Antibiotics, on laying hens (Puthpongsiriporn et al. 2001;

Antibiotics,
used in poultry diets to improve production performance, has been prohibited
because of their negative subsequences in birds and human health (Zulkifli et al. 2000; Apata, 2009; Casewell et al. 2003). Stress has a highly
detrimental influence on laying hens (Puthpongsiriporn et al. 2001; Belloni et al.
2015), which depresses feed intake (FI), body weight, egg production (EP) and
egg quality (Puthpongsiriporn et al.
2001(. Bee glue or propolis, an adhesive, dark
yellow to brown colored balsam, is one of the products from beekeeping which is
found in the hives (Galal et al., 2008)
and usually collected from the buds, leaves and similar parts of trees and
other plants like pine, oak, eucalyptus, poplar, chestnut, and so on by bees
and mixed with their wax (Seven et al.
2010; Valle, 2000). It is composed of 30% wax, 50% resin, 10% of essential
oils, 5% pollen and other substances (Arpášová et al. 2016; Kumova, 2002), but its composition differs depending
on a wide variety of factors including collecting location, time and plant
source. Beneficial effect of dietary supplemental propolis on body weight gain, feed intake, feed
conversion ratio and mortality rate of broilers has been reported
(Havsteen, 2002). The
use of propolis becomes widespread in medical science, apitherapy and in the
bio-cosmetology because of its antiviral, antibacterial, anti-fungal,
anti-inflammatory, anti-ulcer, anti-tumour and immunity stimulative and local
anaesthetic properties (Acikgoz et al.
2005; Seven et al. 2008). Propolis,
has valuable pharmaceutical, antioxidative (Seven et al. 2010) and disinfection effects (Belloni et al. 2015). Cytostatic, anti-mutagenic and immunomodulatory
properties of propolis are based on its rich, flavonoid, phenolic acid and
terpenoid contents (Prytzyk et al.
2003; Wang et al. 2004). It is known
that flavonoids of
propolis show antioxidant characteristics to
the oxidants in the cell membrane like ascorbate (Havsteen, 2002).

Vitamin
C (VitC) assists in reducing the harmful effects of hormones produced by the
adrenal glands during prolonged periods of stress. Besides, it has an important
role in a number of biochemical processes such as synthesis of collagen, which
is an intercellular protein and principal constituent of skin, scales, mucosa,
cartilaginous tissues, bones and conjunctive tissue formation
(Abdel-Tawwab et al. 2004; Seven et al. 2008). Beneficial
effects of dietary supplemental VitC on growth rate, egg production, egg shell
strength and thickness in laying hens and broilers have been observed in
several studies (Bains, 1996; Tatli-Seven
et al. 2006; Tatli-Seven, 2006).

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Keeping
in view the mentioned pharmaceutical advantages of propolis and VitC, the
objective of this study was to evaluate the effects adding propolis and VitC diet,
individually and together, on performance, egg quality characteristics and
blood parameters of laying hens. Assessing the probable synergistic interaction
between dietary propolis and VitC on the mentioned parameters of laying hens
was also investigated.

Materi

Birds

All experimental protocols adhered to the
guidelines of, and were approved by, the Animal Ethics Committee of Razi
University (Kermanshah, Iran) and were in accordance with the guidelines on
animal welfare (The number of approval letter: AD-197-2014). In
this study, 144 Lohman-LSL Lite laying hens (59
weeks of age) were weighed individually and randomly assigned to 6 treatments
with 4 replicates and 6 birds in each replicate in a completely randomized
design. Based on a 3×2 factorial arrangement of treatments, the
birds were supplied with six iso-caloric and iso-nitrogenous diets including 3
levels of EEP (0, 150 and 300 mg/kg) and VitC (0, 250 mg/kg) (Table 1). Feed
and water were ad libitum during 8-week trial period.

Propolis
Origin

Hand-collected propolis samples were kept
dried in the dark until processing. Propolis samples were extracted for two
weeks with 100 ml of 96% ethanol, at room temperature to obtain the extract
(Krell, 1996). After filtration, the ethanol extract was evaporated by using a vacuum
evaporator at 50°C. Afterwards the extract was used in the
experiment.

Performance and Egg traits

The number of eggs laid by birds in each replicate were recorded daily
and summed up at the end of the month to obtain monthly egg production. The
collected eggs were classified as either normal or damaged; the latter included
broken eggs, cracked eggs, and shell-less eggs. Egg production (EP), egg weight
(EW) and feed intake (FI) were daily recorded. Feed conversion ratio (FCR) was
calculated as feed consumed (kg) divided by egg mass. Eggs from each replicate were sampled for quality
characteristics twice a month. All eggs laid by birds in each replicate were
weighed on the day of sampling using an Aculab electronic (sensitivity 0.01g)
top-loading scale. Egg length and diameter were measured and egg shape index
calculated as egg diameter divided by the length. Eggshell thickness was
measured with an Ames micrometer. After cutting the eggs open to obtain yolk
and albumen, the eggshells were carefully washed with water to remove adhering
albumen, dried at room temperature. Subsequently the egg shell was weighed the
nearest 0.01g and eggshell thickness
without inner membranes was measured with the micrometer. Yolk diameter, yolk
and albumen heights were measured. Yolk index was calculated as yolk diameter
divided by yolk height while Haugh unit was determined using USDA Interior Egg
Quality Measure (USDA Chart for scoring broken-out eggs, Catalog 4- 4200
American Instrument Co. Inc. Silver Spring, MD).

Blood Parameters

At the last week of the experiment, four
hens from each treatment were selected at random to collect blood samples via
the wing vein. Then plasma was separated by centrifugation for 10 min at 4000
rpm. The collected plasma was stored at -20°C until the time of
analysis. The frozen plasma was allowed to thaw at room temperature prior to
analysis. Glucose, cholesterol, triglycerides (TG), high density lipoprotein
(HDL), albumin, total protein and cortisal were determined by enzymatic
colorimetric method using available commercial kits (Pars Azmon, Tehran, Iran).