Effect of replacing alfalfa hay with Leucaena leucocephala (L. Leucocephala) leaves on in vitro gas production, digestibility and in situ degradability in buffalo

This study was performed to investigate the effect of replacing alfalfa hay by L. leucocephala leaves in proportions of 25, 50 and 100% on in vitro gas production (GP) parameter, digestibility and in situ degradability in buffalo. Results showed that the volume of GP at 2 to 12 hours after incubation was significantly affected by replacing alfalfa hay with L. leucocephala leaves. In vitro digestibility of organic matter (OMD) differed significantly between treatment as it declined by increasing the alfalfa hay substitution rate from 25 to 100%. The microbial crude protein (MCP) differed significantly between treatments and was the greatest of 589 and 599 mg g of dry matter (DM) when L. leucocephala leaves replaced alfalfa hay at 25 and 50%. The in vitro digestibility of DM (IVDMD) increased significantly at 50% L. leucocephala replacement rate. Moreover, substituting alfalfa hay by L. leucocephala had a significant effect on the in situ degradability parameters. The insoluble but potentially degradable fraction (B) and potential of degradability (A+B) significantly increased for treatment contain 50% L. leucocephala leaves. The effective degradability (ED) was significantly different between dietary treatments and was the greatest when alfalfa hay was replaced by 25 and 50% L. leucocephala. In conclusion, L. leucocephala leaves can substitute 25 to 50% of dietary alfalfa hay in buffalo rations without effect on rumen efficiency.


Introduction
The increase in human populations lead to an increase in food demand (Choct, 1997). The magnitude of animal production is influenced by several factors, in which the role of nutrition represents a major part to ensure that nutrients are supplied to animals adequately. Nutritionists are seeking a practical and scientific solutions to improve animal productivity . Especially in tropical and sub-tropical regions where feeds often are low crude protein (CP) and rich in crude fiber (Wanapat, 2000). Likewise, in arid areas forages like alfalfa hay is either in shortage or imported and formulated at high market prices (Alqaisi, Moraes, Ndambi, & Williams, 2019).
Substituting typically imported feeds with those of feed of native origins reduced feed cost in arid dairy feeding systems (Alqaisi, Hemme, Latacz-Lohmann, & Susenbeth, 2014). Despite of its availability and quality, one of the problems associated with feeding tropical plants to ruminants is that they contain antinutritional substances that reduces animal's feed efficiency. Leucaena leucocephala is a well-known tropical tree. L. leucocephala contains anti-nutritional factors such as mimosine (3-10% of dry matter (DM); Gupta & Atreja, 1999), tannins (total tannin between 2.4 and 3.3%; Islam, Nahar, & Islam, 1995), in addition to saponin, oxalate and alkaloids. The presence of condensed tannin in the L. leucocephala can prevent protein digestion in the rumen (Kang, Wanapat, Pakdee, Pilajun, & Cherdthong, 2012). Tannins are one of the phenolic compounds that are commonly found in some trees, shrubs, legumes as well as in some grains (Sabu, Augur, Swati, & Pandey, 2006). Tannins have anti-nutritional effects which reduces palatability, digestibility, protein availability and digestive disturbances in animals, these effects mostly resulting from the decline in rumen microflora activity by reducing the availability and digestibility of nutrients and minerals (Molan, Attwood, Min, & McNabb, 2001), cell wall destruction (O'Donovan & Brooker, 2001) and interference with extracellular enzyme activity (Frutos, Hervás, Giráldez, & Mantecón, 2004).

Chemical analysis
Before the chemical analysis commence, feed samples were oven-dried at 55 o C for 48 h to evaluate DM content and then were passed through a 1 mm sieve (Wiley mill, Swedesboro, USA). Following the AOAC International procedure, samples were analyzed for CP (N × 6.25;No. 988.05),EE (No. 920.39),Ash (No. 924.05) and acid detergent fiber (ADF) (No. 973.18;Association of Official Analytical Chemists [AOAC], 1998). Furthermore, NDF was analyzed according to Van Soest, Robertson, and Lewis (1991). Acid detergent lignin (ADL) was determined by solubilization of cellulose with a sulfuric acid (Robertson & Van Soest, 1980). Total tannins (TT) of L. leucocephala leaves were measured according to Makkar (2000).

Biogas production parameters and digestibility
To investigate the in vitro GP parameters and the diets digestibility, rumen fluid was collected from four fistulated male buffalos before morning feeding. The animal was offered a diet composed of 60 forage and 40% concentrate a month prior to beginning of the experiment. A pump was used to sample the rumen liquor, which was immediately poured into a thermo-container pre-warmed at 39 o C and pre-flushed with CO 2 . Thereafter it was transferred to the ruminant nutrition laboratory located at the Agricultural Sciences and Natural Resources University of Khuzestan pH of the rumen liquor was immediately measured by portable pH meter (Metrohm model, Swiss). The rumen liquor was filtered by using 4-layer cheesecloth and poured into the plastic tube which was placed in a warm water-bath at 39°C.
The artificial saliva was prepared according to Menke and Steingass (1988) (includes distilled water, buffering solution, resazurin solution, macro mineral solution). Feed samples of 200 mg of each experimental diets with particle size 1 mm were placed in 100 mL glass vial at 39°C (6 replicates for each treatment). Then, 30 mL of mixed ruminal fluid and artificial saliva were added to each vial at a ratio of 1:2 and incubate at 39°C. GP was recorded at 2, 4, 6, 8, 12, 24, 48, 72 and 96 hour after incubation. GP data was fitted to the modified model described by Ørskov and McDonald (1979) as follows Equation 1: where: y is the volume of GP at the time t in mL, b is GP from the fermentable fraction (mL 200 mg -1 of DM), c is the GP rate constant (mL hour -1 ) and t is the incubation time (hour). The organic matter digestibility (OMD) and metabolizable energy (ME) were estimated according to the following Equation 2 and 3: OMD (g kg −1 of DM) = [148.8 + (8.89 × GP 24 ) + (4.5 × CP) + (0.651 × ash)] (Menke et al., 1979) (2) ME (MJ kg −1 of DM) = [2.2 + (0.136 × GP 24 ) + (0.057 × CP)] (Menke et al., 1979) ( The 96 hour apparently degraded substrate (ADS) content (mg g -1 of DM) was calculated as the difference between DM content of substrate and its undegradable DM (Salem et al., 2013). The partitioning factor (PF), microbial protein (MCP) and short chain fatty acids (SCFA) were estimated according Equation 4 to 6. (Blümmel, Steingaβ, & Becker, 1997) (4) where: The 2.2 mg per mL is a stoichiometric factor that expresses milligrams of carbon, hydrogen, and oxygen required for the production of SCFA gas complex associated with production of 1 mL of gas (Blümmel et al., 1997).
The in vitro digestibility of DM (IVDMD) of experimental diets was determined using Tilley and Terry (1963)  0.12 Magnesium Sulfate and 0.16 Calcium Chloride) in a ratio of 1:4. After flushing with CO 2 , tubes were incubated at 39ºC for 48 hour incubation. Thereafter, 6 mL of 20% HCl solution and 5 mL pepsin solution were added and then incubated for 48h simulating post-ruminal degradation. The residual substrates of each tube were filtered and used to determine the digestibility of DM.

In situ degradability of DM
To investigate the in situ degradability of DM, we used the nylon bag method according to (AFRC, 1993). A bags representing the experimental treatments were suspended in the rumen of four rumen-fistulated buffalos (their average body weight was 450 kg and of 3 years old). Animals were fed at 0700 and 1900 hour with a diet composed of 40 concentrate and 60% forage. The concentrate was made of corn and barley grains, wheat bran, vitamin and mineral premix. Whereas the offered forage were alfalfa hay and wheat straw and corn silage. The adaptation period lasted for 14 days. The experimental diets (Table 1) were milled and passed from a 2 mm screen. Then 5 mg of each diet were poured into Dacron bags (10 x 20 cm diameter, and 53 ± 10 μm pore size; R1020, Ankom Technology, Macedon, NY, USA). Different treatment bags were placed in the rumen simultaneously and in 3 replicates each time (96 bags). Nylon bag were incubated for 2, 4, 8, 12, 24, 48, 72 and 96 hour. After incubation, bags were withdrawn from the rumen and washed with cold water. The value of degradability at time zero was obtained by washing three bags per sample with cold water. Difference between primary weight with the weight after incubation, the disappearance rate of DM, was calculated at each of the incubation times. The degradability data obtained for each treatment was fitted to the modified model described by Ørskov and McDonald (1979) as P = A + B(1-e -Ct ) where P is the degradation rate at time t, A is the rapidly soluble fraction, B is the insoluble but potentially degradable fraction; C is the rate of degradation of fraction B; and t is the incubation time. Moreover, the effective degradability (ED) was calculated according to the Equation 7: where: K is the estimated rate of outflow from the rumen. The outflow from the rumen rate was considered 0.02, 0.05 and 0.08 hour.

Statistical analysis
Data on in vitro GP, in vitro digestibility and in situ degradability experiment were analyzed using a mixed model (Mixed procedures) of Statistical Analysis System (SAS, 2008) as follows Equation 8: where: Y ij is the K th observation of the dependent (response) variable for the specific ij th animal, µ is the overall mean, Di is the effect of i th treatment, e ij is the random residual error. Means were compared by the Duncan multiple comparison tests at p < 0.05.

Results and discussion
The effect of replacing dietary alfalfa hay with varying levels of L. leucocephala leaves on the in vitro rumen gas kinetics (b [potential of GP] and c [GP rate constant]) and GP are shown in Table 2.
The values of b and c decreased significantly decreased by increasing the level of L. leucocephala leaves (p < 0.05). Despite of nutrients contents of L. leucocephala leaves (Norton & Poppi, 1995), it's potential of GP (b) was less than the control treatment where Alfalfa hay was a major roughage. The GP volume between 2 and 12 hour differed significantly between treatments. However, the cumulative GP after 12 hour incubation did not differ significantly (p > 0.05) between experimental treatments, indicating a decline in tannins effect in a later incubation period. In our study, we evaluated tannins content of L. leucocephala leaves and was 31.2 g kg -1 of DM. It could be that the presence of anti-nutritional compounds in the L. leucocephala leaves reduced the potential for GP by increasing its dietary inclusion level. Tannins has been reported to reduce the fermentation, digestibility of nutrients and methane production (Beauchemin, McGinn, Martinez, & McAllister, 2007) by reducing of potential of microorganisms to bind to feed particles (McAllister, Bae, Jones, & Cheng, 1994). Furthermore, tannins inhibit the growth of microbial enzymes (McSweeney, Palmer, McNeill, & Krause, 2001). The negative correlation between tannins content and the GP volume found in our study agree with the findings of Khazaal, Boza, and Ørskov (1994) and with Chaji, Direkvandi, and Salem (2020) who found that the presence of tannin reduced GP due to the disruption effect on rumen microorganisms. Table 3 shows the effect of replacing dietary alfalfa hay with different levels of L. leucocephala leaves on in vitro GP parameter and in vitro digestibility of the evaluated diets. Replacing alfalfa hay with varying amounts of L. leucocephala affected the dietary contents of OMD, ME, SCFA, MCP, ADS and the IVDMD. The highest OMD, ME and SCFA contents were observed in the control diet (p < 0.05), than those diets containing L. leucocephala leaves. The low OMD digestibility of L. leucocephala leaves could be in part explained by the negative effect of tannins on fermentation and digestibility. Tannins can be complexed with other nutrients during the in vitro incubation leading to a reduced OMD (Sallam et al., 2010). Whereas the MCP after 24 hour incubation and the ADS content after 96 hour incubation, increased significantly when L. leucocephala leaves replaced alfalfa hay by 50% (p < 0.05). It was reported that the addition of saponin sources to alfalfa hay, increased the in vitro MCP linearly, due to its negative effect on rumen protozoa. Despite we did not evaluate the rumen protaoza in the current study, it was reported that when rumen protozoa decreases, the amount of bacterial predation decreases as well and lead to an increase in the microbial N flow to the duodenum. Furthermore, Barros-Rodríguez et al. (2015) reported that rumen protozoa decreased when sheep were fed with 20 and 40 percent L. leucocephala. Therefore, a decline in rumen protozoa in the L. leucocephala containing diets in the current study could be speculated. The PF shows the rate of substrate digestion to the volume of GP, volatile fatty acids (VFAs) and microbial biomass. There is a negative correlation between GP and PF ratio, as when the GP volume increases the amount of PF decreases (Blümmel et al., 1997). The PF ratio in our study agree with those reported by Soltan, Morsy, Sallam, Louvandini, and Abdalla (2012) who compared between PF value of L. leucocephala treatment and Tifton hay. The later reported that the increase in PF rate in L. leucocephala was associated with the reduction of methane GP. However, in our experiment, although PF numerically increased by including L. leucocephala in the diets, there was no significant difference between treatments (p > 0.05). This could be explained by the presence of tannins that leads to the increase in PF rate. Hence, the higher proportion of digestible nutrients was used to produce MCP compared the synthesis of SCFA as reported by Angaji, Souri, and Moeini (2011) which could explain the increase in MCP and SCFA in treatments that contain 50% L. leucocephala leaves.
There was no significant difference between IVDMD of 25 and 50% L. leucocephala leaves compared to the control diet. Paengkoum (2010) found that using L. leucocephala leaves in the diet decreased the digestibility of DM which agrees with our results at a complete substitution of alfalfa hay by L. leucocephala. Nasimi, Chaji, Mohammadabadi, and Bojarpour (2016) explained the decline in nutrient digestibility due to the greater content of lignin in L. leucocephala leaves compared to alfalfa hay (11 vs. 9%) and due to the anti-nutritional factors that present in L. leucocephala leaves. The level of the IVDMD correlate negatively with the dietary concentration of the anti-nutritional agents which could explain in part the decline in L. leucocephala DM digestibility at full substitution. In situ DM degradability results are presented in Table 4.
Fraction A (the soluble and very rapidly degradable fraction) was not affected by the different L. leucocephala dietary levels (p > 0.05). Fraction B (the insoluble but potentially degradable fraction) and the potential of degradability (A+B) were significantly increased for treatment that contained 50% L. leucocephala leaves (p < 0.05). However, the fraction C increased significantly for treatments contains 25% L. leucocephala leaves (p < 0.05). The effective degradability ED 2 (k = 0.02), ED 5 (k = 0.05) and ED 8 (k = 0.08) increased significantly for treatment that contained 25% L. leucocephala leaves (p < 0.05).
Our results do not agree with Mondal, Walli, and Patra (2008) who found different in situ DM degradability values since they used L. leucocephala leaves as the only dietary component to determine the in situ DM degradability. The variations in the in situ DM degradability of B, A+B and ED could be explained by the nutrients availability of carbohydrates and protein in L. leucocephala pod compared to alfalfa hay (Shahriari, Mohammadabadi, Vakili, Chaji, & Sari, 2017).
In our study, ED 2 , ED 5 and ED 8 decreased when L. leucocephala leaves replaced alfalfa hay at 50 and 100%. This can be due to the presence of anti-nutritional factors such as saponin and tannin in L. leucocephala leaves. Lu and Jorgensen (1987) reported that the presence of saponin in L. leucocephala leaves reduced the digestibility of L. leucocephala leaves. The presence of saponins in L. leucocephala reduce the effect of rumen fibrolytic enzyme thereby it disrupts the fiber digestion in the rumen. This was observed in our study as the effective degradability at rates of 0.02, 0.05 and 0.08 were significantly greater in alfalfa hay based diet at and at 25% L. leucocephala leaves substitution rate compared to higher dietary substitution rates of 50 and 100%. A high concentration of tannins was reported to reduce the digestibility of proteins and carbohydrates and has a negative effect on animal's performance (Reed, Soller, & Woodward, 1990).

Conclusion
Our results showed that substituting dietary alfalfa hay with L. leucocephala leaves at levels of 25 to 50% had no negative effects on the GP parameter and in situ digestibility of DM in buffalo. A full substitution of alfalfa hay by L. leucocephala reduced the in vitro and in situ digestibility parameters which suggest further treatments to reduce their negative effect on animal performance before feeding. Such a treatment needs to be evaluated from economic perspective in comparison with a good quality feeds such alfalfa hay. Overall, L. leucocephala could be an important alternative forage source for ruminants in arid areas.