calothyrsus Leaves

| This study was carried out to determine the effect of plantain leaves ash extract and the source of rumen fluid on in vitro digestibility of rice straw complemented with Calliandra calothyrsus leaves. At the beginning of the trial, the dry leaves of plantain harvested from vicinity of the University of Dschang Research Farm (UDRF), sundried and were ashed on concrete clean surfaces to obtain ash. Subsequently 50, 100 or 150 g of this ash were each dissolved in 1 liter of distilled water to obtain ash extracts solutions. Two sources of rumen fluid (sheep and goat) were collected separately and incubated with four diets (untreated or treated rice straw (70%) and Calliandra calothyrsus leaves (30%) (w/w basis)) each, in triplicate in a two-ways analysis of variance according to the test gas method. The results showed that with the exception of residual nitrogen (N-NDF) and microbial mass (MM) that were similar (p>0.05), all other parameters of the in vitro digestibility of rice straw were significantly (p<0.05) influenced by ash extract treatment irrespective of the rumen fluid source considered. The gas produced (32.06 ± 0.6 and 32.90 ± 0.09 ml), volatile fatty acid (0.70 ± 0.05 and 0.72 ± 0.001 mmol/ml), metabolisable energy (7.09±0.05 and 7.21 ± 0.01 MJ/kg/DM) and in vitro digestibility of organic matter (48.37 ± 0.33 and 55.65 ± 0.13%) obtained with rice straw treated with plantain ash extract from 50 and 100g ash mixed with C. calothyrsus diets respectively were significantly (p<0.05) higher with goat rumen fluid. In contrast, treatment of rice straw with ash extract (86.79 ± 3.85 and 85.27 ± 1.73 mg) significantly (p<0.05) decreased microbial mass compared to untreated straw (103.94 ± 1.64 mg). From the above, therefore it can be concluded that treating rice straw with plantain leaves ash extract improved digestibility in small ruminants.

protein requirement in Cameroon. However, the decrease in the pastoral space on which these animals depend for the benefit of agriculture and competing activities poses a feed problem with negative consequences  on their production and productivity, especially during the dry season. At the same time, this agriculture generates large quantities of crop residues (corn stovers, rice straw, etc.) which can be used to compensate lack of fodder, especially during the dry season. In fact, the digestive characteristics of ruminants enable them to better valorize crop residues, which constitute a potential source of energy for them ( Jami et al., 2014). This use will be more effective if the chemicals treatments processes are used. Indeed, the chemical treatment is effective in breaking down the lignin-polysaccharide membrane bonds contained in the lignified material and releases potentially available carbohydrates to microbial attack ( Jami et al., 2014). For this purpose, several chemicals have often been used including sodium hydroxide, calcium hydroxide and ammonia. However, the unavailability, the high costs, the difficulties of handling and the environmental pollution that some of these substances cause lead to the search of alternative products which are less expensive and have no adverse effect on the environment, as the ashes extracts. Ash (powdery residue left after burning) is readily available in rural areas. It contains minerals that give it alkaline properties (Tiisekwa et al., 1999, Van Ryssen andNdlovu, 2018). These alkaline properties suggest their potential to improve the digestibility of roughage through solubilization of silica and the weakening of the ester bonds between cellulose and lignin (Laswai et al., 2007). Moreover, Ramirez et al. (1992) showed that the dry matter digestibility of 20% ash-treated corn stovers increased by 20% in sheep compared to the control group. Hamed and Elimam (2008) also reported that the in sacco degradation of dry matter, organic matter, and crude proteins of sorghum stovers in Nubian goats receiving a basal ration of groundnut haulms increased with increasing levels (3, 5 and 8%) of Rabaa (Trianthema pentandra). Similarly, Kanyinji et al. (2014) showed that the treatment of Chloris gayana hay with 0, 20, 30 or 40 g of Musa sapientum ash per liter of solution improves its nutritive value in goats. Ingestions and optimal digestibilities were recorded when the hay was treated with 40 g of ash per liter of solution. However, the major shortcoming of crops residue treated with ash extract is their deficiencies in crude proteins (Abdulazeez et al., 2016) that is insufficient to meet microbial proteins needs. Thus, Protein complementation is necessary in addition to this treatment to optimize the digestibility of roughage fodder.
Calliandra calothyrsus adapts perfectly to the Cameroon pedoclimatic conditions, resists drought and is available all year round (Pamo et al., 2006). Its' crude protein content is 28.6% dry matter (DM) (Pamo et al., 2005), and it could be a good complement of rice straws based diets. Little work have been undertaken on the effect of ash extract level of plantain (Musa paradisiaca) leaves on the in vitro digestibility of rice straw complemented with leaves of Calliandra calothyrsus in small ruminants. The present study was therefore initiated to contribute to the valorization of crop residues in ruminants feeding. Its' objective was to evaluate the in vitro digestibility of rice straw treated with plantain leaves ash extract complemented with leaves of C. calothyrsus incubated with goat or sheep rumen fluid.

study AreA
The experiment was carried out at the University of Dschang Research Farm (UDRF) and in the Animal Production and Nutrition Laboratory (LAPRONA) of the same university in West Cameroon. Animals used in this study were provided standardized housing and feeding conditions and their welfare was considered as per international standard.

AnimAl And plAnt mAteriAls
One adult non-gestating West African Dwarf doe and one Djallonke ewe aged 18 month-old weighing 21 and 23 kg, respectively were used as donors of rumen fluid for this study. During the preliminary phase of the evaluation of the in vitro digestibility which lasted two weeks, donor animals received daily 801 g of diets composed of untreated (280 g) or treated rice straw (280 g) and Calliandra calothyrsus leaves (280 g) and water ad libitum.
A month before the study started, animals were administered a preventive drug of oxytetracycline (20%), repeated after two weeks . They were also dewormed with Ivermectine 1% (synthetic broad spectrum anthelmintic active against gastrointestinal nematods and pulmonary adults and larvae). The rumen fluids of ewe and goat were collected separately just after slaughter.
The plant material consisted of rice straw, variety NERI-CA (New Rice for Africa) low lands collected in the plots of the Institute of Agricultural Research for Development in Santchou (Western-Cameroon), transported to the UDRF, then chopped into pieces of 2-5 cm lenghts about with a machete and sun dried before being stored in a shop. Calliandra calothyrsus leaves were harvested before flowering in the UDRF plots and wilted during 2 hours before being served. In vitro digestibilty period lasted 24 hours.

prepArAtion oF plAntAin leAves Ash extrACt And treAtment oF riCe strAW
Plantains leaves (Musa paradisiaca) was ashed on a concrete clean surface at the University of Dschang Research

Journal of Animal Health and Production
September 2020 | Volume 8 | Issue 3 | Page 124  Acid Détergent Lignin ; dMO: digestibility of Organic matter; TN:Total Nitrogen; UTRS + Cc :Untreated rice straw + Calliandra calothyrsus leaves ; RSTPL50 + Cc : Rice straw treated with 50 g of plantains leaves ash extract + C. Calothyrsus leaves ; RSTP100 + Cc: Rice straw treated with 100 g of of plantains leaves ash extract + C. Calothyrsus leaves RSTP150 + Cc : Rice straw treated with 150 g of of plantains leaves ash extract + C. Calothyrsus leaves .
Farm (UDRF) in order to obtain ash. The solutions were prepared by dissolving 0, 50, 100 and 150 g of ash in 1 liter of distilled water and the mixtures were homogenized and allowed to stand for 48 hours. These solutions were filtered using a clean cotton fabric and the resulting filtrates constituted the ash extract solutions. For treatment, 100 g of previously chopped straw was weighed and then dipped into each of the ash extract solutions and kept soaked for 12 hours. At the end of this soaking, the straw was oven-dried at 60 °C up to constant weight, then packaged in envelopes and stored in a well-ventilated place to prevent the growth of mold. The pH of each solutions was measured using a portable EUTECH pH meter (01X244912 Fisher Scientific, France) before and after soaking.

diets
Both untreated and treated rice straw samples were mixed with Calliandra calothyrsus at 70: 30 % (w/w basis) to make up four diets, namely: D0: Untreated rice straw + Calliandra calothyrsus leaves (UTRS + Cc) D1: Rice straw treated with 50 g of plantains leaves ash extract + C. Calothyrsus leaves (RSTPL50 + Cc) D2: Rice straw treated with 100 g of plantains leaves ash extract + C. Calothyrsus leaves (RSTPL100 + Cc) D3: Rice straw treated with 150 g of plantains leaves ash extract + C. Calothyrsus leaves (RSTPL150 + Cc) After the preparation of these diets, 100 g were taken and stored in plastic bags for determination of chemical composition and in vitro digestibility assessment.

sAmples AnAlysis
Mineral composition of untreated or treated rice straw and Calliandra calotyrsus leaves (Table 1) were carried out in the laboratory of Soil Chemistry and Environment of the University of Dschang for the determination mineral contents, following the methods described by Pauwels et al. (1992). Analysis of the chemical composition of experimental diets (Table 2) was done at LAPRONA to determine dry matter, ash, organic matter, crude fiber, cell wall, lipid and total nitrogen content by the methods described by AOAC (2000).

Journal of Animal Health and Production
September 2020 | Volume 8 | Issue 3 | Page 125 evAluAtion oF the In vItro digestibility prepArAtion oF inoCulum And method used The evaluation of the quantity of gas produced and preparation of reagent were done according to the method and procedure described by Menke et al. (1979) modified by Makkar (2002). The day before the test, the samples (weighed in triplicate and introduce into syringes) and two (one for goat rumen fluid and one for sheep rumen fluid) freshly prepared reagent (Menke et al., 1979) were placed in an incubator at 39 °C overnight. Similarly, a water bath was turned on and the temperature was maintained by two thermostats set at 39 °C. The morning before the collection of rumen fluid of goat and sheep, the reagent in which continually arrived a stream of gas (CO 2 ) with a moderately pressure (4 Bars) was placed in a water bath at 39 °C. Then, the Soduim sulfide (417 mg) and 6N NaOH(0.44 ml) were added to this reagent. The rumen fluid collected respectively from goat and sheep just after slaugther was immediately filtered under a stream of CO 2 and 700 ml of each rumen fluid were collected and introduced into each reagent still under CO 2 stream. This mixture (inoculum) was homogenized for 10 minutes using a magnetic rod and 40 ml of each ration with goat inoculum was put into the syringe using a precision dispenser and then placed in the water bath for incubation.

inCubAtion
After 24 hours of incubation, the gas products and corrected by the gas produced by the inoculum in control tubes were used to determine the organic matter digestible (OMD) using the regression equation proposed by Menke and Steingrass, (1988): OMD (%) =14.88+0.889GP+0.45CP+0.0651Ash, where: GP:Amount of gas produced after 24 hours of incubation, CP: Crude protein of the initial sample , Mean while, the content of metabolizable energy (ME), Partitionning Factor (PF) which is the amount of organic matter degraded to produce 1 ml of gas, the microbial mass (MM), the volatile fatty acid (VFA) were calculated using the equations proposed by Makkar (2002): ME (MJ/kgDM) = 2.20+0.136GP+0.057CP, PF (mg/ml) =OMD/GP; VFA(mmol/ml) =0.0239GP-0.0601; MM (mg) =OMD-(GPxSF); OMD (mg) =Amount of organic matter degraded. GP (ml)=Amount of gas produced after 24 hours incubation, SF=Stochiometric factor (2.20 for fodder) After 24 hours of incubation, the contents of each syringe was transferred into a 600 ml beaker and syringe washed twice with two portions of 15 ml Neutral Detergent Solution (NDS) (Van Soest et al.,1991) and emptied into the beaker. The samples were boiled gently for one hour and filtered into pretared filter crucibles. These crucibles were dried at 103°C overnight and weight of the substrate and the weight of the incubated residue after NDS treatment, at the end of the incubation. The residues obtained after treatment with NDS were used to determine the residual nitrogen (N-NDF) by Kjeldahl method.

stAtistiCAl AnAlysis
In vitro digestibility data was subjected to two-ways analysis of variance following General Linear Models using SPSS 20.0 software. When differences existed between treatments, the means were separated by the Duncan's test at 0.05 significance level (Steel and Torrie, 1980).

gAs produCtion
When rice straw was treated with 50 or 100 g ash extract and incubated with sheep or goat rumen fluid, the quantity of gas produced from their incubation was similar to the untreated straw from the third to the 9 th hour (Figure 1). However after the 9 th hour until the end of the incubation (24 hours), gas produced from the treated straw was higher than that of untreated straw (UTRS+Cc) (Figure 2).

Figure 1:
Evolution of the gas produced of different diets incubated with the sheep rumen fluid UTRS + Cc :Untreated rice straw + Calliandra calothyrsus leaves ; RSTPL50 + Cc : Rice straw treated with 50 g of plantains leaves ash extract + C. Calothyrsus leaves ; RSTPL100 + Cc: Rice straw treated with 100 g of of plantains leaves ash extract + C. Calothyrsus leaves RSTPL150 + Cc : Rice straw treated with 150 g of of plantains leaves ash extract + C. Calothyrsus leaves

Journal of Animal Health and Production
September 2020 | Volume 8 | Issue 3 | Page 126 significantly (p<0.05) decreased with ash extract treatment level compared to untreated rice straw. The amount of gas produced after 24 hours of incubation, VFA production, ME and IVDOM of the RSTPL50 + Cc and RSTPL100 + Cc diets incubated with sheep rumen fluid were comparable (p>0.05) and significantly (p<0.05) higher than that of the RSTPL150 + Cc diet. Ash extract treatment levels had no significant effect (p>0.05) on residual nitrogen (N-NDF) and amount of organic matter fermented to produce 1 ml of gas (PF).
Ash extract treatment significantly (p <0.05) increased the amount of gas produced after 24 h of incubation (R 2 =0.58), VFA production (R 2 =0.57), IVDDM (R 2 =0.97), IVDOM (R 2 =0.60) and ME obtained with goat rumen fluid (R 2 =0.57) ( Table 3). In contrast, MM (R 2 =0.69) significantly (p<0.05) decreased with treatment of rice straw with ash extract compared to untreated straw. Gas production, VFA, IVDOM and ME obtained with ash extract treated rice straw incubated with goat rumen fluid was similar (p> 0.05) ( Table 3). With the exception of the RSTPL50+Cc diet, the partitioning factor (PF) also increased with increasing level of ash extract treatment (R 2 =0.76), the highest value was obtained with the RSTPL150 + Cc incubated with goat rumen fluid.
Gas produced after 24 hours of incubation, in vitro digestibility of organic matter (IVDOM), volatile fatty acid (VFA), metabolizable energy (ME) and residual nitrogen (N-NDF) were significantly (p<0.05) higher with goat rumen fluid whatever the level of ash extract treatment considered (Table 3). Conversely, the microbial mass (MM) obtained with the sheep rumen fluid was significantly (p<0.05) greater than that obtained with goat rumen fluid whatever the diet considered. Whatever the rumen fluid considered, the in vitro digestibility of dry matter (IVDDM) and Partitioning factor (PF) has been improved by ash extract treatment. In fact, the IVDDM

Journal of Animal Health and Production
September 2020 | Volume 8 | Issue 3 | Page 127 Figure 2: Evolution of the gas produced of the differents diets incubated with goat rumen fluid UTRS + Cc :Untreated rice straw + Calliandra calothyrsus leaves ; RSTPL50 + Cc : Rice straw treated with 50 g of plantains leaves ash extract + C. Calothyrsus leaves ; RSTPL100 + Cc: Rice straw treated with 100 g of of plantains leaves ash extract + C. Calothyrsus leaves RSTPL150 + Cc : Rice straw treated with 150 g of of plantains leaves ash extract + C. Calothyrsus leaves.
and PF of the treated rice straw incubated with sheep or goat rumen fluid was similar (p>0.05). The residual nitrogen (N-NDF) obtained with the goat rumen fluid was significantly (p<0.05) higher than that recorded with the sheep rumen fluid irrespective of the diet considered. PF and in vitro digestibility of dry matter (IVDDM) obtained with RSTPL100 +Cc and RSTPL150 + Cc diets incubated with goat rumen fluid were similar (p>0.05) and higher than those obtained with sheep rumen fluid for the RST-PL50 + Cc diet. Treatment of rice straw with ash extract resulted in significantly (p<0.05) higher VFA production with goat rumen fluid. There was significant ash extract treatment × rumen fluid source interaction on GP, ME, VFA, IVDDM, IVDOM and PF (p<0.05). Treatment of rice straw with ash extract significantly (p<0.05) influenced all in vitro digestibility parameters irrespective of the rumen fluid source consider with the exception of microbial mass and residual nitrogen which were similar (p>0.05).

dIScuSSIon
The gas produced after 24 hours of incubation was significantly (p<0.05) improved by the treatment of rice straw with 50 or 100 g of ash extract. The values obtained were close to those reported by Pi et al. (2005) with untreated and granulated rice straw (28.7 ml), pretreated (1% NaOH + 4% Ca(OH) 2 ) and non-granulated straw (29.8 ml) combined with a concentrate in Boer goats. However, they were lower than those reported by the same authors following the incubation of pretreated and granulated rice straw associated with a concentrate (33.7 ml). The increase in gas production would be related to the decrease in MM, as noted by (Menke et al., 1979), who observed that forages with high gas production have low microbial mass. Treatment of rice straw with ash extract significantly (p<0.05) improved its ME content, IVDDM, IVDOM and the production of VFA. These results are similar to Guérin's (1999) report increase in ME leads to an increase in the digestibility of organic matter. This could be explained by the improvement of the digestibility of the cell walls fraction of straw by the alkaline treatment resulting in the release of the energy contained in soluble carbohydrates to microorganisms (Pi et al., 2005), the VFA production and microbial cells. Indeed, more the organic matter is degraded, more energy is produced. Treatment of rice straw with ash extract significantly (p<0.05) decreased MM. This decrease could be explained by the contribution of the alkaline treatment modifying the rumen pH resulting in the reduction of the fermentative activity of certain microorganisms. This observation is contrary to that of Pi et al. (2005) who showed that the alkaline treatment of rice straw causes an increase in microbial mass. Treatment of rice straw with different levels of ash extract had no significant effect (p>0.05) on PF values (3.69, 3.35 and 3.29 mg / ml). However, these values were still close to those of conventional feeds (2.74 and 4.65 mg / ml) reported by Blümmel and Bullerdieck (1997). The decline in PF may be related to the significant decrease (p<0.05) in MM (Tendonkeng et al., 2016). The IVDDM of rice straw treated with 50 or 100 g ash extract was significantly (p<0.05) improved compared to untreated straw. The results obtained were higher than that obtained by Nolte et al. (1987) (44%) and  (41.07%) when wheat straw or rice straw was treated respectively with 30 and 10 % ash extract mixed with Calliandra calothyrsus leaves, incubated with goat rumen fluid and less than in vivo digestibility of DM (63.5%) obtained by Abdulazeez et al. (2016) when maize cobs were treated with 50 % urea + 50 % ash extract in sheep feeding .The high value of in vitro of DM digestibility of straw treated with ash extract can be attributed to the use of minerals provided by this treatment, which are essential for rumen microorganisms (Navarro andRodriguez, 1990, Van Ryssen andNdlovu, 2018). In fact, rumen bacteria use minerals for maintenance and growth of their cellulolytic activity ( Jouany et al., 1995). The source of rumen fluid significantly (p<0.05) influenced the in vitro digestibility of rice straw treated with ash extract associated with leaves of Calliandra calothyrsus. The quantity of gas produced, the IVDOM and the metabolizable energy of the diets were significantly (p<0.05) higher with the goat rumen fluid whatever the level of treatment with the ash extract. The same observation was made by Tendonkeng et al. (2016) who showed in a comparative study of two inoculum (goat and sheep) for the study of in vitro digestibility of rice straw treated with 5% urea, that gas produced after 24 hours of incubation was significantly (p<0.05) higher with goat rumen fluid. This result corroborate with that of Leng (1992) and Silanikove (2000) who observed that the goat's

Journal of Animal Health and Production
September 2020 | Volume 8 | Issue 3 | Page 128 rumen fluid has a great ability to tolerate the negative effects associated with increasing concentrations of secondary compounds such as silica and lignin. Microbial mass obtained with sheep rumen fluid was significantly (p<0.05) greater than that obtained with goat rumen fluid whatever the diet considered. This observation confirms that reported by Tendonkeng et al. (2016); this increase explains that the microorganisms of the sheep rumen liquid would have better valorized the nutrients (carbohydrate and protein) resulting from the degradation to ensure their growth. Whatever the rumen fluid considered, the IVDDM has been improved by ash extract treatment. This result is similar to the fundings of Ramirez et al. (1992); Kanyinji et al. (2014) who reported that the high digestibility value of DM, obtained with ash extract treated fodder, can be attributed to its high supply in minerals and their use by rumen microorganisms.

concluSIon
This study shows that the in vitro digestibility parameters of the different diets varied regardless of the source of the ruminal fluid. In fact, the production of gases, VFA, IVDDM, IVDOM and ME obtained with the rice straw treated with the extract of 50 or 100 g of ash incubated with the goat or sheep rumen fluid were similar but significantly higher than those recorded with the straw treated with the extract of 150 g of ash. Ash extract treatment levels had no significant effect on residual nitrogen (N-NDF) and amount of organic matter fermented to produce 1 ml of gas (PF) with sheep rumen fluid.Gas production, VFA, ME, and IVDOM were comparatively higher with goat rumen fluid than sheep. In general, the best results have been obtained with goat rumen fluid. Conversely, the microbial mass obtained with the sheep rumen fluid was greater than that obtained with goat rumen fluid whatever the diet considered.