Effect of Moringa oleifera Seed Extract Administered through Drinking Water on Physiological Responses, Carcass and Meat Quality Traits, and Bone Parameters in Broiler Chickens

Abstract

The efficacy of Moringa oleifera seed extract (MSE) in broiler nutrition is unknown. Thus, this study evaluated the effect of administering MSE through drinking water on physiological responses, carcass and meat quality traits, and bone parameters in Cobb 500 broilers. One-day-old male chicks (n = 250, 58.1 ± 0.23 g live-weight) were randomly allotted into five treatments of five replicates. The treatments were: negative control with drinking water only (NC); a positive control with 5 g probiotic (containing Aspergillus oryzae, Bacillus subtilis, Enterococcus faecium, and Lactobacillus acidophilus; bacterial count of 5 × 10⁸ CFU/g) added into 1 L of drinking water (PC), 60 mL MSE/L drinking water (MSE₆₀); 90 mL MSE/L drinking water (MSE₉₀); and 120 mL MSE/L drinking water (MSE₁₂₀). The MSE groups promoted higher (p < 0.05) carcass weights than the PC and NC control groups. Spleen weights quadratically responded to MSE levels. Gizzard weight, duodenum length, acidic goblet cell count (GCC), and total duodenal and ileal GCC linearly increased with MSE levels. Tibia weight, bone breaking strength, and tibia calcium showed linear increases whereas tibia length and tibiotarsal index showed linear decreases with MSE levels. It can be concluded that the administration of MSE via drinking water improved carcass weights, intestinal morphology, and some meat and bone quality parameters of the birds.

1. Introduction

Sustainable broiler chicken production can contribute to the United Nations’ sustainable development goals to eradicate hunger and ensure good health and wellbeing through the supply of high-quality protein and micronutrients in the form of meat and eggs [1]. It is for this reason that many broiler producers around the world have directed their focus toward optimizing carcass yield and meat quality traits by maintaining high broiler welfare standards, improving gut health, and minimizing locomotion disorders [2]. This has been achieved with prebiotics, probiotics, synbiotics, and phytogenics as viable alternatives to prophylactic and growth-promoting antibiotics. Probiotics are mono or mixed cultures of live beneficial bacteria that are employed in poultry production to create a balanced and diversified gut microbial environment that specifically eliminates pathogens [3]. Optimal dosages of probiotics provide several nutritional and physiological advantages such as improved growth performance, intestinal flora, immune response, and meat quality [4]. However, the rising preference for natural and safe alternatives due to their nonresidual effects and negative effect on the environment has increased the market for phytogenic products as potential alternatives [5]. Phytochemicals are biologically active compounds in plants that elicit physiological changes in farm animals. One of the most recently utilized phytogenic plants in poultry nutrition is Moringa oleifera.

Moringa oleifera seed powder contains biologically active compounds such as alkaloids, flavonoids, phenols, saponins, steroids, and tannins with antimicrobial, antioxidant, and hypocholesterolemic effects [6]. Furthermore, the seed powder is a rich source of crude protein, essential amino acids, dietary fibre, fatty acids, minerals, and vitamins [7], which are essential in maintaining and improving carcass yield, meat quality, gut health, and tibial integrity in broiler chickens. However, high levels of fibre (5.03 mg/g) and condensed tannins (241.67 mg/g) [8] in the seed powder limit its utilization by poultry, especially when included at higher dietary levels [9].

Alternatively, the extraction of water-soluble nutrients and bioactive compounds from moringa seeds could be a strategy to reduce the antinutritional effects of dietary fibre and water-insoluble condensed tannins while allowing the birds full access to moringa’s beneficial bioactive compounds. Water extraction of phytogenic compounds is one affordable strategy that does not require expensive or specialized equipment and can appeal to resource-constrained farmers. However, no studies have attempted to evaluate the effect of administering Moringa oleifera seed extract (MSE) via drinking water in broiler chickens. This study, therefore, investigated the effect of different doses of MSE on carcass characteristics, meat quality, intestinal morphology, goblet cell count (GCC), tibia morphometric parameters, and tibia mineral content in Cobb 500 broilers. It was hypothesized that oral administration of MSE via drinking water would improve carcass characteristics, intestinal morphology, and meat and bone quality parameters of the birds.

2. Materials and Methods

2.1. Treatment Sources and Moringa Extraction Process

The M. oleifera seed powder acquired from Supa Nutri (Johannesburg, South Africa) was soaked in distilled water at a ratio of 1:10 by adding 100 g of the seed powder to 1000 mL of distilled water for 24 h. Thereafter, the extracts were stored in clean containers at 4 °C after being filtered with a muslin cloth to remove the debris from the filtrate.

The extracts were then diluted with fresh drinking water to formulate the treatments that were offered to the birds daily, as follows: (1) a negative control with drinking water only (NC), (2) positive control with 5 g probiotic per litre of drinking water (PC), (3) 60 mL of MSE per litre of drinking water (MSE₆₀), (4) 90 mL of MSE per litre of drinking water (MSE₉₀), and (5) 120 mL of MSE per litre of drinking water (MSE₁₂₀). The multistrain probiotic (with a bacterial count of 5 × 10⁸ CFU/g) was acquired from QBLabs (St. Louis, MO, USA), which contained beneficial bacteria (Aspergillus oryzae, Bacillus subtilis, Enterococcus faecium, and Lactobacillus acidophilus in a proportion of 1:2:1:2). The bioactive compounds, mineral, and vitamin contents of the MSE treatments were as presented in our previous work [10]. Commercial starter (1–21 days) and finisher (22–42 days) diets purchased from De Heus (Pty) Ltd. (Pietermaritzburg, South Africa) were used during the feeding trial. The chemical composition of the commercial starter and finisher diets was as described in our previous work [10].

2.2. Feeding Trial

The feeding trial was conducted between October and November 2020 at Rooigrond Commercial Farm (25°55′0″ S; 25°48′0″ E), located in Rooigrond (North West, South Africa). A total of 250, day-old Cobb 500 male chicks were purchased from Poultry Ranch (Pty) Ltd. (Pretoria, South Africa). In an environmentally controlled room, the birds were allotted 25 pens (experimental units) to which the five experimental treatments were randomly assigned. The treatments were replicated five times, and each pen (2.55 m Length × 1.0 m Width × 5.0 m Height) had 10 birds. The pens were barricaded using galvanized wire net and the floors were covered with dried sunflower husks. Infrared electric lights were used to maintain the house temperature at 34 °C for the first 14 days and thereafter reduced by 2 °C at weekly intervals. The pens were cleaned regularly and a footbath with a disinfectant was used for biosecurity. For the entire 42-d feeding trial, the birds had unlimited access to clean drinking water and feed.

2.3. Slaughter, Carcass, and Meat Quality Characteristics

On day 42 of age, after the feed was withheld for 12 h, all the birds were weighed to determine the slaughter weight (SLWT) and thereafter taken to a nearby abattoir where they were sacrificed by cutting the jugular vein following stunning. All the carcasses were immediately weighed to ascertain hot carcass weight (HCW) and then re-weighed after chilling in a cold room for 24 h to determine cold carcass weight (CCW). Carcass yield was calculated as the proportion of HCW on SLWT. Weights of all carcass parts (breast, drumstick, thigh, wing) and internal organs (cleaned gizzard, liver, and spleen) were weighed with a digital scale (Adam Equipment S.A. PTY, Johannesburg, South Africa).

Breast pH was recorded at 24 (pH_24hr) hours post-mortem with the aid of Corning Model 4 pH–temperature meter (Corning Glass Works, Medfield, MA, USA), and after each experimental unit was calibrated using standard pH solutions. Breast colour coordinates (L* = lightness, a* = redness, and b* = yellowness) were determined 24 h post-mortem with the aid of a colour spectrophotometer (CM 2500c, Konika Minolta, Osaka, Japan) according to the Commission Internationale de I’Eclairage [11]. Breast water holding capacity (WHC) was ascertained by the filter-paper press method by Whiting and Jenkins [12]. Breast cooking loss and drip loss were determined according to the methods of Honikel [13]. Raw breast samples were sheared with the aid of a Meullenet–Owens razor shear blade (A/MORS) installed in a texture analyser (TA.XT plus, Stable Micro Systems, Surrey, UK) to measure the shear force.

2.4. Intestinal Morphometric Parameters

The duodenum (the loop), jejunum (tract before the Meckel’s diverticulum), and ileum (tract before the ileocolic junction) were harvested and prepared according to the methods of Biasato et al. [14]. The lengths of the duodenum, jejunum, and ileum were measured using a tape measure (cm). For the three segments of the small intestine, five villus crypt units with intact lamina propria were selected and placed on slides. The villus height, villus width, crypt depth, villus height:crypt depth ratio, the thickness of lamina propria, muscularis mucosa, and muscularis externa were measured with a light microscope (Olympus CX31, Olympus, Hicksville, New York, NY, USA) at 4× magnification, supported with digitalized live image analysis program (Olympus DP20, Olympus, Bartlett, TN, USA). The crypt depth was measured from the base of the crypt up to the zone of transition between the crypt and the villus. The villus height was measured from the tip of the villus to the villus crypt junction, as described by Sikandar et al. [15].

2.5. Goblet Cell Count (GCC)

Pre-processed slides were exposed to alcian blue periodic acid Shiff (AB-PAS) staining. Three sections were obtained from each intestinal segment and goblet cells were counted in 5 villi per section. Thus, an average of 15 values was measured for each sample. The histochemical differentiation based on acidic and mixed (acidic and neutral) mucin was observed according to the methods described elsewhere by Ashraf et al. [16]. Goblet cells containing acidic mucin were stained blue by the AB, while mixed mucin was stained purple by periodic acid Shiff staining.

2.6. Tibia Morphometric Parameters

The left and right tibia of five carcasses from each replicate were removed as drumsticks, thereafter de-fleshed and dried at room temperature for 24 h. The tibia weight was determined using a weighing scale (Adam Equipment S.A. PTY, Johannesburg, South Africa), while the tibia length (from its proximal to distal end), diaphysis diameter (midpoint of the tibia), and femoral and metatarsal side proximal head thicknesses were measured using an RS PRO digital Vernier caliper (^©RS Components, Midrand, South Africa). The bone breaking strength (BBS) was measured with a force gauge fitted on a texture analyser (TA.XT plus, Stable Micro Systems, Surrey, UK). The MAC AFRIC dial gauge vernier caliper (Adendorff Machinery Mart, Johannesburg, South Africa) was used to measure at midpoint the thickness of the medial and lateral walls. The medullary canal diameter was calculated by subtracting the thicknesses of the medial and lateral walls from the diaphysis diameter (midpoint). The Seedor index was obtained by dividing the tibia weight by its length [17]. The tibiotarsal and robusticity indexes were calculated using the following formulae, respectively:

$$Tibiotarsal index=\frac{diaphysis diameter-medullary canal diameter}{diaphysis diameter}\times 100$$

$$Robusticity index=\frac{tibia length}{cube root of bone weight}$$

2.7. Tibia Mineral Content

The tibia samples were collected in triplicates into crucibles and weighed to obtain their fresh weight. The samples were dried in an oven at 106 °C for 16 h to obtain dry weight and thereafter samples were incinerated in a muffle furnace for 16 h at 800 °C to obtain ash weight [18]. Then, 2 mL of 5 N hydrochloric acid was added to the ash samples and allowed to dissolve, before being transferred to 100 mL volumetric flasks and topped up to the mark with distilled water. After the sediments settled, the fluid was filtered into 14 mL centrifuge tubes and then vortexed before analysis. A solution of 2% nitric acid was used to dilute the samples before being analysed through an inductively coupled plasma mass spectrometer (ICP-MS Nexion 300Q, Perkin Elmer, Johannesburg, South Africa).

2.8. Statistical Analysis

Polynomial contrasts were employed to analyse the data (apart from the PC data) for linear and quadratic effects using response surface regression analysis [19]. Carcass and meat quality characteristics, intestinal morphology, GCC, tibia morphology, and tibia mineral content data were analysed using a one-way analysis of variance by means of general linear model (GLM) procedure of SAS [19], where treatment was considered the only factor. For all the measured parameters, significance was considered at p < 0.05 and the least-squares means were separated using the probability of difference options in SAS.

3. Results

3.1. Carcass and Meat Quality Characteristics

For the entire six-week feeding trial, each chicken consumed a total of 3397–3547 g of the commercial diets and drank between 6099 and 6264 mL of water while accumulating between 1859.9 and 1912.9 g of body mass. The weight gain and feed intake data were used to determine the feed conversion ratio, which ranged between 1.80 and 1.91 g:g. There were linear and quadratic responses (p < 0.05) for spleen weight (R² = 0.882; p = 0.001) and shear force (R² = 0.549; p = 0.039) in response to MSE levels (

Table 1. Carcass and meat quality characteristics (g, unless otherwise stated) of six-week-old broiler chickens (n = 125) orally administered with varying levels of moringa seed extract (MSE) in drinking water.

2 Parameters	1 Treatments					p Value
	NC	PC	MSE60	MSE90	MSE120	3 SEM	GLM	Linear	Quadratic
SLWT	1918	1932	1956	1971	1939	21.00	0.154	0.135	0.517
Carcass yield (%)	69.61 b	69.39 b	71.37 a	71.43 a	71.34 a	0.270	0.002	0.967	0.436
HCW	1335 b	1341 b	1396 a	1408 a	1383 a,b	15.88	0.006	0.296	0.967
CCW	1295 c	1307 b,c	1350 a,b	1365 a	1335 a,b,c	15.00	0.011	0.465	0.980
Breast weight	618.3	694.6	624.4	678.4	633.7	35.40	0.242	0.401	0.386
Drumstick weight	133.0	151.6	135.8	136.2	137.8	6.630	0.325	0.610	0.299
Thigh weight	156.5	142.2	160.2	160.7	161.7	16.12	0.415	0.176	0.510
Gizzard	43.00	44.40	45.90	44.80	45.90	1.140	0.542	0.004	0.174
Liver	55.40	55.70	55.10	55.80	49.50	3.010	0.084	0.399	0.220
Spleen	3.12 b	3.13 b	3.22 a	3.24 a	3.22 a	0.010	0.012	0.001	0.001
pH24hr	5.89 a,b	5.87 b	5.96 a,b	5.89 a,b	6.01 a	0.040	0.022	0.185	0.656
L* (lightness)	51.58	52.43	50.74	51.59	51.67	0.810	0.684	0.861	0.427
a* (redness)	−0.93 a,b	−1.22 b	−0.55 a,b	−0.57 a,b	−0.11 a	0.320	0.021	0.136	0.771
b* (yellowness)	5.60	5.43	4.63	4.86	5.20	0.770	0.542	0.621	0.358
WHC (%)	16.40 a	12.90 a,b	11.20 b	12.80 a,b	12.80 a,b	1.270	0.025	0.057	0.068
Drip loss (%)	4.79	5.82	4.53	5.19	5.44	0.870	0.485	0.534	0.619
Cooking loss (%)	38.14	36.59	32.22	32.46	30.48	2.510	0.842	0.042	0.603
Shear force (N)	3.19 a	3.27 a	2.24 b	2.28 b	2.28 b	0.190	0.005	0.001	0.039

^a,b,c Values within the same row with different subscripts significantly differed (p < 0.05). ¹ Treatments: NC = drinking water only; PC = 5 g probiotic/L of drinking water; MSE₆₀ = 60 mL of moringa seed extract/L of drinking water; MSE₉₀ = 90 mL of moringa seed extract/L of drinking water; MSE₁₂₀ = 120 mL of moringa seed extract/L of drinking water. ² Parameters: SLWT = slaughter weight; HCW = hot carcass weight; CCW = cold carcass weight; pH_24hr = pH at 24 h post-mortem; WHC: water holding capacity. ³ SEM = standard error of the mean.

). A linear decrease was observed for cooking loss (R² = 0.231; p = 0.042), while a linear increase was noted for gizzard weight (R² = 0.432; p = 0.004) as MSE levels increased.

The GLM data showed that the MSE treatments (MSE₆₀, MSE₉₀, and MSE₁₂₀) resulted in a higher carcass yield than the NC and PC treatments. The birds that were orally administered with the MSE (MSE₆₀, MSE₉₀, and MSE₁₂₀) treatments had higher (p < 0.05) HCW than the birds in PC but were statistically similar (p > 0.05) to the birds in NC and MSE₁₂₀. The NC and PC groups had lower CCW compared to all the MSE treatment groups; however, PC was statistically similar (p > 0.05) with the birds in MSE₆₀ and MSE₁₂₀. Treatment MSE₆₀, MSE₉₀, and MSE₁₂₀ promoted heavier spleen weight than the NC and PC groups.

The breast meat pH_24hr and redness (a*) of birds on PC were lower (p < 0.05) than those on NC, MSE₆₀, MSE₉₀, and MSE₁₂₀ treatments. Breast meat from MSE₆₀ had lower WHC than those from NC but was similar (p > 0.05) to those from PC, MSE₉₀, and MSE₁₂₀. The MSE treatments (MSE₆₀, MSE₉₀, and MSE₁₂₀) promoted lower (p < 0.05) shear force than those from the NC and PC groups.

3.2. Intestinal Morphometric Parameters

There were linear and quadratic responses (p < 0.05) for ileal muscularis mucosa thickness (MMT) (R² = 0.853; p = 0.001) in response to MSE levels (

Table 2. Intestinal morphometric parameters of six-week-old broiler chickens (n = 125) orally administered with varying levels of moringa seed extract (MSE) in drinking water.

2 Parameters	1 Treatments					p Value
	NC	PC	MSE60	MSE90	MSE120	3 SEM	GLM	Linear	Quadratic
Duodenum length (cm)	25.81b c	25.34 c	26.55 a	27.54 a	27.15 a	0.440	0.023	0.012	0.515
Jejunum length (cm)	67.87	67.12	68.74	69.15	69.47	0.930	0.845	0.162	0.937
Ileum length (cm)	67.35 b	66.36 b	69.52 a	70.71 a	69.72 a	0.920	0.034	0.064	0.245
Duodenum
VH (µm)	1904 b	1890 b	2009 a	1968 a,b	1955 a,b	27.30	0.018	0.872	0.375
VW (µm)	162.2 b	160.8 b	173.3 a	181.5 a	179.8 a	3.403	0.009	0.003	0.586
CD (µm)	188.4 b	187.4 b	196.2 a	191.2 a,b	192.4 a,b	1.931	0.004	0.138	0.262
LPT (µm)	125.2	121.6	127.8	135.0	136.8	5.487	0.845	0.284	0.526
MMT (µm)	49.00 b	48.80 b	51.70 a	52.40 a	51.90 a	0.958	0.001	0.535	0.527
MET (µm)	247.2	244.2	247.4	256.4	255.4	5.257	0.942	0.241	0.681
VH/CD	10.21	10.08	10.24	10.30	10.17	0.118	0.584	0.572	0.475
Jejunum
VH (µm)	1632 c	1620 c	1739 a	1684 b	1686 b	14.49	0.005	0.091	0.095
VW (µm)	123.4 c	126.2 c	143.4 b	154.6 a	151.4 a	2.160	0.009	0.627	0.296
CD (µm)	163.8 b	158.8 b	171.6 a	169.2 a	169.8 a	2.178	0.025	0.065	0.996
LPT (µm)	118.8 b	118.6 b	121.1 a	121.2 a	121.0 a	0.676	0.005	0.700	0.615
MMT (µm)	39.20 b	38.80 b	40.80 a	41.00 a	40.60 a	0.486	0.006	0.001	0.480
MET (µm)	240.4	226.0	233.6	244.4	239.4	5.986	0.752	0.853	0.746
VH/CD	9.980	10.21	10.13	10.20	10.13	0.120	0.681	0.093	0.198
Ileum
VH (µm)	1209 c	1249 b,c	1333 a	1286 a,b	1278 a,b	20.59	0.011	0.631	0.531
VW (µm)	120.7 c	123.0 c	132.7 b	142.9 a	142.1 a	2.198	0.008	0.036	0.955
CD (µm)	126.2	127.0	127.1	126.4	126.2	1.520	0.142	0.002	0.458
LPT (µm)	127.6	108.4	112.4	119.6	119.4	7.467	0.214	0.457	0.500
MMT (µm)	34.00 c	34.40 b,c	35.20 a,b	35.60 a	35.80 a	0.320	0.001	0.001	0.025
MET (µm)	234.8	235.0	235.4	235.6	235.4	0.410	0.524	0.001	0.789
VH/CD	9.570 c	9.780 b	10.13 a	10.18 a	10.13 a	0.060	0.010	0.763	0.851

^a,b,c Values within the same row with different subscripts significantly differed (p < 0.05). ¹ Treatments: NC = drinking water only; PC = 5 g probiotic/L of drinking water; MSE₆₀ = 60 mL of moringa seed extract/L of drinking water; MSE₉₀ = 90 mL of moringa seed extract/L of drinking water; MSE₁₂₀ = 120 mL of moringa seed extract/L of drinking water. ² Parameters: VH = villus height; VW = villus width; CD = crypt depth; LPT = lamina propria thickness; MMT = muscularis mucosa thickness; MET = muscularis externa thickness; VH/CD = villus height/crypt depth ratio. ³ SEM = standard error of the mean.

) Linear increases were observed for duodenum length (R² = 0.328; p = 0.012), duodenal villus width (VW) (R² = 0.867; p = 0.003), jejunal MMT (R² = 0.845; p = 0.001), ileal VW (R² = 0.491; p = 0.001), ileal crypt depth (CD) (R² = 0.779; p = 0.002), and ileal muscularis externa thickness (MET) (R² = 0.824; p = 0.001) as MSE levels increased.

There were treatment effects (p < 0.05) on the lengths of the duodenum and ileum, duodenal (VH, VW, CD, and MMT), jejunal (VH, VW, CD, LPT, and MMT), and ileal (VH, VW, MMT, and VH/CD). The MSE treatments (MSE₆₀, MSE₉₀, and MSE₁₂₀) resulted in longer duodenum and ileum lengths (p < 0.05) than the NC and PC treatments. The birds that were administered with the MSE treatments (MSE₆₀, MSE₉₀, and MSE₁₂₀) had higher (p < 0.05) duodenal VH and CD than the birds in PC but were statistically similar (p > 0.05) with the birds in NC, MSE₉₀, and MSE₁₂₀. The NC and PC groups had lower duodenal VW and MMT compared to all the MSE treatment groups, whose duodenal VW and MMT values did not differ (p > 0.05). Treatment MSE₆₀ promoted the highest (p < 0.05) jejunal VH, followed by those in MSE90 and MSE120, and the lowest jejunal VH was from the NC and PC groups. Birds on MSE₉₀ and MSE₁₂₀ had the highest (p < 0.05) jejunal VW, followed by those in MSE₆₀, and the lowest was from the NC and PC groups.

Further, birds reared on NC and PC had lower CD, LPT, and MMT than those on MSE₆₀, MSE₉₀, and MSE₁₂₀. Treatments NC and PC promoted lower ileal VH than on the MSE₆₀, MSE₉₀, and MSE₁₂₀ treatments, but PC, MSE₉₀, and MSE₁₂₀ were comparable (p > 0.05). Birds on MSE₉₀ and MSE₁₂₀ had the highest (p < 0.05) ileal VW, followed by those in MSE₆₀, and the lowest ileal VW was from the NC and PC groups. The MSE₆₀, MSE₉₀, and MSE₁₂₀ had higher (p < 0.05) MMT than the NC group. However, PC and MSE₆₀ promoted similar (p > 0.05) ileal MMT. Birds on MSE₆₀, MSE₉₀, and MSE120 had the highest (p < 0.05) ileal VH/CD, followed by those on PC, and the lowest ileal VH/CD was from the NC group.

3.3. Goblet Cell Count (GCC)

There were linear or quadratic responses (p > 0.05) for duodenal and ileal acidic and total GCC as MSE levels increased (

Table 3. Goblet cell count (per 100 µm villus height) of six-week-old broiler chickens (n = 125) orally administered with varying levels of moringa seed extract (MSE) in drinking water.

2 Intestinal Segments	3 Goblet Cell	1 Treatment					p Value
		NC	PC	MSE60	MSE90	MSE120	4 SEM	GLM	Linear	Quadratic
Duodenum	Acidic	46.01 b,c	45.25 c	47.16 b	49.55 a	48.98 a	0.617	0.001	0.001	0752
	Mixed	36.80	36.01	37.67	38.59	38.39	1.011	0.254	0.226	0.823
	Total	82.81 b,c	81.25 c	84.83 b	88.15 a	87.38 a	0.722	0.015	0.003	0.614
Jejunum	Acidic	50.57 b	47.86 c	51.04 a,b	52.33 a	51.84 a,b	0.544	0.003	0.063	0.794
	Mixed	43.71 b	41.29 b	44.71 a	45.92 a	45.29 a	0.827	0.007	0.116	0.591
	Total	94.28 b	89.15 c	95.75 a,b	98.25 a	97.13 a,b	1.183	0.004	0.058	0.630
Ileum	Acidic	50.97 b,c	49.71 c	51.74 a,b,c	52.86 a,b	53.31 a	0.697	0.031	0.009	0.734
	Mixed	44.37 b	43.06 b	45.30 a,b	46.28 a	45.23 a,b	0.754	0.005	0.233	0.339
	Total	95.34 b	92.77 c	97.04 a	99.14 a	98.55 a	1.106	0.002	0.016	0.619

^a,b,c Values within the same row with different subscripts significantly differed (p < 0.05). ¹ Treatments: NC = drinking water only; PC = 5 g probiotic/L of drinking water; MSE₆₀ = 60 mL of moringa seed extract/L of drinking water; MSE₉₀ = 90 mL of moringa seed extract/L of drinking water; MSE₁₂₀ = 120 mL of moringa seed extract/L of drinking water. ² Intestinal segments: duodenum, jejunum, ileum. ³ Goblet cell: acidic, mixed, total. ⁴ SEM = standard error of the mean.

). The duodenal acidic (R² = 0.485; p = 0.001) and total GCC (R² = 0.546; p = 0.001) showed positive linear responses. Additionally, the ileal acidic (R² = 0.338; p = 0.009) and total GCC (R² = 0.304; p = 0.016) linearly increased in response to MSE dosage levels.

Treatment PC promoted lower (p < 0.05) duodenal acidic and total GCC than MSE₉₀ and MSE₁₂₀ treatments, which did not differ (p > 0.05). Birds on MSE₆₀ had similar (p > 0.05) duodenal acidic and total GCC as birds on NC. The PC treatment promoted lower (p < 0.05) acidic, mixed, and total GCC when compared to the MSE treatment (MSE₆₀, MSE₉₀, and MSE₁₂₀) groups. Birds on MSE₁₂₀ had higher acidic GCC than those on PC and NC groups. Treatment MSE₆₀ promoted the same (p > 0.05) acidic GCC as PC and NC treatments. However, treatment PC resulted in lower mixed GCC than treatments MSE₆₀, MSE₉₀, and MSE₁₂₀, which did not differ (p > 0.05). The total GCC of the birds from the MSE treatment (MSE₆₀, MSE₉₀, and MSE₁₂₀) groups was higher (p < 0.05) than those from treatment PC.

3.4. Tibia Morphometric Parameters

There were significant linear and quadratic effects on the thickness of the medial wall (TMW) (R² = 0.747; p = 0.004) as MSE levels increased (

Table 4. Tibia morphometric parameters of six-week-old broiler chickens (n = 125) orally administered with varying levels of moringa seed extract (MSE) in drinking water.

2 Parameters	1 Treatments					p Value
	NC	PC	MSE60	MSE90	MSE120	3 SEM	GLM	Linear	Quadratic
Tibia weight (g)	10.56 c	11.56 b	13.50 a	14.06 a	13.94 a	0.330	0.003	0.003	0.307
Tibia length (mm)	101.5 a,b	103.5 a	97.55 b,c	95.49 c	96.11 c	1.500	0.011	0.001	0.173
Diaphysis diameter (mm)	8.56	8.90	8.64	8.64	8.40	0.180	0.087	0.382	0.167
FPHT (mm)	26.27	26.12	25.51	25.19	25.61	0.410	0.075	0.169	0.720
MPHT (mm)	19.52	19.96	19.51	19.30	19.23	0.450	0.168	0.084	0.397
BBS (N)	242.8 d	246.9 d	261.0 c	273.0 b	282.5 a	2.170	0.001	0.001	0.843
Seedor index (g/mm)	0.10 b	0.11 b	0.14 a	0.15 a	0.15 a	0.010	0.014	0.011	0.116
TMW (mm)	1.33 c	1.38 b,c	1.45 a,b	1.52 a	1.50 a	0.030	0.009	0.002	0.004
TLW (mm)	2.24 b	2.31 b	2.47 a	2.55 a	2.51 a	0.030	0.029	0.021	0.540
MCD (mm)	5.01 a,b	5.21 a	4.720 b,c	4.57 b,c	4.39 c	0.160	0.020	0.004	0.494
Tibiotarsal index	41.61 c	41.64 c	45.48 b	47.12 a,b	47.81 a	0.740	0.003	0.001	0.176
Robusticity index	21.76 a,b	22.03 a	21.19 b,c	20.89 c	20.98 c	0.210	0.005	0.001	0.457

^a,b,c,d Values within the same row with different subscripts significantly differed (p < 0.05). ¹ Treatments: NC = drinking water only; PC = 5 g probiotic/L of drinking water; MSE₆₀ = 60 mL of moringa seed extract/L of drinking water; MSE₉₀ = 90 mL of moringa seed extract/L of drinking water; MSE₁₂₀ = 120 mL of moringa seed extract/L of drinking water. ² Parameters: FPHT = Femoral side proximal head thickness; MPHT = metatarsal side proximal head thickness; BBS = bone breaking strength; TMW = thickness of the medial wall; TLW = thickness of the lateral wall; MCD = medullary canal diameter. ³ SEM = standard error of the mean.

). Oral administration of MSE linearly increased tibia weight (R² = 0.651; p = 0.003), bone breaking strength (BBS) (R² = 0.877; p = 0.001), Seedor index (R² = 0.470; p = 0.011), medullary canal diameter (MCD) (R² = 0.636; p = 0.004), and robusticity index (R² = 0.859; p = 0.001). However, it resulted in linear decreases for tibia length (R² = 0.736; p = 0.001), thickness of the lateral wall (TLW) (R² = 0.359; p = 0.021), and tibiotarsal index (R² = 0.735; p = 0.001).

The tibial weight of birds in MSE (MSE₆₀, MSE₉₀, and MSE₁₂₀) groups was heavier (p < 0.05) than those on PC. Treatment PC promoted the longest tibia than MSE₆₀, MSE₉₀, and MSE₁₂₀ treatments, which did not differ (p > 0.05). The MSE₁₂₀ promoted the highest BBS (282.5 N) compared to PC (246.9 N). Birds on PC had a lower Seedor index than those from the MSE treatment (MSE₆₀, MSE₉₀, and MSE₁₂₀) groups. Likewise, PC had lower TMW than the MSE administered groups but had the same (p > 0.05) TMW as the birds on MSE₆₀. Birds on NC had a higher TLW than birds on MSE₆₀, MSE₉₀, and MSE₁₂₀, which had statistically similar TLW values. Birds on PC had a higher (p < 0.05) MCD and robusticity index than those on MSE₆₀, MSE₉₀, and MSE₁₂₀. Birds on PC had a lower tibiotarsal index compared to those on MSE₉₀ and MSE₁₂₀.

3.5. Tibia Mineral Content

Oral administration of MSE resulted in linear increases for tibia ash (R² = 0.941; p = 0.001) and calcium (R² = 0.983; p = 0.001) (

Table 5. Tibia ash and mineral content of six-week-old broiler chickens (n = 125) orally administered with varying levels of moringa seed extract (MSE) in drinking water.

2 Parameters	1 Treatments					p Value
	NC	PC	MSE60	MSE90	MSE120	3 SEM	GLM	Linear	Quadratic
Ash (g)	43.36 d	45.34 c	47.63 b	49.88 a	50.86 a	0.460	0.001	0.001	0.227
Calcium (mg/g)	214.0 e	222.5 d	238.6 b	236.0 b	240.5 a	0.830	0.001	0.001	0.208
Phosphorus (mg/g)	114.0 b	118.7 a	119.0 a	117.6 a	118.4 a	2.080	0.005	0.195	0.296
Ca:P (mg/mg)	1.88 b	1.87 b	2.01 a	2.01 a	2.03 a	0.040	0.010	0.070	0.220
Magnesium (mg/g)	4.66 c	6.09 b	6.90 a	7.02 a	6.95 a	0.680	0.007	0.060	0.984
Iron (µg/g)	44.51	46.34	41.51	48.37	50.42	3.480	0.174	0.209	0.701
Zinc (µg/g)	1.45 c	1.60 b	1.75 a	1.77 a	1.80 a	0.100	0.011	0.489	0.187

^a,b,c,d,e Values within the same row with different subscripts significantly differed (p < 0.05). ¹ Treatments: NC = drinking water only; PC = 5 g probiotic/L of drinking water; MSE₆₀ = 60 mL of moringa seed extract/L of drinking water; MSE₉₀ = 90 mL of moringa seed extract/L of drinking water; MSE₁₂₀ = 120 mL of moringa seed extract/L of drinking water. ² Parameters: Ca:P = calcium/phosphorus ratio. ³ SEM = standard error of the mean.

). There were significant treatment effects on tibia ash and mineral content except for iron. The tibia ash of birds on PC was lower (p < 0.05) than those on MSE₉₀ and MSE₁₂₀ treatments. Birds on PC had lower tibia calcium levels than those on MSE₁₂₀, but not comparable (p < 0.05) to MSE₆₀ and MSE₉₀. The tibia phosphorus of birds in the PC group was similar (p > 0.05) to those in the MSE treatment groups. Birds reared on MSE₆₀, MSE₉₀, and MSE120 had higher (p < 0.05) Ca:P than those reared on NC and PC, for which the Ca:P did not differ (p > 0.05). Birds reared on MSE₆₀, MSE₉₀, and MSE₁₂₀ had the highest (p < 0.05) tibial magnesium and zinc contents followed by those on PC, and the lowest tibial magnesium and zinc concentration were from those on NC.

4. Discussion

4.1. Carcass and Meat Quality Characteristics

Carcass yield is important for determining the growth performance and economic returns of broiler meat. The noted increase in carcass yield, HCW, and CCW from MSE-supplemented birds indicates an improvement in muscle and bone development than in the control groups. This was expectable given that moringa has growth-stimulating properties. Similarly, Tariq et al. [20] noted an increase in the carcass yield and breast weight of chickens fed diets containing 1% clove (Syzygium aromaticum) seed meal. Contrary to this study, Ashom et al. [21] noted a similarity among dietary treatments for carcass yield when roselle (Hibiscus sabdariffa) seed meal was supplemented (50%) in the diets of finishing broilers. The increased CCW observed in this study among the MSE-administered group could be translated to more cuts available for sale, signifying high profitability.

Furthermore, the lack of differences in gizzard and liver weights among the treatments might be a result of the elimination of the nonstarch polysaccharides of moringa seed through water extraction, as it has been noted by Matshogo et al. [22] that a direct relationship exists between dietary fibre levels and internal organ sizes. The liver is generally responsible for the secretion of alkaline phosphatase, alanine transaminase, and gamma-glutamyl transferase enzymes, and high levels indicate a liver disorder. The similarity in the values of these enzymes among treatments obtained in our previous work [10] justifies the lack of differences in liver weight.

The observed increase in spleen weights indicates that there was a positive immune response to the nutraceutical content of MSE. Thus, MSE nutraceutical can be stated to operate as an immune system booster by increasing blood flow and boosting the body’s immunological function. Hajati et al. [23] noted no variation in the spleen weight when grape seed extracts were administered up to 300 mg/Kg diet to birds.

The oral administration of MSE increased ultimate meat pH, which is supported by the findings of Liu et al. [24] who administered chestnut wood extract to broilers for 42 days. The 24 h pH values generally demonstrate the rate of carcass glycolysis and are linked with meat shelf life. Hence, oxidation tends to occur rapidly in meat with a reduced pH_24hr value. The PC treatment had the lowest pH_24hr and redness (a*) values, which supports previous studies that noted that light-coloured broiler meat samples have reduced pH_24hr values [25].

Meat producers value the WHC because it plays an essential part in determining the ultimate weight of the meat. The increased WHC through oral administration of MSE₉₀ and MSE₁₂₀ agrees with the findings of Shen et al. [26], who administered bamboo leaf extracts to broilers.

Present findings show that shear force values declined with MSE administration, suggesting that the meat became tenderer with the supplementation of MSE. This was not consistent with the report of Park et al. [27] who reported no effect on shear force of broiler meat when Saposhnikovia divaricata, Lonicera japonica, or Chelidonium majus extracts were administered for 35 days. However, Shen et al. [26] noted variations among treatments for shear force, which agrees with this present study. Nonetheless, the observed differences among these trials can be attributed to differences in various plant species, the concentration of the phytochemicals, and the route of administration of the phytogenics.

4.2. Intestinal Morphometric Parameters

Moringa oleifera seed powder has nutraceuticals that can be utilised to improve intestinal morphometric parameters, goblet cell count, tibia morphometric parameters, and tibia mineral content in chicken diets. The current study demonstrated that oral administration of MSE enhanced duodenal and ileal lengths. In contrast, Hajati et al. [28] found no effect on the lengths of the duodenum or ileum when broiler diets were supplemented with grape seed extract or vitamin C for 42 days. Brenes et al. [29] showed a decrease in the duodenal and ileal length of broilers supplemented with grape seed extracts, while Thomas et al. [30] found that birds fed with diets containing 0.5% green tea high in flavonoids had shorter intestinal lengths. The disparity in results can be ascribed to the difference in the solvents used in the extraction, concentration of the phytochemicals, and the route of administration.

Nutrient absorption from the small intestine is facilitated by specialized structures known as the villi. The increased VH suggests an increased surface area for efficient nutrient absorption. Deep crypts suggest rapid tissue turnover and a requirement for new tissue that promotes nutrient absorption. Therefore, the increased CD implies acceleration in the replacement of enterocytes, which make up the walls of the villi. The increased duodenal VH, VW, and CD are consistent with the findings of Carboni et al. [31], who administered teff (Eragrostis tef) seed extracts to broilers and observed improved VH, VW, and CD. The increased jejunal VH, VW, and CD disputed the report by Ao and Kim [32], that grape seed extract (0.02%) increased jejunal VH and decreased CD. The increased ileal VH and VW corroborated the findings of Rafeeq et al. [33] who found that the ileal VH and VW were enhanced by cumin (Cuminum cyminum) seed and jir (Artemisia scoparia) seed extracts, respectively.

The absorption capacity of the small intestine is determined by the VH/CD ratio, suggesting that optimum digestion and absorption are attained when the VH/CD ratio is increased. The increased ileal VH/CD ratio implies that MSE improved nutrient digestion and absorption, which is not consistent with the observations of Li et al. [34]. Li et al. [34] noted the VH/CD ratio was decreased at the duodenum, increased at the ileum, and had no effect on the jejunal section. The increased VH/CD ratio induced an intestinal structure more oriented to digestion, with improved absorptive and hydrolysis potential, requiring fewer nutrients to be directed towards intestinal maintenance. Therefore, with MSE administration, the intestinal structure of the small intestine was enhanced. Furthermore, the administration of MSE elevated MMT in the three segments of the small intestines and lamina propria thickness in the jejunum. The lamina propria and muscularis mucosa are reliable in moving digested and absorbed nutrients into the bloodstream.

4.3. Goblet Cell Count

Maximizing broiler physiological performance requires consideration of their digestive tract health, which is a complex, multifaceted system that takes several small intestine features into consideration (such as the microbiota and mucin dynamics). Apart for the mixed GCC in the duodenum, the noted increase in the intestinal GCC disputes an earlier report by Carboni et al. [31] who reported that teff seed extract increased duodenal neutral and mixed GCC. However, Tan et al. [35] noted an increased GCC when dandelion (Taraxacum officinale) root extract was administered to gold pompanos fish. Pereira da Silva et al. [36] recorded increased acidic, neutral, and mixed GCCs in all three segments of the small intestine. These findings imply that MSE administration promoted goblet cell production, which protects the intestinal lumens. The increase in acidic goblet cells, containing acidic mucin due to the administration of MSE, possibly contributed to the reduction in intestinal pH, which, in the long term, may lead to increased solubilisation and uptake of nutrients and improved intestinal microbial profile.

4.4. Tibia Morphometric Parameters

The structure of bones and their morphometric parameters together with their structural properties are critical parameters in ascertaining bones’ ability to perform their basic functions, which are to give structural support and ensure normal locomotion. The observed increase in tibia morphometric parameters and BBS is, however, not consistent with the report of Leskovec et al. [37] who reported a negative effect of olive leaf and marigold extracts on tibia morphometric parameters and BBS. Abbas and Khauoon [38] noted increased tibia length but no effect on other tibia morphometric parameters when grape seed extract was administered in broiler diets. The elevated bone weight, BBS, and tibiotarsal index in the current study may possibly be attributed to the presence of flavonoids, minerals, vitamins, and antioxidants in MSE. Similarly, Hohman and Weaver [39] reported an increase in tibia morphometric parameters and BBS when grape seed extracts were administered to Sprague Dawley rats. Shen et al. [40] explained that the presence of antioxidants in plants could lead to low oxidative stress in the bones and potentially benefit bone health. Shah et al. [41] noted differences in the thickness of the wall, medullary canal diameter, and tibiotarsal index, which supports the findings of this study. Moringa polyphenols can positively affect tibia morphometric parameters and strength by protecting the bone through various mechanisms. Through their antioxidative and anti-inflammatory activities, polyphenols can improve osteoblast genesis, suppress osteoclast genesis, and increase osteoimmunological action [37].

4.5. Tibia Mineral Content

The obtained tibia ash and mineral composition results are not in agreement with the report of Liu et al. [42] who administered extracts of Herba epimedii and Ligustri lucidi in diets of Wistar rats and observed a lack of dietary effects in ash content and mineral contents (Ca, P, K, Mg, Mn, and Zn). Calcium and phosphorus are the most abundant minerals in bones, and their distribution influences the formation and mineralization of bone [43]. The observed increase in tibia ash and mineral composition could point to better mineral bioavailability due to the increased absorption of water-soluble nutrients from MSE. The increased concentration of tibia Ca, P, Ca:P ratio, magnesium, and zinc of birds administered with MSE may possibly be due to the increased mineral absorption. Abdullah et al. [44] stated that magnesium enriches bone formation by activating osteoclasts, which increases the Ca:P ratio. The increase in zinc concentration could indicate the stimulation of DNA production in osteoblasts, thereby increasing bone weight and the absorption of calcium ions in the bones.

5. Conclusions

The results showed that oral administration of Moringa oleifera seed extracts up to 90 mL per litre of drinking water improved carcass yield, some internal organ weight, meat quality, intestinal morphometric parameters, goblet cell count, tibia weight, breaking strength, and calcium and phosphorus. We concluded that the administration of Moringa oleifera seed extracts through drinking water has the potential to enhance dressing percentage, organ sizes, intestinal morphology, and some bone and meat traits in broiler production.