applied
sciences
Article
Effect of Moringa oleifera Seed Extract Administered through
Drinking Water on Physiological Responses, Carcass and Meat
Quality Traits, and Bone Parameters in Broiler Chickens
Chidozie Freedom Egbu 1,2,3, * , Lebogang Ezra Motsei 1,2 , Azeez Olanrewaju Yusuf 4
and Caven Mguvane Mnisi 1,2
Citation: Egbu, C.F.; Motsei, L.E.;
Yusuf, A.O.; Mnisi, C.M. Effect of
Moringa oleifera Seed Extract
Administered through Drinking
Water on Physiological Responses,
Carcass and Meat Quality Traits, and
Bone Parameters in Broiler Chickens.
Appl. Sci. 2022, 12, 10330. https://
doi.org/10.3390/app122010330
Academic Editor: Monica Gallo
Received: 22 August 2022
Accepted: 10 October 2022
Published: 13 October 2022
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4.0/).
Appl. Sci. 2022, 12, 10330. https://doi.org/10.3390/app122010330
1 Department of Animal Science, Faculty of Natural and Agricultural Science, North-West University,
Mmabatho 2735, South Africa
2 Food Security and Safety Focus Area, Faculty of Natural and Agricultural Sciences, North-West University,
Mmabatho 2745, South Africa
3 Department of Agricultural Education, School of Vocation and Technical Education, Alvan Ikoku Federal
College of Education, Owerri P.M.B. 1033, Nigeria
4 Department of Animal Production and Health, Federal University of Agriculture,
Abeokuta P.M.B. 2240, Nigeria
* Correspondence: egbuchidozie@gmail.com; Tel.: +27-83-690-2706
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.
Keywords: bioactive compounds; broilers; carcass characteristics; feed additives; moringa
1. Introduction
Sustainable broiler chicken production can contribute to the United Nations’ sustain-
able 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
https://www.mdpi.com/journal/applsci
Appl. Sci. 2022, 12, 10330 2 of 13

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 afford-
able 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 charac-
teristics, 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 probi-
otic (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◦ 550 000 S; 25◦ 480 000 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
Appl. Sci. 2022, 12, 10330 3 of 13

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 (pH24hr ) 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 drum-
sticks, 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 (mid-
Appl. Sci. 2022, 12, 10330 4 of 13

point of the tibia), and femoral and metatarsal side proximal head thicknesses were mea-
sured 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:
diaphysis diameter − medullary canal diameter
Tibiotarsal index = × 100
diaphysis diameter

tibia length
Robusticity index =
cube root o f 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). 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.
Appl. Sci. 2022, 12, 10330 5 of 13

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.

1 Treatments p Value
2 Parameters
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; pH24hr = pH at 24 h post-mortem; WHC: water holding capacity. 3 SEM = standard error of the
mean.

The breast meat pH24hr 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) 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.
Appl. Sci. 2022, 12, 10330 6 of 13

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

1 Treatments p Value
2 Parameters
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
a 181.5 a 179.8 a
VW (µm) 162.2 b 160.8 b 173.3 3.403 0.009 0.003 0.586
a
CD (µm) 188.4 b 187.4 b 196.2 191.2 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.

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). 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.
Appl. Sci. 2022, 12, 10330 7 of 13

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 1 Treatment p Value
Intestinal 3 Goblet
Segments Cell NC PC MSE60 MSE90 MSE120 4 SEM GLM Linear Quadratic
Acidic 46.01 b,c 45.25 c 47.16 b 49.55 a 48.98 a 0.617 0.001 0.001 0752
Duodenum 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
51.04 51.84
Acidic 50.57 b 47.86 c a,b 52.33 a a,b 0.544 0.003 0.063 0.794
Jejunum Mixed b b 44.71 a 45.92 a 45.29 a 0.827 0.007 0.116 0.591
43.71 41.29
b c 95.75 a 97.13
Total 94.28 89.15 a,b 98.25 a,b 1.183 0.004 0.058 0.630

51.74 52.86
Acidic 50.97 b,c 49.71 c a,b,c a,b 53.31 a 0.697 0.031 0.009 0.734
Ileum 45.30 45.23
Mixed 44.37 b 43.06 b a,b 46.28 a a,b 0.754 0.005 0.233 0.339
c a a a
Total 95.34 b 92.77 97.04 99.14 98.55 1.106 0.002 0.016 0.619
a,b,cValues within the same row with different subscripts significantly differed (p < 0.05). 1 Treat-
ments: 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.

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). 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 .
Appl. Sci. 2022, 12, 10330 8 of 13

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.

1 Treatments p Value
2 Parameters
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,dValues within the same row with different subscripts significantly differed (p < 0.05). 1 Treat-
ments: 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.

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). 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.

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.

1 Treatments p Value
2 Parameters
NC PC MSE60 MSE90 MSE120 3 SEM GLM Linear Quadratic
Ash (g) d 45.34 c b 49.88 a 50.86 a 0.460 0.001 0.001 0.227
43.36 47.63
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 Treat-
ments: 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.
Appl. Sci. 2022, 12, 10330 9 of 13

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 rela-
tionship 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 pH24hr
value. The PC treatment had the lowest pH24hr and redness (a*) values, which supports
previous studies that noted that light-coloured broiler meat samples have reduced pH24hr
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 intesti-
nal 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
Appl. Sci. 2022, 12, 10330 10 of 13

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 ad-
ministration, 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 diges-
tive 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 neu-
tral 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 struc-
tural properties are critical parameters in ascertaining bones’ ability to perform their basic
functions, which are to give structural support and ensure normal locomotion. The ob-
served 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 in-
creased 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, miner-
als, vitamins, and antioxidants in MSE. Similarly, Hohman and Weaver [39] reported an
Appl. Sci. 2022, 12, 10330 11 of 13

increase in tibia morphometric parameters and BBS when grape seed extracts were admin-
istered 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, break-
ing 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.

Author Contributions: Conceptualization, C.F.E., L.E.M. and A.O.Y.; methodology, C.F.E., L.E.M.
and A.O.Y.; software, C.F.E. and C.M.M.; validation, L.E.M., A.O.Y. and C.M.M.; formal analysis,
C.F.E. and C.M.M.; investigation, C.F.E., L.E.M. and A.O.Y.; resources, L.E.M.; data curation, C.F.E.
and L.E.M.; writing—original draft preparation, C.F.E. and A.O.Y.; writing—review and editing,
L.E.M., A.O.Y. and C.M.M.; visualization, C.F.E., L.E.M., A.O.Y. and C.M.M.; supervision, L.E.M. and
A.O.Y.; project administration, L.E.M.; funding acquisition, L.E.M. All authors have read and agreed
to the published version of the manuscript.
Funding: The first author is grateful to the North-West University Doctoral bursary for the financial
support received during this study.
Institutional Review Board Statement: The study was conducted according to the guidelines of the
Declaration of Helsinki and approved by the Animal Production Research Ethics Committee of the
North-West University (approval no. NWU-02002-20-A5: 1 October 2020).
Informed Consent Statement: Not applicable.
Data Availability Statement: The first author is willing to provide the study’s data on such a request.
Acknowledgments: We are grateful to V. Mlambo (University of Mpumalanga) for his technical
support of the manuscript development.
Conflicts of Interest: The authors declare no conflict of interest.
Appl. Sci. 2022, 12, 10330 12 of 13

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