Slov Vet Res 2023  |  Vol 60 No 3  |  135
Influence of Feed Restriction and Zinc Oxide 
Nanoparticles Supplementation on Growth 
Performance, Blood Biochemistry, Intestinal 
Morphology and Cecal Fermentation Parameters 
of Growing Rabbits 
Key words
feed restriction;
growth;
rabbits;
Zn oxide nanoparticles
Karima El-Naggar1*, Abeer M. El-Shenawy2, Sabreen E. Fadl3 
1Department of Nutrition and Veterinary Clinical Nutrition, Faculty of Veterinary Medicine, Alexandria 
University, Egypt, 2Unit of Biochemistry, Nutritional Deficiency diseases and Toxicology, Animal Health 
Research Institute, Kafr El-Sheikh Branch, Agricultural Research Center, Egypt,  3Biochemistry Department, 
Faculty of Veterinary Medicine, Matrouh University, Egypt.
*Corresponding author: karima.muhammad@alexu.edu.eg    
Abstract: The present study investigated the response of growing rabbits in terms 
of growth performance, serum biochemical, intestinal morphology, and caecal 
fermentation parameters to feed restriction and zinc oxide nanoparticles (ZnO-NPs) 
supplementation. A total of 60 New Zealand male rabbits were randomly distributed 
into 6 groups: AL-0 (fed ad libitum + fresh water as control); AL-15 and AL-30 (ad libitum + 
water supplemented with ZnO-NPs in water, 15 and 30 mg/L, respectively); and R-0, R-15 
and R-30 were the same as the first 3 groups but with restricted feeding regime. Rabbits 
fed ad libitum and supplemented with ZnO-NPs (15 mg/L) showed the highest body 
weight with no significant difference from AL- fed groups or R-0. Feed conversion ratio 
(FCR) showed no difference among the different experimental groups (P > 0.05). ZnO-
NPs supplementation reduced the serum lipid profile parameters, catalase enzyme in 
R-30, superoxide dismutase in AL-15 and AL-30 while increased serum malondialdehyde 
(MDA) in both ad libitum and restricted rabbits. ZnO-NPs administration resulted in 
lower caecal ammonia in AL-30 compared to its control (AL-0) as well as the content of 
individual volatile fatty acids (VFAs) (acetate, butyrate and propionate) (P < 0.05). Ileum 
morphological parameters (mucosal length, villi length, and goblet cell number) were 
modified in response to the feed restriction and ZnO-NPs addition. In conclusion, feed 
restriction program applied in this experiment altered rabbit growth performance (final 
body weight and weight gain with no differences in FCR), improved ileum morphology 
while had no significant effect on caecal fermentation (VFAs profile) or microbiological 
parameters. ZnO-NPs supplementation in both levels (15 and 30 mg/L) differently 
modulated serum lipid profile, antioxidant enzymes and MDA, VFAs profile in cecum 
and ileal morphology with no differences in rabbit growth performance. 
Received: 3 September 2022
Accepted: 30 May 2023
DOI 10.26873/SVR-1564-2023
UDC 636.92:591.53:577:591.4:616.15
Pages: 135–47
Original Research Article
Introduction 
In mammals, the peri-weaning period is considered one 
of the main stress periods in animal life as the young ani-
mal shifts from mother’s milk to the solid feed. During this 
critical period, fast growth occurs accompanied by many 
problems such as high incidence of metabolic disorders, 
high morbidity and mortality, and digestive disturbances, 
which have deleterious effects (1), causing economic losses 
in the commercial rabbit production. Antibiotics have been 
used to control these disturbances, but the emergence of 
136  |  Slov Vet Res 2023  |  Vol 60 No 3
antibiotic resistance has heightened the need to find other 
solutions to this problem. Several approaches have been 
considered to protect rabbit health during this stressful 
period, such as nutritional modulation and hygienic con-
trol. One of these approaches is the feed restriction, which 
can be done in two ways: quantitatively or qualitatively. 
Qualitative restriction involves limiting the amount of nu-
trients like protein and energy in feed (2). The quantitative 
restriction of feed can be done by limiting the feeder access 
time or reducing the amount of offered feed (2). Previous 
trials informed beneficial effects of feed restriction as it 
stimulated compensatory growth, improved feed efficiency 
utilization (3), improved digestibility of nutrients, lowered 
fat accumulation in carcasses (4, 5), and reduced the post-
weaning digestive disturbance as the epizootic rabbit enter-
opathy (6). Beside these beneficial effects, also some nega-
tive effects such as reduced final weight and dressing out 
percentage in feed restricted rabbits, were reported (7, 8).
Another different approach is through the supplementation 
of additives in feed or water. Zinc (Zn) is an essential trace 
element which plays a vital role in cell division, synthesis, 
and stabilization of DNA (9). Moreover, it has many 
beneficial effects on different physiological functions 
such as acid-base balance, nutrient metabolism, and 
immune response (10, 11). MacDonald (12) reported that 
Zn improved feed utilization through participating in the 
metabolism and assimilation of different nutrients such as 
carbohydrates, proteins, and lipids. Recently, manufactured 
nanoparticles (NP) have shown new characteristics such 
as great specific surface area, high surface activity, a lot 
of surface-active centers, and high catalytic efficiency 
(13). Due to the advantage of small size and high surface 
reactivity, nanoparticles showed higher transport, uptake 
and increased absorption efficiencies, enhancing their 
bioavailability inside the animal body (14). The inclusion of 
nano additives especially nano minerals in animal nutrition 
has been widely adopted to enhance the growth and 
production of livestock. Zinc nanoparticle supplementation 
was found to be beneficial in rabbits (15, 16), piglets (17) 
and poultry (18, 19). Therefore, the aim of the present 
study was to investigate the effect of feed restriction and 
ZnO-NPs supplementation in the drinking water on the 
growth performance, some blood biochemical parameters, 
intestinal morphology, caecal microbiology, and volatile 
fatty acids fermentation in growing rabbits. 
Materials and methods
Ethical statement
Animal management procedures were undertaken in 
accordance with the requirements of the Animal Care and 
Ethics Committee of the Faculty of Veterinary Medicine, 
Alexandria University, Egypt (AU 013-2022/10/12-3-149).
Rabbit care and experimental design
Sixty New Zealand male weaned rabbits; 33-35 days 
old (average body weight 770 ± 5.96 g) were randomly 
distributed into 6 experimental groups (10 rabbits/group) 
with 3 replicates/group (3-4 rabbits/each). Experimental 
groups were arranged as follows: AL-0 (rabbits were fed ad 
libitum + fresh water as control); AL-15 and AL-30 (rabbits 
fed ad libitum + water supplemented with ZnO-NPs, 15 and 
30 mg/L respectively); R-0 (restricted feed + fresh water); 
R-15 and R-30 (restricted feed + water supplemented with 
ZnO-NPs, 15 and 30 mg/L respectively). The restriction 
program applied throughout a 2-month experimental 
period was done as rabbits were fed ad libitum (AL) at the 
first day, next day they were fed at a level of 93% of AL, 
then again AL and next day 93% of AL of the day before 
(20). The zinc oxide water dispersion nanoparticles used 
were <100 nm particle size (TEM), (Sigma-Aldrich, catalog 
No.721077). Growing rabbits were kept on a commercial 
pelleted diet illustrated in table 1. The commercial diet used 
was formulated to meet the nutrient requirements of the 
growing rabbits according to (21). Rabbits were kept in 
wire-galvanized batteries that had feeders and drinkers. All 
the animals were kept under the same management and 
hygienic conditions. 
Growth performance: To implement the feed restriction 
program and ZnO-NPs supplementation, rabbits were 
weighed every 2 weeks, and feed intake (FI) was measured 
daily for all the experimental groups. Feed conversion ratio 
(FCR) and weight gain (WG) were calculated in accordance. 
Sample collection: At the end of the experiment, three 
rabbits from each group were chosen at random and used 
for sample collection. Blood was drawn from the rabbit 
ear vein for serum separation and biochemical parameter 
analysis. Rabbits were then sacrificed with an overdose 
of pentobarbital sodium at 60 to 70 mg/kg live weight. 
The ileum specimen was preserved in 10% formalin for 
histopathological examination. Caecal content (one gram) 
was collected under aseptic conditions for microbiological 
examination, while the remainder was used for VFAs 
fermentation analysis.
Blood biochemical parameters: Blood samples were 
collected in clean vials without anticoagulant. The serum 
was separated by centrifugation at 3000 rpm for 10 
min. Samples were used for the analysis of some serum 
biochemical parameters including total protein, albumin, 
glucose, triglycerides (TG), total cholesterol (TC), low 
and high-density lipoproteins (LDL and HDL), some liver 
function enzymes as aspartate amino transferase (AST) 
and alanine amino transferase (ALT), some kidney function 
parameters (uric acid and creatinine), some antioxidant 
enzymes as superoxide dismutase (SOD) and catalase, 
serum zinc concentration, and malondialdehyde (MDA) 
using commercial kits produced by Bio-diagnostic Co. 
(Diagnostic and Research reagents).
Slov Vet Res 2023  |  Vol 60 No 3  |  137
Intestinal morphology: The fixed ileal tissue was 
embedded in paraffin blocks, sectioned (5 µm), stained 
with haematoxylin and eosin (H&E) as previously described 
by Bancroft et al. (22), and then examined using a light 
microscope. The morphometric measurement of various 
parameters of intestinal villi and their associated crypt 
was performed quantitatively using image J software 
(Bethesda, MD, USA) according to Abràmoff et al. (23). 
Caecal microbiology and volatile fatty acids: According 
to Mountzouris et al. (24), the collected cecal contents were 
serially diluted ten times. The total bacterial and total coli-
form counts were calculated using the method described 
by Bivolarski et al. (25). The remaining cecal contents were 
immediately collected, squeezed with sterile gauze to ob-
tain the caecal filtrate, and the residues were discarded. 
The caecal filtrate was diluted with an equal volume of 
diluted sulfuric acid before the pH was determined (using 
a digital pH meter). The prepared filtrate samples were 
frozen at -20°C for subsequent analysis of ammonia and 
VFAs concentrations according to the methods described 
by Alvarenga et al. (26).
Statistical analysis: The obtained data were subjected 
to two-way ANOVA to test the effects of different levels 
of ZnO-NPs, feed restriction as well as their interaction. 
Statistical analysis was conducted using GraphPad Prism 
6 (GraphPrism Software, La Jolla, CA, USA). The obtained 
data were presented as mean ± standard error (SE) and 
significance was considered at P< 0.05.
Results
Growth performance of growing rabbits
Table 2 illustrates the rabbit’s growth performance in re-
sponse to ZnO-NPs supplementation and feed restriction. 
Applying the feed restriction significantly affected the rab-
bit’s growth performance in terms of final body weight 
(BW), weight gain (WG), and feed intake (FI) (P < 0.05). The 
final BW and WG of rabbits under restriction and supple-
mented with ZnO-NPs (15 mg/L) were significantly reduced 
(P < 0.05) compared with the same group of rabbits freely 
fed and receiving the same dose of ZnO-NPs. The ZnO-
NPs administration in the water had no significant effect 
(P > 0.05) on BW or WG either in the ad libitum or restricted 
rabbits. However, rabbits fed ad libitum and supplemented 
with ZnO-NPs (15 mg/L) showed the highest BW and WG 
(P > 0.05). 
A non-significant reduction of FI (P > 0.05) was obtained 
in restricted rabbits compared with their relative control 
rabbits. The effect of the restriction regime was clear in 
those supplemented with ZnO-NPs (30 mg/L) as FI was 
significantly reduced (P < 0.05) compared with their control 
group, which received the same dose but fed freely. Neither 
feed restriction nor Zn supplementation affected the FCR (P 
> 0.05) of growing rabbits, although it was non-significantly 
improved in ad libitum fed rabbits supplemented with the 
lower dose of ZnO-NPs (15 mg/L). 
Blood biochemical parameters
Blood serum concentrations of total protein and glucose 
showed no difference (P > 0.05) among different 
experimental groups; however, glucose concentration 
Table 1: Diet ingredient composition used during the experiment
Ingredients g/kg
Berseem hay 305
Yellow corn 90
Barely 203.5
Wheat bran 180
Soybean Meal (44%)  160
Wheat straw 20
Molasses 20
Dicalcium phosphate 8
Calcium carbonate 7
Common Salt  3
Mineral and vitamin premix1 3
Dl-methionine 0.5
Chemical composition (%)
Dry matter 87.32
Crude protein 17.01
Ether extract 2.60
Crude fiber 12.18
Nitrogen free extract 49.02
Ash 6.51
Digestible energy (DE) MJ/kg diet 10.46
1Mineral and vitamin premix composition (per 3kg): Vitamin A = 12,000,000 
IU, D3 = 2,000,000 IU, E = 10,000 mg, K3 = 1,000 mg, B1 = 1,000 mg, B2 
= 5,000 mg, B6 = 1,500 mg, B12 = 10 mg, Niacin = 30,000 mg, Biotin = 50 
mg, Folic acid = 1,000 mg, Pantothenic acid = 10,000 mg, Choline chloride = 
500,000 mg, Zinc = 50,000 mg, Manganese = 60,000 mg, Iron = 30,000 mg, 
Copper = 10,000 mg, Iodine = 1,000 mg, Selenium = 100 mg, Cobalt = 100 
mg, Calcium carbonate to 3kg
138  |  Slov Vet Res 2023  |  Vol 60 No 3
was numerically increased in feed restricted rabbits 
irrespective of ZnO-NPs supplementation (Table 3). The 
interaction between the feed restriction regime and ZnO-
NPs supplementation significantly altered the serum 
albumin and globulin concentrations (P < 0.05). Albumin 
concentration was significantly reduced in ad libitum-fed 
rabbits supplemented with 15 mg/L of ZnO-NPs, while 
showed no differences (P > 0.05) in the restricted ones. 
Reducing the amount of feed significantly increased (P 
< 0.05) the globulin concentration when compared with 
rabbits fed freely without added ZnO-NPs (control). Unlike 
albumin, serum globulin concentration was significantly 
increased in full-fed rabbits supplemented with 15 mg/L 
of ZnO-NPs, while showed no differences (P > 0.05) in the 
restricted ones. 
Table 2: Effect of feed restriction and Zinc nanoparticles (ZnO-NPs) supplementation on rabbit growth performance
ZnO-NPs (mg/L) Initial body weight(g/ rabbit)
Final body weight
(g/ rabbit)
Total weight gain
(g/ rabbit)
Feed intake
(g/ rabbit)
Feed conversion 
ratio
AL-0
Ad libitum
774.50±14.92 2142.50±88.40AB 1368.00±83.98AB 7148.29±53.10A 5.23±0.36
AL-15 763.50±16.33 2251.00±44.90A 1487.50±39.99A 7104.00±60.58AB 4.77±0.10
AL-30 766.50±17.51 2065.56±71.18AB 1299.06±73.12AB 7239.85±190.42A 5.57±0.36
R-0
Restricted
776.50±17.65 2077.50±69.99AB 1301.00±72.38AB 6924.10±212.14AB 5.32±0.39
R-15 775.00±14.26 2042.50±105.37B 1257.50±110.61B 6970.32±247.79AB 5.54±0.39
R-30 764.00±17.43 1971.50±21.63B 1207.50±25.82B 6697.78±108.04B 5.54±0.16
Two-way Anova (P-value)
Feed offering regime 0.785 0.039 0.031 0.026 0.230
ZnO-NPs level 0.821 0.187 0.246 0.886 0.544
Interaction 0.909 0.573 0.482 0.414 0.164
Values shown in table are mean ± SE. Uppercase letters correspond to statistical significance (P < 0.05) between ad libitum and restricted fed groups
Table 3: Effect of feed restriction and Zinc nanoparticles (ZnO-NPs) supplementation on some blood serum biochemical parameters of growing rabbits
ZnO-NPs (mg/L) Total Protein(g/dL)
Albumin
(g/dL)
Globulin
(g/dL)
Albumin/ globulin 
ratio
Glucose
(mg/dL)
AL-0
Ad libitum
5.96±0.05 5.20±0.09aA 0.76±0.14bB 7.49±1.77 93.07±2.41
AL-15 6.01±0.04 4.85±0.11bB 1.16±0.14aA 4.30±0.56 99.56±1.14
AL-30 6.03±0.02 4.97±0.03abAB 1.06±0.05aA 4.69±0.25 102.90±5.12
R-0
Restricted
6.01±0.03 4.97±0.12aAB 1.04±0.09aA 4.84±0.49 104.36±6.38
R-15 6.02±0.01 5.12±0.01aA 0.90±0.00aAB 5.71±0.02 107.35±4.39
R-30 6.02±0.04 5.08±0.02aAB 0.94±0.04aAB 5.41±0.25 106.06±3.77
Two-way Anova (P-value)
Feed offering regime 0.538 0.422 0.654 0.795 0.053
ZnO-NPs level 0.444 0.482 0.367 0.292 0.378
Interaction 0.667 0.019 0.029 0.055 0.639
Values shown in table are mean ± SEM. Lowercase letters correspond to the statistical differences (P < 0.05) between different ZnO-NPs levels, while 
uppercase letters correspond to statistical significance (P < 0.05) between ad libitum and restricted fed groups
Slov Vet Res 2023  |  Vol 60 No 3  |  139
Table 4 shows the effects of experimental treatments on 
the serum concentrations of antioxidant enzymes (SOD 
and catalase), MDA, and serum Zn concentration. Catalase 
enzyme activity was significantly altered with ZnO-NPs ad-
dition, feed restriction applied, and the interaction between 
them (P < 0.05). Lower enzyme activity was observed in 
restricted rabbits compared with those freely fed. With 
ZnO-NPs supplementation, reduced catalase activity was 
found in restricted rabbits received 30 mg/L. SOD enzyme 
activity nearly followed the same trend of reduction which 
Table 4: Effect of feed restriction and Zinc nanoparticles (ZnO-NPs) supplementation on the serum concentrations of antioxidant enzymes (catalase, 
superoxide dismutase), malondialdehyde and serum zinc concentration in growing rabbits
ZnO-NPs (mg/L) Catalase (U/ml)
SOD
(U/ml)
MDA
(nmol/ml)
Zinc 
(mg/dL)
AL-0
Ad libitum
145.16±8.69aA 427.67±36.67a 9.56±0.40b 3.59±0.22bB
AL-15 124.37±12.15aAB 304.37±12.40b 12.69±0.38a 5.11±0.14aA
AL-30 107.52±1.50aA 292.60±9.05b 14.37±0.81a 4.81±0.32abAB
R-0
Restricted
89.95±6.97aB 346.31±38.28a 10.66±0.36b 3.94±0.16bB
R-15 120.92±4.98aAB 314.10±14.64a 12.98±0.52a 4.28±0.20bAB
R-30 42.24±18.32bB 274.00±19.21a 13.75±0.24a 5.43±0.22aA
Two-way Anova (P-value)
Feed offering regime 0.0004 0.160 0.519 0.781
ZnO-NPs level 0.001 0.003 <0.0001 0.0001
Interaction 0.023 0.207 0.247 0.013
Values shown in table are mean ± SEM. Lowercase letters correspond to the statistical differences (P < 0.05) between different ZnO-NPs levels, while 
uppercase letters correspond to statistical significance (P < 0.05) between ad libitum and restricted fed groups. 
Table 5: Effect of feed restriction and Zinc nanoparticles (ZnO-NPs) supplementation on some serum liver and kidney function related parameters of 
growing rabbits
ZnO-NPs (mg/L) AST
1
(U/L)
ALT2
(U/L)
Uric acid
(mg/dL)
Creatinine
(mg/dL)
AL-0
Ad libitum
38.33±2.60aB 27.00±4.00 6.17±0.03 1.97±0.04
AL-15 43.00±1.00aAB 31.00±4.58 6.08±0.07 1.95±0.04
AL-30 40.67±1.45aAB 29.00±1.73 6.09±0.05 1.91±0.04
R-0
Restricted
47.33±3.84aA 32.67±2.03 6.14±0.02 1.98±0.01
R-15 37.67±0.88bB 32.00±1.73 6.00±0.06 1.98±0.04
R-30 39.00±1.73bB 27.00±1.53 6.14±0.02 1.94±0.06
Two-way Anova (P-value)
Feed offering regime 0.714 0.519 0.625 0.511
ZnO-NPs level 0.367 0.496 0.084 0.437
Interaction 0.017 0.430 0.396 0.920
Values shown in table are mean ± SEM. Lowercase letters correspond to the statistical differences (P < 0.05) between different ZnO-NPs levels, while 
uppercase letters correspond to statistical significance (P < 0.05) between ad libitum and restricted fed groups. 1AST: aspartate amino transferase, 2ALT 
alanine amino transferase.
140  |  Slov Vet Res 2023  |  Vol 60 No 3
was observed also with increasing ZnO-NPs level in the 
water especially in the ad libitum rabbits (P < 0.05). MDA 
as an important indicator of oxidative stress, was signifi-
cantly increased with ZnO-NPs supplementation (P< 0.05) 
compared to rabbits received fresh water without Zn. ZnO-
NPs supplementation altered the serum Zn concentration, 
which was significantly increased (P < 0.05) in either ad 
libitum or restricted rabbits. The highest serum Zn concen-
tration was shown in the 15 mg/L ZnO-NPs supplemented 
ad libitum rabbits, and the 30 mg/L restricted fed rabbits.
As presented in table 5, kidney function-related parameters 
such as uric acid and creatinine were not affected by the feed 
offering regime or ZnO-NPs administration or the interaction 
Table 6: Effect of feed restriction and Zinc Nanoparticles supplementation (ZnO-NPs) on serum lipid profile of growing rabbit
ZnO-NPs (mg/L) Cholesterol(mg/dL)
Triglyceride 
(mg/dL)
HDL1
(mg/dL)
LDL2
(mg/dL)
VLDL3
(mg/dL)
AL-0
Ad libitum
86.63±3.50aA 110.03±1.92a 21.37±0.77a 43.26±2.36a 22.01±0.38a
AL-15 70.87±1.86bAB 92.97±1.65b 17.13±0.38b 35.14±2.26b 18.59±0.33b
AL-30 72.77±2.10bAB 96.10±4.37b 19.30±0.93ab 34.25±3.66b 19.22±0.87b
R-0
Restricted
80.70±1.81aA 108.33±2.19a 20.23±0.3a 38.80±2.45a 21.67±0.44a
R-15 78.27±1.40abAB 99.00±1.74ab 18.57±0.70a 39.90±1.40a 19.80±0.35b
R-30 69.50±1.92bB 89.97±2.64b 17.00±0.86b 34.51±1.24a 17.99±0.53c
Two-way Anova (P-value)
Feed offering regime 0.736 0.781 0.263 0.092 0.781
ZnO-NPs level 0.0002 <0.0001 0.002 0.048 <0.0001
Interaction 0.020 0.098 0.054 0.192 0.098
Values shown in table are mean ± SEM. Lowercase letters correspond to the statistical differences (P < 0.05) between different ZnO-NPs levels, while 
uppercase letters correspond to statistical significance (P < 0.05) between ad libitum and restricted fed groups. 1HDL: high density lipoprotein, 2LDL: low 
density lipoprotein, 3VLDL: very low- density lipoprotein
Table 7: Effect of feed restriction and Zinc Nanoparticles (ZnO-NPs) supplementation on volatile fatty acids in caecum of growing rabbits
ZnO-NPs (mg/L) Ammonia(mg/dL)
Total volatile fatty 
acids (mM)
Acetate 
(% of total VFAs)
Propionate
(% of total VFAs)
Butyrate 
(% of total VFAs)
AL-0
Ad libitum
14.90±0.38a 50.40±3.72 71.73±1.16aA 6.03±0.33a 24.23±1.19a
AL-15 13.00±0.42ab 45.67±1.17 63.47±0.81bAB 4.80±0.35b 20.80±0.80b
AL-30 12.20±0.29b 40.67±6.23 63.80±1.00bAB 4.90±0.42b 19.17±0.73b
R-0
Restricted
13.67±0.64a 49.93±3.44 67.07±0.86aAB 5.33±0.03a 22.57±0.55a
R-15 13.23±0.24a 40.50±4.65 67.27±0.80aAB 5.03±0.24a 23.07±1.23a
R-30 12.30±0.74a 41.90±1.05 62.90±1.50bB 4.93±0.20a 20.03±0.41b
Two-way Anova (P-value)
Feed offering regime 0.462 0.613 0.505 0.552 0.505
ZnO-NPs level 0.004 0.077 0.0003 0.031 0.003
Interaction 0.284 0.962 0.006 0.274 0.114
Values shown in table are mean ± SEM. Lowercase letters correspond to the statistical differences (P < 0.05) between different ZnO-NPs levels, while 
uppercase letters correspond to statistical significance (P < 0.05) between ad libitum and restricted fed groups. 
Slov Vet Res 2023  |  Vol 60 No 3  |  141
between them. The same response was obtained with 
the ALT enzyme activity. On the other hand, AST enzyme 
activity was significantly modified by the interaction of 
our treatments (P < 0.05). Zn supplementation lowered 
the AST concentration (P < 0.05) in feed restricted rabbits 
compared with their control rabbits without Zn addition. As 
illustrated in table 6, all the measured parameters of the 
serum lipid profile were significantly (P < 0.05) modified by 
ZnO-NPs addition in water. ZnO-NPs administration (both 
levels) to the full-fed rabbits resulted in lower (P < 0.05) TC, 
TG, LDL, VLDL, as well as reduced serum HDL only with 15 
mg/L when compared to their control without Zn addition. 
Additionally, the same trend of reduction was observed 
in TC, TG, LDL, and VLDL in feed restricted rabbits which 
received the higher dose of ZnO-NPs (30 mg/L).
Volatile fatty acid concentration and caecal 
microbiology
In the freely fed rabbits, ZnO-NPs supplementation 
significantly reduced (P < 0.05) ammonia (AL-30) and 
individual VFAs concentrations (acetate, propionate, and 
butyrate) in the cecal contents at both levels (AL-15 and 
AL-30) compared to AL-0 (Table 7). The same response of 
Figure 1: Effect of feed restriction and Zinc Nanoparticles supplementation on cecal microbiology (total bacterial count and total coliform count) in 
growing rabbits. Results expressed as means ± SEM
Table 8: Effect of feed restriction and Zinc Nanoparticles (ZnO-NPs) supplementation on morphology of small intestine (ileum) of growing rabbits
ZnO-NPs (mg/L) Mucosal length(µm)
Villi length
(µm)
Villi width
(µm)
Crypt depth
(µm)
Goblet cell
(number/mm2)
AL-0
Ad libitum
556.44±27.22bB 290.57±19.27bB 171.14±37.22aA 72.04±10.62b 2.78±0.25cD
AL-15 781.54±22.05aAB 527.21±54.64aAB 114.15±9.61bAB 111.39± 4.46a 8.71±0.04bB
AL-30 826.11±12.33aAB 239.07±115.45bB 111.67±1.85bAB 115.85±5.04a 11.41±0.34aA
R-0
Restricted
673.19±32.19bB 380.08±21.23bB 85.30±4.80bB 86.44±3.36b 4.81±0.18cC
R-15 936.33±25.92aA 709.36±35.20aA 122.23±13.48bAB 117.07±2.33a 10.48±0.62bAB
R-30 1000.93±83.95aA 567.18±115.64abA 126.37±10.03bAB 104.89±2.44a 11.48±0.32aA
Two-way Anova (P-value)
Feed offering regime 0.0021 0.015 0.162 0.565 0.0014
ZnO-NPs level <0.0001 0.009 0.815 0.0002 <0.0001
Interaction 0.821 0.547 0.022 0.167 0.052
Values shown in table are mean ± SEM. Lowercase letters correspond to the statistical differences (P < 0.05) between different ZnO-NPs levels, while 
uppercase letters correspond to statistical significance (P < 0.05) between ad libitum and restricted fed groups
142  |  Slov Vet Res 2023  |  Vol 60 No 3
reduction was obtained with cecal acetate and butyrate 
content in the feed restricted rabbits supplemented with 30 
mg/L (R-30) when compared to control restricted rabbits 
(R-0). 
The total bacterial count showed no differences among 
groups (P > 0.05) (Figure 1). On the other hand, the total 
coliform count was significantly altered (P < 0.05) with 
ZnO-NPs administration. In the freely fed rabbits, ZnO-NPs 
addition (15 mg/L) increased the total coliform count while 
reduced it (P > 0.05) when a higher dose of Zn was used 
(30 mg/L). In the restricted fed rabbits, non-significant 
difference was obtained (P > 0.05) when compared with the 
control receiving fresh water.
Intestinal Morphology
Intestinal morphometry parameters of the ileum are shown 
in Table 8 and Figure 2. Feed restriction and ZnO-NPs 
supplementation significantly affected the mucosal length, 
villi length, and goblet cell number. When compared to the 
control group, it increased (P < 0.05) mucosal length, villi 
length, crypt depth, and goblet cell number while decreasing 
villi width. Villi length was significantly increased in both 
rabbits (freely fed or restricted) and supplemented with 15 
mg/L. On the other hand, villi length was reduced (P < 0.05) 
in the ad libitum fed rabbits supplemented with 30 mg/L 
of ZnO-NPs compared to rabbits supplemented with 15 
mg/L ZnO-NPs. Goblet cells showed the highest number 
with the highest level of Zn added to the water. Besides, 
villi width was significantly changed in response to the 
interaction of the two factors (P < 0.05). In ad libitum rabbits, 
Zn administration reduced the villi width (P < 0.05), while 
showed no difference in the restricted rabbits (P > 0.05) 
compared with their control group without Zn addition. 
Discussion
Feeding strategy for growing animals considers an 
important factor affecting their growth performance, 
lean body mass obtained and could help in avoiding 
problems associated with the early life fast growth rate 
such as increased body fat deposition, high incidence of 
metabolic disorders and high mortality. In recent years, 
global interest in using nanotechnology has been increased 
Figure 2: Morphology of rabbit ileum in response to different feeding regimes and ZnO-NPs supplementation As shown in 1) normal mucosal lining with 
no pathological changes; 2) an increase in the villi length; 3) an increase in mucosal length; 4) increase in the villi branches; 5) increase in the villi length; 
and 6) marked increase in the mucosal villi number and branches
Slov Vet Res 2023  |  Vol 60 No 3  |  143
as nanoparticles including Zn-NPs demonstrated a great 
potential as mineral supplements in livestock diets. 
In the present study, feed restriction reduced rabbit growth 
performance in terms of final weight and gain while showed 
no effect on FCR.  Different result was reported with Yakubu 
et al. (27), who found that feed restriction (8 hrs. per day 
feeding (7.00 a.m-3.00 p.m.) or a skip-a-day system) of 
weaned rabbits for five weeks resulted in no difference in 
their weight. Also, Meo et al. (5) found that both restricted 
(90 % of the ad libitum) and ad libitum rabbits showed simi-
lar weight at the slaughter time however, reported improved 
FCR in the restricted group. Birolo et al. (20) found that the 
application of 93% of ad libitum intake during the first grow-
ing period (from weaning, 37 d until slaughter (at 73 d and 
80 d of age) enhanced rabbit health status without affect-
ing growth performance. Moreover, feed restriction nega-
tively affected the WG of rabbits, which could be associated 
with the reduced amount of feed consumed per day, which 
in turn resulted in inadequate intake of nutrients needed to 
support the rapid growth and development of the growing 
rabbit (28). No statistical difference was observed in the 
FCR between the different experimental groups. The re-
ported results were consistent with Tumova et al. (29) who 
documented that feed restriction systems for growing rab-
bits had no effect on the feed efficiency. As reported before, 
the impact of feed restriction depends on its intensity, dura-
tion, digestive physiology, and age of rabbits when applied 
(30). Our results suggested that the restriction was not se-
vere (mild restriction) to affect rabbit growth, resulting in 
restricted rabbit weight nearly approaching the weight of 
a full fed rabbit and reflected on the FCR at the end of the 
experiment. 
Moreover, ZnO-NPs administration had no significant effect 
on rabbit growth performance, however, freely fed rabbits 
received 15 mg/L showed the highest body weight among 
groups. This improvement could be attributed to the better 
bioavailability and absorption of Zn, which has an important 
role in the metabolism of nutrients, in addition to the 
enhanced intestinal villi length. Unlike the obtained results, 
Hassan et al. (16) found that dietary supplementation of 
rabbits with nano-Zn at 30 and 60 mg /kg diet increased 
BW and FI. In support, Tag-El Din (15) reported that BW 
was insignificantly larger in rabbits that received different 
Nano-Zn compared with their control. Inconsistency in the 
obtained results between experiments could be associated 
with the difference in the experimental design, such as the 
route of Zn administration, nanoparticle size, number of 
samples used for measurements and the feeding regime 
applied in the current experiment.
 Serum biochemical parameters are considered important 
diagnostic tools which help in assessing the metabolic 
condition of an animal and reflect its physiological 
response to different external and internal conditions, 
including nutrition (31). The interaction between the feed 
offering regime and Zn supplementation altered the 
albumin and globulin serum concentrations. The current 
results showed lower serum albumin concentration in the 
freely fed rabbits supplemented with ZnO-NPs (15 mg/L) 
compared to the same group subjected to feed restriction. 
In the same regard, Hassan et al. (16) found that diet 
fortification with nano Zn elevated total protein and globulin 
with no clear changes in albumin concentration of growing 
rabbits. Increased serum globulin in freely fed rabbits 
supplemented with ZnO-NPs was consistent with weaning 
piglets supplemented with nano Zn (32) and  (33) in broiler 
chickens. Differences in the obtained results in terms of 
these parameters between restricted and freely fed rabbits 
could be associated with the reduced FI, which is correlated 
with the water consumption and consequently affected the 
dose of Zn administrated. 
In the current experiment, a non-significant difference in 
glucose concentration in response to feeding restriction and 
ZnO-NPs addition was obtained. Likewise, Van Harten and 
Cardoso (34) found that the limitation of voluntary intake 
didn’t reduce glucose concentration. They explained that 
animals under restriction require no more catabolism of 
glucose, and this was supported by the level of the glucose-
6-phosphate, which didn’t show any changes. Similarly, 
Ebeid et al. (35) stated that plasma glucose concentration 
showed no difference in growing rabbits subjected to feed 
restriction.
Regardless of the feed offering regime applied, serum 
MDA concentration was increased with increasing ZnO-
NPs level added in water, suggesting that the addition of 
ZnO-NPs could have an oxidative stress effect on the 
growing rabbits. This finding was supported by Sharma et 
al. (36), who found that ZnO-NPs induced oxidative stress 
effects and increased the liver function enzymes, AST 
and ALT. In agreement, Ismail and El-Araby (37) found a 
significant increase in hepatic MDA levels in rabbits that 
received ZnO-NPs in their diet. Zn plays a significant role 
in the antioxidant defense system as an important part of 
the antioxidant enzyme SOD (38). The antioxidant defense 
system is formed of three levels of defense with SOD, CAT, 
and glutathione peroxidase forming the first level, as their 
main function is the prevention of free radical formation 
through scavenging their precursors (39). In our study, SOD 
and catalase serum activities were reduced by increasing 
the ZnO-NPs level, with the lowest concentration recorded 
at 30 mg/L, suggesting that the supplemented Zn failed 
to enhance the antioxidant system. Likewise, Ismail and 
El-Araby (37) found depressed renal and hepatic catalase 
activity in ZnO NPs supplemented rabbits, which could be 
associated with oxidative stress induced by nanoparticles. 
While this contradicts the findings of Hassan et al. (16) who 
reported elevated SOD activities in growing rabbits given 30 
and 60 mg nano-Zn /kg diet.
 Moreover, the interaction between the 2 factors affected 
the activity of the liver enzyme, AST. In ad libitum fed rabbits, 
the activity of this enzyme was increased with increasing Zn 
144  |  Slov Vet Res 2023  |  Vol 60 No 3
levels, while the opposite result was obtained in restricted 
rabbits. Fazilati (40) reported that liver enzyme activity was 
increased in rat serum treated with ZnO nanoparticles (50 
ppm, 100 ppm and 200 ppm). The measured kidney function 
related parameters, creatinine and uric acid, showed no 
changes between the different groups, suggesting no 
adverse effects of experimental treatments in the present 
trial. These results are consistent with Peris and Abd El-
Latif (41), who found that ALT, urea-N, and creatinine serum 
concentrations showed no significant difference between 
restricted and ad libitum fed rabbits. Furthermore, ZnO-
NPs supplementation at both levels was associated with a 
significantly increased serum Zn concentration in the Zn-
supplemented rabbits, which suggests high availability and 
absorption of ZnO-NPs. Similar results were reported by 
Hassan et al. (16).
Regarding the lipid profile, it was altered with ZnO-NPs 
supplementation. In the full-fed rabbits, different lipid 
profile parameters, including TC, TG, HDL, LDL, and 
VLDL, were significantly reduced with Zn addition. The 
response of reduction in these parameters appeared with 
administration of ZnO-NPs (30mg/L) in the restricted 
rabbits.  In the same direction, Tag-El Din (15) detected 
an insignificant reduction in plasma TG and TC of rabbits 
supplemented with Nano-Zn (30 and 60 mg/kg diet). Also, 
Ismail and El-Araby (37) who found a significant decrease 
in serum TG, TC, and VLDL-C in ZnO-NPs supplemented 
rabbits. Zinc consider as an important component of several 
enzymes (metalloenzymes) involved in lipid digestion and 
absorption (42). Additionally, cholesterol concentration 
responded to the interaction between the Zn added and 
the feed restriction regime as it showed the lowest level 
in restricted rabbits supplemented with 30 mg/L of ZnO-
NPs. In support, El-Speiy et al. (30) documented lower TC, 
total lipids, and TG in rabbits under feed limitation. Our 
results suggested that ZnO-NPs added had an important 
regulatory role in the lipid metabolism, which was reflected 
by changes in the lipid profile parameters measured and 
growth performance of growing rabbits. The obtained lipid 
profile in response to ZnO-NPs administration needs further 
studies to understand the mechanism behind especially at 
the genetic level. 
The fermentation process occurs in rabbit cecum and their 
end products as VFAs plays an essential role in feed uti-
lization efficiency and rabbit performance. Gidenne (43) 
reported that the VFA covers about 30-50 % of the mainte-
nance energy requirements of the adult rabbit. In the freely 
fed rabbits, ZnO-NPs addition in water altered the concen-
tration of ammonia and different individual VFAs (acetic, 
butyric, and propionic) in the cecal content, as they were 
reduced compared with the control rabbits which received 
fresh water. A different result was obtained by Chrastinová 
et al. (44), who found no differences in VFAs profile be-
tween control and experimental groups supplemented with 
different sources of Zn. The obtained results confirm the 
importance of Zn as an essential microelement involved in 
metabolic processes in the body.  
Rabbits subjected to a feed limitation regime showed a 
non-significant change in the total bacterial count or coli-
form count in the cecal content compared with the freely 
fed rabbits. Similarly, Martignon et al. (45) found that the 
FI level had no effect on the bacterial community structure 
(the number of bacterial 16S rDNA copies per gram of ce-
cum). The constant composition of ingested feed material 
as well as the buffering capacity of the cecal contents were 
suggested by Michelland et al. (46) as the main explanation 
for the lack of effect of different FI levels on the cecal bac-
terial profile. In either full fed or restricted rabbits, ZnO-NPs 
administration showed a numerical increase in total bacte-
rial count, with the higher count in the full fed ones. In sup-
port, Diao et al. (47) found a similar result when 100 mg/
kg of zinc was supplemented in the diet of weaned piglets. 
Besides, the total coliform count was increased with 15 
mg/L ZnO-NPs administration, while it was reduced with 
30 mg/L ZnO-NPs in the full-fed rabbits.  In the restricted 
fed rabbits, non-significant difference was obtained when 
compared with the control receiving fresh water. In the 
same regard, Mahmoud et al. (18) found a significant re-
duction in the coliform count in the cecal content of broilers 
with different levels of Zn nanoparticles. The alteration in 
the cecal microbiota in response to the ZnO-NPs supple-
mentation could be associated with its antibacterial activ-
ity (48). The latter author documented that the antibacterial 
activity of nanoparticles is dependent on their surface area 
and concentration, as the smaller the size, the larger the 
surface area, which becomes more reactive against bac-
teria, increasing their antibacterial activity. Therefore, the 
inconsistency obtained between the current study and the 
previous studies could be associated with the characteris-
tics of nanoparticles used as size, concentration, form, and 
duration of exposure (18), species under the study, tech-
niques that could give a complete picture of the microbial 
profile in the cecum and the host gut health and number of 
samples taken during the experiment. In the current study, 
the limited number of samples taken could contribute to 
the unclear picture of the experimental treatment response. 
Nutrition including the amount of feed offered, is considered 
an important factor affecting the physiological function of 
the gastrointestinal tract as it relates to absorption (49). 
Gidenne et al. (2) assumed that the limitation of voluntary 
intake could change the morphology of the intestinal 
mucosa. In the present study, the ileal morphology of 
growing rabbits in terms of mucosal length, villi length, crypt 
depth, and goblet cell number were increased in response to 
feeding restriction and ZnO-NPs administration in water. In 
the same regard, Tůmová et al. (50) revealed that one-week 
feed restriction of growing rabbits modified the intestinal 
morphology as it showed longer small intestine villi with 
deeper crypts. In contrast, Martignon et al. (45) found that 
a three-week intake limitation applied (25% reduction of the 
Slov Vet Res 2023  |  Vol 60 No 3  |  145
intake) did not affect the morphometric development of the 
ileal mucosa. The discrepancy in experimental outcomes 
might be associated with the restriction regime applied, 
duration, and intensity. Moreover, Oliveira et al. (51) stated 
that young animals have higher nutrient requirements and 
the intestinal mucosa recovers rapidly during refeeding. Dou 
et al. (52) reported that duodenal morphometric changes 
that occurred in response to starvation were normalized 
by refeeding on the second day. This could explain our 
finding of increased intestinal morphological parameters 
in restricted rabbits as they were freely fed in the first day 
then they were restricted in the next day (fed at the level of 
93 % of ad libitum fed the day before), then again ad libitum 
followed by restriction.
Izadi et al. (53) reported that the increased intestinal 
absorptive capacity was associated with longer intestinal 
villi, and consequently beneficial effects on growth 
performance. This finding was shown in the present study 
as ZnO-NPs supplementation at the lower dose (15 mg/L) 
increased the intestinal villi length as well as deeper crypt, 
which was reflected by increased rabbits’ final weight in the 
freely fed rabbits. In the same direction, El-Katcha et al. (33) 
reported that nano- Zn addition to the broiler diet increased 
the jejunal villi length. Increasing the Zn dose to 30 mg/L 
had a negative impact as it reduced the intestinal villi 
length in the freely fed rabbits or showed non-significant 
changes in the restricted rabbits when compared with the 
lower dose of ZnO-NPs (15 mg/L). These changes with 
the higher dose of Zn might be associated with reduced 
intestinal absorptive capacity and reduced body weight 
of rabbits obtained at the end of the experiment. Goblet 
cells contribute to the maintenance of the intestinal barrier 
by secreting mucin. In the present study, increasing ZnO-
NPs levels were associated with an increased goblet cell 
number in the intestine, which could have a favorable 
protective effect on rabbit intestine. This result is in quite 
agreement with studies, which documented higher goblet 
cell numbers with Zn supplementation in mouse (54) and 
weaned piglets (55).
Conclusion 
Under the conditions of the current experiment, it could be 
summarized that the feed restriction program applied in this 
experiment (rabbits were fed ad libitum for one day, then on 
the next day, they were fed at the level of 93 % of the intake 
fed the day before) altered rabbit growth performance (final 
body weight and weight gain with no differences in feed 
conversion ratio), improved the ileum morphology while 
had no effect on caecal fermentation (VFAs profile) or 
microbiological parameters. ZnO-NPs supplementation in 
both levels (15 and 30 mg/L water) differently modulated 
serum lipid profile, antioxidant enzymes and MDA, VFAs 
profile in cecum and ileal morphology with no differences 
in rabbit growth performance.  
Disclosure statement
No conflicts of interest, financial, or otherwise, are declared 
by the authors.
Acknowledgements
Authors would gratefully thank Prof. Mosaad A. Soltan, 
Professor and head of Nutrition and Veterinary Clinical 
Nutrition Department, Faculty of Veterinary Medicine, 
Alexandria University, Egypt for his help during the course 
of the experiment.
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Vpliv omejitve krme in dodajanja nanodelcev cinkovega oksida na rastno 
zmogljivost, biokemijo krvi, črevesno morfologijo in parametre cekalne 
fermentacije rastočih kuncev
K. El-Naggar, A. M. El-Shenawy, S. E. Fadl 
Izvleček: V tej študiji smo proučevali odziv rastočih kuncev na omejitev krme in dodajanje nanodelcev cinkovega oksida 
(ZnO-NP) v okviru uspešnosti rasti, biokemičnih parametrov v serumu, morfologije črevesja in fermentacije v slepem 
črevesu. Skupno 60 samcev novozelandskih kuncev je bilo naključno razdeljenih v 6 skupin: AL-0 (krmljenje ad libitum 
+ sladka voda kot kontrola); AL-15 (krmljenje ad libitum + voda z dodatkom 15 mg/l ZnO-NP) in AL-30 (krmljenje ad 
libitum + voda z dodatkom 30 mg/l ZnO-NP). Skupine R-0, R-15 in R-30 so bile enake prvim trem, vendar z omejenim 
režimom krmljenja. Kunci, hranjeni ad libitum z dodatkom ZnO-NP (15 mg/L), so imeli največjo telesno maso brez 
statistično značilnih razlik v primerjavi s skupinami AL-0, AL-30 in R-0. Razmerje pretvorbe krme (FCR) se med različnimi 
poskusnimi skupinami ni razlikovalo (P > 0,05). Dodajanje ZnO-NP je vplivalo na zmanjšanje parametrov lipidnega profila 
v serumu, tj. encim katalaza pri R-30 in superoksid dismutaza pri AL-15 in AL-30, medtem ko se je vsebnost serumskega 
malondialdehida (MDA) povečala tako pri kuncih, krmljenih ad libitum, in kuncih z omejenim režimom krmljenja. Dajanje 
ZnO-NP je pri AL-30 v primerjavi s kontrolo (AL-0) povzročilo znižanje vsebnosti amonijaka v slepem črevesu ter vsebnosti 
posameznih hlapnih maščobnih kislin (acetata, butirata in propionata) (P < 0,05). Kot odgovor na omejitev krme in 
dodajanje ZnO-NP so se spremenili morfološki parametri ileuma (dolžina sluznice, dolžina resic in število čašastih celic). 
Ključne besede: omejitev krme; rast; kunci; nanodelci Zn oksida