Effect of Moringa oleifera Leaf Powder Supplementation on Growth Performance, Digestive Enzyme Activity, Meat Quality, and Cecum Microbiota of Ningdu Yellow Chickens

Abstract

This study aimed to investigate the impact of dietary supplementation with Moringa oleifera leaf powder (MOLP) on the growth performance, digestive enzyme activity, meat quality, and cecum microbiota of Ningdu yellow chickens. A total of 300 78-day-old Ningdu yellow chickens with similar initial body weights were randomly distributed into five treatments consisting of six replicates of 10 birds. The control group (M0) was fed a basal diet, and the experimental groups were fed diets supplemented with 0.5% (M0.5), 1% (M1), 2% (M2), and 4% (M4) of MOLP, respectively. Our results showed that dietary supplementation with 2% MOLP significantly (p < 0.05) decreased the feed to gain (F/G) and showed a quadratic (p < 0.05) decrease with the level of MOLP. Dietary supplementation with 1~4% MOLP resulted in a significant increase (p < 0.05) in serum total superoxide dismutase (T-SOD) activity and total antioxidant capacity (T-AOC). Furthermore, both serum T-SOD and T-AOC exhibited linear and quadratic increases (p < 0.01) in response to the supplementation with MOLP in the diets. Dietary supplementation with 1~4% MOLP significantly (p < 0.05) decreased serum uric acid (UA) level. Additionally, 4% MOLP significantly (p < 0.05) decreased triglycerides (TG), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels, and showed linear and quadratic effects. The activity of lipase in the duodenum showed a linear decreasing trend (p < 0.05) with the level of MOLP, while the activities of α-amylase (both in duodenum and jejunum) showed a linear and quadratic increasing trend (p < 0.05). In addition, there was a linear decrease response in abdominal fat (p < 0.05) to MOLP supplementation levels in the diets. In terms of meat quality, dietary supplementation with 4% MOLP significantly reduced (p < 0.05) the L*45 min and L*24 h values of the breast muscle, and drip loss had a linear decreasing trend (p < 0.05). In terms of cecum microbiota, dietary supplementation with 1~4% MOLP significantly increased the Bacteroidota abundance but decreased the Firmicutes abundance (p < 0.05). Overall, dietary supplementation with MOLP improved the growth performance and meat quality of Ningdu yellow chickens through improving the antioxidant function, intestinal digestive enzyme activity, and the cecal microbial structure. The optimum level of MOLP in the diet of Nindu yellow chicken is recommended to be 2.59%.

1. Introduction

Moringa oleifera is a perennial tree of the genus Moringa in the family Moringaceae [1]. The nutritive organs of Moringa oleifera, such as leaves, pods, buds, flowers, stems, and roots, are rich in minerals, vitamins, and pharmacologically active ingredients. Moringa oleifera leaf powder (MOLP) has not only numerous bioactive substances but also exhibits a variety of biological substances, including flavonoids, polyphenols, phenylpropanoids, terpenoids, alkaloids, isothiocyanates, organic acids, and more. MOLP has biological properties of antioxidant, antibacterial, anti-inflammatory, and immune-enhancing activities [2,3,4]. Additionally, MOLP is also rich in nutrients. The apparent metabolizable energy of MOLP in broilers is 11.4 MJ/kg, the crude protein is 25~31%, the fat is 6~7%, and the crude fiber is less than 12% [5,6]. Furthermore, MOLP is a kind of plant-based feed ingredient with high protein content and balanced amino acids, and there are up to 19 kinds of amino acids in Moringa leaves, including lysine, methionine, threonine, tryptophan, histidine, isoleucine, leucine, valine, phenylalanine, arginine, and glycine, which are the 11 kinds of essential amino acids required by poultry [7]. Because of its functional and nutritional characteristics, MOLP can be used as one of the feed ingredients for broilers, replacing part of the protein feed.

Nkukwana et al. [6] pointed out that the inclusion of MOLP from 0.1% to 2.5% in broiler diets improved the feed utilization efficiency and augmented the growth potential of broiler chicks. The study also found that adding MOLP (1.2%) could enhance the small intestine length and the surface area of duodenal villi, as well as improve microarchitecture and acidic mucin production [8]. In addition, MOLP was beneficial to improving the antioxidant status of broilers. Dietary MOLP (1~5%) increased the activities of glutathione peroxidase, catalase, and total superoxide dismutase while decreasing the level of thiobarbituric acid reactive substances of broilers [9]. Gadzirayi et al. [10] examined the impact of MOLP as an alternative protein source to soybean meal in poultry farming. These authors showed that inclusion of MOLP as a protein supplement in broiler diets at 25% inclusion level produces broilers of similar weight and growth rate compared to those fed under conventional commercial diets. Similarly, MOLP as a protein source replacing 5% rapeseed cake in the diet could improve the growth performance, carcass characteristics, and cecal microbial structure of broilers [11]. Rehman, et al. [12] revealed that the addition of MOLP to broilers’ diets significantly increased the pH, pectoral muscle fiber diameter, and water holding capacity of broiler pectoral muscles. MOLP has the potential to serve as a feed component for broiler chickens to enhance the levels of polyunsaturated fatty acids (PUFAs), improve the oxidative capacity and color of breast muscle, and reduce abdominal fat. Dietary supplementation with MOLP increased the contents of n − 3 PUFA and n − 6 PUFA, improved meat color of a* (redness) values, and decreased the malondialdehyde level in breast muscle during storage [13]. MOLP also was conducive to improving the pectoral muscle tenderness and meat color of white-feather broilers [14].

Current research on MOLP as a non-conventional feed ingredient for poultry has been focused on white-feathered broilers, while reports on yellow-feathered chicken breeds were rare. In recent years, the yellow-feathered chicken industry has developed rapidly and plays an important role in the development of China’s poultry industry, its production ranked after the white-feathered broilers [15]. Additionally, the yellow-feathered chicken has longer growth cycles and higher production costs than white-feathered broilers. Accordingly, it is also necessary to further study MOLP as an unconventional feed resource in yellow-feathered chicken diets. Ningdu yellow chicken is one of the representative breeds of local yellow-feathered chicken in China and is widely recognized by consumers for its good meat quality and high nutritional value. Moreover, its annual slaughtering capacity has already reached around 100 million birds [16]. Consequently, this study aimed to examine the effects of dietary MOLP supplementation on growth performance, digestive enzyme activity, meat quality, and cecum microbiota and to further explore the optimal requirements of moringa leaf powder for Ningdu yellow chicken. These findings would enhance the utilization of MOLP as an unconventional feed resource in Ningdu yellow chicken production.

2. Materials and Methods

The animals used in this experiment received approval from the Animal Care and Use Committee of the Jiangxi Academy of Agricultural Sciences under approval number 2022-JXAAS-XM-08.

2.1. Animal Management and Experimental Design

A total of 300 female Ningdu yellow chickens of 78 days old with an initial weight about 1218 g were selected and were randomly distributed into five groups with six replicates of 10 chickens through a completely randomized design. The control group (M0) was administered a basal diet, while the experimental groups were fed diets diet complemented with 0.5% (M0.5), 1% (M1), 2% (M2), and 4% (M4) of MOLP, respectively. All experimental diets were formulated to contain the same nutrient levels. The MOLP was provided by Jiangxi University of Traditional Chinese Medicine and was crushed and passed through an 80-mesh screen. The MOLP (dry basis) in this experiment contained the following nutrients: gross energy 16.86 MJ/kg, crude protein 20.22%, crude fat 6.10%, crude fiber 5.90%, calcium 3.70%, and total phosphorus 0.20%. The experiment lasted 28 d. The chicken house was kept under 18 h continuous light and 6 h darkness. Throughout the entire testing phase, the temperature was set at 25 °C. Three-layer, three-dimensional netting was used, and the procedures of immunization, sanitation, and disinfection of the test chickens were carried out in accordance with the standard breeding system.

The Ningdu yellow chickens were given access to feed and water ad libitum. The diets were devoid of antibiotics and coccidiostats and were pelleted prior to feeding. All nutrients contained in the experimental diets met the nutritional requirements of yellow chickens (NY/T3645-2020). The formulations of the experimental diets and corresponding nutrient levels are detailed in

Table 1. Composition and nutrient levels of experimental diets (fed basis) %.

Items	M0	M0.5	M1	M2	M4
Ingredients (% w/w)
Corn	67.08	66.88	66.69	65.91	64.82
Soybean meal	20.16	20.07	19.97	19.77	19.36
Wheat middling and reddog	4.70	4.42	4.13	3.85	3.02
Moringa oleifera leaf	0.00	0.50	1.00	2.00	4.00
Soybean oil	4.28	4.37	4.46	4.75	5.19
L-Lys·HCl, 79%	0.12	0.12	0.12	0.12	0.11
DL-Met, 99%	0.14	0.14	0.14	0.14	0.14
Limestone	1.13	1.11	1.09	1.05	1.03
CaHPO4	1.39	1.39	1.40	1.41	1.33
Premix 1	1.00	1.00	1.00	1.00	1.00
Tatal	100	100	100	100	100
Nutrient levels 2
Metabolizable energy (Mcal/kg)	3.06	3.06	3.06	3.06	3.06
CP (%)	15.48	15.50	15.47	15.45	15.48
Lys (%)	0.85	0.85	0.85	0.85	0.85
Met (%)	0.39	0.39	0.39	0.39	0.39
Ca (%)	0.80	0.79	0.78	0.79	0.79
Available P (%)	0.36	0.36	0.36	0.36	0.36
Total P (%)	0.56	0.56	0.58	0.57	0.58

¹ The premix provided the following per kg of the diets: VA 12 500 IU, VD 2 500 IU, VE 25 mg, VK₃ 3 mg, VB_l 3 mg, VB₂ 8 mg, VB₆ 7 mg, VB_l2 0.03 mg, D-pantothenic acid 20 mg, niacin 50 mg, biotin 0.1 mg, folic acid 1.5 mg, Cu (as copper sulfate) 8 mg, Fe (as ferrous sulfate) 100 mg, Mn (as manganese sulfate) 100 mg, Zn (as zinc sulfate) 100 mg, I (as potassium iodide) 0.6 mg, Se (as sodium selenite) 0.16 mg. ² Metabolizable energy, available P, Lys and Met were calculated values, while the others were measured values.

.

2.2. Growth Performance and Carcass Characteristic

After 12 h of fasting, the final body weight (BW) of the Ningdu yellow chickens was recorded for each replicate at the age of 105 days. The feed consumption of each replicate during the experimental period was recorded. Subsequently, the average daily gain (ADG), average daily feed intake (ADFI), and ratio of feed to gain (F/G) were calculated.

At the age of 105 days, two Nindu yellow chickens around the average weight were picked from each replicate and subsequently slaughtered to determine carcass yield, breast yield, leg yield, and abdominal fat percentage.

2.3. Determination of Biochemical Parameters

Before slaughter of chickens, 4 mL blood samples were collected from the wing vein. After 30 min of standing, the blood samples were centrifuged at 1500 rpm for 10 min by centrifugation (LC-LX-L50C, Shanghai LiChen Instrument Technology, Ltd., Shanghai, China). Then, a total of 60 serum samples were stored at −20 °C until analysis.

The concentrations of serum total protein (TP), albumin (ALB), globulin (GLB), uric acid (UA), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total cholesterol (TC), and triglycerides (TG) were analyzed by a Myriad BS-420 Automatic Biochemistry Instrument (Shenzhen Myriad Bio-Medical Electronics Co., Ltd., Shenzhen, China). The concentrations of serum immunoglobulin [17] A, IgM, and IgG were determined using chicken-specific ELISA kits (Jiangsu Enzyme Immunity Institute, Yancheng, China). The activities of total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC), and the concentrations of malondialdehyde (MDA) were measured in serum using their respective commercial assay kits (Jiancheng Bioengineering Research Institute, Nanjing, China).

2.4. Intestinal Mucosal Collection and Digestive Enzyme Activity Assay

At the age of 105 days, the chickens were fasted for 12 h. Six yellow chickens per group (one bird from each replicate) were randomly selected and slaughtered. Subsequently, the duodenum, jejunum, and ileum were removed on ice, and the intestinal mucosa were scraped with a slide and stored in a refrigerator at −80 °C for the detection of α-amylase, lipase, and trypsin activities.

The intestinal mucosal tissue was weighed accurately and to added nine times the volume of saline according to the ratio of weight (g):volume (mL) = 1:9; the 10% homogenate was mechanically homogenized under ice-water bath conditions, centrifuged at 2500 rpm for 10 min, and the supernatant was collected for determination. The activities of α-amylase, trypsin, and lipase were determined with the corresponding assay kits (Shanghai Enzyme-linked Biotechnology Co., Ltd., Shanghai, China).

2.5. Meat Quality Measurements

At the age of 105 days, two chickens from each replicate group were randomly taken (a total of 60 chickens), and the right breast muscles were isolated and used for meat quality measurements.

Meat color: The color of the inner surface of breast meat was evaluated by measuring the brightness (L*), redness (a*), and yellowness (b*) values. The measurements were taken 45 min after slaughter and again after 24 h of storage at 4 °C to observe any changes in color.

Drip loss: The right breast muscle sample (2 × 2 × 2 cm³) was weighed (recorded as W1); the meat sample was suspended in a hollow disposable paper cup and was placed in a refrigerator at 4 °C, and the meat sample was taken out after 24 h. The juices on the surface of the sample were gently wiped away with a pre-prepared filter paper, and the meat sample was then weighed (W2). Drip loss (%) = (W2 − W1)/W1 × 100%.

Cooking loss: The samples (5 × 3 × 3 cm³) were cut via cubic membrane and then weighed (W1); then, the meat samples were put into self-sealing bags, the air inside the self-sealing bags was evacuated, and they were then put into a water bath at 80 °C for 30 min. The meat samples were taken out from the bag, put on filter paper for 30 min, then cooled down to room temperature, and then the meat samples were weighed after wiping off the moisture on the surface with filter paper (W2). Cooking loss (%) = 100% × (W1 − W2)/W1.

Shear force: Three pieces of raw breast muscle of the same size were cut off with scissors. All samples were placed perpendicular to the muscle fiber axis on a C-LM3 digital muscle tenderness instrument for cutting, and the values were recorded.

pH value: A piece of meat sample was taken from each of the right breast muscle, and the pH value of broiler breast muscle was determined by pH meter for 45 min and 24 h pH values. Each meat sample was measured three times.

2.6. DNA Extraction and 16S rRNA Gene Sequencing

At the end of the test, a total of 30 birds (one bird per replicate) were randomly chosen and slaughtered for isolation of the cecum. Approximately 1 g of contents from the cecum was collected and gently squeezed into sterile tubes for microbiological analysis. Subsequently, all samples were flash frozen in liquid nitrogen and stored at −80 °C until being used for DNA collection. Total DNA was extracted from the frozen fecal materials using a PowerSoil DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA, USA) following the manufacturer’s instructions. The extracted DNA concentration and purity were assessed by measuring the absorbance ratios at 260 nm/280 nm and 260 nm/230 nm. Immediately afterwards, all the DNA samples were stored at −80 °C until subsequent processing. Finally, The DNA samples underwent amplification of the V3–V4 hypervariable region of 16S rDNA, followed by sequencing on the Illumina NovaSeq platform. The analysis of cecal microbiota richness and diversity was carried out by Novogene Co., Ltd. (Beijing, China).

2.7. Statistics Analysis

All data were analyzed by one-way analysis of variance (ANOVA) through SPSS 19.0 statistical software, and the Tukey test was used for multiple comparisons, with p ≤ 0.05 indicating significant differences. Both the linear and quadratic regression analyses were used to evaluate impacts of dietary MOLP supplementation dosage. Data were expressed as mean and pooled SEM.

3. Results

3.1. Growth Performance

The effects of dietary MOLP supplementation on the growth performance of Ningdu yellow chickens are shown in

Table 2. Effects of Moringa oleifera leaf powder supplementation on growth performance of Ningdu yellow chickens ¹.

Items 2	Groups					SEM	p-Value
	M0	M0.5	M1	M2	M4		ANOVA	Linear	Quadratic
BW (g)	1714	1767	1748	1775	1744	7.28	0.55	0.19	0.05
ADFI (g/d)	74.43	77.70	75.57	75.31	72.68	0.86	0.49	0.29	0.05
ADG (g/d)	17.91 b	19.71 ab	18.97 ab	20.00 a	18.71 ab	0.25	0.05	0.34	0.25
F/G (g/g)	4.16 b	3.94 ab	3.98 ab	3.77 a	3.88 ab	0.04	0.01	0.05	0.01

¹ Different lowercase letters represent significant differences (p ≤ 0.05), and data are the mean of six replicates with 10 chickens each. ² BW, body weight; ADG, average daily gain; ADFI, average daily feed intake; F/G, feed to gain.

. Compared with the control group (M0), dietary addition of 2% MOLP significantly (p < 0.05) reduced the F/G and pronouncedly (p < 0.05) increased ADG. Furthermore, dietary supplementation with MOLP quadratically increased BW, ADG and reduced F/G (p ≤ 0.05).

3.2. Serum Biochemical Parameters

Table 3. Effects of Moringa oleifera leaf powder supplementation on serum biochemical indices of Ningdu yellow chickens ¹.

Items 2	Groups					SEM	p-Value
	M0	M0.5	M1	M2	M4		ANOVA	Linear	Quadratic
TG (mg/mL)	0.79 b	0.74 b	0.65 b	0.68 b	0.52 a	0.02	<0.01	<0.01	<0.01
TC (μmol/dL)	30.14	34.10	32.07	29.07	31.32	0.72	0.26	0.42	0.45
UA (umol/L)	262.60 b	233.16 ab	202.49 a	215.37 a	199.56 a	5.17	<0.01	0.01	0.01
ALP (IU/L)	19.14 a	21.06 ab	21.78 ab	23.72 b	23.91 b	0.53	0.04	0.02	0.06
AST (IU/L)	520.98 b	526.69 b	431.42 a	471.23 ab	429.37 a	8.89	<0.01	0.03	0.05
ALT (IU/L)	51.78 b	53.33 b	41.86 a	43.90 a	41.37 a	1.19	<0.01	0.01	0.04
TP (mg/mL)	26.02	26.09	25.05	27.50	26.46	0.40	0.40	0.06	0.02
ALB (mg/mL)	14.65	15.17	13.53	13.74	12.68	0.31	0.18	0.10	0.20
GLB (mg/mL)	11.18	10.26	11.51	13.75	13.44	0.52	0.17	0.01	0.01

¹ Different lowercase letters represent significant differences (p ≤ 0.05), and data are the mean of six replicates with 10 chickens each. ² TG, triglycerides; TC, total cholesterol; UA, uric acid; TP, total protein; ALB, albumin; GLB, globulin; AST, aspartate aminotransferase, ALT, alanine aminotransferase; ALP, alkaline phosphatase.

shows the impact of dietary MOLP addition on the serum biochemical indices of Ningdu yellow chickens. The serum level of TG in the M4 group was significantly (p < 0.01) lower than those in the M0, M0.5, M1, and M2 groups. The UA concentrations in the M1, M2, and M4 groups were significantly (p < 0.01) lower than the M0 group. And the TG and UA concentrations showed linear decreases with increasing levels of MOLP in the diets (p < 0.05). The ALP activities in the M2 and M4 groups were significantly (p < 0.01) higher than the M0 group. And the activity of ALP showed linear improvement as the inclusion level of MOLP increased. The activities of AST and ALT in the M1 and M4 groups were significantly (p < 0.01) lower than the M0 group. And the activity of AST and ALT showed linear (p < 0.05) decreases with increasing levels of MOLP in the diets. In addition, the concentrations of GLB showed linear and quadratic increases (p < 0.05) with increasing levels of MOLP in the diets. No linear or quadratic effects were shown in TC (p > 0.05) as a result of varying levels of dietary MOLP supplementation.

3.3. Serum Antioxidant Indexes

As shown in

Table 4. Effects of Moringa oleifera leaf powder supplementation on serum antioxidant indices of Ningdu yellow chickens ¹.

Items 2	Groups					SEM	p-Value
	M0	M0.5	M1	M2	M4		ANOVA	Linear	Quadratic
T-SOD (U/mL)	90.86 a	91.92 a	106.10 b	106.22 b	124.74 c	2.33	<0.01	<0.01	<0.01
GSH-Px (U/mL)	245.18 a	271.12 ab	281.72 ab	316.53 bc	360.06 c	7.93	<0.01	0.01	0.01
T-AOC (μmol Trolox/mL)	1.28 a	1.41 ab	1.71 bc	1.69 bc	1.99 c	0.15	<0.01	<0.01	<0.01
MDA (nmol/mL)	7.33 ab	8.01 b	6.66 a	6.68 a	6.54 a	0.05	0.01	0.01	0.04

¹ Different lowercase letters represent significant differences (p ≤ 0.05), and data are the mean of six replicates with 10 chickens each. ² T-SOD, total superoxide dismutase; GSH-Px, glutathione peroxidase; T-AOC, total antioxidant capacity; MDA, the concentrations of malondialdehyde.

, with increasing levels of MOLP in the diets, the activities of T-SOD, GSH-Px, and T-AOC showed linear and quadratic (p < 0.05, p < 0.05, respectively) increases with increasing levels of MOLP in the diets, while MDA concentrations showed a linear (p < 0.05) reduction.

3.4. Intestinal Digestive Enzyme Activity

As shown in

Table 5. Effects of Moringa oleifera leaf powder supplementation on digestive enzyme activity of Ningdu yellow chickens ¹.

Items	Groups					SEM	p-Value
	M0	M0.5	M1	M2	M4		ANOVA	Linear	Quadratic
duodenum
Trypsin (U/g·pro)	82.57	99.89	99.78	103.66	105.47	3.37	0.23	0.36	0.32
α-Amylase (U/g·pro)	118.27	125.39	131.92	134.35	144.96	4.89	0.55	0.01	0.03
lipase (U/g·pro)	4.78	4.95	4.32	4.36	4.03	0.15	0.27	0.02	0.07
jejunum
Trypsin (U/g·pro)	95.27 a	97.87 ab	123.47 b	111.93 ab	114.66 ab	3.44	0.01	0.07	0.17
α-Amylase (U/g·pro)	99.80	117.76	130.31	142.76	125.33	5.06	0.07	0.04	0.02
lipase (U/g·pro)	3.42	3.42	4.20	5.48	4.44	0.27	0.10	0.19	0.25
ileum
Trypsin (U/g·pro)	82.45	98.60	96.13	93.38	84.07	3.43	0.50	0.48	0.15
α-Amylase (U/g·pro)	103.00	128.53	128.99	114.14	107.86	4.77	0.35	0.85	0.04
lipase (U/g·pro)	3.38	3.66	3.23	4.05	3.60	0.16	0.64	0.14	0.29

¹ Different lowercase letters represent significant differences (p ≤ 0.05), and data are the mean of six replicates with 10 chickens each.

, the MOLP levels dramatically increased the activities of α-amylase (both in duodenum and jejunum) and showed linear and quadratic increases (p < 0.05, p < 0.05, respectively) with increasing levels of MOLP in the diet. Additionally, the activity of lipase in the duodenum showed a linear decrease (p < 0.05) with the increasing level of MOLP in the diet. Moreover, the activity of trypsin in the jejunum was significantly higher in the M1 group compared to the M0 group. In addition, the activity of α-amylase in the ileum exhibited a quadratic decline in response to MOLP supplementation in the diets.

3.5. Carcass Characteristics and Meat Quality

As shown in

Table 6. Effects of Moringa oleifera leaf powder supplementation on carcass quality of Ningdu yellow chickens ¹.

Items	Groups					SEM	p-Value
	M0	M0.5	M1	M2	M4		ANOVA	Linear	Quadratic
Carcass (%)	71.65	71.44	70.87	73.78	71.24	0.39	0.14	0.40	0.69
Breast muscle (%)	15.22	15.35	15.58	15.61	15.03	0.21	0.90	0.59	0.18
Leg muscle (%)	20.58	20.83	21.50	22.08	21.18	0.22	0.20	0.05	0.05
Abdominal fat (%)	7.39 b	7.45 b	7.37 b	5.56 a	6.09 a	0.21	0.01	0.03	0.08

¹ Different lowercase letters represent significant differences (p ≤ 0.05), and data are the mean of six replicates with 10 chickens each.

, the percentages of abdominal fat in the M2 and M4 groups were significantly lower than in the M0 groups. And a statistically significant linear relationship (p < 0.05) was observed between the levels of MOLP in the diets and the abdominal fat percentage. Furthermore, there was both a linear and quadratic response in leg muscle (p = 0.05, p = 0.05, respectively) to MOLP supplementation levels in the diets. No linear or quadratic effects (p > 0.05) were observed in carcass and breast muscle in response to the dietary MOLP addition levels.

As can be seen from

Table 7. Effects of Moringa oleifera leaf powder supplementation on meat quality of Ningdu yellow chickens ¹.

Items 2	Groups					SEM	p-Value
	M0	M0.5	M1	M2	M4		ANOVA	Linear	Quadratic
L*45 min	60.38 a	56.89 b	59.87 ab	58.50 ab	57.12 b	0.37	<0.01	0.29	0.58
a*45 min	11.87	10.75	10.84	10.96	11.62	0.18	0.19	0.72	0.85
b*45 min	7.94	8.70	9.29	8.78	9.38	0.20	0.10	0.67	0.14
pH45 min	5.71	5.80	5.62	5.64	5.62	0.04	0.61	0.27	0.58
L*24 h	61.11 a	58.44 b	58.61 b	58.31 b	58.80 b	0.27	<0.01	0.01	0.01
a*24 h	10.32	9.16	9.87	9.59	10.37	0.23	0.43	0.68	0.40
b*24 h	8.82	9.25	9.29	10.13	10.71	0.27	0.17	0.31	0.56
pH24 h	5.47	5.40	5.60	5.53	5.52	0.02	0.06	0.88	0.98
Cooking loss, %	27.38	26.27	27.00	26.10	25.96	0.19	0.10	0.12	0.30
Drip loss, %	2.18	1.53	1.84	1.51	1.57	0.09	0.06	0.05	0.16
Shear force, N	3.22	2.77	3.05	2.78	2.95	0.10	0.64	0.89	0.45

¹ Different lowercase letters represent significant differences (p ≤ 0.05), and data are the mean of six replicates with 10 chickens each. ² L*, lightness; a*, redness; b*, yellowness.

, compared with the M0 group, the breast muscle color of L*45 min values in M0.5 and M4 groups were significantly decreased (p < 0.01), and L*24 h values were decreased in all MOLP supplementation groups. Moreover, cooking loss had a decreasing trend (p = 0.10), while there was no significant difference in shear force and pH (p > 0.05).

3.6. Cecum Microflora

As shown in Figure 1, in the M2 group, the Chao1 indexes were significantly higher than those in the M0, M0.5, M1, and M4 groups (Figure 1A). There were no significant effects on the Shannon index, Simpson index, and Dominance index with increasing levels of MOLP.

At the phylum level (Figure 2A), the most abundant phyla were Bacteroidetes and Firmicutes, accounting for 88.15% on average in the M0 group, 89.39% in the M0.5 group, 85.12% in the M1 group, 89.10% in the M2 group, and 89.11% in the M4 group, respectively. Compared with the M0 group, the M1, M2, and M4 group increased the Bacteroidota abundance (Figure 2B, p < 0.05) and decreased the Firmicutes abundance (Figure 2C, p < 0.05).

At the genus level (Figure 3A), the top 10 dominant genera were Rikenellaceae_RC9, Bacteroides, Prevotellaceae_UCG-001, Ruminococcaceae_UCG-004, Methanobrevibacter, Clostridiales bacterium_CHKCI001, Phascolarctobacterium, Megamonas, Olsenella, and Faecalibacterium, which comprised 41~52% of the total genera. Compared to the M0 group, the M1 and M4 group increased the Rikenellaceae_RC9 abundance (Figure 3B, p < 0.05). Compared to the M0 group, the M1, M2, and M4 group decreased the CHKCI001 abundance (Figure 3C, p < 0.05). Other genera had no difference between the experimental groups.

4. Discussion

Moringa is rich in nutrients, especially proteins, vitamins, and polyphenols and flavonoids, all of which are beneficial to animal growth performance. Previous studies have reported that the addition of suitable amounts of Moringa leaves to diets could enhance the production of broilers and swine [8,18]. Cui et al. [13] reported that dietary MOLP supplementation quadratically decreased BW, ADG and increased F/G of Arbor Acres broilers, and the optimal percentage of MOLP added to the diet was 1.56%. Abu Hafsa et al. [9] reported that broilers fed a diet with the inclusion of 1% MOLP recorded the highest final body weight and best feed conversion, but MOLP inclusion at 5% dietary level resulted in lower daily weight gain and feed conversion. Our study showed that dietary supplementation with MOLP quadratically reduced F/G, which was basically consistent with the results of the above studies. And the impact of different levels of dietary MOLP supplements was analyzed using quadratic regression, resulting in the following equation for F/G: y = 0.056x² − 0.29x + 4.15 (p < 0.05, R² = 0.605). It was determined that the optimal proportion of MOLP in diet was 2.59%. The increased growth performance may be attributed to higher digestive enzyme activity, as well as the improvement of endogenous digestive enzyme secretion which can improve nutrient utilization [6]. Our study also found that the activities of α-amylase (in both duodenum and jejunum) showed a quadratic increase with increasing levels of MOLP in diet. Moreover, there was an improvement in trypsin in the jejunum.

UA is an important indicator of protein metabolism in animals and can also be used to assess liver function and amino acid balance [19]. In this study, the concentration of UA showed linear decreases with increasing levels of MOLP in the diets, suggesting that dietary supplementation with MOLP (0.5–4%) does not adversely affect protein utilization. ALT, AST, and ALP are the main indicators of liver function, and serum AST and ALT activities are significantly increased in hepatic insufficiency [20]. In this experiment, the activities of AST and ALT showed linear decreases with increasing levels of MOLP, indicating that MOLP (0.5–4%) has no adverse effects on liver functions in Ningdu yellow chickens. Our results of liver function tests were consistent with those of Abu Hafsa et al. [9], who found that there was a significant reduction in AST and ALT levels of MOLP-treated (0.5–5%) white-feather broilers compared to the control group. Also, Abdel-Wareth et al. [21] demonstrated that diets supplemented with MOLP (3–9%) promoted liver health by decreasing the serum ALT and AST concentrations of Hy-Line brown hens.

Cellular redox homeostasis is mainly maintained by an elaborate endogenous antioxidant defense system, which includes endogenous antioxidant enzymes such as T-SOD, GSH-Px, and T-AOC [22]. In our study, dietary supplementation with MOLP showed a linear increase in the serum antioxidant parameters of GSH-Px, T-SOD, and T-AOC. Meanwhile, dietary supplementation with MOLP linearly reduced the MDA content. The above results suggest the positive impact of MOLP on Ningdu yellow chickens’ antioxidant defense system. Similarly, M. Shen, T. Li, L. Qu, K. Wang, Q. Hou, W. Zhao, and P. Wu [17] reported that diets supplemented with MOLP (2.5%) significantly enhanced serum T-SOD concentrations and the mRNA expression levels of SOD1 and SOD2. Previous studies have also demonstrated that MOLP could increase the antioxidant capacity of breast muscle [13]. Furthermore, dietary supplementation with MOLP could increase tissue SOD and CAT activities and lowered hepatic MDA level in Nile tilapia [23]. The enhanced antioxidant capacity may be associated with chlorogenic acid, quercetin, and vitamin E in MOLP, all of which exhibit significant free radical scavenging properties and inhibit lipid peroxidation [2,24].

In terms of carcass characteristics, the abdominal fat was linearly reduced in response to dietary MOLP supplementation. The result was coherent with findings by Cui et al. [13], who indicated that diet supplementation with MOLP linearly decreased abdominal fat on d 21 and 42. Moreover, our study found that the serum level of TG decreased in response to dietary MOLP inclusion, which indicated a variation in lipid metabolism induced by dietary MOLP supplementation. In agreement with our results, Abdel-Wareth et al. [21] reported that dietary MOLP supplementation decreased the serum total cholesterol and TG levels of laying chickens. Similarly, Shen et al. [17] observed that serum total cholesterol and low-density lipoprotein cholesterol contents were lowered with the level (2.5% and 10%) of MOLP fed in laying chickens’ diets. It was explained that the higher amount of natural fiber in MOLP may regulate serum lipid metabolism.

Meat color can directly influence consumers’ purchasing decisions and is an important indicator for determining the freshness of meat. The value of the L* value is inversely related to meat quality, while the size of the b* value reflects the freshness of the meat [25]. Our study showed that 4% MOLP supplementation significantly reduced L*45 min and L*24 h value, whereas the b*45 min showed an increasing trend. Moreover, diet supplementation with MOLP linearly decreased the drip loss of breast muscle. The above results indicated that supplementation with MOLP could improve the meat quality of broilers. Similar studies by Cui et al. [13] have reported indications that dietary MOLP supplementation enhanced the color of meat during storage. Those drip-loss results were in agreement with those of Rehmanet al. [12], who observed that 15 g/kg MOLP supplementation had a lower drip loss. Myoglobin is a critical protein that significantly influences the coloration of meat. Enhancements in meat quality may be caused by the fact that the myoglobin oxidation processes were changed due to the improved antioxidant capacity of the meat [13].

Gut microbial communities play an important role in digestion, physiological function, gut health, nutrient metabolism, and immune regulation in poultry. In terms of alpha diversity in the cecal microbiota, there were no significant differences in Shannon index, Simpson index, and Dominance index between the treatment groups, which indicated that the addition of MOLP could maintain the homeostasis of intestinal flora and have no negative effects on the health status of chickens, while the 2% MOLP addition group had a higher Chao1 index than the other treatment groups, suggesting that MOLP addition could improve the α-diversity of intestinal microbiota. The index of intestinal microbial community can reflect the stability of the intestinal microbial environment, and, the higher the index, the more stable it tends to be, and the stronger the level of resistance to stress [26]. Regarding the structural changes of microbial communities, Bacteroidetes and Firmicutes were the main phyla in each treatment at the phyla level. Firmicutes are associated with dietary energy uptake, and Bacteroidetes are associated with the degradation of proteins and carbohydrates for metabolism [27]. In this study, the relative abundance of Bacteroidetes in the 1–4% MOLP groups was higher than the control group. This can be explained by the fact that Moringa leaves are rich in polysaccharides, which makes the Bacteroidetes more competitive in the chicken intestinal tract. Moreover, the relative abundance of Bacteroidetes in the 1–4% Moringa-leaf-added groups were lower than control group, which suggested that the addition of Moringa leaves is beneficial in reducing energy over-absorption leading to the deposition of abdominal fat. This result is consistent with a previous investigation that demonstrated that the genus Rikenellaceae_RC9 exhibited enhancements in response to a high-fiber diet [28]. In this study, we also noted a substantial increase in the abundance of Rikenellaceae_RC9 within the 1% and 4% experimental groups. As the level of dietary MOLP leaves increased, the level of fiber in the diet was necessarily increasing. Consequently, the elevation of Rikenellaceae_RC9 may be correlated with the level of dietary fiber. In addition, our study also found that the relative abundance of CHKCI001 was reduced in the 1~4% groups. However, CHKCI001 is a relatively new bacterial family and therefore its metabolic function in the chicken has not yet been defined.

5. Conclusions

In conclusion, dietary supplementation with MOLP could improve growth performance, intestinal digestive enzyme activity, antioxidant function, meat quality, and the cecal microbial structure, and is beneficial to reducing abdominal fat deposits. This study provides further evidence that MOLP could be used as a feed ingredient for Ningdu yellow chickens to improve growth performance, with an inclusion of 2.59% in the diets.