INTRODUCTION

Feed and fodder are key nutritional input to supply carbohydrate, protein, vitamins and mineral for efficient performance of growing dairy animals (Pulina et al., 2020). India faces a net deficit of green fodder (35.6%), dry crop residues (10.95%) and concentrate feed ingredients (44%). The feeding systems based on forage are mandatory to lower the livestock rearing cost as the feed account 60-70% of the total livestock rearing cost (Anonymoys, 2013). The cheap and easily available unconventional feedstuffs are imperative to livestock production system (Oduro et al., 2008). Moringa is well known to have nutritional and pharmacological properties (Soliva et al., 2005). Moringa leaves are non-expensive, easily available and unconventional source of nutrients (Dong et al., 2019) and are abundant in protein, amino acids, fatty acids, minerals, vitamins, calcium, potassium which are the basic to growth of animal body (Deshmukh, 2014) along with various phenolic and oxycaroteniod compounds. The high biological value of nutrients of leaves have potential for adoption as feed for ruminant (Pradhan, 2016). This high valued Leaves’ protein and other nutrients need to evaluate for growth of dairy heifer calves replacing the concentrate mixture. An in vitro experimentation outside living animals to evaluate Moringa oleifera meal incorporation in total mixed ration on digestibility is cheap, short duration and having more control condition (Makkar, 2004) as compared to an in vivo evaluation on livestock which is costly and time-consuming event.

It was hypothesized that incorporation of Moringa oleifera meals in total mixed ration will improve in vitro fermentation kinetics. The study was planned to assess the effect of incorporation of various levels of Moringa oleifera meals in TMR on in vitro digestibility, total gas production (TGP) and metabolizable energy (ME) content for growing crossbred heifer calves.

MATERIALS AND METHODS

The study was undertaken at Animal Nutrition Research Department, College of Veterinary Science and A. H., Anand Agricultural University, Anand, Gujrat, India  during 2020–2021 after an ethical approval (LRS/322/2020) from the Institutional Animal Ethics Committee of College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand.

Green hybrid napier, Moringa oleifera fodder, groundnut straw and compound concentrate mixture were procured from Livestock Research Station, Anand Agricultural University, Anand was used for this experiment. Green hybrid napier was chopped 5-6 cm length and sun dried on cemented floor for three days. PKM 1 variety of Moringa oleifera fodder was harvested after 75 days showing at 1.5 feet above the ground level was used for study. Moringa fodder was sun dried for 3 days on cemented yard. Leaves and stems were separated after drying moisture level near 10 %. Leaves and soft twigs were used for this study. The soft twigs of Moringa fodder were ground using grinder with 2 mm sieve. Eighty four percent leaves and sixteen percent soft twigs of Moringa oleifera was mixed to prepare Moringa oleifera meal (MOM). After sun drying of green ingredients, all these ingredients were oven dried for twenty-four hours at 70°C and finely ground in hammer mill using 1 mm sieve. These ingredients were mixed in ratios shown in Table 1 to prepare different total mixed ration (TMR). The TMR was prepared to meet nutritional requirement of crossbred dairy heifer calves weighing 100 kg and growing at 700 g day^-1 as per NRC (2001) standard. The calculated nutritional values of TMRs was 17.46±0.004% crude protein (CP), 2.54±0.005 Mcal ME kg^-1 DM and 65.43±0.225% TDN.

Table 1: Levels of various ingredients (%) in different TMR

The ingredients were analyzed for proximate constituents (Anonymous, 1995) and fiber fractions (Van Soest, 1991) and that of different TMRs were calculated based on actual parameters of analysis.  The compounded concentrate mixture of TMR replaced with 0, 2.5, 5.0, 7.5, 10.0, 12.5, 15.0, 17.5, 20.0 and 22.5 percent of MOM, was designated as T₁, T₂, T₃, T₄, T₅, T₆, T₇, T₈, T₉ and T₁₀, respectively to ascertain the effect on in vitro dry matter digestibility, in vitro TGP, and ME content. Three crossbred calves of same age and breed was selected as donor of rumen inoculums for in vitro study. The crossbred heifer calves used to collect rumen liquor, were fed to meet nutrients requirement as per NRC (2001) with free access to clean and wholesome potable water. Rumen liquor was collected using negative pressure pump with stomach tube from three crossbred heifer calves. The rumen liquor from three crossbred heifer calves was strained through four-layer muslin cloth and mixed. This fluid was termed strained rumen liquor (SRL). 500 mg of each TMR with various levels of compounded concentrate mixture and MOM were incubated at 39±1°C for 48 h in a shaker twin water bath with 40 ml of fresh McDougall buffer and 10 ml SRL as described by Menke et al.  (1979) in triplicate. Fresh macro and micro buffer solutions to be use in incubation was prepared and kept in incubator at 39 ⁰C. After incubation of 48 hours, the content of each syringe was filtered through dried and pre-weighed Gooch crucible, which was again dried and weighed. The blank without TMR also incubated in triplicate to arrive at net gas production. Gas production was measured at 1, 2, 3, 4, 5, 8, 10, 12, 20, 24, 26, 30, 44 and 48 hours after incubation and line chart plotted. The metabolizable energy (ME) content of TMR was calculated as per Elghandour, (2015) and the metabolizable energy content of TMR was converted in Mega-calorie (Mcal). 

ME (MJ/kg DM) = 2.20+0.136 Gp+0.057 CP%, (R²=0.94)

Where, CP is crude protein% and Gp is ml of net gas production from 200 mg dry sample.

The digestibility of dry matter, TGP and ME were analyzed (Snedecor and Cochran, 1994) using online WASP- Web Agri State Package software developed by Central Coastal Agricultural Research Institute, Goa.

RESULTS AND DISCUSSION

The proximate and fiber fractions composition of ingredients and TMR used in experiment are presented in Table 2. The total gas production, in vitro dry matter digestibility (IVDMD) and metabolizable energy of different TMRs are presented in Table 3. An average content of crude protein (CP), ether extract (EE), crude fibre (CF), nitrogen free extract (NFE), total ash (TA), neutral detergent fibre (NDF), acid detergent fibre (ADF) and total digestible nutrients was 17.46±0.004, 3.22±0.006, 24.53±0.102, 43.98±0.032, 10.87±0.144, 50.14±0.266, 39.82±0.158 and 65.43±0.225%, respectively. An average metabolizable energy (ME) of TMRs was 2.54±0.005 Mcal/kg DM. Dey et al. (2014) had reported comparable crude protein (26.34±0.67%) of Moringa leaves. However, Nouala et al.  (2006) had reported slight lower CP (23.27%), NDF (18.74%) and ADF (16.07%) of Moringa oleifera leaves. Seradja et al.  (2019) also reported lower crude protein as 20.1, 21.0 and 20.4% of DM of Moringa forage consist of leaves, twigs and new buds harvested at 30, 40 and 50 days after pruning, this variation may be due to different agro-climatic zone and variety of Moringa.

Table 2: Average proximate composition, fiber fractions (% on DM basis) and energy value of feeds and fodder

Table 3: Total gas and IVDMD during in vitro fermentation and ME content of ration

3.1. In vitro total gas production (IVTGP)

An in vitro gas production of TMRs incorporated with different levels of MOM was significantly (p<0.05) higher compared to control TMR. However, incorporation of MOM in total mixed ration at 5.0 percent had revealed significant (p<0.05) higher IVTGP, and further higher incorporation of MOM in TMR was without improvement in total gas production. Results of in vitro fermentation shown significantly (P<0.05) higher total gas production when compounded concentrate mixture was replace with MOM at 5% and higher levels. A gas production characteristic was presented in Figure 1. The lag phase time was about one hour and similar in all the TMRs. 

Figure 1: In vitro gas production (ml) at different time interval

The Moringa leaves containing feed mixture produce significant effect on fermentation which resulted in higher (p<0.001) gas production (up to +2.30%) and the values of gas production were 144.13, 194.13, 149.15, 152.56 and 155.96 ml g^-1 DM on in vitro incubation of Moringa oleifera leaves (ML) replacing the wheat straw (WS) @ 0, 100, 5, 10 and 20%, respectively for buffalo as revealed the study of Dey et al.  (2014). Similarly, linearly higher (p<0.0001) in vitro gas production of feed mixture at 24 hours incubation was noted by Nouala et al. (2006) when concentrate (40%) part replaced with 0, 25, 50 and 100% Moringa oleifera, and roughage part of feed mixture was ground nut hay (60%). In contrast to the present study on initiation of gas production, the discrete lag phase reduction (P=0.04) was observed from 1.1 to 0.0 hours when in vitro incubation of alfalfa hay to Moringa oleifera forage cut at 40 days after pruning, respectively (Seradja et al., 2019).  

In present experiment, higher in vitro gas production might be due to higher limiting amino acids like lysine and Methionine (14.06 and 4.97 g kg^-1 DM, respectively) in the protein of M. oleifera leaves (Henuk, 2018) and optimal composition of amino acids with higher digestible protein content (Babiker et al., 2017) which is essential for rumen microbial multiplication and growth. The early gas production was not observed (no leg phase difference) on replacement of compounded concentrate mixture with Moringa oleifera meal in present study may be due to high protein concentrate mixture was replace with Moringa oleifera meal and both have readily degradable carbohydrate and other nutrients for initiation of microbial activity during substrate fermentation.

3.2.  In vitro DM digestibility (IVDMD)

An in vitro rumen fermentation data revealed significant (p<0.05) effect of incorporation of MOM in total mixed ration at 7.5% on in vitro dry matter digestibility (64.678±0.679%) in comparison to control TMR (57.459±0.512%) and TMR containing 2.5% MOM (56.898±1.322%). Trend on in vitro digestibility of dry matter was observed to be variable, however effect of incorporation of MOM @ 7.5 and 20.0% in TMR was significant (p<0.05). The replacement of compounded concentrate mixture with MOM at 7.5% in total mixed ration was found beneficial.

An in vitro higher (p<0.001) true digestibility was observed when feed mixture, (30 and 40% concentrate and 70 and 60% ground nut hay- roughage, respectively) incubated for 24 hours with 50, 75 and 100% concentrate replaced by Moringa oleifera as reported by Nouala et al. (2006). The study of Dey et al.  (2014) revealed higher (p<0.001) true degradability of dry matter (up to +11.47%) on in vitro incubation of Moringa oleifera leaves replacing the wheat straw (WS) @ 0, 100, 5, 10 and 20%, respectively for buffalo. The true dry matter degradability was 39.82, 86.40, 45.63, 49.58 and 52.72%, respectively. The higher in vitro dry matter degradability in study might be owing to roughage portion was 50% of total mixed rations and improvement in dry matter degradability suggest strong candidate to replace high protein concentrate mixture for growing crossbred heifers. The higher degradability of TMRs with various levels of Moringa oleifera meal might be due to higher rumen microbial growth and multiplication as Moringa oleifera meal contains higher digestible protein with optimal composition of amino acids (Babiker et al., 2017) and negligible tannin and other anti-nutritive compounds (Makkar and Becker, 1996).   

3.3.  Metabolizable energy (ME) content of TMR

The ME content of TMR obtained in present experiment was ranges from 1.685±0.512 to 3.223±0.057 Mega calories per kilogram of TMR dry matter. The value observed in present experiment was both lower and higher than calculated value of metabolizable energy of TMR. The metabolizable energy of TMR (3.137±0.056 Mcal kg^-1 DM) significantly improved at 5.0% MOM incorporation in comparison to control TMR (1.685±0.047 Mcal kg^-1 DM). Further higher level of MOM incorporation was without significant effect on improvement of energy levels. An incorporation of 5% and higher level was found sufficient to improve energy content of TMR.

Nouala et al.  (2006) reported that the ME content of Moringa oleifera forage harvested at 30, 40 and 50 days post pruning was higher (10.97, 10.98 and 9.92 MJ kg^-1 DM, respectively; P<0.01) in comparison to second cut alfalfa hay (8.33 MJ kg^-1 DM) at 24 hour in vitro incubation utilizing ewe rumen fluid. In present study, ME content of TMRs with Moringa oleifera meal was higher than reported by Nouala et al.  (2006) owing to TMR has 50% concentrate ingredients and concentrate component was replace with 2.5 to 22.5 Moringa oleifera meal on protein equivalent basis. The incorporation of Moringa oleifera meal 5% and above up to 22.5% in TMR to replace high protein concentrate mixture was found suitable to improve ME content of rations.       

An in vitro investigation (Lunagariya et al., 2017) of exogenous fibrolytic enzymes (EFE) @ 0, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380 and 400 mg kg^-1 TMR in the ration of dairy cows weighing 500 kg and producing 12 kg 4% fat corrected milk (FCM) per day, resulted in the significantly (p<0.05) higher and optimum in vitro digestibility of DM (63.03%) and OM (63.62%) as well as Total Gas Production (72.35 ml 500 mg^-1 TMR) at supplementation of 240 mg EFE kg^-1 TMR, along with better ME (7.16 MJ kg^-1 DM) and MBP (97.63 mg 500 mg^-1 TMR) content of ration. An in vivo investigation on optimum EFE (240 mg kg^-1 TMR) in crossbred cows yielded significantly higher 4% FCM and milk fat with improved feed conversion efficiency and better economic returns in HF crossbred cows (Lunagariya et al., 2019a) and improved digestibility of dry matter and nutrients as well as higher body weight gain in dry non-pregnant Gir and crossbred dairy cows (Lunagariya et al., 2019b). An application of in vitro investigation to reduce numbers of treatments and animals for in vivo investigation was found beneficial along with economically effective, less time consuming and allows more control over experiment as reported by Makkar (2004). Looking to these investigations and present study, it can be suggested that an incorporation of Moringa oleifera meal @ 5.0 with increment levels of 2.5% and higher levels up to 22.5% to replace high protein concentrate mixture in total mixed ration found suitable for in vivo investigation.  

CONCLUSION

An incorporation of Moringa oleifera meal @ 5.0 with increment levels of 2.5% up to 22.5% to replaced high protein concentrate mixture (50%) in TMR improved (p<0.05) dry matter digestibility (60.879±0.251 to 64.678±0.679%), gas production (81.000±9.018 to 95.667±1.764 ml 500 mg^-1 TMR) and ME content (2.356±0.068 to 3.223±0.057 Mcal ME kg^-1 TMR) of total mixed ration which was suitable for in vivo growth experiment.

ACKNOWLEDGEMENT

Author(s) acknowledge the financial and logistic supports from Anand Agricultural University, Anand.

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