This study was conducted to determine the effects of methionine
(Met) supplementation on productive and reproductive performance, immune
response and antioxidant status in breeder quails reared under heat stress
(HS). A total of 125 breeder quails were divided into five groups. One group
was kept in an environmentally controlled room at 22
Heat as a stressor for birds has been studied extensively for many years. Heat stress (HS) occurs when the amount of heat produced by an animal exceeds the animal's capacity to dissipate the heat to its surrounding environment. Birds experiencing HS tend to reduce their heat production by limiting feed intake, with subsequent negative effects on live weight gain, egg production, egg quality and feed efficiency (Donkoh, 1989), as well as a negative effect on the productive performance of poultry. Several techniques are available to alleviate the adverse effect of HS on poultry performance. Because of the high costs associated with cooling structures that house animals, solutions have been aimed primarily at dietary manipulation (Konca et al., 2009). Nutritional strategies have included the following: adjusting feeding times or temporary feed withdrawal (Francis et al., 1990), dietary energy and protein levels manipulation (Austic, 1985), diet supplementation with vitamin C (Carmina and Cristina, 2013), Se or Cr (Habibian et al., 2014; Ghazi et al., 2012), probiotics and prebiotics (Berrin, 2011), essential oils (Dalkilici et al., 2015) and water supplementation with electrolytes (Lara and Rostagno, 2013).
Recently, the connection between HS and oxidative stress has received much interest (Mujahid et al., 2005, 2009). Oxidative stress is the presence of reactive species in excess of the available antioxidant capacity animal cells. The effects of HS are possibly due to acceleration in the rate of reactive oxygen species (ROS) formation and/or an increase in ROS reactivity (Bai et al., 2003). The defenses of organisms against ROS may be mediated by nonenzymatic or enzymatic antioxidants, mainly represented by the enzymes superoxide dismutase (SOD), catalase (CAT) and the glutathione (GSH) defense system (Abrashev et al., 2008). Antioxidant mechanisms are more often attributed to vitamins (e.g., vitamin E and vitamin C) than to amino acids. However, sulfur amino acids (SAAs) play a major role in the antioxidant systems of the cell in different ways.
Methionine (Met) is a methyl group donor and a precursor of cysteine, cystathionine, homocysteine, S-adenosylhomocysteine and glutathione (Rubin et al., 2007). Diets with Met deficiency result in an increase in heat production (Sekiz et al., 1975), followed by an increase in adverse effects of HS when the environmental temperature is high (Bunchasak and Silapasorn, 2005). Balancing the amino acid composition in the diet with Met supplementation improves production performance through pathways of polyamine metabolism (Gonzalez-Esquerra and Leeson, 2005) and glutathionine (derived from methionine) and may reduce the damage from oxidative stress. Also, the Met requirement determined by NRC (1994) is for laying quails and this may be insufficient for supporting the reproductive performance and hatchability in breeder quails (especially under hyper-thermoneutral conditions). It has been shown that dietary supplementation of Met above NRC (1994) recommendation enhanced cellular immunity in birds (Tsiagbe et al., 1987). Therefore, dietary supplementation of breeder diets with Met above the NRC recommendations may increase the transmission of antibodies to offspring in breeders.
There is very limited information in the literature on the use of Met in breeder quails. Therefore, the aim of the present study was to determine whether dietary supplementation with Met could reduce the adverse effect of HS on the performance, antioxidant status and immune response, as well as the transfer rate of maternal IgY to offspring (no report in the literature) in heat-stressed breeder quails.
All experimental protocols adhered to the guidelines of, and were approved by, the Animal Ethics Committee of the University of Kurdistan (approval code 32155/1395; Sanandaj, Iran).
A total of 200 Japanese quails at 45 d of age were obtained from a local
supplier. The obvious runts and birds with extreme weight were eliminated.
Overall, 125 Japanese quails at 45 d of age were distributed
in a completely randomized design, consisting of five treatments, five
replicates and five (four females and one male) birds per replicate. A
period of 20 d was provided for the birds to adapt to the basal diets, cages
and a thermoneutral environmental temperature (22
Ingredients and chemical composition of the basal diet fed to breeder quails.
Diets were formulated as shown in Table 1. The control diet was formulated mainly based on corn, soybean meal and corn gluten meal, and satisfied all NRC (1994) requirements. The birds had unlimited access to water and feed throughout the entire experiment period.
Egg production, feed intake, average egg weight (total weight of eggs laid/number
of eggs laid, per experimental unit), feed conversion ratio (grams of feed
per grams of eggs) and eggshell, yolk and albumen percentage were evaluated. Egg
production was determined by daily egg collection, which was recorded on one
spreadsheet per replicate. At 2, 4, 6 and 8 weeks of assay, all eggs
produced in a single day were collected for quality evaluation (average
weight, albumen and yolk percentages, eggshell thickness and Haugh unit).
Average albumen weight per replicate was determined to be the difference
between average egg weight and average yolk weight plus eggshell weight per
replicate. In order to determine eggshell weight, eggs were cracked, and the
shells were washed and dried at room temperature for 48 h prior to being
weighed on an analytical balance. Shell thickness was measured by a
micrometer as an average of three points (top, medial and base). The Haugh unit was
calculated according to Brant et al. (1951), (100 log (
Approximately 250 eggs (50 eggs per treatment) were collected over a
3 d period in week 8 and stored at 15
Unhatched eggs were analyzed for embryo mortality causes, which were
classified as early, middle and late death. In order to determine offspring
serum IgG (five blood samples per treatment), all 1 d old chicks were
hatched from each replicate, bled by cardiac puncture, and blood was
collected. The serum was then separated by centrifugation at
IgY was isolated from egg yolks using the Polson et al. (1980) method.
Briefly, an equal volume of buffer (0.01 M sodium phosphate, 0.1 M NaCl,
pH 7.5) was added to yolk and stirred. Solid polyethylene glycol 6000
(Sigma-Aldrich) was added to a concentration of 3.5 %, stirred until
dissolved, and the resultant protein precipitate was pelleted by
centrifugation at
The total IgG levels in the maternal serum, IgY in the egg yolks and day-old
chick's serum IgG were determined using sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions
according to Laemmli (1970). The resultant from the IgY precipitation steps
was dissolved in the sample buffer with 2 % 2-mercaptoethanol and run on a
5 % stacking gel and 10 % separating gel. Samples and standards were
loaded on the gel at a concentration of 20
The cellular immune response was assessed by a cutaneous basophilic
hypersensitivity test using phytohaemagglutinin (PHA). At day 30 and day 45
of the experiment, one quail from each replicate was selected, and the toe
thickness of both the left and right feet at the third and fourth interdigital
spaces was measured by micrometer. Immediately following the measurements,
0.05 mL of phosphate-buffered saline (PBS) and 0.05 mL PHA (1 mg PHA mL
Humoral immune response was evaluated by hemagglutination (HA) antibody
titer estimation. A suspension of sheep red blood cell (SRBC) (1 % and
2.5 %
Blood samples were collected from the wing vein of each bird into a bottle
containing ethylene diamine tetra acetic acid (EDTA, 2 mg mL
At day 60, one quail from each replicate was randomly selected, and blood
samples were taken and transferred to the animal house slaughter facility.
Liver samples were taken immediately after slaughter and homogenized in
freezing isotonic physiological saline to form homogenates at a
concentration of 0.1 g mL
Blood samples were collected from the wing vein into EDTA-coated syringes,
and the plasma was separated by centrifugation at
Malondialdehyde levels and SOD, CAT and GPx were measured using spectrophotometric methods (Hitachi U-2001 spectrophotometer, Tokyo, Japan) as described in the literature. SOD activity was measured by the xanthine oxidase method, which monitors the inhibition of nitro blue tetrazolium reduction and the change of absorbance at 560 nm (Sun et al., 1988). CAT activity was measured following the decrease in absorbance at 240 nm due to hydrogen peroxide decomposition (Aebi, 1984). GPx activity was measured at 412 nm by quantifying the rate of oxidation of reduced glutathione to oxidized glutathione (Hafeman et al., 1974). The thiobarbital method (Placer et al., 1966) was used to determine the MDA level with a wavelength of 532 nm to determine absorbance.
Plasma levels of triglycerides, total cholesterol, low-density lipoprotein (LDL)-cholesterol and high-density lipoprotein (HDL)-cholesterol were analyzed using a spectrophotometer (Hitachi U-2001, Tokyo, Japan) per the instructions on the corresponding reagent kit (Pars Azmun, Tehran, Iran).
The obtained data were submitted to analysis of variance using the general
linear model (GLM) procedure of the SAS statistical package (SAS Institute,
2002). Significant difference among means of treatments was detected by
Duncan's multiple range test procedures. The differences were considered
significant at
Effect of methionine on egg weight, egg production, daily feed
intake and FCR
The effects of HS on breeder quails were observed as a decrease in egg weight
(EW), egg production (EP) and daily feed intake (DFI) in the HS group as
compared to the thermoneutral (TN) conditions group (Table 2). Met
supplementation increased EW, EP and DFI compared to the basal group exposed
to HS. The feed conversion ratio (FCR) was significantly (
Effect of methionine on yolk and albumen percentage, shell thickness and Haugh unit in breeder quails.
The effects of dietary supplementation with Met during HS on egg quality
parameters in Japanese breeder quails are presented in Table 3. There were
no significant (
Effect of methionine on fertility, hatchability and embryonic mortality in breeder quails.
As shown in Table 4, fertility and hatchability decreased (
There was no significant (
Effect of methionine on maternal serum IgG, egg yolk IgY, offspring
serum IgG (mg mL
Results cited in Table 5 show that the maternal serum IgG, egg yolk IgY,
offspring serum IgG and IgG transfer to offspring in breeder quails fed
diets supplemented with Met under hot environmental stress. Results
indicated that maternal serum IgG, egg yolk IgY, offspring serum IgG and IgG
transfer to offspring significantly (
Effect of methionine on humoral (log
The data in Table 6 represent the humoral immune responses and
cell-mediated immune responses of birds. Birds reared under HS conditions
showed a significant (
The cell-mediated immune response to PHA was reduced (
Effect of methionine on heterophil, lymphocyte, monocyte,
eosinophil and
The effects of dietary treatments on WBC are shown in Table 7. HS led to an
increase in heterophil and
Effect of methionine on antioxidant parameters and malondialdehyde in the plasma and liver of breeder quails.
Table 8 shows the effects of dietary treatments on plasma and liver
antioxidant indices. The plasma and liver activities of SOD, CAT and GPx
were lower (
Effect of methionine on plasma lipid parameters in breeder quails.
Table 9 shows the effects of dietary treatments on plasma levels of
HDL-cholesterol, LDL-cholesterol, total cholesterol and triglycerides.
Results indicated that the HDL-cholesterol was lower (
In the current study, high environmental temperature reduced the DFI, EP, EW and egg quality, which is in agreement with previous findings (Tables 2, 3). The reduced EP and EW could be attributed in part to the decrease in feed intake and to an impairment in the utilization of nutrients. Heat stress could also negatively impact nutrient utilization in poultry via changes in the intestinal morphology, where different intestinal segments (duodenum, jejunum and ileum) exhibit lesions that vary in their degree, as well as differences in their relative weight, villus height, villus surface area, crypt depth (Al-Fataftah and Abdelqader, 2014), immunoglobulin A-secreting cell area and epithelial cell area (Lambert et al., 2002) and by decreasing the activity of digestive enzymes (Ruan and Niu, 2001; Yi et al., 2016).
When the ambient temperature exceeds the thermoneutral zone for birds, feed intake is decreased in order to minimize the production of metabolic heat (Swennen et al., 2007), which adversely affects productive performance (Quinteiro-Filho et al., 2010).
To minimize these adverse effects of HS, many practical approaches have been
suggested to enhance the thermotolerance of birds in order to support
productivity (Del Vesco et al., 2014). In our study, we investigated Met
as a way to protect quails against HS. Diet supplementation with 1.15, 1.30
and 1.45 times the Met requirements restored the impairment in DFI, EP, EW and
egg quality in quails submitted to HS. The reduced EP and egg quality in
heat-exposed quails might be due to the reduction in feed intake and an
impairment in the utilization of nutrients. In addition, diet
supplementation with Met improves productive performance through pathways of
polyamine metabolism (Gonzalez-Esquerra and Leeson, 2006). Glutathionine
derived from Met may also reduce damage from oxidative stress. Therefore,
the TSAA requirement would be higher under hyper-thermoneutral conditions
than under thermoneutral conditions. The inclusion of a high level of Met
(1.30 and 1.45 times the Met requirement) improved productive performance in
the HS group compared to the TN group. Dietary supplementation with Met for
breeder quails exposed to HS significantly improved the egg parameters
including egg weight and HU scores to levels similar to those of quails
reared under TN conditions. Several investigators also reported significant
increases in egg production with higher Met
The internal quality of eggs can be evaluated particularly by HU. Birds exposed to HS and fed the basal diet had the lowest HU score compared to other HS groups. Thus, it can be deduced that Met concentrations of 1.15, 1.30 and 1.45 times the NRC (1994) recommendations in breeder quail diets under a heat-stressed environment were sufficient to improve the quality of albumen as evidenced by HU scores to a level similar to that of quails reared under TN conditions.
Heat stress can affect the reproductive function of poultry in different ways. Heat-stressed birds had depressed egg production due to imponderables in the calcium–estrogen relationship, so we can infer that HS decreases albumin consistency, yolk size and calcium deposits in the egg shell. The depression in the ovarian blood flow is a possible mechanism for the reduction in ovarian function as a differential ovarian blood flow pattern was detected in laying hens subjected to HS (Mashaly et al., 2004). Dietary supplementation with Met in breeder quails exposed to HS significantly improved the egg parameters including egg weight, eggshell thickness, albumen percentage and yolk percentage. Consequently, Met could eliminate the negative effects of HS with regard to hatchability.
The results of this study showed that the embryonic mortality of breeder quails decreased by dietary supplementation with Met in the HS group. Tolba et al. (2015) reported that Met supplementation in the diet of quails can improve the infertility egg ratio, early embryonic mortality and later embryonic mortality. In this context, the reduction in mortality in Met group quails may also be attributed to improved immunity as discussed below.
In addition to impairing performance, HS also affects the immune response in birds. In the current study, Met improved the maternal serum IgG, egg yolk IgY and offspring serum IgG in comparison to the basal group exposed to thermal stress. The levels of supplementation can also strongly affect immunomodulation. In the present study, higher levels of Met fed to heat-stressed quails resulted in higher levels of maternal serum IgG, egg yolk IgY and offspring serum IgG compared to similar parameters in TN quails. The highest antibody transmission rate from breeder quails to offspring also belonged to quails that received 1.45 times the Met requirement. The percentage of IgY transfer can be expressed as the percentage of the dam's plasma IgY levels circulating in the blood of 1 d old chicks (approximately 33.60 %). In agreement with our results, Loeken and Roth (1983) reported that the amount of IgY transported is independent of egg size and known to be proportional to the maternal serum IgY concentration.
In this study, the humoral and cell-mediated immune responses were suppressed
under HS conditions. It is well known that environmentally stressed poultry
generally have a depressed humoral immune response (Gharib et al., 2005). In
agreement with the present study, Met supplementation optimized the response
to PHA as well as total antibody response to SRBC as a
The heterophil-to-lymphocyte ratio has been used as a sensitive indicator of
stress, including HS, in poultry (Gross and Siegel, 1983; Mashaly et al.,
2004). An increased
Heat stress could also induce oxidative stress resulting in an imbalance in antioxidant status in birds (Lin et al., 2006). Under HS conditions, as the bird's body attempts to maintain its thermal homeostasis, increased levels of ROS occur. Consequently, tissues and cells possess defense mechanisms to detoxify ROS by radical scavengers such as SOD, CAT and GPx (Wu et al., 2004). The results of our study are in agreement with data available in the literature. In the present study, the activities of SOD, CAT and GPx were lower and MDA concentration in serum was higher in quails exposed to HS compared to the TN group. Dietary Met supplementation increased the serum and liver activity of antioxidant enzymes (SOD, CAT and GPx) and reduced the concentrations of MDA, an indirect parameter of lipid peroxidation and overproduction of ROS, in heat-stressed quails. Inclusion of the higher level (1.45 times) of Met in the diet resulted in higher levels of antioxidant enzymes and lower MDA than in the TN group. Synthesizing antioxidant enzymes such as SOD and GPx is an important regulation with regard to animal responses to stress conditions. Glutathione is a tripeptide synthetized from glutamate, glycine and cysteine. Cysteine can be synthetized from homocysteine from the precursor Met (Shoveller et al., 2005); therefore, when dietary Met is available in adequate amounts, larger quantities can be directed towards cysteine synthesis via the transsulfuration pathway.
With regard to HDL and LDL plasma levels, the present study showed a positive trend for an increase in HDL and a decrease in LDL in supplemented Met (1.15, 1.30 and 1.45 times the Met requirements as NRC (1994) recommended) vs. the HS group fed a basal diet. This indicates the beneficial and protective effects of these methyl donors on bird health in particular and on productive performance in general. In addition, administration of Met to the diet restored plasma levels of HDL-cholesterol, LDL-cholesterol, total cholesterol and triglycerides near to those of the TN group.
Possible mechanisms for the lipid-lowering effects of Met might be related to its antioxidant properties. In our study, Met supplementation reduced serum peroxide content. The maintenance of the normal structure of lipoprotein receptors is necessary for their function, improving the cellular uptake of serum lipids from the blood. Reactive oxygen species produced during oxidative stress react with lipoproteins to produce oxidation states, decreasing the cellular uptake of lipids from the blood (Diniz et al., 2006; Schaffer, 2003; Brizzi et al., 2003). Thus, the antioxidant action of Met might contribute to elevated cellular lipid uptake, resulting in a decrease in serum cholesterol levels.
Breeder quails subjected to the stress of a high thermal environment exhibited deteriorated productive performance, reproduction, egg quality, antioxidant status and immune response. Dietary supplementation with either 1.15, 1.30 and 1.45 times the Met requirements (as NRC recommendation) in breeder quails reared under HS conditions can alleviate the adverse effects of HS and improve the performance, reproduction, antioxidant status and immunity, as well as maternal antibody transmission. In addition, administration of Met to the diet restored most parameters nearer to the TN group. In some cases, supplementation of the diet with 1.45 times the Met requirement resulted in better responses compared to quails under TN conditions. Therefore, it was concluded that Met in higher levels of recommended NRC (1994) could be used as a feed additive in breeder quails for the alleviation of the adverse effects of HS.
The original data of the paper are available upon request from the corresponding author.
OK performed the research. OK and AS performed the data analyses and wrote the manuscript. AK revised the manuscript.
The authors declare that they have no conflict of interest.
The financial support provided by the University of Kurdistan is gratefully acknowledged. The authors are grateful to Salam Ibrahim at the University of North Carolina at Greensboro for his constructive reading of the manuscript.
This paper was edited by Manfred Mielenz and reviewed by two anonymous referees.