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Poult Sci 2008. 87:41-49. doi:10.3382/ps.2007-00306
© 2008 Poultry Science Association
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IMMUNOLOGY, HEALTH AND DISEASE

Effect of Selection for Phagocytosis in Dwarf Chickens on Immune and Reproductive Characters

H. Li*,{dagger}, Y. Zhang*,{dagger}, Z. H. Ning*,{dagger}, X. M. Deng*,{dagger}, Z. X. Lian*,{dagger},1 and N. Li{dagger},1

* State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing 100094, P. R. China; and {dagger} Laboratory of Animal Genetics and Breeding, Ministry of Agriculture, College of Animal Science and Technology, China Agricultural University, Beijing 100094, P.R. China

1 Corresponding author: lianzhx{at}cau.edu.cn or ninglbau{at}public3.bta.net.cn


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In current study, phagocytosis product (PP) of peripheral blood monocytes was detected among 920 dwarf chickens (460 per sex) at 20 wk of age, and based on discrepancies of PP, the flock was grouped (the highest group, the medium group, and the lowest group). Then serum hemagglutination inhibition antibody titers and subpopulations of T-lymphocytes of each group were examined after inoculations of avian influenza virus H5N2 inactivated vaccine (20 wk of age), avian influenza virus H9 inactivated vaccine (24 wk of age), and Newcastle disease virus-egg drop syndrome virus bigeminal inactivated vaccine (28 wk of age), respectively, to study the relationship between PP and immune response. To gain insight into effects of selection for PP on number of eggs, mean egg weight, fertilization rate, hatchability, and rate of healthy chicks, 9 (3 x 3) mating combinations were conducted. The results showed that (1) selection for higher PP in both sexes benefited to humoral immunity but not CD8+ T-lymphocyte mediated immunity in dwarf chickens; (2) there were effects of selection for higher PP in hens on fertilization rate (P < 0.05), hatchability (P < 0.05), rate of healthy chicks (P < 0.05), and level of IgY antibody (P < 0.0001); however, hens’ PP had no effects on number of eggs (P ≥ 0.05) or egg weight (P ≥ 0.05) and cocks’ PP had no effect (P ≥ 0.05) on any trait mentioned above. The results indicated that phagocytosis of peripheral blood monocytes might be an indicator of humoral immunity in dwarf chickens; furthermore, selection of hens with higher PP was not only beneficial to fertilization rate, but also benefited to hatchability and rate of healthy chicks in that the hens had stronger humoral immunity, which might contribute to maternal antibody in eggs.

Key Words: monocyte • phagocytosis product • humoral immunity • production trait • dwarf chicken


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Monocytes, effectors and modulators of immune system, derive from bone marrow monoblasts and are released into the peripheral blood where they circulate for several days in noninflammatory condition before entering tissues to replenish macrophage populations (Volkman and Gowans, 1965; Mosser, 2003). During proinflammatory state, metabolic and immune stimuli can elicit recruitment of monocytes to peripheral sites (Van Furth et al., 1973) where monocytes regulate inflammatory processes via chemotaxis, pinocytosis/phagocytosis, and release of cytokines (Kedzierska and Crowe, 2002; Stevenson, 2003). Besides being a main member of mononuclear phagocyte system, which plays a pivotal role in a variety of homeostatic processes including host defense, immunoregulation, and tumor surveillance (Fedorko and Hirsch, 1970; Johnston, 1988; Taylor and Gordon, 2003; Hume, 2006), monocytes are also a member of professional antigen presenting cells (APC), which play critical roles in resisting infections. The APC activate and modulate adaptive immune response by expressing different kinds of MHC molecules, which are the bases of T-lymphocyte activation and specific recognition to antigens in adaptive immune response (Watts, 1997).

Phagocytosis is an important activity for monocytes to exert immune functions. During phagocytic process, monocytes exhibit multilevel activations to impart differing immune functions. On one hand, changes of phagocytic ability of monocytes/macrophages are linked to levels of innate immune response in that the changes are correlated with levels of effector molecules such as reactive nitrogen intermediates and reactive oxygen intermediates (H2O2 and O2; Higginbotham et al., 1992; Higginbotham and Pruett, 1994). On the other hand, changes of phagocytic ability of monocytes are linked to adaptive immune response, as they are accompanied by different levels of MHC molecules, secretion of immune-related cytokines, and proliferation of peripheral blood T-lymphocytes (McAdams and Leonard, 1993; Hallwirth et al., 2002). And the relationship between monocyte phagocytosis and adaptive immune response has been suggested to be utilized in medical treatments. Hallwirth et al. (2002) and El-Gamal et al. (2005) pointed out that phagocytosis of monocytes was a reliable parameter to predict early-onset sepsis and a stage marker of sepsis because ones with sepsis had decreased phagocytic capacity of monocytes and lower level of MHC class II molecules. And before that, changes of monocyte phagocytosis and reduction of T-lymphocytes had already been deemed an indirect indicator of depression (McAdams and Leonard, 1993).

In chickens, some researches also focus on the relationship between phagocytosis of monocytes and disease resistance. Previous studies suggested that chickens with B19B21 and B2B2 genotypes have enhanced resistances to Marek’s disease (Briles et al., 1980) and Rous sarcoma virus (Heinzelmann et al., 1981), respectively, compared with those with B19B19 and B5B5 genotypes, whereas what is worth considering is that monocytes/macrophages from individuals with B19B21 and B2B2 genotypes had stronger phagocytosis than those from individuals with B19B19 or B5B5 genotypes (Qureshi and Taylor, 1993; Qureshi et al., 2000). There was also a study suggesting that selection for high serum immunoglobulin level to certain disease resistance was beneficial for enhancing phagocytosis of macrophages (Sarker et al., 2000). Besides that, Li et al. (2001) reported that selection for increased body weight in turkeys had no effect on phagocytosis of monocytes. Previous studies are mainly about effects of selection for resistances or production traits on phagocytic capacity of monocytes, whereas studies on effects of selection for phagocysis of monocytes on immunity or production in chickens are seldom set forth.

Therefore, the experiment was conducted with 2 objectives. The first objective was to investigate effects of selection for phagocytosis of peripheral blood monocytes on humoral immunity and cell-mediated immunity in chickens. The second objective was to study the effects of selection based on phagocytosis product of monocytes on some production traits of breeder chickens. The animals used in this study were CAU 3 brown-egg dwarf chickens, a line of brown-egg dwarf layers developed by 4 repeated backcrosses of the meat type dwarf ISA-Vedette to the female CAU brown egg layer, whose BW at 20 wk of age was about 1,200 g, total egg number to 72 wk was approximately 285, and average egg weight was 56 g (Yang et al., 1996).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Experimental Animals

Nine hundred twenty healthy unmated dwarf chickens (CAU 3 brown-egg dwarf chickens, 460 per sex) were obtained from China Agricultural University. They were hatched on the same day and were reared under the same management practices to minimize the environmental effects.

Cell Preparation

Heparinized blood (final concentration: 100 IU/mL) samples were collected from the brachia wing veins of 460 hens and 460 cocks at 20 wk of age. Procedures of isolation, purification, and culture of peripheral blood monocytes were optimized based on previous publications (Schenkel et al., 2004; Satoh et al., 2006). Briefly, peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation over Ficoll-hypaque (1.077 ± 0.001 g/mL, Sigma, St. Louis, MO) at 500 x g for 30 min, and the middle layer, which mainly included thrombocytes, lymphocytes, and monocytes was collected. The cells were then washed with RPMI 1640 medium (Gibco, Paisley, PA49RF, Scotland, UK) by centrifugation at 800 x g for 10 min twice. The number and viability of PBMC were determined by Trypan blue (Sigma) exclusion (viability > 99%). The PBMC at a concentration of 2 x 107 cells/mL were planted in 24-well tissue culture plates (Corning Inc., Corning, NY) at the rate of 1 mL/well with RPMI 1640. The plates were incubated at 41°C and 5% CO2 for 1.5 h, and then the nonadherent cells (most of lymphocytes) were washed out with RPMI1640. After 24 h of incubation and washing away nonadherent cells, approximately 90% of adherent cells were monocytes. The aim was to enrich monocytes by sloughing off residual thrombocytes. Monocytes were digested with 0.02% EDTA-0.25% Trypsin in PBS (without Ca2+ and Mg2+) for 5 min, and then the cells were harvested and replanted in 96-well tissue culture plates (Corning Inc.) at 5 x 105 cells/mL with 200 µL RPMI1640 and 10% fetal bovine serum (Hyclone, Logan, Utah; 3 wells per sample).

Preparation of Enzyme-Labeled Escherichia coli

Escherichia coli-HRP was prepared by labeling horseradish peroxidase (HRP; Sigma) to E. coli, consulting sodium meta-periodate (NaIO4) oxidation method, which was optimized by Tsang et al. (1995). Briefly, bacteria were cultivated and harvested at OD625 nm = 0.77 and were killed with formalin. The conditions were sodium meta-periodate (NaIO4): HRP was 40:1; duration of oxidation was 20 min at 25°C, pH 4.4; HRP: E. coli was 1 mg of HRP: 1010 cfu E. coli; duration of conjugation was 2 h at 25°C, pH 9.5.

Assays of Monocyte Phagocytosis Product

After 6 h of cell culture, E. coli-HRP was added into each well of 96-well tissue culture plates. Ratio of E. coli-HRP to monocytes was 20:1, and the plates were incubated at 41°C and 5% CO2 for 1 h. Then each plate was washed thrice with RPMI1640 to remove noncell-associated bacteria and incubated for another 10 min, allowing monocytes to ingest bacteria bound to their surfaces. Then samples were developed with 0.1 mg/mL of 3,3'5,5'-tetramethyl benzidine (TMB; Sigma) solution for 30 min at 25°C. The plates were read at a primary wavelength of 450 nm and a reference wavelength of 630 nm by model 550 Microplate Reader (BioRad). Phagocytosis product (PP) was calculated as following:


Formula

where OD (cells) is the absorbance of a well with cells, E. coli-HRP, and TMB solution; and OD (blank) is the absorbance of a well with medium, E. coli-HRP, and TMB solution, without cells.

Group Design

Then the 460 hens and 460 cocks, respectively, were divided into 5 groups of equal numbers orderly according to PP. For both hens and cocks, ones with the highest PP (HPPG), the medium PP (MPPG), and the lowest PP (LPPG) were reserved for the following experiments, and other 2 groups whose PP were between HPPG and MPPG or MPPG and LPPG were eliminated.

Inoculation

Chickens were inoculated with one dose (0.5 mL) of inactivated vaccines of avian influenza virus H5N2 [no.2004602282, Weike BioTechnology Corp., Harbin, China] at 20 wk of age, one dose (0.5 mL) of inactivated vaccines of avian influenza virus H9 [no.2005080012076, Zhaoqing Dahua Agricultural Medicine Corp., Guangdong, China] at 24 wk of age, and one dose (0.5 mL) of bigeminal inactivated vaccines of Newcastle disease (ND) virus-egg drop syndrome (EDS) virus [no.2005101836, Weike BioTechnology Corp.] at 28 wk of age.

Assays of Immune Response

On d 14 postvaccination with H5N2 and H9 and on d 10 postvaccination with ND-EDS, serum was extracted from brachia wing vein blood and used for microtiter hemagglutination inhibition test to assay serum hemagglutination inhibition (HI) antibody titers. Briefly, 100 µL of serum diluted to 1:20 was put in the first well of 96-well V-bottomed microtitre plates and serially diluted in PBS. Transfers were made to all wells through well 11, which contained 1:20,480 dilution of the original specimen. Then, 4 U of virosis antigen (1 haemagglutinating activity unit (1U) was defined as the highest dilution that gave rise to hemagglutination) was added to all wells including well 12, which served as the antigen control, and all wells were mixed with 50 µL 2% suspension of chicken red blood cells. The results were read 30 min later by elevating the plates to an angle of 45° for 1 min (Cotter and Van Eerden, 2006; Dalloul et al., 2006).

Flow cytometry analyses of T-lymphocytes were performed as previously described (Eicher-Pruiett et al., 1992; Boeker et al., 1999) with some modifications. Heparinized blood samples were collected from the brachia wing veins of chickens on d 14 or 10 postvaccination and PBMC suspension were obtained as mentioned above. The concentration of cells was modulated at 1 x 107 cells/mL, and after blocking Fc{gamma} receptor by incubating 0.35 mL cell suspension with 35 µL of normal mouse serum (Southern Biotechnology Associates Inc., Birmingham, AL) for 45 min at 4°C, 100 µL of cell suspension was incubated with 5 µL of mouse anti-chicken CD4 monoclonal antibody conjugated to FITC (Southern Biotechnology Associates Inc., Birmingham, AL) and 5 µL of mouse anti-chicken CD8{alpha} monoclonal antibody conjugated to phycoerythrin (Southern Biotechnology Associates Inc.) for 35 min at 4°C. Cell suspensions incubated with mouse IgG1{kappa} jugated to FITC and phycoerythrin (Southern Biotechnology Associates Inc.), respectively, were used as isotype controls. After washing 3 times with 0.1 M PBS containing 0.3% BSA, the stained cells were used for Flow Cytometric Analysis (FACS Analysis; FACSCalibur, BD Biosciences, San Jose, CA), and data were analyzed by Cell QuestPro Software (BD Biosciences).

Mating Combinations

The experiment was performed with 9 (3 x 3) mating combinations. The mating combinations (hen x cock) were as follows: LPPG male x LPPG female; LPPG male x MPPG female; LPPG male x HPPG female; MPPG male x LPPG female; MPPG male x MPPG female; MPPG male x HPPG female; HPPG male x LPPG female; HPPG male x MPPG female; HPPG male x HPPG female. Specifically, 29 cocks at 42 wk of age were selected randomly from each group (HPPG, MPPG, and LPPG). Until then, the numbers of hens belonging to HPPG, MPPG, and LPPG were 87, 81, and 84, respectively. Cocks from each group were randomly mated to hens from 3 groups (29 hens from HPPG, 27 hens from MPPG, and 28 hens from LPPG).

Determination of Total IgY Level and Production Traits

When hens were at the age of 43 wk, eggs were collected for 11 consecutive days. Eggs within the first 3 d (2 to 4 eggs/hen) were collected and stored at 4°C for examining levels of egg yolk and egg white IgY antibodies. The method to extract antibodies and the protocol to determine IgY antibodies were according to previous publications (Hamal et al., 2006). Eggs of each mating combination within the following 8 d were collected and hatched on the same day under the same conditions. Total number of eggs per hen and mean egg weight within the 11 d and fertilization rate, hatchability, and rate of healthy chicks (rate of healthy chicks = number of healthy chicks/number of fertile eggs) in the 8 d of each mating combination were recorded.

Statistical Analysis

The SPSS software package (SPSS for Windows, Release 10.0.1, SPSS Inc., Chicago, IL) was used for statistical analysis in this study.

A model for a 3 x 3 factorial design was constructed as follows:


Formula

where Yij was the ijth average phenotypic record of the trait, µ was the common mean, {alpha}i was the effect of the ith cock’s PI, βj was the effect of the jth hen’s PI, {alpha}i x βj was the interactive effect of the ith cock’s and the jth hen’s PI, and eij was the residual error. A P-value of <0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Distribution of Phagocytosis Product

The PP differed among the flock in both sexes, and the distribution was shown in Figure 1Go. The mean of hens’ PP was 1.333 ± 0.409 (ranging from 0.215 to 2.464, CV = 30.683%), and the mean of cocks’ PP was 1.130 ± 0.431 (ranging from 0.234 to 2.189, CV = 38.142%).


Figure 1
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  		Figure 1. Distribution of phagocytosis product (PP) of peripheral blood monocytes in dwarf chickens (460 hens and 460 cocks were used in this study). Monocytes harvested from peripheral blood of chickens at 20 wk of age were planted in 96-well tissue culture plates at 1 x 105 cells/well, and then were incubated with 20 folds horseradish peroxidase labeled Escherichia coli for 1 h. After ingestion of bacteria bound to cell surfaces, monocytes were developed with 0.1 mg/mL of 3,3'5,5'-tetramethyl benzidine solution for 30 min at 25°C. Absorbance was measured at 450 and 630 nm. Results were shown as the value of optical density (OD), PP = OD (cells) – OD (blank), and data were means ± SEM, 3 wells/chicken.

 
Considering the further studies, hens and cocks, respectively, were divided into 5 groups of equal numbers orderly according to PP, and hens whose PP were below 0.994 were defined as LPPG, whose PP were between 1.264 and 1.421 were defined as MPPG, and whose PP were above 1.649 were defined as HPPG; cocks whose PP were below 0.765 were defined as LPPG, whose PP were between 0.959 and 1.231 were defined as MPPG, and whose PP were above 1.511 were defined as HPPG. There were 92 chickens in each group reserved. Other chickens whose PP were between HPPG and MPPG or MPPG and LPPG were eliminated (184 chickens of each sex).

Serum HI Antibody Titers and T-Lymphocyte Subset Analysis

Serum HI antibody titers of hens and cocks in response to 3 vaccines were shown in Figure 2Go and Figure 3Go, respectively. As to anti-H5N2, anti-H9, and anti-ND antibody titers, there were effects of selection for PP of monocytes in both sexes; namely, there were significant differences (P < 0.05) among LPPG, MPPG, and HPPG, and antibody titers of HPPG (hens/cocks: H5N2, 5.612/5.565; H9, 7.779/6.855; ND, 5.899/4.696) were higher (P < 0.05) than those of LPPG (hens/cocks: H5N2, 5.062/5.065; H9, 7.286/6.341; ND, 5.058/4.123). There were no differences (P ≥ 0.05) of antiEDS antibody titers among 3 groups, and antiEDS antibody titers were low (P < 0.05, data were not shown) compared with other 3 titers.


Figure 2
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  		Figure 2. Plasma levels of anti-avian influenza virus (H5N2), anti-avian influenza virus (H9), anti-Newcastle disease virus (ND), and anti-egg drop syndrome virus (EDS) antibodies in hens. Hens were vaccinated against H5N2, H9, and ND and EDS, respectively, at the end of 20, 24, and 28 wk. On d 14 or 10 postvaccination, serum samples were serially diluted in PBS in 96-well V-bottomed microtiter plates and 4 U of virosis antigen (1 hemagglutinating activity unit (1U) was defined as the highest dilution that gave rise to hemagglutination) was added, followed by adding into 2% suspension of chicken red blood cells. The data were gained after 30 min by elevating the plates to an angle of 45° for 1 min. LPPG = lowest phagocytosis product group; MPPG = medium phagocytosis product group; HPPG = highest phagocytosis product group. Data were means ± SEM, 92 hens in each group, n = 3. a–cValues with different superscripts differ significantly (P < 0.05).

 

Figure 3
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  		Figure 3. Plasma levels of anti-avian influenza virus (H5N2), anti-avian influenza virus (H9), anti-Newcastle disease virus (ND), and anti-egg drop syndrome virus (EDS) antibodies in cocks. Cocks were vaccinated against H5N2, H9, and ND and EDS, respectively, at the end of 20, 24, and 28 wk. On d 14 or 10 postvaccination, serum samples were serially diluted in PBS in 96-well V-bottomed microtiter plates and 4 U of virosis antigen (1 hemagglutinating activity unit (1 U) was defined as the highest dilution that gave rise to hemagglutination) was added, followed by adding into 2% suspension of chicken red blood cells. The data were gained after 30 min by elevating the plates to an angle of 45° for 1 min. LPPG = lowest phagocytosis product group; MPPG = medium phagocytosis product group; HPPG = highest phagocytosis product group. Data were means ± SEM, 92 cocks in each group, n = 3. a,bValues with different superscripts differ significantly (P < 0.05).

 
As shown in Table 1Go, whichever vaccine was inoculated, there were effects (P < 0.05) of selection for PP of monocytes on percentage of CD4+ T-lymphocytes and ratio of CD4+ T-lymphocyte to CD8+ T-lymphocytes in both sexes, and percentage of CD4+ T-lymphocytes and CD4+/CD8+ ratio in HPPG (CD4+ hens/cocks: H5N2, 0.432/0.427; H9, 0.430/0.460; ND/EDS, 0.442/0.434; CD4+/CD8+ hens/cocks: H5N2, 1.514/1.520; H9, 1.555/1.594; ND/EDS, 1.503/1.463) were higher (P < 0.05) than those in LPPG (CD4+ hens/cocks: H5N2, 0.411/0.407; H9, 0.399/0.418; ND/EDS, 0.409/0.415; CD4+/CD8+ hens/cocks: H5N2, 1.395/1.408; H9, 1.488/1.419; ND/EDS, 1.422/1.357). There were no effects (P ≥ 0.05) of selection on the percentage of CD8+ T-lymphocytes among groups.


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  		Table 1. Percentage of T-lymphocytes subpopulation and ratio of CD4+ T-lymphocytes to CD8+ T-lymphocytes in peripheral blood of dwarf chickens1,2
 
Production Traits

The 3 x 3 factorial design for estimating variance components suggested that there were no effects of selection for PP of monocytes on total number of eggs per hen (F(2, 247) = 2.075, P = 0.128) and mean egg weight per hen (F(2, 247) = 0.301, P = 0.741). Hens’ PP significantly affected fertilization rate (F(2, 247) = 5.841, P = 0.003), hatchability (F(2, 247) = 4.158, P = 0.017), and rate of healthy chicks (F(2, 247) = 4.058, P = 0.016). Among the groups classified according to the levels of hens’ PP without regarding the levels of cocks’ PP, fertilization rate, hatchability, and rate of healthy chicks of LPPG were the lowest (P < 0.05) compared with those of HPPG (Table 2Go).


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  		Table 2. Classification of production traits according to phagocytosis products of 252 hens, 87 cocks, and 9 mating groups, respectively, in draft chickens1
 
There was no significant effect of either cocks’ PP or interaction of hens’ PP and cocks’ PP on any production trait we detected (P < 0.05). Table 2Go also showed that there were no differences in the production traits among 9 mating combination, except fertilization rate, which was higher (P < 0.05) in the combination of MPPG male and HPPG female than in the combination of LPPG male and LPPG female.

So considering the results of variance components estimation of the effects of hens’ PP on fertilization rate, hatchability, and the rate of healthy chicks and multiple comparisons of fertilization rate, hatchability, and the rate of healthy chicks among the groups classified based on levels of hens’ PP, there were significant effects of selection for hens in HPPG on them.

Maternal IgY Antibody

There were significant discrepancies (P < 0.0001) of IgY antibody levels in both egg yolks and egg whites among 3 groups, which were classified according to levels of hens’ PP. The IgY antibody levels of egg yolk (mg/mL), egg yolk total (mg), egg white (ug/mL), and egg white total (µg) in HPPG were the highest (P < 0.0001); however, the IgY antibody levels in LPPG were the lowest (P < 0.0001; Table 3Go).


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  		Table 3. Levels of IgY in egg yolk and egg white from LPPG, MPPG, and HPPG of hens in dwarf chickens1
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Because serum HI antibody titer is a valid indicator of humoral immunity in chickens (Kong et al., 2004), antibody titers against H5N2, H9, ND, and EDS were detected via microtiter hemagglutination inhibition test. Results suggested that the groups with higher PP had higher levels of humoral immune response, although the result of the anti-EDS antibody titer was not positive. However, there were significant differences of percentages of CD4+ T-lymphocytes, ratios of CD4+ T-lymphocytes to CD8+ T-lymphocytes and anti-ND titers among groups, which suggested that there were differences in humoral immunity between HPPG and LPPG after inoculating with ND-EDS bigeminal inactivated vaccine. But why was not it embodied by the anti-EDS titer? What should be taken into consideration was that compared with H5N2, H9, and ND, anti-EDS titer was much lower, so larruping performance of anti-EDS titer might be caused by antigenic interference or antibody suppression when ND antigen and EDS antigen were inoculated simultaneously (Wehrle, 1968), and even H5N2 or H9 antigen. The study indicated that effect of selection for higher PP of monocytes on humoral immunity was positive, which was, to some extent, similar to that reported by Heller et al. (1992), whose report showed that chickens selected for high level of antibody response to E. coli vaccination had greater phagocytic ability. Studies on chicken lines selected for serum immunoglobulin M or G level showed that the high serum immunoglobulin M line had higher (P < 0.01) phagocytic ability than the low serum immunoglobulin M line at 25 and 30 wk of age, and the high serum immunoglobulin G line had higher (P < 0.01) phagocytic ability than the low serum immunoglobulin G line at 25 wk of age (Sarker et al., 2000). As monocytes/macrophages initiate adaptive immune responses via antigen processing and presentation (Powell et al., 1983; Vainio et al., 1988) and cytokines releasing (Sekellick et al., 1994; Sundick and Gill-Dixon, 1997; Schneider et al., 2000; Lillehoj et al., 2001). These studies suggest that monocytes/macrophages with high phagocytic ability are more efficient in antigen processing and presentation than their low phagocytic counterparts.

It has been established that T-lymphocytes are the most pivotal part of vertebrate immune system, which widely participate in immune responses. In poultry, CD4+ T-lymphocytes play an important role in regulating adaptive immunity (including cellular immunity and humoral immunity), although dichotomy within the Th lymphocyte compartment has not been fully established (Erf, 2004). Level of CD8+ T-lymphocytes reflects T-lymphocyte mediated cytotoxicity of organisms, and the ratio of CD4+ T-lymphocytes to CD8+ T-lymphocytes after vaccination is used to estimate the trend of adaptive immune response. To further understand the relationship between phagocytosis of monocytes and immune responses in dwarf chickens, peripheral blood T-lymphocyte subsets were analyzed. In our previous study, percentages of CD4+ and CD8+ T-lymphocytes returned to original levels around d 30 postinoculation, and on d 14 postinoculation of H5N2 or H9 inactivated vaccine, percentages of CD4+ and CD8+ T-lymphocytes reached the maximums, whereas on d 10 postinoculation of ND – EDS bigeminal inactivated vaccine, percentages of CD4+ and CD8+ T-lymphocytes reached the maximums, which was coincident with former studies (Bing et al., 2000; Fu et al., 2001).

Results of percentage of CD4+ T-lymphocytes indicated individuals with high phagocytic ability of monocytes had strong helper function to regulate immune responses, but we could not point out if cytotoxic T-lymphocyte response contributed to the discrepancy, so percentage of CD8+ T-lymphocytes was tested, and the results suggested that there were no effects of selection for monocytes with high phagocytic ability on cytotoxic T-lymphocyte response, and CD4+/CD8+ ratio also showed that humoral immunity contributed more to the discrepancy between HPPG and LPPG. Generally, humoral immune response and cellular immune response are stimulated simultaneously, so what can be inferred is that cytotoxic T-lymphocyte immunity, different from humoral immunity, is not reflected by phagocytic ability of monocytes, which might be because MHC II molecules that modulate humoral immunity are only expressed by APC, but MHC I molecules that activate CD8+ T-lymphocytes can be expressed by nearly all nucleated cells.

Production performances are negatively related to certain disease resistance in poultry production (Miller et al., 1992; Liu et al., 1995); thus, we performed 3 x 3 design and examined the relationship of phagocytosis of monocytes and some production traits. One cock from each group (HPPG, MPPG, and LPPG) was mated to 3 hens, respectively, from HPPG, MPPG, and LPPG, so hens from each group had the same cock effect, whereas the importance of information derived from hens and cocks was different, which might have contributed to different accuracies between hens and cocks. Statistical analysis showed that selecting a specific mating combination benefited the fertilization rate; however, cocks’ PP and interaction of hens’ and cocks’ PP had no effect on fertilization rate, so the difference among mating combinations might be caused by different selection for hens’ PP. Phagocytosis of monocytes/macrophages has been reported to play important roles in hens’ ovarian events, such as follicular growth, ovulation, and regression of postovulatory follicles (Barua et al., 1998). The effect of maternal PP of monocytes on fertilization rate might be due to the effect of monocytes on follicular development and regression of postovulatory follicles, which are beneficial to fertilization. But why could maternal PP affect health of chicks? In fact, embryo and newly hatched chicks are susceptible to pathogens, which determine livability of chicks, because of their incompletely developed immune systems (Desmidt et al., 1998; Chadfield and Hinton, 2004; Hamal et al., 2006). The primary means of antigen-specific protection before 2 wk is maternal antibody (Leslie and Martin, 1973; Martin and Leslie, 1973; Leslie, 1975; Lawrence et al., 1981), which is transferred to offspring by eggs (Orlans, 1967; Ogata et al., 1970). Chickens have 3 classes of antibodies: IgY, IgA, and IgM, of which IgY is predominant Ig isotype transferred to eggs as well as to newly hatched chicks (Hamal et al., 2006). We detected levels of IgY in eggs, and based on the data, we found maternal phagocytosis was positively related to levels of IgY in eggs. It has been well established that levels of IgY deposited in eggs and transferred to offspring are directly related to the circulating levels of IgY in hens (Loeken and Roth, 1983; Hamal et al., 2006), so the relationship between maternal phagocytosis and maternal IgY might stem from the relationship between phagocytosis and humoral immunity, and this kind of maternal protection contributes to hatchability and rate of healthy chicks in chicks.

When phagocytizing pathogens, monocytes present antigens of the pathogens to T-lymphocyte to stimulate immune response, so to some extent, phagocysis of monocytes can reflect disease resistances of organisms. Our study suggests that selection for high phagocytosis of monocytes can improve humoral immunity in dwarf chickens and is important to certain production traits of breeder chickens. It is known that phagocytosis of monocytes reflects innate immunity (Weiss, 1989; Beckerman et al., 1993; Higginbotham and Pruett, 1994), and in this study, we find that phagocytosis of monocytes is also related to adaptive immunity in dwarf chickens, indicating that selecting chickens with high phagocytosis of monocytes as breeders might enhance innate and adaptive immunities. The feasibility to improve immunity and performances of chicken lines by continuously selecting high phagocytosis of monocytes needs further studies on effector mechanisms and transmissibility of monocyte phagocytosis.


   ACKNOWLEDGMENTS
 
This research was supported by the National Key Basic Research ‘973’ Program of China (No. 2006CB102100) and National Natural Science Foundation of China (No. 30471234). We thank Qingcai Zhang (China Agricultural University, China) for supplying experimental animals.

Received for publication July 21, 2007. Accepted for publication September 8, 2007.


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