INTRODUCTION
In
young growing animal species, such as broiler chickens their fast growing
process imposes some degree of stress on the animals during production,
especially at the commercial setting where poultry diets are fortified with
high density nutrients to maximize growth. Based on the findings of Herbert et al. (2005) and those of Stahly et al. (2007), one of the nutritional
means of supporting the animals for enhanced growth and performance is to fortify
poultry diets with antioxidant vitamins, such as vitamin C (Johnson et al., 2019).
The fore-stated is primarily true due
to the fact that there are inherent measures that animals, including poultry
have developed through which they are capable of avoiding oxidative stress. In
other words, animals possess a complex oxidation defense
system that are modulated by special molecules known as antioxidants whereas
the byproduct of oxidative stress is MDA. According to Hyman (2011),
antioxidant molecules are glutathione (GSH), superoxide dismutase (SOD),
catalase (CAT) and glutathione peroxidase (GSH-Px). The byproducts of the
reactions involving these antioxidants are mainly malondialdehyde (MDA). Therefore,
MDA levels in the blood are indicators of the level of oxidative stress an
animal has been exposed to.
Amongst the antioxidants, GSH is the
major or master antioxidant as it is also responsible in preserving its cohort
antioxidants. This has earned GSH being referred to as ‘mother of all
antioxidants’, master detoxifier and master of the immune system (Hyman, 2011).
SOD catalyzes the dismutation of superoxide radical into oxygen or hydrogen
peroxide thereby protecting cellular components from oxidation by reactive
oxygen species (ROS). CAT converts hydrogen peroxide to carbon dioxide and also
uses hydrogen peroxide to breakdown harmful toxins in the body (Sharma and
Dubey, 2005). GSH-Px scavenges and inactivates ROS thereby protecting the
animal from oxidative damage by acting as an efficient quencher of ROS
(Vangronsveld and Clijsters, 1994). Johnson et
al. (2019) demonstrated that vitamins C and E upregulated GSH, SOD, CAT and
GSH-Px and simultaneously reduced MDA in the pig. There is paucity of
information on these parameters in the broiler chicken. Therefore, the objectives
of this study are: to investigate the effects of feeding graded levels of
vitamin C on antioxidants and to also evaluate the effects of grade levels of
vitamin C on oxidants levels in broiler chickens.
MATERIALS AND METHODS
Experimental site
This
study was carried out at the poultry unit of the Teaching and Research Farm,
Rivers State University, Nkpolu-Oroworukwo, Port Harcourt. The farm is situated
at latitude 40 48’N and longitude 60 48’E at the Rivers
State University campus.
Animals
One
hundred and twenty (120) Agrited day-old
chicks were acquired from a reputable commercial poultry dealer in
Port-Harcourt, Rivers State. The animals on arrival at the Rivers State
University Teaching and Research Farm were brooded to properly pre-condition
them to their new environment. The animals by the fourth week were observed to
have properly adapted to their environment and thus were randomly assigned into
four dietary treatment groups of 30 birds/treatment group with 3 replications of 10 birds/replicate. The pens were properly cleaned and
disinfected before the birds’ arrival. Feeders and drinkers were also properly
cleaned to also ensure that the animals’ environment were “pathogen-free”.
During the brooding period all protocols, including the necessary medications
were provided. Animals were fed similar diets from day one through the end of
the 4th week. Water was provided ad
libitum. The experiment lasted for 8 weeks and thus animals received their
respective experimental diets for 4 weeks.
Experimental Diets
Hybrid
feedTM grower mash were used in the study. In other words, the diets fed
to the animals during the last four weeks of the experimental period were
similar in all nutrients except their dietary vitamin C levels as: control or
treatment 1 (T1, contained only basal level), treatment 2 (T2, contained
200mg of vitamin C), treatment 3 (T3, contained 300mg of vitamin C)
and treatment 4 (T4, contained 400mg of vitamin C)/kg of diet,
respectively. The animals were fed these graded levels of vitamin C-based diets
for 4 weeks.
Blood Sample
Collection
At
the end of the study period, birds were killed for blood collection. 9 birds
were randomly collected from each treatment group consisting of 3 birds from
each replicate of the four treatment groups, respectively. The blood was collected from each bird into treated tubes with ethylene
diamine tetra-acetic acid (EDTA) and immediately snap frozen for later
antioxidants and oxidants analyses.
Antioxidants
Antioxidants
molecules analyzed for were: CAT, SOD, GSH and GSH-Px, whereas, the oxidants
analyzed for was MDA. CAT was analyzed for according to the method of Aebi et al.
(1974). SOD was analyzed for according to the method of Misra and Fridorich
(1972). GSH and GSH-Px were measured according to the method of Agerganrd and
Jensen (1982). MDA was measured according to the method of Varsney and Kale
(1990).
Experimental Design
and Statistical Analyzes
The
study was designed and conducted as a completely randomized design (CRD). Data
obtained were subjected to analysis of variance (ANOVA) using general linear
model (GLM) procedure of SAS. Treatment means were compared using Tukey’s test.
Therefore, the model was: Yij =
µ + Xi + Eij,
where Yij =
individual observation of the treatment, µ = population mean, Xi = effect of the ith treatment and Eij = the error term. An α-level of
0.05 was used for all statistical
comparisons to represent significance.
RESULTS
The
results of the antioxidants’ molecules are shown in Table 1.
For
the oxidant MDA, there were significant (P
< 0.05) differences amongst the treatment groups. For instances in the
MDA concentrations, animals in the T1 group had the highest level
and linearly significantly reduced as the level of vitamin C dietary increased with
the T4 group had significantly (P
< 0.05) the lowest level.
DISCUSSION
Vitamin
C is an antioxidant vitamin as it is actively involved in the regulation of
molecules that scavenge for ROS and convert them to less harmful products;
implying that they significantly contribute to the overall health status of the
animal (Michael et al., 2010: NRC,
2012). Some of these molecules are GSH, GSH-Px, CAT and SOD whereas the production
levels of MDA are indicators of the degree of the effectiveness of the
antioxidant vitamin activities on the antioxidant molecules.
Antioxidants protect cells, tissues and
animals’ organs by reacting with free radicals (Traber et al., 2011; NRC, 2012). These antioxidant molecules are produced
endogenously in the body of the animal, such as GSH and its cohorts in the GSH
defense system. In animal nutrition, these antioxidant molecules productions
can also be triggered by inclusion of antioxidant vitamins to aid in
stimulating their productions in maintaining normal health status of the animal
(Bourne et al., 2000). Furthermore,
it is worthy of note that antioxidants work synergistically in the animal body
to elicit effective defense functions (Ursini, 2000: Johnson et al., 2019). Therefore, one
antioxidant molecule synergizes with another team member for the system to be
very efficient. SOD is widely perceived as the first level of antioxidant
defense probably due to it being more resident in the cytosol (Surai, 2002). It
converts superoxide radical into hydrogen peroxide and oxygen. GSH-Px and CAT
convert hydrogen peroxide to water. GSH acts as co-enzyme by a variety of
crucial life processes, such as detoxification of xenobiotic, removal of
hydro-peroxides and other free radicals (Surai, 2002). However, there were no differences
in the antioxidant molecules investigated in the plasma concentrations of the
birds in all treatment groups in the current study. This probably might be
related to the fact that there was no known attack on the animal, including
pathogens since the animal environments were maintained at high level of
sanitation (NRC, 2012) throughout the experimental period.
Conversely, while we found no differences in
the antioxidant molecules amongst treatment groups, those of oxidants, namely MDA
were significantly reduced in the T2, T3 and T4
animals compared with the T1 group. This is a clear indication that
the ingestion of the vitamin C-based diets reduced oxidative stress in the
animals of T2, T3 and T4 groups. MDA is a
major by-product of oxidative stress (Guo et
al., 2006). Therefore, the significantly reduced levels of MDA in the T2,
T3 and T4 animals compared with the T1
animals was a further confirmation indicating that the animals that received
the vitamin C-based diets experienced lesser activities of oxidative stress compared
with the T1 animals. This observation is in tandem with those of Hyman
(2011), Achuba and Otuya, (2006)
and Johnson et al. (2019). This is
again supported by the findings of improved performance of the animals on the
vitamin C-based diets that was part of this present study.
CONCLUSION
The
ingestion of vitamin C-based diets had no effect on antioxidant molecules;
however, it reduced oxidative stress as evidenced by significant reduction
levels of MDA in broiler chickens that ingested the vitamin C-based diets.
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