Veterinary Immunology and Immunopathology 160 (2014) 266–272

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Veterinary Immunology and Immunopathology

j our na l ho me pag e: www.elsev ier .com/ loca te /vet imm

Chronobiological studies of chicken IgY: Monitoring ofinfradian, circadian and ultradian rhythms of IgY in blood andyolk of chickens

Jin-Xin Hea, Diraviyam Thirumalaia, Rüdiger Schadea,b, Xiao-Ying Zhanga,∗

a College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, Chinab Institute of Pharmacology, Charité – Universitätsmedizin Berlin, Germany

a r t i c l e i n f o

Article history:Received 24 December 2013Received in revised form 14 May 2014Accepted 26 May 2014

Keywords:ChickenSerum IgY

a b s t r a c t

IgY is the functional equivalent of mammalian IgG found in birds, reptiles and amphibians.Many of its biological aspects have been explored with different approaches. In order toevaluate the rhythmicity of serum and yolk IgY, four chickens were examined and rearedunder the same conditions. To monitor biological oscillations of IgY in yolk and serum, theeggs and blood samples were collected over a 60 day period and the rhythm of yolk andserum IgY was determined by direct-ELISA. Results indicated that, there is a significantcircaseptan rhythm in yolk IgY and circaquattran rhythm in serum IgY. The serum IgYconcentration reached a peak in the morning, decreased to a minimum during the daytime

Yolk IgY

Circadian rhythm and increased again at night revealing a significant circadian rhythm was superimposedby an ultradian rhythm. These data are suited to address the controversies concerning theIgY concentration in egg yolk and blood of laying hens. In addition, this study raised newquestions, if the different rhythms in yolk and serum are concerned.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

IgY antibodies found in birds, reptiles and amphibiansare the avian counterpart of IgG, and are transferred fromthe serum to egg yolks in females to confer natural pas-sive immunity to their embryos and offspring (Leslie andClem, 1970; Liou et al., 2010). Significant and rapid growthof IgY literature reflects increasing interest of the scien-

tific community in chicken egg yolk antibody productionand the extraction of antigen-specific antibodies from eggyolk. Chicken IgY is widely used in immunodiagnostics

∗ Corresponding author at: College of Veterinary Medicine, NorthwestAgriculture and Forestry University, Yangling 712100, China.Tel.: +86 29 8709 1239; fax: +86 29 8709 1239.

E-mail address: zhang.xy@nwsuaf.edu.cn (X.-Y. Zhang).

http://dx.doi.org/10.1016/j.vetimm.2014.05.0160165-2427/© 2014 Elsevier B.V. All rights reserved.

and immunotherapy due to its cost-effectiveness, highyield and convenience (Sun et al., 2013; Duan et al., 2009;Schade et al., 2005). In terms of biochemical properties, IgYpossesses several advantages compared with mammalianIgG. For example, IgY does not activate the mammaliancomplement system, does not bind to mammalian rheuma-toid factors, protein A or G. In addition, the phylogeneticdistance between mammals and avian species make itpossible to prepare antibodies in chicken against highlyconserved mammalian proteins with increased affinity andavidity (Cai et al., 2012; Kovacs-Nolan and Mine, 2004;Larsson et al., 1991, 1993). Chickens are usually immu-nized with the target antigen, that induces a humoral

immune response that results in the production of spe-cific IgY in the blood that is then transported to the yolk(Marcq et al., 2013; Bollen and Hau, 1997). The concentra-tion of total IgY in the yolk is obviously related to that in

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he circulating blood (Hamal et al., 2006). Regarding theevel of IgY in serum and yolk, inconsistent results haveeen reported; some researchers reporting that yolk titersimilar or slightly higher than serum titers (Patterson et al.,962a,b; Bar-Joseph and Malkinson, 1980; Schmidt et al.,989); some reports indicating that IgY level is lower inolk than in serum (Fraser et al., 1934; Dohms et al., 1978);nd others reporting no difference between IgY concentra-ion in yolk and serum (Heller, 1975). These inconsistentesearch data make it difficult to identify a suitable timeeriod to ensure high yield of specific antibodies. A cir-aseptan rhythm (within a short time period of 7 days)f yolk IgY was first described by Schade et al. (1991); inhe recent past, it was studied in detail and observed theomplex biological oscillation for the total IgY titer (Paulyt al., 2008). From this finding, it could be understandablehat such biological oscillation (rhythm) of serum and yolkgY in chicken might be the reason for the aforementionediscrepancies.

A recent study reported that, the lineage specificationf a key immune cell named TH17 is under direct circa-ian control (Yu et al., 2013). The concentration of serum

mmunoglobulin from lymphocytes also manifests biolog-cal oscillations (Casale and de Nicola, 1984). In hens, likether animals, the lymphocyte population in peripherallood also undergoes some fluctuations over time. Biolog-

cal rhythms in multiple frequencies have been found inhe immune system, which has important clinical conse-uences and applications to medical practice including thestablishment and interpretation of reference values. In allomponents of the immune system, it shows rhythmic vari-tions in various frequencies, and the periodic changes inmmune responses in hens have also been studied. In ordero address the discrepancies of previous reports regardingotal IgY concentrations in serum and yolk, the goal of theurrent study was to monitor the rhythmicity of IgY anti-odies for a 60 day study period and to determine whetherhe production of IgY antibodies exhibits any significantiological rhythms in serum and yolk.

. Materials and methods

.1. Experimental animals

All animal experimental protocol was reviewed andpproved by the Ethics Committee of Northwest A&F Uni-ersity for the use of Laboratory Animals. Four 29-week-oldrown Leghorn Hens were procured from a local poultry

arm and maintained in individual cages, exclusively fab-icated for the maintenance of chickens (Pinhengli, Henan,hain). The food and water were available ad libitum. Thereas no any modification in the ideal conditions for regu-

ar chicken maintenance in terms of light and dark, roomemperature and humidity.

.2. Reagents and chemicals

Skim milk was obtained from Becton, Dickinson andompany, USA. Tween-20, 3,3′,5,5′-tetramethylbenzidineTMB) and polyethylene glycol (PEG) 6000 were purchased

munopathology 160 (2014) 266–272 267

from Sigma USA. Goat anti-chicken was bought fromAbcam, Canada.

2.3. Blood sample collection and processing

In order to determine the biological rhythm of IgY inblood serum, 200 �l of blood samples from the wing veinof each chicken was drawn on every alternate days by orbefore 8 a.m. using retention needle. The collected bloodsamples were incubated at room temperature for 2 h andthen at 4 ◦C for 1 h. After incubation the samples were cen-trifuged (Heraeus Fresco 17, Thermo, Germany) at 5000×gfor 10 min at 4 ◦C, supernatant was collected and storedat 4 ◦C for further analysis. In addition, a total of 18 bloodsamples (200 �l of each) from 4 chickens were drawn at 2 hinterval for 36 h and processed as aforementioned methodto determine the circadian rhythm of serum IgY.

2.4. Extraction of yolk IgY

Eggs were collected daily and stored at 4 ◦C. The yolkIgY antibodies were extracted using the method reportedby Polson et al. (1980). Briefly, the egg yolk was diluted 1:2with sterile phosphate buffer solution (PBS) (pH 7.4). Forelimination of lipids and lipoprotein, 3.5% (w/v) polyeth-ylene glycol (PEG) 6000 was added. After gentle shakingfollowed by centrifugation (HC-3018R, Anhui USTC ZonkiaScientific instruments, China) (10,000×g for 20 min at4 ◦C), the supernatant was filtrated and powder PEG 6000was added to a final concentration of 12% (w/v) andsubsequently the solution was centrifuged. Finally, the pre-cipitated pellet was dissolved in 1.2 mL of PBS, transferredinto a micro-dialysis device and dialyzed against PBS at 4 ◦C.

The concentration of protein was measured photomet-rically at 280 nm using UV-spectra (Specord 50 UV–VISspectrophotometer, Analytik Jena AG, Germany) and wascalculated according to the Lambert–Beer law with anextinction coefficient of 1.33 for IgY.

2.5. Determining the optimal sample dilutions usingELISA

Total IgY in both serum and yolk samples was mea-sured using an ELISA protocol (Sun et al., 2013) with minormodifications. In brief, serum and yolk IgY samples (tripli-cate) were diluted to multiples of 10-fold dilutions from101 to 1012 times with carbonate buffer solution (CBS).Microtiter plates (Maxisorp F96, Thermo Fisher Scientific,Langenselbold, Germany) were coated with serum and yolkIgY by overnight incubation at 4 ◦C. After emptying thewells, plates were washed with 0.5% PBS containing 0.05%Tween-20 (PBST) three times (3 min/time), incubated withPBS containing 5% skim milk (PBSM) for 1 h at 37 ◦C, andagain washed with 0.5% PBST three times (3 min/time).Subsequently, an alkaline phosphatase conjugated goatanti-chicken IgY (1 mg mL−1, 1:5000 diluted in PBS) wasadded. Plates were incubated for 1 h at 37 ◦C. Finally, 100 �L

of 3,3′,5,5′-tetramethylbenzidine (TMB, 1%, w/v) was addedto each well as substrate and incubated for 15 min at roomtemperature, The reaction was stopped by adding 50 �Lof 2 mol/L H2SO4 to the wells. The optical density (OD) at

and Immunopathology 160 (2014) 266–272

Fig. 1. The optimal dilutions of yolk and serum IgY samples. Note: Each

268 J.-X. He et al. / Veterinary Immunology

450 nm was measured on a microtiter plate reader (ELx800,BioTek, USA). By detection of absorbance OD450 a standardcurve was calculated in order to determine the midpointregion of the optimal dilution of the sample. The slight vari-ation of IgY in yolk and serum can be sensitively detectedunder this ranges.

2.6. Monitoring of the biological rhythms in serum andyolk IgY

Blood samples were drawn on every alternate day fromall experimental hens for a 60-day study period and theeggs were collected every day. Yolk and serum samples(triplicate) were diluted with CBS to the optimal dilution(106-standardized in the present study). The titer of IgY inboth serum and yolk was determined by ELISA as describedabove. The biological oscillations of IgY concentration inserum and yolk IgY were analyzed using the OD450 absorp-tion values.

2.7. Statistical analysis

The GraphPad Prism software (GraphPad, San Diego, CA)was used to analyze the OD values, whereas the Chronos-Fit software version 1.05 (Zuther and Lemmer, 2004) wasused to perform chronobiological study of chicken serumand yolk IgY, i.e. the biological rhythm of serum and yolkIgY over 60 days, the serum IgY during a day and the yolkIgY concentration.

3. Results

3.1. Determination of the optimal sample dilutions forIgY by ELISA

The dilutions of IgY (yolk and serum) as determined by450 nm absorbance was found to be in the linear range(Fig. 1). The concentration specified and the absorbancewas linear in the dilution range of 106–107. The dilution106 was selected as the optimal sample dilution for bothyolk and serum samples. Laying performance of the exper-imental chickens during the study period is summarized inTable 1.

3.1.1. The biological rhythm of serum and egg yolk IgYThe OD450 values of total IgY in serum and yolk were

obtained using an optimal dilution of 106, and graph of

serum and yolk IgY fluctuations was drawn by GraphPadPrism software (Fig. 2). The OD450 values of serum IgY wasdramatically increased after the first blood sample (Fig. 2,chicken 1 and 4), the reason for this enhancement was

Table 1Laying performance during the experimental period.

Parameters Chicken 1

Weight gain (g) 115.5

Number of laying eggs 54

Weight of egg (g) 52.5 ± 2.28

Volume of yolk (mL) 14.6 ± 1.12

Volume of yolk supernatant (mL)a 27.5 ± 2.34

aRepresents the total volume of the IgY preparation after processing of each yolk

value represents the mean ± SD of triplicate analysis of three independentexperiments.

unclear. Crossed serum and yolk IgY values were seen, butthe yolk IgY value was higher than that of serum IgY. Somedata from serum (5–33 days) and yolk (10–47 days) IgYwere chosen to evaluate IgY rhythm. It was found that,there was a 4-day rhythm in chicken serum (Fig. 2) andthere was a 7 day rhythm in yolk IgY concentration. Thesame rhythm of yolk and serum IgY was also displayedby the Chronos-Fit program (Fig. 3A and B). Furthermore,a circadian rhythm could be observed in chicken serumIgY, with a peak in the morning and decreased values dur-ing the day and again increased at night (Fig. 3C-grayedzone of graph). The total IgY concentration was foundto be from the lowest level 31.33 mg egg−1 to highestlevel 57.05 mg egg−1 within the circadian rhythm by theChronos-Fit program (Fig. 3D).

4. Discussion

IgY is transferred from mother hens to chicks to conferpassive immunity, and these antibodies are accumulatedin egg yolk from the blood and transported across yolk sacmembranes during the developmental period (Kowalczyket al., 1985). It has been reported that only 10% of the yolkIgY has importance in the passive immunity of the newlyhatched chickens. It seems that the biological oscillationsof IgY might not be a risk factor for passive immunity. Thereis a discrepancy concerning the IgY concentration in yolkand serum. Some researchers have stated there is no dif-

ference between them, whereas others demonstrated thatIgY concentration in yolk is higher than in serum (Schadeet al., 2005).

Chicken 2 Chicken 3 Chicken 4

147.5 167 138.551 48 5255.7 ± 2.45 47.2 ± 2.56 51.9 ± 2.8515.2 ± 1.01 15.01 ± 0.83 14.2 ± 1.0828.6 ± 2.87 28.4 ± 3.12 26.6 ± 3.07

by the PEG method.

J.-X. He et al. / Veterinary Immunology and Immunopathology 160 (2014) 266–272 269

Fig. 2. IgY levels in serum and yolk over a 60 day period. Panel: (A) chicken 1, (B) chicken 2, (C) chicken 3 and (D) chicken 4. CS and CY indicated serumand yolk IgY, respectively. Each value represents the mean ± SD of triplicate analysis of two independent experiments.

Fig. 3. The biological rhythms of IgY in serum and yolk. Panel: a, b, c, and d represented chicken 1, 2, 3 and 4, respectively. (A) The rhythm of yolk IgY from10 to 47 days (significant period length around 7 days: a83.86%; b86.06%; c87.99% and d85.63%). (B) The serum IgY rhythm from 5 to 35 days (significantperiod length around 4 days; a67.63%; b67.06%; c65.41% and d77.75% rhythms). (C) The circadian rhythm of chickens for 36 h, 65.01%, 95.33%, 88.53% andrhythm 85.01% respectively were analyzed. Grayed zone = 12-hour dark span. (D) The data obtained from ultraviolet spectrophotometry with the significantperiod length around 7 days. a81.63%; b73.06%; c85.41% and d82.75% circadian rhythms.

270 J.-X. He et al. / Veterinary Immunology and Immunopathology 160 (2014) 266–272

Table 2IgY concentration in the serum and yolk.

Antigen Blood IgY (mg mL−1) Yolk IgY (mg mL−1) References

NDV and BDV 3.26 ± 0.22 1.15 ± 0.03a Hamal et al. (2006)NDV and BDV 6.02 ± 0.40 2.26 ± 0.03a Hamal et al. (2006)Escherichia coli 4.6–7.1 3.9–6.3 Kariyawasam et al. (2004)Non-immunization 9.4 10.1 Erhard et al. (2000)Non-immunization 5.9 5.1 Erhard et al. (2000)Non-immunization 15.7 ± 7.9 7.9 Kowalczyk et al. (1985)HIL-6 – 1.23 ± 0.07 times than serum IgY Woolley et al. (1995)

1.23

DV and

of IgY. On the whole, these biological oscillations of IgY arenot fully dependent upon the immunization protocol withthe target antigen. These data are indispensable while IgY

Tricyclic antidepressants –

Notes: HIL-6 – human interleukin-6.a PEG-8000 was added to make a final concentration of 3.5% (wt/vol). N

In the current study, it was found that there were dis-tinct biological fluctuations in serum and yolk IgY levelsover 60 days of the study period. The finding was evidentfor each experimental chicken (Fig. 2, chicken 1–4). It isspeculated, due to the difference in the rhythms of serumand yolk IgY, these fluctuations influence the concentrationdiscrepancies of total IgY and the relationship between IgYin serum and yolk, including positive, negative and no cor-relation. The concentration of IgY in yolk and serum havebeen extensively reported by many researchers (Table 2);there are huge variations in IgY levels among different stud-ies and thus possibly influenced by different factors suchas genetic lines or breeds of chickens, purification meth-ods and assessment techniques. At present it is unclear,which biological mechanism predominantly regulates theserum and yolk IgY levels. But, it is not surprising that theIgY Ab is a component of the immune system and the IgYconcentration is under the control of the circadian clock.

IgY is selectively transferred from blood to the yolk(Loeken and Roth, 1983) and the concentration of total IgYin the yolk is directly related to the circulating IgY levelin hen’s blood (Hamal et al., 2006). In this study, we haveinvestigated the biological rhythm of serum and yolk IgY fora period length of 60 days. Significant differences in rhythmfor serum and yolk were observed (Fig. 2). A detailed anal-ysis of a part of the data (days 5–33 for serum and days10–47 for yolk) clearly showed that, there was a 4 dayrhythm (circaquattran) in serum IgY level and a 7 daysrhythm (circaseptan) in yolk IgY concentration (Fig. 3A andB); this was almost consistent throughout the study period.The finding was marked with the data of a single chicken(Fig. 4). Circaseptan (or weekly) rhythm of yolk IgY hasbeen confirmed and already reported (Schade et al., 2001).This finding indicates that, the rhythmicity of IgY in yolkand serum might be interlinked and the serum IgY rhythminfluences the yolk IgY rhythm. Thus means the IgY transferis related to the serum IgY level.

The results of current study suggest that the delaybetween the appearance of IgY in serum and yolk couldbe 3–4 days. The concentration of IgY in the yolk is invari-ant throughout maturation of the oocyte (∼8 mg/ml) andthe rate of uptake could be as maximum as during the last3 days of growth before ovulation (Kowalczyk et al., 1985).There is controversy concerning the time delay between

the appearance of serum IgY and their detection in the eggyolk, a delay of 3–4 days (Woolley and Landon, 1995) or 5–6days (Patterson et al., 1962a) may take place according tothe previous reports. This delay is possibly affected by the

± 0.135 times than serum IgY Woolley et al. (1995)

BDV mean newcastle disease virus and infectious bronchitis virus.

complex biological association among the number of eggsundergoing their final processing, serum IgY rhythm andhalf-life of the circulating IgY. The half-life of the circulatingIgY of an adult chicken is approximately 36–65 h (Pattersonet al., 1962b) and 30–40% of them are catabolized (Klasing,1998).

Melatonin functions as an immunoregulator in thedevelopment and maturation of immune system and alsoin the progression of the immune response, and its rhythmin chicken pineal glands lasts for 5 days (Takahashi et al.,1980). The serum IgY concentration could be correlatedwith melatonin that characteristically increases noctur-nally or during darkness (Skwarlo-Sonta et al., 2002).Perhaps this is the reason for the increased peak concentra-tion during the morning and gradual decreases in daytimethen again increases after sunset in the ultradian rhythm ofIgY. The data of a single chicken illustrated to evident thisfinding (Fig. 5 grayed zone of graph). The diurnal rhythmin particular parameters of the chicken immune system arein different degrees, depending on the pineal gland and itshormone melatonin (Skwario-Sonta et al., 1992). At thisjuncture, it is understandable that the significant circadianrhythm is superimposed by an ultradian rhythm.

In summary, our data indicates that the concentration ofyolk IgY level increases in non-immunized hens. There is asignificant circaseptan rhythm in yolk IgY and circaquat-tran rhythm in serum IgY and in addition a noticeableultradian rhythm is superimposing the circadian rhythm

Fig. 4. Biological rhythm of IgY in serum and Yolk. Note: the data was fromchicken 1, because four chickens showed the same IgY rhythm in serumand yolk.

J.-X. He et al. / Veterinary Immunology and Immunopathology 160 (2014) 266–272 271

hicken

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B

B

Fig. 5. Circadian rhythm of IgY in serum. Note: the data was from c

ntibodies are used to monitor and evaluate the vaccine.urther research is necessary to clarify the physiologi-al background of the differences between the biologicalhythms in blood and yolk.

onflict of Interest

We have no conflict of interests to disclose and theanuscript has been read and approved by all named

uthors.

cknowledgements

This work was supported by Key Program for Inter-ational S & T Cooperation Projects of Shaanxi Province2012KW-20), The Research Fellowship for Internationaloung Scientists (31350110512), National Natural Scienceoundation, and Ministry of Education and State Admin-stration of Foreign Experts Affairs “Overseas Teacher”roject (No.MS2011XBNL057), China.

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