Extract of Moringa oleifera leaves improves feed utilization of lactating Nubian goats

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Research Paper

Extract of Moringa oleifera leaves improves feed utilization of lactating Nubian goats ⁎

A.E. Kholifa, , G.A. Goudaa, U.Y. Aneleb, M.L. Galyeanc a b c

Dairy Science Department, National Research Centre, 33 Bohouth St. Dokki, Giza, Egypt North Carolina Agricultural and Technical State University, Greensboro, NC, 27411, USA Office of the Provost, Texas Tech University, Lubbock, TX, 79409, USA

A R T I C L E I N F O

A B S T R A C T

Keywords: Feed utilization Moringa oleifera Nubian does Phytogenic extracts Ruminal fermentation

The present experiment aimed to assess the effect of providing oral doses of Moringa oleifera leaf extract on feed intake, nutrient digestion, and ruminal and blood serum measurements in goats. Sixteen lactating Nubian does (36.5 ± 0.6 kg) were used in a quadruplicated 4 × 4 Latin square design over an 88-day period. An aqueous M. oleifera extract was supplemented orally to each doe at doses of 0 (Control treatment), 10 (ME10 treatment), 20 (ME20 treatment) or 40 mL (ME40 treatment). Compared with control, M. oleifera extract linearly increased (P < 0.01) nutrient intake and digestibility of dry matter, organic matter, and neutral detergent fiber, without affecting digestibility of crude protein and ether extract. Without affecting ruminal pH and ammonia-N, M. oleifera extract increased (P < 0.05) total short-chain fatty acids (SCFA), branched-chain SCFA, and propionic acid concentrations; however, the extract linearly decreased (P < 0.01) acetic/propionic ratio and calculated methane production. Increased (P < 0.01) serum albumin and glucose concentrations, and decreased (P < 0.05) cholesterol, triglycerides, glutamic oxaloacetic transaminase and glutamic pyruvic transaminase concentrations were noted with the inclusion of M. oleifera extract. It is concluded that an oral dose of M. oleifera extract enhanced feed intake and digestibility and ruminal fermentation in lactating Nubian does. Although further research is needed, performance responses associated with increasing the dose of M. oleifera extract to 40 mL/doe were not large; thus, the 20 mL dose is recommended for practical use.

1. Introduction Increasing feed utilization and productive performance of ruminants through improving animal health and feed utilization and by altering the microbial ecosystem and ruminal function are the main goals of animal nutritionists and microbiologists. The inclusion of antibiotics and ionophores enhances feed efficiency and nutritive value; however, increased regulations and public concerns about their metabolites and residues, as well as anti-microbial resistance, have compelled researchers to explore alternative strategies to improve performance (Matloup et al., 2017). Potential alternative strategies to replace antibiotics include the use of exogenous enzymes (Morsy et al., 2016), live yeast (Hassan et al., 2016), herbal plants (Kholif et al., 2017a), phytogenic extracts (Valdes et al., 2015), and essential oils (Matloup et al., 2017) to enhance nutrient utilization and animal productivity. Moringa oleifera Lam (syns. Moringa pterygosperm, family

Moringaceae) is a tree distributed almost worldwide. M. oleifera is a good source of protein (Kholif et al., 2015, 2016) with an excellent fatty acid and amino acid profiles (Sánchez-Machado et al., 2010). Moreover, M. oleifera is rich in bioactive compounds such as essential oils, saponins, and tannins, which are present in different parts of the plant (Mendieta-Araica et al., 2011a; Salem et al., 2014; Kholif et al., 2015, 2016). These compounds often have some antimicrobial and anthelmintic properties, which can improve feed utilization by ruminants and animal performance (Valdes et al., 2015). Phytogenic extracts and plant parts containing these bioactive compounds might provide a low-cost alternative for improving feed utilization and lactational performance (Mendieta-Araica et al., 2011a; Kholif et al., 2015), and experimental evidence suggests that phytogenic extracts in diets of ruminants can improve feed efficiency and animal productivity (Valdes et al., 2015). The positive effects of such phytogenic extracts are mainly a result of the secondary metabolites such as tannins and saponins, which have the

Abbreviations: ADF, acid detergent fiber expressed exclusive of residual ash; BW, body weight; CH4, methane; CF, crude fiber; CP, crude protein; DE, digestible energy; DM, dry matter; EE, ether extract; GOT, glutamate-oxaloacetate transaminase; GPT, glutamate-pyruvate transaminase; ME, metabolizable energy; NDF, neutral detergent fiber expressed exclusive of residual ash; NFE, digestible nitrogen free extract; NSC, non-structural carbohydrates; OM, organic matter; SCFA, short-chain fatty acids; TDN, total digestible nutrients ⁎ Corresponding author. E-mail address: [email protected] (A.E. Kholif). http://dx.doi.org/10.1016/j.smallrumres.2017.10.014 Received 13 April 2017; Received in revised form 4 August 2017; Accepted 31 October 2017 0921-4488/ © 2017 Elsevier B.V. All rights reserved.

Please cite this article as: Kholif, A.E., Small Ruminant Research (2017), http://dx.doi.org/10.1016/j.smallrumres.2017.10.014

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Table 1 Composition of ingredients and experimental basal diet fed to the lactating Nubian goats (g/kg DM basis). Item

Crushed yellow corn

Soybean meal

Wheat bran

Berseem clover (Trifolium alexandrinum)

Basal (Control) diet1

Dry matter (g/kg wet material) Organic matter Crude protein Ether extract Non-structural carbohydrates Neutral detergent fiber Acid detergent fiber Lignin Cellulose Hemicellulose

866 890 91 45 540 214 89 10 79 125

889 928 408 21 356 143 96 9 87 47

871 852 130 56 204 462 131 38 93 331

141 882 133 25 301 423 324 48 276 99

564 874 173 32 370 299 186 27 159 113

1 The control diet consisted of (per kg DM): 400 g of Egyptian berseem clover (Trifolium alexandrinum), 300 g crushed corn, 200 g soybean meal, 80 g wheat bran, 10 g limestone, 5 g salt, and 5 g mineral and vitamin mixture [containing per kg: 141 g Ca, 87 g P, 45 g Mg, 14 g S, 120 g Na, 6 g K, 944 mg Fe, 1613 mg Zn, 484 mg Cu, 1748 mg Mn, 58 mg I, 51 mg Co, 13 mg Se, 248000 IU vitamin A, 74000 IU vitamin D3, and 1656 IU vitamin E].

using the percent relative peak area. A tentative identification of the compounds was performed based on the comparison of their relative retention time and mass spectra with those of the NIST, WILLY library data of the GC–MS system. A total of 30 peaks from the leaf extract of M. oleifera were detected in the GC–MS chromatograms, with the retention time ranging from 16.64 to 61.11 min; all were identified as C10 to C43 compounds.

ability to alter ruminal fermentation (Salem et al., 2014). To the best of our knowledge, no information is available on the effect of an aqueous M. oleifera extract on feed efficiency; however, several studies on the inclusion of M. oleifera leaves or silages in diets of lactating cows, sheep and goats have been published (Sultana et al., 2015; Babiker et al., 2016; Zeng et al., 2017). More recently, inclusion of M. oleifera leaves in ruminant diets resulted in quantitative and qualitative improvement in animal performance (Mendieta-Araica et al., 2011b; Cohen-Zinder et al., 2016; Kholif et al., 2016). We hypothesized that the bioactive compounds in an aqueous M. oleifera extract will enhance feed utilization and ruminal fermentation of does. Therefore, the present study aimed to investigate the effect of providing the extract orally at different doses on nutrient utilization, digestibility, and ruminal fermentation in lactating does.

2.2. Does, feeding and experimental design Does were cared and handled in accordance with the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching (FASS, 2010). During the first week of lactation, sixteen lactating Nubian does, weighing 36.5 ± 0.6 kg body weight (BW), were randomly assigned to four experimental treatments in a quadruplicated 4 × 4 Latin square design, with four treatments, four periods and four does per treatment within each period, resulting in 16 replicates per treatment for the experiment. The experimental treatments were assigned randomly to the four groups in the first period, after which a predetermined sequence was followed that allowed each doe to receive each treatment. Does were individually housed in soil-surfaced pens (1.5 m2/doe) under shade without bedding and with free access to water. They were offered the experimental diets to meet their nutrient requirements according to NRC (2007) recommendations. Adjustments were made to the feed offered to ensure collection of orts. Does were weighed at the beginning and at the end of each experimental period. The basal diet fed to the does contained (per kg, DM basis): 400 g of Egyptian berseem clover (Trifolium alexandrinum), 300 g crushed yellow corn, 200 g soybean meal, 80 g wheat bran, 10 g limestone, 5 g minerals/vitamins mixture, and 5 g table salt. The chemical composition of the ingredients and basal diet is shown in Table 1. Does were fed the basal diet supplemented with the extract at (per doe daily): 0 mL (Control treatment), 10 mL (ME10 treatment), 20 mL (ME20 treatment), or 40 mL (ME40 treatment). Diets were offered to each doe individually at 08:00 and 16:00 h in two equal portions. The extract was administered orally to individual does, once daily, with a 20-mL syringe before the morning feeding at 08:00 h to ensure the full dose was received. Each experimental period lasted 22 days; 15 days of adaptation to the new diet and 7 days for sample collection (sampling of feed and orts, feces, ruminal fluid, and blood). Milk yield, composition and fatty acids profile also were measured during the last 7 days of each period, but those data are not reported in the present manuscript.

2. Materials and methods 2.1. Moringa oleifera extract preparation Plant leaves of M. oleifera were collected randomly from several young and mature trees, freshly chopped into 1- to 2-cm lengths, and immediately extracted at 1 kg dry matter (DM) leaf/8 L of water. Plant materials were soaked and incubated in water at 25–30 °C for 72 h in closed 25-L jars. After incubation, jars were heated to 39 °C for 1 h, and then immediately filtered with gauze, discarding the solid fraction and retaining the liquid fraction for further use. The extract was prepared weekly and stored at 4 °C for daily use. The concentration of total tannins in M. oleifera leaves was determined according to Makkar (2003), and total phenolic content was determined chromatographically as described by Meier et al. (1988). Assuming 100% extraction efficiency, 8 L of the extract would contain 22 g total tannins and 48 g total phenolics or 2.75 g of tannins and 6 g of total phenolics per liter. As previously reported in Kholif et al. (2017b), for determination of chemical constituents of the extract, M. oleifera leaves (100 g) were soaked in 150 mL of methanol, acetone, and hexane (1:1:1 v/v; HPLC grade) solvent at room temperature. After 24 h of soaking, the extract was filtered through Whatman No.1 paper and over active charcoal to remove chlorophyll. The extract was then concentrated to 20 mL and lyophilized with a freeze dryer (Alpha 1–4 LDplus, Martin Christ, Osterode am Harz, Germany) to obtain dried extract (Valdes et al., 2015). At the Central Laboratory of National Research Centre (Egypt), 10 mL of the extract was analyzed using GC–MS (Thermo Scientific, Trace GC Ultra/ISQ Single Quadrupole MS, TG-5MS fused silica capillary column (30 m long, 0.25 mm internal diameter, and 0.1-mm film thickness)). For GC–MS detection, an electron ionization system with ionization energy of 70 electron volts (eV) was used, and helium was the carrier gas at a constant flow rate of 1 mL/min. Injector and MS transfer line temperatures were set at 280 °C. Quantification of all the identified components was investigated

2.3. Nutrient digestibility, and chemical analyses During the collection period (i.e., the last 7 days of each period), feed intake was recorded daily by weighing the offered diets and 2

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at 4000 × gfor 20 min. Serum was separated into 2-mL clean dried Eppendorf tubes and frozen at −20 °C until analysis. Concentrations of total protein, albumin, urea-N, glutamate-oxaloacetate transaminase (GOT), glutamate-pyruvate transaminase (GPT), glucose, creatinine, cholesterol and triglycerides were analyzed in blood serum samples using specific kits (Stanbio Laboratory, Boerne, TX) following manufacturer instructions. Globulin concentration was calculated as the difference between total protein and albumin.

refusals from the previous day. In addition, all feces excreted were totally collected twice daily from each doe at 07:00 and 15:00 h, according to the methodology proposed in Kholif et al. (2016), and stored at −10 °C for later analysis. A sample of about 100 g (as-is basis) of feces from each doe was taken daily and pooled by doe within period. The composited samples were dried in a forced-air oven at 65 °C for 72 h then ground along with feed and orts samples to pass a 1-mm screen using a Wiley mill grinder (Arthur H. Thomas, Philadelphia, PA) and retained for later determination of compositional analyses. Diets nutritive and energy values included total digestible nutrients (TDN), digestible crude protein (CP), digestible energy (DE), metabolizable energy (ME), and net energy for lactation (NEL), which were calculated using NRC (2001) equations: TDN (%) = digestible CP + digestible EE × 2.25 + digestible crude fiber (CF) + digestible nitrogen free extract (NFE); DE (Mcal/kg) = 0.04409 × TDN (%); ME (Mcal/kg) = 1.01 × DE (Mcal/kg) − 0.45; NEL (Mcal/kg) = 0.0245 × TDN (%) − 0.12; digestible CP (%) = digestible CP × CP% (Mcal/ kg = 4.19 MJ/kg; data for CF and NFE are not shown in tables). Feed, orts, and fecal samples were analyzed for DM (method 930.15), ash (method 942.05), nitrogen (method 954.01), and ether extract (EE; method 920.39), according to AOAC (1997) official methods. Neutral detergent fiber (NDF) was determined by the procedure of Van Soest et al. (1991) without the use of alpha amylase but with sodium sulfite. Acid detergent fiber (ADF) was analyzed according to AOAC (1997. Lignin was analyzed by solubilization of cellulose with sulfuric acid in the ADF residue according to Van Soest et al. (1991). Non-structural carbohydrates (NSC; = 1000 – (NDF + crude protein (CP) + EE + ash); cellulose (NDF – lignin); hemicellulose (NDF – ADF); and organic matter (OM; 1000 – ash) were calculated.

2.6. Statistical analyses Data were analyzed using a quadruplicated 4 × 4 Latin square design, with four periods and four treatments. Individual goats were the experimental units. The statistical model included the fixed effect of square and treatment and the random effects of period and goat nested within square: Yijkl = μ + Si + Tj + Pk + Gl(Si) + Eijkl, where Yijkl is each individual observation for a given variable, μ is the overall mean, Si is the square effect, Tj is the treatment effect, Pk is the period effect, Gl(Si) is the effect of goat within square, and Eijkl is the residual error. Statistical analyses were performed using PROC MIXED of SAS (SAS Inst., Inc.; Cary, NC). The probability of difference option of the least squares means statement was used for multiple comparisons of means, and polynomial (linear and quadratic) contrasts (adjusted for the unequal spacing of treatments) were used to examine dose responses to increasing doses of M. oleifera extract and for comparison between control vs. the average of M. oleifera treatments. Significance was declared at a level of P ≪ 0.05. 3. Results

2.4. Sampling and analyses of ruminal fluid

3.1. Weight changes, feed intake, nutrient digestibility, and diet nutritive value

On the last day of each experimental period, ruminal contents were sampled at 0, 3, and 6 h after the morning feeding to determine the pH and concentration of fermentation end-products. Because the fermentation process reached its peak activity at approximately 3 h of after the morning feeding, only data of ruminal fermentation at 3 h are presented. Ruminal contents (approximately 100 mL) were collected by using a stomach tube and hand pump, and then composite samples taken from each doe were strained through 4 layers of cheesecloth. The pH of ruminal fluid was measured immediately using a pH meter (HI98127 pHep®4 pH/Temperature Tester, Hanna® Instruments, Italy). A subsample of 5 mL was preserved in 5 mL of 0.2 M HCl for ammonia-N analysis, and 0.8 mL of ruminal liquor was mixed with 0.2 mL of a solution containing 250 g of metaphosphoric acid/L for total short chain fatty acids (SCFA) analysis. All samples were stored at −20 °C until analyzed in the laboratory. The concentration of ruminal ammonia-N was determined according to AOAC (1997). Individual SCFA were measured by gas-liquid chromatography (Varian 3700; Varian Specialties Ltd, Brockville, Ontario, Canada). The separation process was carried out with a capillary column (30 m × 0.25 mm internal diameter, 1-mm film thickness, Supelco Nukol; Sigma–Aldrich, Mississauga, ON, Canada) with flame ionization detection. The column temperature was adjusted to 100 °C for 1 min, then increased by 20 °C/ min to 140 °C, followed by 8 °C/min to 200 °C, and held at this temperature for 5 min. The injector temperature was 200 °C, with the detector temperature set at 250 °C, and helium was the carrier gas. Methane (CH4; mmol/L) was calculated using the equation of Moss et al. (2000) from SCFA composition as: CH4 = 0.45 (acetic) − 0.275 (propionic) + 0.4 (butyric).

No differences were observed for initial (P = 0.162), final (P = 0.135), and BW changes (P = 0.916) among the different treatments (Table 2). M. oleifera extract increased (P ≤ 0.001) nutrient intake compared with the control. Increasing the dose of extract also resulted in a positive linear (P ≪ 0.001) effect on nutrient intake, with the greatest values noted for ME40 treatment. Compared with the control treatment, the ME10, ME20, and M40 treatments increased (P ≪ 0.001) digestibilities of DM, OM, NSC, NDF, hemicellulose, cellulose (P = 0.004), and ADF (P ≪ 0.019). The greatest digestibilities were noted (linear [P ≪ 0.01] and quadratic [P ≪ 0.05] effects) in the ME20 and ME40 treatments (Table 2). As a result of the increased nutrient digestibility for the various dietary components, greater (P ≪ 0.001) calculated values for TDN, DE, ME, and NEL were observed with supplementation of M. oleifera extract (Table 2). 3.2. Ruminal fermentation and blood serum measurements Feeding ME10, ME20, and ME40 diets did not affect (P ≫ 0.05) ruminal pH, and concentrations of ammonia-N, butyric acid, valeric, and branched SCFA (Table 3). Greater total SCFA (P = 0.003), and propionic acid (P ≪ 0.001) were observed with the M. oleifera extract treatments. A linear effect (P = 0.005) of supplementing the extract was noted for total branched SCFA concentration, with the ME40 treatment being greater (P ≪ 0.05) than the control. The treatments ME20 and ME40 had the highest (linear effect, P ≪ 0.001) concentrations of total SCFA, acetic acid, and propionic acid compared with control treatment. Acetic acid concentration responded in a linear manner (P = 0.003) to the dose of M. oleifera extract, with the with ME40 treatment being greater than the control and intermediate values for the ME10 and ME20 treatments. Decreases (P ≤ 0.001) in acetic/ propionic ratio, calculated CH4, and CH4/total SCFA ratio were observed with ME10, ME20, and ME40 treatments compared with the

2.5. Sampling and analyses of blood serum On the last day of each experimental period, blood samples (10 mL) were taken 4 h after feeding from the jugular vein of each doe into a clean dry tube without anticoagulants. Blood samples were centrifuged 3

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Table 2 Body weight, feed intake, nutrient digestibility and nutritive value of diets fed to lactating Nubian goats (n = 16) supplemented with different doses of Moringa oleifera extract. Treatments1

SEM

Control

ME10

ME20

ME40

Body weight Initial (kg) Final (kg) Change (g/d)

36.4 35.0 −6.3

36.1 34.6 −6.8

36.7 35.6 −5.0

36.6 35.4 −5.4

0.20 0.31 2.01

Intake (g/d) Dry matter Organic matter Crude protein Ether extract Non-structural carbohydrates Neutral detergent fiber Acid detergent fiber Cellulose Hemicelluloses

751a 656a 130a 24.3a 278a 224a 140a 119a 84.6a

788b 689b 136b 25.6b 292b 235b 146b 125b 88.9b

808bc 706bc 139bc 26.2bc 299bc 242bc 150bc 128bc 91.2bc

828c 723c 143a 26.8c 306c 247c 154c 132c 93.4c

Digestibility (g absorbed/kg ingested) Dry matter Organic matter Crude protein Ether extract Non-structural carbohydrates Neutral detergent fiber Acid detergent fiber Cellulose Hemicellulose

541a 556a 570 586 557a 552a 538a 567a 551a

567b 583b 568 593 573b 567b 553ab 590b 569b

586c 588b 561 591 587c 594c 567b 591b 581b

Digestible nutrients and energy value2 Total digestible nutrients (g/kg DM) Digestible crude protein (g/kg DM) Digestible energy (MJ/kg DM) Metabolizable energy (MJ/kg DM) Net energy of lactation (MJ/kg DM)

510a 98.3 2.25a 2.27a 1.13a

533b 98.0 2.35b 2.38b 1.19b

538b 96.8 2.37b 2.40b 1.20b

P-value

Contrast Control vs. Moringa

Linear

Quadratic

0.162 0.135 0.916

0.628 0.592 0.806

0.364 0.323 0.710

0.187 0.156 0.696

10.7 9.4 1.9 0.36 4.0 3.2 2.0 1.7 1.21

≪ 0.001 ≪ 0.001 ≪ 0.001 0.001 ≪ 0.001 ≪ 0.001 ≪ 0.001 ≪ 0.001 ≪ 0.001

≪ 0.001 ≪ 0.001 ≪ 0.001 ≪ 0.001 ≪ 0.001 ≪ 0.001 ≪ 0.001 ≪ 0.001 ≪ 0.001

≪ 0.001 ≪ 0.001 ≪0.001 ≪ 0.001 ≪ 0.001 ≪ 0.001 ≪ 0.001 ≪ 0.001 ≪ 0.001

0.103 0.103 0.120 0.086 0.105 0.099 0.114 0.094 0.098

588c 590b 558 596 589c 595c 565b 592b 583b

6.4 4.1 5.6 5.7 4.2 5.4 7.0 5.5 4.6

≪ 0.001 ≪ 0.001 0.418 0.684 ≪ 0.001 ≪ 0.001 0.019 0.004 ≪ 0.001

≪ 0.001 ≪ 0.001 0.257 0.305 ≪ 0.001 ≪ 0.001 0.005 0.004 ≪ 0.001

≪ 0.001 ≪ 0.001 0.110 0.294 ≪ 0.001 ≪ 0.001 0.009 0.008 ≪ 0.001

0.005 0.005 0.794 0.854 0.008 0.013 0.073 0.020 0.008

539b 96.3 2.38b 2.40b 1.20b

3.7 0.96 0.016 0.016 0.009

≪0.001 0.418 ≪0.001 ≪0.001 ≪0.001

≪0.001 0.257 ≪0.001 ≪0.001 ≪0.001

≪0.001 0.110 ≪0.001 ≪0.001 ≪ 0.001

0.005 0.794 0.005 0.005 0.005

Means in the same row with different superscripts differ, P ≪ 0.05. P-value is the observed significance level of the F-test for treatment; SEM = standard error of the mean. 1 The goats were fed a basal diet and given an oral dose of Moringa oleifera extract at 0 mL (Control treatment), 10 mL (ME10 treatment), 20 mL (ME20 treatment), or 40 mL (ME40 treatment)/goat daily. 2 Calculated according to NRC (2001).

Table 3 Ruminal fermentation and blood serum measurements in lactating Nubian goats (n = 16) supplemented with different doses of Moringa oleifera extract. Treatments1 Item Ruminal fermentation measurements pH Ammonia-N (g/L) Total short-chain fatty acids (SCFA; mmol/L) Acetic (mmol/L) Propionic (mmol/L) Butyric (mmol/L) Valeric (mmol/L) Iso-butyric (mmol/L) Iso-valeric (mmol/L) Acetic/propionic ratio Total branched SCFA (mmol/L) Branched SCFA (mmol/100 mmol of total SCFA) Methane (mmol/L)2 Methane/SCFA ratio

SEM

Control

ME10

ME20

ME40

6.00 29.2 112a 69.6a 22.7a 13.5 1.73 1.23 1.84 3.06a 3.07a 2.78 27.5a 25.0a

5.97 28.5 119b 75.2ab 27.0b 13.1 2.01 1.44 1.99 2.78b 3.43ab 2.84 26.2b 21.9b

5.92 28.5 124bc 75.5ab 27.9b 12.8 1.93 1.49 2.01 2.70bc 3.50ab 2.88 25.8bc 21.4b

5.90 28.5 127c 81.3b 30.9b 13.6 2.12 1.59 2.18 2.64c 3.77b 2.87 25.5c 19.4c

0.055 0.52 2.4 2.31 0.80 0.54 0.175 0.106 0.112 0.034 0.148 0.108 0.17 0.66

3.65b 75.0b 127b 154b 17.4b 26.1b

3.72b 76.2bc 128b 150b 17.0b 24.9b

3.73b 77.4c 127b 146b 16.1b 24.3b

0.047 0.75 2.1 3.2 0.95 0.93

Blood serum measurements (g/L, unless stated otherwise) Albumin 3.49a Glucose 70.8a Cholesterol 137a Triglycerides 164a Glutamic-pyruvic transaminase (Units/L) 20.7a Glutamate-oxaloacetate transaminase (Units/L) 28.8a

P-value

Contrast Control vs. Moringa

Linear

Quadratic

0.515 0.704 0.003 0.020 ≪ 0.001 0.739 0.457 0.145 0.255 ≪ 0.001 0.030 0.900 ≪ 0.001 0.001

0.262 0.242 0.001 0.010 ≪ 0.001 0.675 0.159 0.037 0.116 ≪ 0.001 0.010 0.497 ≪ 0.001 ≪ 0.001

0.150 0.404 ≪ 0.001 0.003 ≪ 0.001 0.776 0.181 0.035 0.054 ≪ 0.001 0.005 0.563 ≪ 0.001 ≪ 0.001

0.745 0.471 0.102 0.695 0.086 0.300 0.698 0.406 0.862 0.009 0.465 0.640 0.001 0.125

0.003 ≪ 0.001 0.006 0.003 0.009 0.007

0.004 ≪ 0.001 0.005 0.006 0.001 0.001

0.002 ≪ 0.001 0.017 0.006 0.004 0.002

0.031 0.007 0.022 0.124 0.100 0.108

Means in the same row with different superscripts differ, P ≪ 0.05. P-value is the observed significance level of the F-test for treatment; SEM = standard error of the mean. 1 The goats were fed a basal diet and given an oral dose of Moringa oleifera extract at 0 mL (Control treatment), 10 mL (ME10 treatment), 20 mL (ME20 treatment), or 40 mL (ME40 treatment)/goat daily. 2 Methane production (mmol/L) = 0.45 (acetic) − 0.275 (propionic) + 0.4 (butyric); Moss et al. (2000).

4

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supplemented diets. The positive effects observed in nutrient digestion with increasing levels of the extract suggest that the delivered levels of tannins, phenolic, and other secondary metabolites in the extract were within a range that did not impair the ruminal environment and microbial activity. It is well documented that ruminal microorganisms can degrade and utilize secondary metabolites (Frutos et al., 2004) at low levels, using them as energy sources without any negative effects on rumen fermentation. In their review, Frutos et al. (2004) noted that the level of tannins should be less than 5% in the feed to minimize any negative effects on nutrient digestibility. Much lower levels of bioactive compounds (secondary metabolites) were delivered to does in the present study. Similarly, previous studies (Mendieta-Araica et al., 2011b; Kholif et al., 2015) showed that feeding M. oleifera plant materials containing high levels of secondary metabolites had no negative effects on nutrient digestibility or ruminal fermentation activity, which also was observed by Cohen-Zinder et al. (2016) when they fed M. oleifera at 18% of the fed diet. Crude protein digestibility was unaffected by the administration of the extract; however, CP digestibility was numerically less, particularly with the ME20 and ME40 treatments. This effect might reflect the ability of tannins to bind protein and decrease ruminal protein degradability (Bodas et al., 2012), again suggesting that the levels of tannins and other secondary metabolites dosed in the present study were not sufficiently high to negatively alter fermentation and digestion. In agreement with the present results, but with a different extract, Valdes et al. (2015) reported an increased feed intake and enhanced feed digestion when lambs fed Salix babylonica extract at 30 mL daily. With feeding M. oleifera leaves, Kholif et al. (2015) also reported greater intake by lactating does.

control treatment, reflecting changes in propionate. The ME40 treatment had the lowest values for these calculated variables (linear effect, P≪0.001 for all variables and quadratic effect, P ≪ 0.01) for acetic/ propionic ratio and CH4. No effects were observed (P ≫ > 0.05) from supplementing the does with M. oleifera extract on the concentrations of blood serum total protein, globulin, urea-N, and creatinine (data not shown). Greater (P ≤ 0.003) albumin and glucose concentrations were observed with ME10, ME20, and ME40 treatments compared with the control treatment (Table 3). The ME20 and ME40 treatments had the highest concentrations (linear effect, P ≪ 0.01; quadratic effect, P ≪ 0.05) compared with the control treatment. In contrast to results for albumin and glucose, the ME10, ME20, and ME40 treatments decreased (P ≪ 0.05) cholesterol, triglycerides, GOT, and GPT concentrations in the serum. 4. Discussion 4.1. Principal chemical constituents As previously reported by Kholif et al. (2017b), the GC/MS results identified 30 compounds, and there were three major antimicrobial compounds representing 65.6% of total identified compounds: hexadecanoic acid, methyl ester at 236.7 mg/g DM (Rajeswari et al., 2013), 9,12,15-octadecatrienoic acid, methyl ester, (Z,Z,Z) at 313.5 mg/g DM (Rahman et al., 2014), and 9,12-octadecadienoic acid (Z,Z)-, methyl ester at 104.7 mg/g DM (Wei et al., 2011). Almost no information is available about the biological activity of most of the identified compounds; however, 9,12,15-octadecatrienoic acid, methyl ester, (Z,Z,Z) has been shown to have antibacterial, anti-inflammatory, hypocholesterolemic, hepatoprotective, nematicide, insectifuge, and 5-alpha reductase inhibitor activity (Devi and Muthu, 2014). Hexadecanoic acid, methyl ester has some antibacterial and antifungal properties and possesses antioxidant activity (Chandrasekaran et al., 2011). Other compounds, with antimicrobial activities were detected at somewhat lower concentrations: 10-methoxy-9-phenylphenanthrene at 64.4 mg/g DM, and octadecanoic acid, methyl ester at 43.8 mg/g DM (Meechaona et al., 2007). Octadecanoic acid methyl ester has antioxidant and antimicrobial activities (Rahman et al., 2014). Such biological activities may be responsible for the positive effects of feeding M. oleifera leaves extract observed in the present experiment. Further study of the concentrations of these and other components in extract is essential to explore the bioactive phyto-constituents of pharmaceutical importance.

4.3. Ruminal fermentation measurements With no difference between treatments, ruminal pH at 3 h after feeding ranged between 5.9 and 6.0. The reported range is within the acceptable range for fiber digestion (Ørskov and Ryle, 1990). Kholif et al. (2015) observed that ruminal pH was not affected by feeding M. oleifera leaves to lactating does. Ruminal ammonia-N concentration ranged from 28.5 to 29.2 g/L, which fails within the range reported by Satter and Slyter (1974) for maximal microbial growth and activity. Lack of effect in ruminal ammonia-N concentration with feeding M. oleifera extract is further evidence that the levels of M. oleifera extract used were optimal for ruminal fermentation. Kholif et al. (2016) observed a decrease in ruminal ammonia-N concentration with feeding M. oleifera leaves fresh, as hay or silage compared with a control diet. Greater SCFA concentrations with the administration of M. oleifera extract is likely a result of improved nutrient digestion, as their concentrations depend on feed digestibility, rate of absorption, and ruminal microflora activity (Kholif et al., 2016). The concentration of SCFA followed a similar trend to NSC digestibility, revealing that greater NSC digestibility could be a primary reason for greater SCFA concentrations, especially propionate. Furthermore, inclusion of M. oleifera extract increased the proportion of ruminal propionate, which is considered beneficial in dairy production (Kholif et al., 2016) because propionic acid is the primary gluconeogenic SCFA required for lactose biosynthesis (Linn, 1988). Other studies in which animals were fed secondary metabolites containing plants or extracts showed greater propionate and sometimes acetate concentrations (Bodas et al., 2012). Conversely, the greater acetic acid noted with the highest level of the extract (ME40 treatment) could have a beneficial effect on the concentration of milk fat (Linn, 1988), as will be discussed later. An increased acetic acid concentration could have resulted from enhanced fiber digestibility (Kholif et al., 2016) with the inclusion of the extract. Altered ruminal individual SCFA might be related to the positive effects of secondary metabolites in the extract potentially inhibiting Gram-positive bacteria and favoring propionate-producing bacterial species, thereby resulting in greater accumulation of propionate in the rumen (Wallace et al.,

4.2. Feed intake and nutrient digestibility As a result of the negative effects of high levels of secondary metabolites present in phytogenic extracts and the binding of salivary proteins by feed tannins causing a bitter or sour sensation that decreases palatability, it was expected that negative effects on feed intake would be observed with increasing levels of M. oleifera extract. Negative effects were absent with increasing levels of administered extract, and instead, feed intake was increased by about 5, 8, and 10%, for ME10, ME20 and ME40 treatments, respectively. Mendieta-Araica et al. (2011b) fed Brown Swiss cows with diets based mainly on fresh M. oleifera, and reported higher nutrient intake and digestibility. Conversely, Cohen-Zinder et al. (2016) and Zeng et al. (2017) observed no difference in intake when cows were fed M. oleifera silage-based diet, revealing that the form in which M. oleifera is fed to animals affects intake. In addition, a significantly lower levels of secondary metabolites were delivered to does in the present study compared with this studies. Results indicate that the doses of extract and associated intake of secondary metabolites used in the present study were within the range required for optimal effects on the ruminal environment and digestion without negative effects on feed intake. Improved ruminal fermentation with feeding M. oleifera extract is likely the main reason for enhanced nutrient digestibility of the 5

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2002). As direct measurements of the microbial population were not made in the present study, additional research will be needed to address this possibility. Decreased calculated CH4 production with M. oleifera extract by about 4.7–7.3% also could be related to the secondary metabolites present in the extract (Bodas et al., 2012), as these compounds can have an inhibitory effect on ruminal CH4-producing bacteria (Bodas et al., 2012). Decreasing their population will decrease the amount of available hydrogen for methanogensis. Moreover, suppression of ruminal ciliate protozoa can significantly decrease production of CH4 (Bodas et al., 2012). In their review, Bodas et al. (2012) reported a decrease of 8–14% in methanogenesis with flavonoid-rich extracts in continuous culture of rumen microbes. Because CH4 was calculated from concentrations of SCFA in the present study, further direct measurements of CH4 production and changes in the microbial population will be required to fully assess the effects of the M. oleifera extract on these variables. All doses of M. oleifera extract used in the present study generally had positive effects on ruminal fermentation. Recent reports have shown that low, and sometimes medium, levels of secondary metabolites in phytogenic extracts have positive effects on ruminal fermentation and productivity in vivo and in vitro compared with high levels (Salem et al., 2014). Greater concentrations of secondary metabolites, like tannins can negatively affect ruminal protein and fiber degradability through binding microbial enzymes (McSweeney et al., 2001) or by slowing their digestion rates (Bodas et al., 2012). The variation among results of studies might be related to the nature and concentration of secondary metabolites in different extracts.

(2016), and Zeng et al. (2017) reported that feeding does diets containing M. oleifera decreased serum cholesterol and triglycerides concentrations. Decreasing serum concentrations of cholesterol and triglycerides from a nutritional point of view is nutritionally desirable for lactating animals especially during the peripartum period (Katoh, 2002; Zeng et al., 2017). Greater serum glucose concentrations with feeding M. oleifera extract is potentially important from nutritional point of view for lactating does (Kholif et al., 2016). Serum glucose generally varies within a narrow range, and the values we observed across all treatments would be considered clinically normal. Although reasons for the increase with M. oleifera extract cannot be determined from our results, one might speculate that because the concentration of serum glucose followed a similar trend to OM digestibility and ruminal propionic acid concentration, the higher glucose concentration could reflect changes in these variables associated with supplementing the extract. In addition, unique characteristics of some chemical constitutes observed in the extract, as shown in the GC-identified compounds, might have affected serum glucose, but this possibility would require further study.

4.4. Blood chemistry measurements

Conflict of interest

All blood metabolites determined in the present study were within the reference ranges (Boyd, 2011) for goats. Lack of effects on serum total protein, urea-N, and creatinine concentrations suggest minimal protein catabolism and normal kidney function in the goats in the present study. Higher serum albumin concentration with the inclusion of M. oleifera extract is an indicator of improved nutritional and physiological status of the does. The positive effect of M. oleifera extract on serum albumin might be a result of increased CP intake and increased OM digestibility (Kholif et al., 2015). Reported values of GOT and GPT concentrations were within the normal physiological range, suggesting no pathological lesions in the liver (Stanek et al., 1992). Decreased GOT and GPT concentrations are important indicators of normal or enhanced liver function suggesting the absence of pathological lesions in the liver (Stanek et al., 1992). Kholif et al. (2015, 2016) observed greater GPT concentration with feeding M. oleifera leaf meal. In their experiments, does were fed diets containing 150 and 200 g M. oleifera leaf meal/kg DM of diet, which means that greater amounts of secondary metabolites were delivered to does compared with the present study. Moreover, Rivero et al. (2016) showed that secondary metabolites at high doses for a considerable time of consumption can cause anemia, damage to the liver and kidney, and sometimes death. Zeng et al. (2017) reported that feeding M. oleifera silage did not affect serum concentrations of GOT, GPT, glucose, total protein, and albumin, revealing no effects on hepatic metabolism and immune response of lactating cows. The mechanism by which M. oleifera extract deceased cholesterol and triglycerides is not clear. Lowered blood cholesterol and triglyceride concentrations observed with oral supplementation of M. oleifera extract could be a result of a functional effect of the phenolic acids in the extract (Babiker et al., 2016). Saxena et al. (2013) showed that phytochemicals in the medicinal plants or their phytogenic extracts can decrease the synthesis and absorption of cholesterol and triglycerides. Moreover, Jain et al. (2010) observed that M. oleifera inhibits endogenous cholesterol biosynthesis by reducing the activity of HMG-CoA reductase. Similar to present results, Kholif et al. (2015), Babiker et al.

All authors declare that there are no present or potential conflicts of interest among the authors and other people or organizations that could inappropriately bias their work.

5. Conclusions Providing an oral dose of the extract of M. oleifera to lactating Nubian does increased feed intake and enhanced nutrient digestibility. Moreover, enhanced ruminal fermentation was observed with the supplementation of M. oleifera extract. The levels of 20 and 40 mL/goat daily had similar effects; therefore, the dose of 20 mL is recommended for practical use in lactating does.

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