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

Tahereh Mohammadabadi Morteza Chaji Ehsan Direkvandi Othman Alqaisi About the authors

ABSTRACT.

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-1 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.

Keywords:
buffalo; in situ digestibility; gas production; leucaena leucocephala leaves

Introduction

The increase in human populations lead to an increase in food demand (Choct, 1997Choct, M. (1997). Feed non-starch polysaccharides: chemical structures and nutritional significance. Feed Milling International, 191, 13-26.). 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 (Direkvandi et al., 2020Direkvandi, E., Mohammadabadi, T., Chaji, M., Elghandour, M. M., Barbabosa-Pleigo, A., & Salem, A. Z. M. (2020). Effect of sulfuric acid and molasses on the chemical composition, ruminal fermentation, and digestibility of silage of Conocarpus erectus L. tree leaves and branches. Agroforestry Systems, 94(4), 1601-1609. doi: 10.1007/s10457-020-00495-5
https://doi.org/10.1007/s10457-020-00495...
). Especially in tropical and sub-tropical regions where feeds often are low crude protein (CP) and rich in crude fiber (Wanapat, 2000Wanapat, M. (2000). Rumen manipulation to increase the efficient use of local feed resources and productivity of ruminants in the tropics. Asian-Australasian Journal of Animal Sciences, 13, 59-67.). Likewise, in arid areas forages like alfalfa hay is either in shortage or imported and formulated at high market prices (Alqaisi, Moraes, Ndambi, & Williams, 2019Alqaisi, O., Moraes, L. E., Ndambi, O. A., & Williams, R. B. (2019). Optimal dairy feed input selection under alternative feeds availability and relative prices. Information Processing in Agriculture, 6(4), 438-453. doi: 10.1016/j.inpa.2019.03.004
https://doi.org/10.1016/j.inpa.2019.03.0...
).

Substituting typically imported feeds with those of feed of native origins reduced feed cost in arid dairy feeding systems (Alqaisi, Hemme, Latacz-Lohmann, & Susenbeth, 2014Alqaisi, O., Hemme, T., Latacz-Lohmann, U., & Susenbeth, A. (2014). Evaluation of food industry by-products as feed in semi-arid dairy farming systems: the case of Jordan. Sustainability Science, 9(3), 361-377. doi: 10.1007/s11625-013-0240-6
https://doi.org/10.1007/s11625-013-0240-...
). Despite of its availability and quality, one of the problems associated with feeding tropical plants to ruminants is that they contain anti-nutritional 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, 1999Gupta, H. K., & Atreja, P. P. (1999). Influence of feeding increasing levels of leucaena leaf meal on the performance of milch goats and metabolism of mimosine and 3-hydroxy-4 (1H) pyridone. Animal Feed Science and Technology, 78(1-2), 159-167. doi: 10.1016/S0377-8401(98)00263-6
https://doi.org/10.1016/S0377-8401(98)00...
), tannins (total tannin between 2.4 and 3.3%; Islam, Nahar, & Islam, 1995Islam, M., Nahar, T. N., & Islam, M. R. (1995). Productivity and nutritive value of Leucaena leucocephala for ruminant nutrition - review. Asian-Australasian Journal of Animal Sciences, 8(3), 213-217. doi: 10.5713/ajas.1995.213
https://doi.org/10.5713/ajas.1995.213...
), 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, 2012Kang, S., Wanapat, M., Pakdee, P., Pilajun, R., & Cherdthong, A. (2012). Effects of energy level and Leucaena leucocephala leaf meal as a protein source on rumen fermentation efficiency and digestibility in swamp buffalo. Animal Feed Science and Technology, 174(3-4), 131-139. doi: 10.1016/j.anifeedsci.2012.03.007
https://doi.org/10.1016/j.anifeedsci.201...
). 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, 2006Sabu, A., Augur, C., Swati, C., & Pandey, A. (2006). Tannase production by Lactobacillus sp. ASR-S1 under solid-state fermentation. Process Biochemistry, 41(3), 575-580. doi: 10.1016/j.procbio.2005.05.011
https://doi.org/10.1016/j.procbio.2005.0...
). 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, 2001Molan, A. L., Attwood, G. T., Min, B. R., & McNabb, W. C. (2001). The effect of condensed tannins from Lotus pedunculatus and Lotus corniculatus on the growth of proteolytic rumen bacteria in vitro and their possible mode of action. Canadian Journal of Microbiology, 47(7), 626-633. doi: 10.1139/w01-060
https://doi.org/10.1139/w01-060...
), cell wall destruction (O'Donovan & Brooker, 2001O’Donovan, L., & Brooker, J. D. (2001). Effect of hydrolysable and condensed tannins on growth, morphology and metabolism of Streptococcus gallolyticus (S. caprinus) and Streptococcus bovis. Microbiology, 147(Part 4), 1025-1033. doi: 10.1099/00221287-147-4-1025
https://doi.org/10.1099/00221287-147-4-1...
) and interference with extracellular enzyme activity (Frutos, Hervás, Giráldez, & Mantecón, 2004Frutos, P., Hervás, G., Giráldez, F. J., & Mantecón, A. R. (2004). Review. Tannins and ruminant nutrition. Spanish Journal of Agricultural Research, 2(2), 191-202. doi: 10.5424/sjar/2004022-73
https://doi.org/10.5424/sjar/2004022-73...
).

Despite of its anti-nutritional contents, the L. leucocephala is an evergreen legume contains leaves and pods that are rich in CP and neutral detergent fiber (NDF) being 235 and 386 g kg-1 of DM, respectively (Clavero & Razz, 2003Clavero, T., & Razz, R. (2003). The performance of goats browsing Leucaena leucocephala in the semi arid areas of Northwest Venezuela. Revista Científica de Veterinária, 13(6), 460-463.). Garcia, Ferguson, Neckles, and Archibald, (1996Garcia, G. W., Ferguson, T. U., Neckles, F. A., & Archibald, K. A. E. (1996). The nutritive value and forage productivity of Leucaena leucocephala. Animal Feed Science and Technology, 60(1-2), 29-41. doi: 10.1016/0377-8401(95)00922-1
https://doi.org/10.1016/0377-8401(95)009...
) reported a high CP content of L. leucocephala leaves and forages of 292 and 220 g kg-1 of DM. Furthermore, L. leucocephala seed and leaves are a good source of minerals such as potassium, phosphorus, nitrogen, calcium, magnesium, zinc, copper and manganese (Alabi & Alausa, 2006Alabi, D. A., & Alausa, A. A. (2006). Evaluation of the mineral nutrients and organic food contents of the seeds of Lablab purpureus, Leucaena leucocephala and Mucuna utilis for domestic consumption and industrial utilization. World Journal of Agricultural Sciences, 2(1), 115-118.). Feeding a mixture of L. leucocephala and forage increased milk production in lactation cows (Harrison, McSweeney, Tomkins, & Eckard, 2015Harrison, M. T., McSweeney, C., Tomkins, N. W., & Eckard, R. J. (2015). Improving greenhouse gas emissions intensities of subtropical and tropical beef farming systems using Leucaena leucocephala. Agricultural Systems, 136, 138-146. doi: 10.1016/j.agsy.2015.03.003
https://doi.org/10.1016/j.agsy.2015.03.0...
). Moreover, supplementing a L. leucocephala in a total ration has increased feed intake (Islam et al., 1995Islam, M., Nahar, T. N., & Islam, M. R. (1995). Productivity and nutritive value of Leucaena leucocephala for ruminant nutrition - review. Asian-Australasian Journal of Animal Sciences, 8(3), 213-217. doi: 10.5713/ajas.1995.213
https://doi.org/10.5713/ajas.1995.213...
). In view to its nutritional value, L. leucocephala might be used as a substitute feeds for ruminants in arid areas. Therefore, the objective of this study is to investigate the effect of substituting alfalfa hay with varying levels of L. leucocephala leaves on in vitro gas production (GP) parameter, digestibility and in situ degradability in buffalo.

Material and methods

This study was carried out at the research farm of Agricultural Sciences and Natural Resources University of Khuzestan (Mollasani, Iran).

Forage preparation and experimental diets

The L. leucocephala leaves were prepared from L. leucocephala trees available on Mollasani city (Iran, Khuzestan). Sampling was performed by plot method from 10 trees. Then the equal amount of samples mixed, air-dried in the shadow and grounded. Leaves were then chopped into approximately 20 mm particle length and DM was evaluated. Alfalfa hay was grown in Iran on irrigated land and was supplied to the university farm monthly. Alfalfa hay samples were collected from the university farm. In this experiment, alfalfa hay was replaced by L. leucocephala leaves in proportions of 0 (without L. leucocephala; control), 25, 50 and 100%.

The diets were formulated according to National Research Council (NRC, 2001National Research Council [NRC]. (2001). Nutrient requirements of dairy cattle (7th ed., rev.). Washington, DC: National Academies Press.) and were homogenized in their metabolizable energy (ME) and CP contents. The control diet reflects a typical feed ration offered to buffalo cattle in Iran. Table 1 shows the ingredients and the chemical composition of the experimental diets used for the in vitro GP, digestibility and the in situ DM degradability.

The contents of DM, Ash, CP, NDF, ether extract (EE) and total tannin (TT) of L. leucocephala leaves evaluated and were found to be 329, 70, 235, 298, 107 and 31.2 g kg-1 of DM, respectively. Likewise, the contents of DM, Ash, CP, NDF and EE of alfalfa hay were evaluated and found to be 900, 100, 170, 425 and 16 g kg-1 of DM, respectively.

Table 1
Ingredients and chemical composition of experimental diets used for the in vitro gas production parameter, digestibility and the in situ DM degradability evaluation.

Chemical analysis

Before the chemical analysis commence, feed samples were oven-dried at 55oC 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], 1998Association of Official Analytical Chemists [AOAC]. (1998). Official methods of analysis of AOAC international (16th ed.). Gaithersburg, MD: AOAC International.). Furthermore, NDF was analyzed according to Van Soest, Robertson, and Lewis (1991Van Soest, P. J., Robertson, J. B., & Lewis, B. A. (1991). Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. Journal of Dairy Science, 74(10), 3583-3597. doi: 10.3168/jds.S0022-0302(91)78551-2
https://doi.org/10.3168/jds.S0022-0302(9...
). Acid detergent lignin (ADL) was determined by solubilization of cellulose with a sulfuric acid (Robertson & Van Soest, 1980Robertson, J. B., & Van Soest, P. J. (1980). The detergent system of analysis and its application to human foods. In W. P. T. James & O. Theander (Eds.), The analysis of dietary fiber in food (p. 123-158). New York, NY: Marcel Dekker.). Total tannins (TT) of L. leucocephala leaves were measured according to Makkar (2000Makkar, H. P. S. (2000). Quantification of tannins in tree foliage. A laboratory manual for the FAO/IAEA Co-ordinated research project on ‘Use of nuclear and related techniques to develop simple tannin assays for predicting and improving the safety and efficiency of feeding ruminants on tanniniferous tree foliage’. Vienna, AT: FAO/IAEA.).

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 39oC and pre-flushed with CO2. 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 (1988Menke, K. H., & Steingass, H. (1988). Estimation of energetic feed value obtained from chemical analysis and in vitro gas production using rumen fluid. Animal Research and Development, 28, 7-55.) (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 (1979Ørskov, E. R., & McDonald, I. (1979). The estimation of protein degradability in the rumen from incubation measurements weighted according to rate of passage. The Journal of Agricultural Science, 92(2), 499-503. doi: 10.1017/S0021859600063048
https://doi.org/10.1017/S002185960006304...
) as follows Equation 1:

y = a + b 1-e-ct(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×GP24)+(4.5×CP)+0.651×ash)(Menke et al., 1979Menke, K. H., Raab, L., Salewski, A., Steingass, H., Fritz, D., & Schneider, W. (1979). The estimation of the digestibility and metabolizable energy content of ruminant feedingstuffs from the gas production when they are incubated with rumen liquor in vitro. The Journal of Agricultural Science, 93(1), 217-222. doi: 10.1017/S0021859600086305
https://doi.org/10.1017/S002185960008630...
) (2)

ME MJ kg-1 of DM=[2.2 +0.136×GP24+0.057×CP](Menke et al., 1979Menke, K. H., Raab, L., Salewski, A., Steingass, H., Fritz, D., & Schneider, W. (1979). The estimation of the digestibility and metabolizable energy content of ruminant feedingstuffs from the gas production when they are incubated with rumen liquor in vitro. The Journal of Agricultural Science, 93(1), 217-222. doi: 10.1017/S0021859600086305
https://doi.org/10.1017/S002185960008630...
) (3)

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., 2013Salem, A. Z., Zhou, C.-S., Tan, Z.-L., Mellado, M., Salazar, M. C., Elghandopur, M. M., & Odongo, N. E. (2013). In vitro ruminal gas production kinetics of four fodder trees ensiled with or without molasses and urea. Journal of Integrative Agriculture, 12(7), 1234-1242. doi: 10.1016/S2095-3119(13)60438-4
https://doi.org/10.1016/S2095-3119(13)60...
). The partitioning factor (PF), microbial protein (MCP) and short chain fatty acids (SCFA) were estimated according Equation 4 to 6.

PF (mg mL-1) = ADS (mg)GP96 (mL) (Blümmel, Steingaβ, & Becker, 1997Blümmel, M., Steingaβ, H., & Becker, K. (1997). The relationship between in vitro gas production, in vitro microbial biomass yield and 15N incorporation and its implications for the prediction of voluntary feed intake of roughages. British Journal of Nutrition, 77(6), 911-921. doi: 10.1079/BJN19970089
https://doi.org/10.1079/BJN19970089...
) (4)

MCP (mg g-1 of DM)= ADS-(mL GP24×2.2 mg mL-1) (Blümmel et al.,1997Blümmel, M., Steingaβ, H., & Becker, K. (1997). The relationship between in vitro gas production, in vitro microbial biomass yield and 15N incorporation and its implications for the prediction of voluntary feed intake of roughages. British Journal of Nutrition, 77(6), 911-921. doi: 10.1079/BJN19970089
https://doi.org/10.1079/BJN19970089...
) (5)

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., 1997Blümmel, M., Steingaβ, H., & Becker, K. (1997). The relationship between in vitro gas production, in vitro microbial biomass yield and 15N incorporation and its implications for the prediction of voluntary feed intake of roughages. British Journal of Nutrition, 77(6), 911-921. doi: 10.1079/BJN19970089
https://doi.org/10.1079/BJN19970089...
).

SCFA (mmol 200 mg-1 of DM)=(0.0222×GP24)-0.00425 (Getachew, Makkar, & Becker, 2002Getachew, G., Makkar, H. P. S., & Becker, K. (2002). Tropical browses: contents of phenolic compounds, in vitro gas production and stoichiometric relationship between short chain fatty acid and in vitro gas production. The Journal of Agricultural Science, 139(3), 341-352. doi: 10.1017/S0021859602002393
https://doi.org/10.1017/S002185960200239...
) (6)

where:

GP96 is the volume of GP at the 96 hour incubation, GP24 is the volume of GP at the 24 hour incubation and CP is crude protein (g kg-1 of DM).

The in vitro digestibility of DM (IVDMD) of experimental diets was determined using Tilley and Terry (1963Tilley, J. M. A., & Terry, R. A. (1963). A two‐stage technique for the in vitro digestion of forage crops. Grass and Forage Science, 18(2), 104-111. doi: 10.1111/j.1365-2494.1963.tb00335.x
https://doi.org/10.1111/j.1365-2494.1963...
) method. Therefore, rumen liquor was collected and mixed with McDougall buffer solution (includes [g L-1] 9.8 Sodium Bicarbonate, 2.44 Sodium Phosphate Dibasic, 0.57 Potassium Chloride, 0.47 Sodium Chloride, 0.12 Magnesium Sulfate and 0.16 Calcium Chloride) in a ratio of 1:4. After flushing with CO2, 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, 1993Agricultural and Food Research Council [AFRC]. (1993). Energy and protein requirements of ruminants. Wallingford, GB: CAB International.). 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 (1979Ørskov, E. R., & McDonald, I. (1979). The estimation of protein degradability in the rumen from incubation measurements weighted according to rate of passage. The Journal of Agricultural Science, 92(2), 499-503. doi: 10.1017/S0021859600063048
https://doi.org/10.1017/S002185960006304...
) 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:

ED = A +(B×CC+K) (Ørskov & McDonald, 1979Ørskov, E. R., & McDonald, I. (1979). The estimation of protein degradability in the rumen from incubation measurements weighted according to rate of passage. The Journal of Agricultural Science, 92(2), 499-503. doi: 10.1017/S0021859600063048
https://doi.org/10.1017/S002185960006304...
) (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, 2008Statistical Analysis System [SAS]. (2008). SAS/STAT 9.2 user’s guide. Cary, NC: SAS Institute Inc.) as follows Equation 8:

Yij = µ + Di + eij (8)

where:

Yij is the Kth observation of the dependent (response) variable for the specific ijth animal, µ is the overall mean, Di is the effect of ith treatment, eij 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, 1995Norton, B. W., & Poppi, D. P. (1995). Composition and nutritional attributes of pasture legumes. In J. P. F. D'Mello & C. Devendra (Eds.), Tropical legumes in animal nutrition (p. 23-47). Wallingford, GB: CAB International.), 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, 2007Beauchemin, K. A., McGinn, S. M., Martinez, T. F., & McAllister, T. A. (2007). Use of condensed tannin extract from quebracho trees to reduce methane emissions from cattle. Journal of Animal Science, 85(8), 1990-1996. doi: 10.2527/jas.2006-686
https://doi.org/10.2527/jas.2006-686...
) by reducing of potential of microorganisms to bind to feed particles (McAllister, Bae, Jones, & Cheng, 1994McAllister, T. A., Bae, H. D., Jones, G. A., & Cheng, K. J. (1994). Microbial attachment and feed digestion in the rumen. Journal of Animal Science, 72(11), 3004-3018. doi: 10.2527/1994.72113004x
https://doi.org/10.2527/1994.72113004x...
). Furthermore, tannins inhibit the growth of microbial enzymes (McSweeney, Palmer, McNeill, & Krause, 2001McSweeney, C. S., Palmer, B., McNeill, D. M., & Krause, D. O. (2001). Microbial interactions with tannins: nutritional consequences for ruminants. Animal Feed Science and Technology, 91(1-2), 83-93. doi: 10.1016/S0377-8401(01)00232-2
https://doi.org/10.1016/S0377-8401(01)00...
). The negative correlation between tannins content and the GP volume found in our study agree with the findings of Khazaal, Boza, and Ørskov (1994Khazaal, K., Boza, J., & Ørskov, E. R. (1994). Assessment of phenolics-related antinutritive effects in Mediterranean browse: a comparison between the use of the in vitro gas production technique with or without insoluble polyvinylpolypyrrolidone or nylon bag. Animal Feed Science and Technology, 49(1-2), 133-149. doi: 10.1016/0377-8401(94)90087-6
https://doi.org/10.1016/0377-8401(94)900...
) and with Chaji, Direkvandi, and Salem (2020Chaji, M., Direkvandi, E., & Salem, A. Z. M. (2020). Ensiling of Conocarpus erectus tree leaves with molasses, exogenous enzyme and Lactobacillus plantarum impacts on ruminal sheep biogases production and fermentation. Agroforestry Systems, 94(1), 1611-1623. doi: 10.1007/s10457-019-00436-x
https://doi.org/10.1007/s10457-019-00436...
) 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.

Table 2
Effect of alfalfa hay replacing with different levels of L. leucocephala leaves on in vitro rumen gas kinetics and cumulative gas production (mL 200 mg-1 of DM).
Table 3
Effect of alfalfa hay replacing with replacing alfalfa hay with different levels of L. leucocephala leaves on in vitro gas production parameter and in vitro digestibility.

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., 2010Sallam, S. M. A. H., Bueno, I. C. S., Godoy, P. B., Nozella, E. F., Vitti, D. M. S. S., & Abdalla, A. L. (2010). Ruminal fermentation and tannins bioactivity of some browses using a semi-automated gas production technique. Tropical and Subtropical Agroecosystems, 12(1), 1-10.). 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. (2015Barros-Rodríguez, M. A., Solorio-Sánchez, F. J., Sandoval-Castro, C. A., Klieve, A., Rojas-Herrera, R. A., Briceño-Poot, E. G., & Ku-Vera, J. C. (2015). Rumen function in vivo and in vitro in sheep fed Leucaena leucocephala. Tropical Animal Health and Production, 47(4), 757-764. doi: 10.1007/s11250-015-0790-y
https://doi.org/10.1007/s11250-015-0790-...
) 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., 1997Blümmel, M., Steingaβ, H., & Becker, K. (1997). The relationship between in vitro gas production, in vitro microbial biomass yield and 15N incorporation and its implications for the prediction of voluntary feed intake of roughages. British Journal of Nutrition, 77(6), 911-921. doi: 10.1079/BJN19970089
https://doi.org/10.1079/BJN19970089...
). The PF ratio in our study agree with those reported by Soltan, Morsy, Sallam, Louvandini, and Abdalla (2012Soltan, Y. A., Morsy, A. S., Sallam, S. M. A., Louvandini, H., & Abdalla, A. L. (2012). Comparative in vitro evaluation of forage legumes (prosopis, acacia, atriplex, and leucaena) on ruminal fermentation and methanogenesis. Journal of Animal and Feed Sciences, 21(4), 759-772. doi: 10.22358/jafs/66148/2012
https://doi.org/10.22358/jafs/66148/2012...
) 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 (2011Angaji, L., Souri, M., & Moeini, M. M. (2011). Deactivation of tannins in raisin stalk by polyethylene glycol-600: Effect on degradation and gas production in vitro. African Journal of Biotechnology, 10(21), 4478-4483.) 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 (2010Paengkoum, P. (2010). Effects of neem (Azadirachta indica) and Leucaena (Leucaena leucocephala) fodders on digestibility, rumen fermentation and nitrogen balance of goats fed corn silage. Journal of Animal and Veterinary Advances, 9(5), 883-886. doi: 10.3923/javaa.2010.883.886
https://doi.org/10.3923/javaa.2010.883.8...
) 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. Esfahani, Chaji, Mohammadabadi, and Bojarpour (2016Esfahani, S. N., Chaji, M., Mohammadabadi, T., & Bojarpour, M. (2016). The investigation of digestion and fermentation of diets containing different parts of subabul tree (Leucaena leucocephala). Journal of Animal Production, 18(1), 39-49. doi: 10.22059/JAP.2016.54271
https://doi.org/10.22059/JAP.2016.54271...
) 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 ED2 (k = 0.02), ED5 (k = 0.05) and ED8 (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 (2008Mondal, G., Walli, T. K., & Patra, A. K. (2008). In vitro and in sacco ruminal protein degradability of common Indian feed ingredients. Livestock Research for Rural Development, 20(4), 63.) 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, 2017Shahriari, Z., Mohammadabadi, T., Vakili, S. T., Chaji, M., & Sari, M. (2017). Effect of replacing alfalfa with subabul (Leucaena leucocephala) pod on digestibility, in vitro fermentation and in situ degradability in cow and buffalo. Journal of Animal Production Research, 6(3), 63-72. doi: 10.22124/AR.2017.2366
https://doi.org/10.22124/AR.2017.2366...
).

In our study, ED2, ED5 and ED8 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 (1987Lu, C. D., & Jorgensen, N. A. (1987). Alfalfa saponins affect site and extent of nutrient digestion in ruminants. Journal of Nutrition, 117(5), 919-927. doi: 10.1093/jn/117.5.919
https://doi.org/10.1093/jn/117.5.919...
) 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, 1990Reed, J. D., Soller, H., & Woodward, A. (1990). Fodder tree and straw diets for sheep: intake, growth, digestibility and the effects of phenolics on nitrogen utilisation. Animal Feed Science and Technology, 30(1-2), 39-50. doi: 10.1016/0377-8401(90)90050-I
https://doi.org/10.1016/0377-8401(90)900...
).

Table 4
In situ DM degradability of diets with replacing alfalfa hay with different levels of L. leucocephala leaves.

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.

Acknowledgements

The authors gratefully acknowledge the Agricultural Sciences and Natural Resources University of Khuzestan for their financial support

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Publication Dates

  • Publication in this collection
    14 June 2021
  • Date of issue
    2021

History

  • Received
    08 Feb 2020
  • Accepted
    17 Sept 2020
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