IMMUNOLOGY AND MOLECULAR BIOLOGY
Melatonin and the Enhancement of Immune Responses
in Immature Male Chickens1
C. P. Brennan,* G. L. Hendricks III,* T. M. El-Sheikh,† and M. M. Mashaly*,2
*Department of Poultry Science, The Pennsylvania State University, University Park, Pennsylvania 16802;
and †Department of Animal and Poultry Production, South Valley University, Egypt
ABSTRACT Understanding the role of melatonin in affecting different physiological functions, especially immune responses, is becoming increasingly important in
the basic and applied sciences. Enhancing the immune
response will result in increasing disease resistance and,
therefore, improve production efficiency. The purpose of
the present study was to investigate the effects of melatonin, administered during the light or dark period, on
BW, feed consumption (FC), and immune responses of
immature chickens. Eight-week-old Cornell White Leghorn males were used in this study. The doses of melatonin were 0, 5, 10, 20, and 40 mg/kg BW. Melatonin was
administered s.c. every 24 h for 7 consecutive d. The
chicks were randomly divided into two groups; one
group received injection during the middle of the light
period, and the other group received injection during the
middle of the dark period. All birds received 16 h light
and 8 h darkness during a 24-h period. Body weights
were measured before and after melatonin treatment, and
FC was also measured. After the seven injections, blood
samples were collected from the brachial vein, and total
white blood cell (WBC) counts, differential cell counts,
and activities of T and B lymphocytes were measured.
Body weight was not significantly affected by dose of
melatonin or time of injection. Furthermore, melatonin
did not significantly affect FC; however, FC was significantly lower in the group that was injected in the dark
vs. light period. The WBC counts of birds injected with
40 mg melatonin/kg BW were significantly higher than
the WBC counts of saline-injected birds. The heterophil/
lymphocyte (H/L) ratios of birds injected during the light
period were significantly higher than those of birds injected during the dark period. T- and B-lymphocyte proliferation were significantly higher in birds injected with
40 mg melatonin/kg BW compared to saline-injected
birds. These results indicate that melatonin in vivo is
important in enhancing not only circulating WBC but
also activities of B and T lymphocytes of immature male
chickens without adversely affecting BW.
(Key words: chicken, melatonin, body weight, feed consumption, immunity)
2002 Poultry Science 81:371–375
intake in domestic fowl was reduced. On the contrary,
Apeldoorn et al. (1999) reported that melatonin supplemented in the diets of chickens had no effect on feed
intake. This contradiction could be due to a difference
in photoperiod used, dosage of hormone, or the type
of bird used. In studying the effect of melatonin on
energy metabolism, Apeldoorn et al. (1999) found that
there were no significant interactions between lighting
schedule and melatonin on energy metabolism in chickens. However, they found that the heat production related to activity was reduced when melatonin was supplemented to the diet.
The effects of melatonin on immune responses have
also been investigated. In rats, melatonin has been
shown to inhibit the development of mammary tumors
(Tamarkin et al., 1981; Kothari et al., 1997). Melatonin
added to the drinking water of quail resulted in an
INTRODUCTION
Melatonin is secreted during darkness by the pineal
gland and sets the internal biological clock that governs
different daily and seasonal cycles or rhythms in various
physiological systems, including the cardiopulmonary,
reproductive, excretory, thermoregulatory, behavioral,
immune, and neuroendocrine systems in birds (Pang et
al., 1996). Melatonin has different physiological functions in the body; it can affect feed consumption, energy
metabolism, and immune responses. Bermudez et al.
(1983) found that following melatonin injection, feed
2002 Poultry Science Association, Inc.
Received for publication April 12, 2001.
Accepted for publication October 18, 2001.
1
This work was partially supported by the College of Agricultural
Sciences, Undergraduate Research Program, Pennsylvania State University and Cultural Attaché, Egyptian Embassy.
2
To whom correspondence should be addressed: [email protected].
Abbreviation Key: FC = feed consumption; H/L = heterophil/lymphocyte; IFN-γ = interferon-γ; WBC = white blood cell.
371
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BRENNAN ET AL.
increase in total white blood cells (WBC), an increase
in the percentage of lymphocytes, a decrease in the percentage of heterophils, and a decrease in the heterophil/
lymphocyte (H/L) ratio (Moore and Siopes, 2000). In
Syrian hamsters, chronic daily administration of melatonin increased cytokine production (Champney et al.,
1998) and splenic lymphoproliferative responses to concanavalin A (Champney et al., 1997). Previously, we
reported that melatonin in vitro enhanced the mitogenic
response of T and B lymphocytes in chickens (Kliger et
al., 2000).
Length of photoperiod also affects immune responses.
Moore and Siopes (2000) found that decreasing the photoperiod significantly increased lymphocyte percentage, decreased heterophil percentage, and decreased the
H/L ratio in quail. A review of studies that examined
the effects of photoperiod on immune function showed
that short photoperiod enhances immune function in a
variety of species (Nelson and Demas, 1996).
It seems that the effect of melatonin may depend on
the time of administration. In chickens, Skwarlo-Sonta
et al. (1991) found that the effect of exogenous melatonin
on the diurnal rhythm of total WBC and serum agglutinins is more dependent on time of treatment than on
the dose of hormone used.
It is clear that more studies are needed in chickens to
further investigate the effects of melatonin on immune
responses. Therefore, the present study was conducted
to examine the effects of different doses of melatonin
administered at different times on immune parameters
as well as on BW and feed consumption (FC).
MATERIALS AND METHODS
Animals and Experimental Design
Eight-week-old Cornell White Leghorn male chickens
were used in this study. The chickens were housed in
batteries and feed and water were supplied ad libitum.
The same light treatment was provided for all the birds,
16 h light:8 h darkness, with lights on at 0600 h. The
birds were randomly divided into two groups of 40
birds each. One group was injected with different doses
of melatonin3 in the middle of the light period (Light
group), the other injected in the middle of the dark
period (Dark group). The 40 birds from each group were
divided into five subgroups (two replicates each) and
received daily s.c. injection with different dosages of
melatonin dissolved in absolute ethanol. The doses
were: 0, 5, 10, 20, and 40 mg melatonin/kg BW and
were injected for 7 consecutive d. Individual BW, at the
beginning and end of the experiment, and feed con-
3
Sigma Chemical Co., St. Louis, MO 63178-9916.
Fisher Scientific, Hanover Park, IL 60103.
5
ICN Biomedicals, Irvine, CA 92626.
6
Skatron Instruments, Sterling, VA 20167.
7
#1209 Rackbeta, LKB Instruments, Gaithersburg, MD 20877.
4
sumption per replicate per week were measured. After
the last injection, blood was taken from the brachial
vein, and the immune parameters were measured in the
blood samples as described below.
Immunological Parameters
Total WBC. Total WBC counts were made using brilliant cresyl blue dye (Haddad and Mashaly, 1990).
Differential Cell Counts. Differential counts were
prepared using Hema 3 stain.4 The different types of
WBC were counted, and percentages of heterophils and
lymphocytes were calculated. Heterophil/lymphocyte
ratio was also calculated.
Proliferation Assay for T and B Lymphocytes. The
mitogen cell-proliferation assay used was described
previously (Kliger et al., 2000). Briefly, leukocytes were
separated from red blood cells using histopaque-1077.3
Leukocytes were plated at 5 × 105 cells per well, and 50
µL of Con A3 at 12.5 µg/mL or 50 µL of pokeweed
mitogen3 at 25 µg/mL was added.
Cells were then incubated at 37 C in a humidified
atmosphere of 5% CO2 for 48 h. One µCi of 3H-thymidine5 was added to each well. Cells were again incubated at 37 C under 5% CO2 for 18 h to allow for 3Hthymidine uptake. Cells were harvested onto glass-fiber
filters using a cell harvester.6 The filters were placed
in scintillation vials with scintillation fluid and were
counted using a scintillation counter.7 Counts per minute were determined for each well. Control counts
from wells with no mitogen were subtracted from treatment counts.
Statistical Analysis
SAS威 software general linear models procedure (SAS
Institute, 1996) was used to analyze data with two-way
ANOVA with dose of melatonin and time of injection as
the main effects. Means were separated using Duncan’s
multiple-range test with significance set at P < 0.05.
RESULTS AND DISCUSSION
Overall, the change in BW was not significantly affected by dose of melatonin or time of injection (data
not shown). These results are similar to those of Apeldoorn et al. (1999) who found that melatonin in broiler
feed did not affect BW. However, the results are different from those of Injidi and Forbes (1983) who found
that melatonin inhibited growth of 1-d-old male chicks
when they were injected s.c. for 28 d. Differences in the
results could be due to age of birds used (1 d vs. 9 wk)
and duration of injection (7 d vs. 28 d). Furthermore,
Clark and Classen (1995) found that adding melatonin
to the diets of broiler chicks inhibited growth between
0 to 14 d of age.
In our study, we found no effect of melatonin on FC.
These results agree with those of Apeldoorn et al. (1999)
who showed that adding melatonin to a broiler diet
MELATONIN AND IMMUNITY
FIGURE 1. Effect of dose and time of melatonin injection for 7 consecutive d on circulating total white blood cells (× 106/mL) in immature
male White Leghorn chickens. a-bMeans within dose or time without a
common letter differ significantly (P < 0.05). For dose of injection, n =
10 to 16; for time of injection, n = 33 to 34.
did not affect feed intake. Other researchers found that
melatonin inhibited FC (Bermudez et al., 1983; Clark
and Classen, 1995). Differences in results could be due
to the age of birds used and duration of treatment. In the
present study, even though different doses of melatonin
did not affect FC, the birds that were injected during
the dark period consumed significantly less feed than
birds injected in the light period (304 g/bird per wk
light period vs. 263 g/bird per wk dark period). This
reduction in FC could be caused by an increase in melatonin levels due to an additive effect of exogenous and
endogenous melatonin during the dark period compared to the light period. Endogenous melatonin is
higher at night compared to during the day (Leung,
1991).
Data for the total WBC counts and T- and B-lymphocyte activities are shown in Figures 1, 2, and 3, respectively. Melatonin was found not only to significantly
FIGURE 2. Effect of dose and time of melatonin injection for 7 consecutive d on peripheral leukocytes stimulated by concanavalian A in
immature male White Leghorn chickens. a-cMeans within dose or time
without common letters differ significantly (P < 0.05). For dose of injection, n = 10 to 16; for time of injection, n = 33 to 34.
373
FIGURE 3. Effect of dose and time of melatonin injection for 7 consecutive d on peripheral leukocytes stimulated by pokeweed mitogen in
immature male White Leghorn chickens. a-bMeans within dose or time
without common letters differ significantly (P < 0.05). For dose of injection, n = 10 to 16; for time of injection, n = 33 to 34.
increase total WBC but also to significantly increase Tand B-lymphocyte activities. The overall total WBC of
birds injected with 40 mg melatonin/kg BW were 90.5
× 106/mL compared to 70.7 × 106/mL for saline-injected
birds. The counts per minute for T-lymphocyte proliferation of birds injected with 40 mg/kg BW were 48,308
vs. 10,652 for saline-injected birds, whereas the counts
per minute for B-lymphocyte proliferation of birds injected with 40 mg/kg BW were 14,060 vs. 4,279 for saline-injected birds. These results indicate the importance of melatonin administered in vivo on enhancing
the proliferative activity of T and B cells assayed in
vitro. These results reemphasize our previous findings
in chickens in which we found that melatonin in vitro
enhances the activities of T and B lymphocytes (Kliger
et al., 2000). Furthermore, melatonin receptors were
found in the chicken bursa of Fabricius (Liu and Pang,
1993) and duck thymus (Liu and Pang, 1992). B and T
lymphocytes are involved in humoral and cell-mediated
immunities. Enhancing these immunities might assist
in increased disease resistance.
It seems that melatonin is not only important in chickens but has also been reported to be an immunomodulator in mammalian species as well. Champney et al.
(1997), using Syrian hamsters, found that s.c. injection
of melatonin increased splenic lymphoproliferative responses to concanavalin A. Furthermore, Caroleo et al.
(1992) determined that melatonin injected s.c. restored
the impaired T-helper cell activity in immunodepressed
mice. Giordano and Palermo (1991) showed that melatonin in vivo enhanced the capacity of murine splenocytes
to mediate antibody-dependent cellular cytotoxicity.
The immunomodulatory role of melatonin could be due
to the presence of binding sites in blood lymphocytes
from humans; granulocytes, thymus, and spleen from
rodents; and bursa of Fabricius from birds (Calvo et
al., 1995).
The melatonin influence on immune responses could
be through the stimulation of cytokine production (Mae-
374
BRENNAN ET AL.
TABLE 1. Effect of time of melatonin injection for 7 consecutive d
on percentage heterophils, lymphocytes, and heterophil/
lymphocyte (H/L) ratio in immature
male White Leghorn chickens
Treatment
Light
Dark
Lymphocyte
b
56.1
64.8a
Heterophil
a
28.3
23.7b
H/L ratio
0.532a
0.391b
a,b
Means within a column and with no common superscript differ
significantly (P < 0.05); n = 33 to 34.
stroni, 1995), which in turn enhances lymphocyte activities. Garcia-Maurino et al. (1997) suggested that melatonin in vitro enhanced production of interleukin-2, interleukin-6, and interferon-γ (IFN-γ) by human
peripheral blood mononuclear cells and Colombo et al.
(1992) determined that IFN-γ was produced in vitro by
mice splenocytes after melatonin stimulation. Furthermore, Champney et al. (1998) found increased serum
IFN-γ levels in Syrian hamsters after melatonin injection. Different cytokines are involved in the growth and
activation of different mononuclear cells (Cruse and
Lewis, 1995). It has also been suggested that melatonin
influences the immune response through opiatergic
mechanisms (Maestroni et al., 1987).
Moore and Siopes (2000) found that total WBC counts
in Japanese quail increased significantly during a
shorter photoperiod compared to a longer photoperiod
and constant lighting, indicating that darkness increases
total WBC numbers, which could be due to increased
melatonin levels in darkness as reported by Leung
(1991). However, in our study, we found that total WBC
counts were significantly reduced during darkness. This
reduction cannot be explained physiologically.
In general, different doses of melatonin did not affect
the percentage of heterophils or lymphocytes or the H/L
ratio (data not shown). However, there were significant
differences in cell type percentages and H/L ratios between the light and dark periods (Table 1). The percentage of lymphocytes was highest during the dark period,
and the percentage of heterophils was lowest during
the dark period. The H/L ratio was significantly lower
during the dark period compared to the light period.
Our results are in agreement with those of Moore and
Siopes (2000), who found that decreasing the photoperiod or adding melatonin to the drinking water of Japanese quail significantly increased lymphocyte percentage, decreased heterophil percentage, and decreased the
H/L ratio. They concluded that constant lighting was
more stressful than shorter daily photoperiods, as indicated by the H/L ratio, and that exogenous melatonin
has an immuno-reconstituting effect on quail placed in
constant lighting.
In summary, our results demonstrate that melatonin
in vivo is important in enhancing not only circulating
WBC but also activities of B and T lymphocytes of immature male chickens. In addition, melatonin may also
reduce stress, as indicated by the reduction of the H/
L ratio during the dark period.
REFERENCES
Apeldoorn, E. J., J. W. Schrama, M. M. Mashaly, and H. K.
Parmentier, 1999. Effect of melatonin and lighting schedule
on energy metabolism in broiler chickens. Poultry Sci.
78:223–229.
Bermudez, F. F., J. M. Forbes, and M. H. Injidi, 1983. Involvement
of melatonin and thyroid hormones in the control of sleep,
food intake and energy metabolism in domestic fowl. J. Physiol. 337:19–27.
Calvo, J. R., M. Rafii-el-Idrissi, D. Pozo, and J. M. Guerrero,
1995. Immunomodulatory role of melatonin: Specific binding
sites in human and rodent lymphoid cells. J. Pineal Res.
18:119–126.
Caroleo, M. C., D. Frasca, G. Nistico, and G. Doria, 1992. Melatonin as immunomodulator in immunodeficient mice. Immunopharmacology 23:81–89.
Champney, T. H., G. C. Allen, M. Zannelli, and L. A. Beausang,
1998. Time-dependent effects of melatonin on immune measurements in male Syrian hamsters. J. Pineal Res. 25:142–146.
Champney, T. H., J. Prado, T. Youngblood, K. Appel, and D.
N. McMurray, 1997. Immune responsiveness of splenocytes
after chronic daily melatonin administration in male Syrian
hamsters. Immunol. Lett. 58:95–100.
Clark, W. D., and H. L. Classen, 1995. The effects of continuously
or diurnally fed melatonin on broiler performance and
health. Poultry Sci. 74:1900–1904.
Colombo, L. L., G. J. Chen, M. C. Lopez, and R. R. Watson, 1992.
Melatonin induced increase in gamma-interferon production
by murine splenocytes. Immunol. Lett. 33:123–126.
Cruse, J. M., and R. E. Lewis, 1995. Illustrated Dictionary of
Immunology. CRC Press, New York, NY.
Garcia-Maurino, S., M. G. Gonzalez-Haba, J. R. Calvo, M. RafiiEl-Idrissi, V. Sanchez-Margalet, R. Goberna, and J. M. Guerrero, 1997. Melatonin enhances IL-2, IL-6, and IFN-γ production by human circulating CD4+ cells: A possible nuclear
receptor-mediated mechanism involving T helper type 1 lymphocytes and monocytes. J. Immunol. 159:574–581.
Giordano, M., and M. S. Palermo, 1991. Melatonin-induced enhancement of antibody-dependent cellular cytotoxicity. J. Pineal Res. 10:117–121.
Haddad, E. E., and M. M. Mashaly, 1990. Effect of thyrotropinreleasing hormone, triiodothyronine, and chicken growth
hormone on plasma concentrations of thyroxine, triiodothyronine, growth hormone, and growth of lymphoid organs
and leukocyte populations in immature male chickens. Poultry Sci. 69:1094–1102.
Injidi, M. H., and J. M. Forbes, 1983. Growth and food intake
of intact and pinealectomised chickens treated with melatonin and triiodothyronine. Br. Poult. Sci. 24:463–469.
Kliger, C. A., A. E. Gehad, R. M. Hulet, W. B. Roush, H. S.
Lillehoj, and M. M. Mashaly, 2000. Effects of photoperiod and
melatonin on lymphocyte activities in male broiler chickens.
Poultry Sci. 79:18–25.
Kothari, A., A. Borges, A. Ingle, and L. Kothari, 1997. Combination of melatonin and tamoxifen as a chemoprophylaxis
against N-nitroso-N-methylurea-induced rat mammary tumors. Cancer Lett. 111:59–66.
Leung, F. C., 1991. Circadian rhythms of melatonin release from
chicken pineal in vitro: Modified melatonin radioimmunoassay. Proc. Soc. Exp. Biol. Med. 198:826–832.
Liu, Z. M., and S. F. Pang, 1992. Binding of [125I]-labelled iodomelatonin in the duck thymus. Biol. Signals 1:250–256.
Liu, Z. M., and S. F. Pang, 1993. [125I] iodomelatonin-binding
sites in the bursa of Fabricius of birds: Binding characteristics,
subcellular distribution, diurnal variations and age studies.
J. Endocrinol. 138:51–57.
Maestroni, G. J., 1995. T-helper-2 lymphocytes as a peripheral
target of melatonin. J. Pineal Res. 18:84–89.
Maestroni, G. J., A. Conti, and W. Pierpaoli, 1987. Role of the
pineal gland in immunity: II. Melatonin enhances the anti-
MELATONIN AND IMMUNITY
body response via an opiatergic mechanism. Clin. Exp. Immunol. 68:384–391.
Moore, C. B., and T. D. Siopes, 2000. Effects of light conditions
and melatonin supplementation on the cellular and humoral
immune responses in Japanese quail Coturnix coturnix japonica. Gen. Comp. Endocrinol. 119:95–104.
Nelson, R. J., and G. E. Demas, 1996. Seasonal changes in immune function. Q. Rev. Biol. 71:511–548.
Pang, S. F., C. S. Pang, A. M. S. Poon, Q. Wan, Y. Song, and G.
M. Brown, 1996. An overview of melatonin and melatonin
receptors in birds. Poult. Avian Biol. Rev. 7:217–228.
375
SAS Institute, Inc., 1996. SAS User’s Guide: Statistics. (version
6.12) SAS Institute, Cary, NC.
Skwarlo-Sonta, K., M. Thaela, B. Gluchowska, D. Stepien, and
M. Jagura, 1991. Effect of dose and time of melatonin injections on the diurnal rhythm of immunity in chickens. J. Pineal
Res. 10:30–35.
Tamarkin, L., M. Cohen, D. Rossell, C. Reichert, M. Lippman,
and B. Chabner, 1981. Melatonin inhibition and pinealectomy
enhancement of 7,12-dimethylbenz(a)anthracene-induced
mammary tumors in the rat. Cancer Res. 41:4432–4436.