Differential Modulation of 25-hydroxycholecalciferol on Innate Immunity of Broiler Breeder Hens

doi:10.3390/ani11061742

. 2021 Jun 10;11(6):1742.

doi: 10.3390/ani11061742.

Differential Modulation of 25-hydroxycholecalciferol on Innate Immunity of Broiler Breeder Hens

Pao-Chia Chou¹, Pei-Chi Lin², Shu-Wei Wu², Chien-Kai Wang^{2

3}, Thau-Kiong Chung⁴, Rosemary L Walzem⁵, Lih-Shiuh Lai¹, Shuen-Ei Chen^{2

3

6

7}

Affiliations

¹ Department of Food Science and Biotechnology, National Chung Hsing University, Taichung 40227, Taiwan.
² Department of Animal Science, National Chung Hsing University, Taichung 40227, Taiwan.
³ The iEGG and Animal Biotechnology Center, National Chung Hsing University, Taichung 40227, Taiwan.
⁴ DSM Nutritional Products Asia Pacific, Singapore 117440, Singapore.
⁵ Department of Poultry Science, Texas A&M University, College Station, TX 77843, USA.
⁶ i-Center for Advanced Science and Technology (iCAST), National Chung Hsing University, Taichung 40227, Taiwan.
⁷ Innovation and Development Center of Sustainable Agriculture (IDCSA), National Chung Hsing University, Taichung 40227, Taiwan.

PMID: 34200930
PMCID: PMC8230489
DOI: 10.3390/ani11061742

Differential Modulation of 25-hydroxycholecalciferol on Innate Immunity of Broiler Breeder Hens

Pao-Chia Chou et al. Animals (Basel). 2021.

. 2021 Jun 10;11(6):1742.

doi: 10.3390/ani11061742.

Authors

Pao-Chia Chou¹, Pei-Chi Lin², Shu-Wei Wu², Chien-Kai Wang^{2

3}, Thau-Kiong Chung⁴, Rosemary L Walzem⁵, Lih-Shiuh Lai¹, Shuen-Ei Chen^{2

3

6

7}

Affiliations

¹ Department of Food Science and Biotechnology, National Chung Hsing University, Taichung 40227, Taiwan.
² Department of Animal Science, National Chung Hsing University, Taichung 40227, Taiwan.
³ The iEGG and Animal Biotechnology Center, National Chung Hsing University, Taichung 40227, Taiwan.
⁴ DSM Nutritional Products Asia Pacific, Singapore 117440, Singapore.
⁵ Department of Poultry Science, Texas A&M University, College Station, TX 77843, USA.
⁶ i-Center for Advanced Science and Technology (iCAST), National Chung Hsing University, Taichung 40227, Taiwan.
⁷ Innovation and Development Center of Sustainable Agriculture (IDCSA), National Chung Hsing University, Taichung 40227, Taiwan.

PMID: 34200930
PMCID: PMC8230489
DOI: 10.3390/ani11061742

Abstract

Past immunological studies in broilers focused on juveniles within the rapid pre-slaughter growth period and may not reflect adult immune responses, particularly in breeders managed with chronic feed restriction (R). The study aimed to assess innate immune cell functions in respect to R vs. ad libitum (Ad) feed intake in breeder hens with and without dietary 25-hydroxycholecalciferol (25-OH-D₃) supplementation. Ad-feed intake consistently suppressed IL-1β secretion, respiratory burst, and cell livability in peripheral heterophils and/or monocytes along the feeding trial from the age of 51 to 68 weeks. Supplemental 25-OH-D₃ repressed IL-1β secretion and respiratory burst of both cells mostly in R-hens, but promoted monocyte phagocytosis, chemotaxis, and bacterial killing activity in Ad-hens in accompany with relieved hyperglycemia, hyperlipidemia, and systemic inflammation. Overnight cultures with leukocytes from R-hens confirmed the differential effects of 25-OH-D₃ to rescue immune functions altered by glucose and/or palmitic acid exposure. Studies with specific inhibitors further manifested the operative mechanisms via glucolipotoxicity in a cell type- and function-dependent manner. The results concluded no predominant changes between R- vs. Ad-feed intake on leukocyte defense against pathogens despite some differential differences, but supplemental 25-OH-D₃ exerts more pronounced effects in Ad-hens.

Keywords: 25-hydroxycholecalciferol; broiler breeder hens; feed restriction; glucolipotoxicity; innate immunity.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

**Figure 1**
Plasma glucose, insulin, TG, NEFA, and IL-1β concentrations of broiler breeder hens provided with restricted (R) or ad libitum (Ad) feed intake. At age of 58 and 65 weeks (wks), 6 hens from each group were randomly selected for blood collection for plasma glucose, insulin, triacylglycerol (TG), non-esterified fatty acid (NEFA), and interleukin-1β (IL-1β) analysis (A–E, respectively, n = 6). #; significant difference by Ad-feed intake (vs. corresponding R hens, p < 0.05), *; significant difference by 25-OH-D₃ (vs. R- or Ad-hens, p < 0.05). 25-OH-D₃: 25-hydroxycholecalciferol.

**Figure 2**
Effects of dietary supplementation of 25-OH-D₃ on IL-1β secretion, phagocytosis, and the respiratory burst of leukocytes in broiler breeder hens provided with restricted (R) or ad libitum (Ad) feed intake. Peripheral leukocytes (2 × 10⁶ cells) isolated from hens at age 59–60 and 67–68 weeks (wks) were cultured for 3 h and medium were then collected for interleukin-1β (IL-1β) analysis (A) by Western blot method based on equivalent amounts of protein (n = 6). Freshly prepared cells were used for phagocytosis analysis (B) and respiratory burst analysis (C) (n = 6). Chemiluminescence results of respiratory burst analysis were expressed as integrated counts over 45 min. Results of Western blots were expressed as rations relative to R-hens. *; significant difference by 25-OH-D₃, p < 0.05, #; significant difference by Ad-feed intake. 25-OH-D₃; 25-hydroxycholecalciferol.

**Figure 3**
Effects of dietary supplementation of 25-OH-D₃ on chemotaxis, bacterial killing and cell livability of leukocytes in broiler breeder hens under restricted (R) or ad libitum (Ad) feed intake. Freshly prepared cells (2 × 10⁵) from hens at age of 66 weeks were used for chemotaxis analysis through trans-well method (A), incubated with *Salmonella Typhimurium* (ST) at a 1:2 ratio for 45 min for bacterial killing analysis (B), or suspended in RPMI for cell livability analysis (C) (n = 6). Cell viability was defined as the percentage of total cells present that were viable in a cell death analysis with FITC-Annexin-V/propidium iodide (PI) staining and cytometry sorting. Results of bacterial killing were expressed as ratios relative to R-hens. *; significant difference by 25-OH-D₃, p < 0.05, #; significant difference by Ad-feed intake. 25-OH-D₃; 25-hydroxycholecalciferol.

**Figure 4**
Effects of dietary supplementation of 25-OH-D₃ on VDR protein amounts and p65 activation of leukocytes in broiler breeder hens provided with restricted (R) or ad libitum (Ad) feed intake. Freshly prepared cells from hens at the age of 66 weeks were used for total protein extraction for vitamin D receptor (VDR) expression (A) and nuclear extracts were used for p65 (a subunit of nuclear factor kappa B, NFκB) translocation (B) through Western blot method. Results were normalized to β-actin or H2AX and expressed as ratios relative to R-hens (n = 6). *; significant difference by 25-OH-D₃, p < 0.05, #; significant difference by Ad-feed intake. 25-OH-D₃; 25-hydroxycholecalciferol.

**Figure 5**
Effects of 25-OH-D₃ on leukocyte functions. Peripheral heterophils and monocytes (2 × 10⁶ cells) isolated from R-hens at age 61–65 weeks were treated with various levels of 25-hydroxycholecalciferol (25-OH-D₃) overnight (16 h). Media were collected for interleukin-1β (IL-1β) analysis by Western blot method based on protein equivalency (A); cells were collected for phagocytosis (B) and respiratory burst analysis (C) (n = 4). Chemiluminescence results of respiratory burst analysis were expressed as integrated counts over 45 min. Results of Western blots were expressed as ratios relative to control (0 nM of 25-OH-D₃). Means with different letters differ significantly (p < 0.05).

**Figure 6**
Effects of glucose and palmitic acid on leukocyte functions. Peripheral heterophils and monocytes (2 × 10⁶ cells) isolated from R-hens at age 61–65 weeks were treated with various levels of glucose or palmitic acid (PA) overnight (16 h). Media were collected for interleukin-1β (IL-1β) analysis by Western blot method based on equivalent amounts of protein (A,D) and collected cells were used for phagocytosis analysis (B,E) and respiratory burst analysis (C,F) (n = 4). Chemiluminescence results of respiratory burst analysis were expressed as count per second (CPM) over 45 min. Results of Western blots were expressed as rations relative to control (no glucose or PA supplementation). Means with different letters differ significantly (p < 0.05).

**Figure 7**
Effects of 25-OH-D₃ on leukocyte functions challenged with glucose and/or palmitic acid. Peripheral heterophils and monocytes (2 × 10⁶ cells) isolated from R-hens at age 61–65 weeks were treated with vehicle, 25-hydroxycholecalciferol (25-OH-D₃, 50 nM), palmitic acid (PA, 1.5 mM), and/or glucose (200 mg/dL) overnight (16 h). Media were collected for interleukin-1β (IL-1β) analysis (A) based on the equivalent amounts of protein and cells used for phagocytosis (B) and respiratory burst analysis (C) (n = 4). Chemiluminescence results of respiratory burst analysis were expressed as integrated counts over 45 min. Results of Western blots were expressed as rations relative to control (no PA, glucose and 25-OH-D₃ supplementation). *, significant difference vs. corresponding control (p < 0.05). + or − indicates with or without PA, glucose, or 25-OH-D₃.

**Figure 8**
Mechanisms of glucolipotoxicity of leukocyte functions. Peripheral leukocytes (2 × 10⁶ cells) from R-hens at age 61–65 weeks were pre-treated with TC (Triacsin C, 5 μM), FB1 (fumonisin B1, 25 μM), DPS (Desipramine, 10 μM), or nMPG (N-mercaptopropionyl-glycine, 0.3 mM). After being replaced with the medium, cells were treated with the vehicle, palmitic acid (PA, 1.5 mM), or glucose (200 mg/dL) overnight (16 h). Media were collected for interleukin-1β (IL-1β) analysis (A) by Western blot method based on equivalent amounts of protein and collected cells used for phagocytosis (B) and respiratory burst analysis (C) (n = 4). Chemiluminescence results of respiratory burst analysis were expressed as integrated counts over 45 min. Results of Western blots were expressed as rations relative to control (Ctl). *; significant difference vs. corresponding control (vehicle), p < 0.05, #; significant difference vs. vehicle, p < 0.05.

**Figure 9**
Effects of 25-OH-D₃ on glucose or palmitic acid challenged leukocyte survival. Peripheral leukocytes (1 × 10⁶ cells) isolated from R-hens at age 61–65 weeks were cultured with indicated levels of glucose or palmitic acid (PA) in the presence or absence of 25-hydroxycholecalciferol (25-OH-D₃, 50 nM) overnight (16 h). Collected cell were used for cell death analysis (n = 4). Means with different letters differ significantly among different levels of glucose or PA (p < 0.05). Means with different letters differ significantly (p < 0.05). *, significant difference vs. corresponding control (p < 0.05). Means with different letters differ significantly (p < 0.05). + or − indicates with or without PA, glucose, or 25-OH-D₃. Figure 9 is for cell death analysis, (A,B) mean different cell types, (A) heterophils, (B) mpnocytes.

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References

1. Griffin H.D., Goddard C. Rapidly growing broiler (meat-type) chickens: Their origin and use for comparative studies of the regulation of growth. Int. J. Biochem. 1994;26:19–28. doi: 10.1016/0020-711X(94)90190-2. - DOI - PubMed
1. Yu M.W., Robinson F.E., Etches R.J. Effect of feed allowance during rearing and breeding on female broiler breeders. 3. Ovarian steroidogenesis. Poult. Sci. 1992;71:1762–1767. doi: 10.3382/ps.0711762. - DOI - PubMed
1. Chen C.Y., Lin H.Y., Chen Y.W., Ko Y.J., Liu Y.J., Chen Y.H., Walzem R.L., Chen S.E. Obesity-associated cardiac pathogenesis in broiler breeder hens: Pathological adaption of cardiac hypertrophy. Poult. Sci. 2017;96:2428–2437. doi: 10.3382/ps/pex015. - DOI - PubMed
1. Chen C.Y., Huang Y.F., Ko Y.J., Liu Y.J., Chen Y.H., Walzem R.L., Chen S.E. Obesity-associated cardiac pathogenesis in broiler breeder hens: Development of metabolic cardiomyopathy. Poult. Sci. 2017;96:2438–2446. doi: 10.3382/ps/pex016. - DOI - PubMed
1. Zou A., Nadeau K., Wang P.W., Lee J.Y., Guttman D.S., Sharif S., Korver D.R., Brumell J.H., Parkinson J. Accumulation of genetic variants associated with immunity in the selective breeding of broilers. BMC Genet. 2020;21:5. doi: 10.1186/s12863-020-0807-z. - DOI - PMC - PubMed

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[1] Griffin H.D., Goddard C. Rapidly growing broiler (meat-type) chickens: Their origin and use for comparative studies of the regulation of growth. Int. J. Biochem. 1994;26:19–28. doi: 10.1016/0020-711X(94)90190-2. - DOI - PubMed

[2] Griffin H.D., Goddard C. Rapidly growing broiler (meat-type) chickens: Their origin and use for comparative studies of the regulation of growth. Int. J. Biochem. 1994;26:19–28. doi: 10.1016/0020-711X(94)90190-2. - DOI - PubMed

[3] Yu M.W., Robinson F.E., Etches R.J. Effect of feed allowance during rearing and breeding on female broiler breeders. 3. Ovarian steroidogenesis. Poult. Sci. 1992;71:1762–1767. doi: 10.3382/ps.0711762. - DOI - PubMed

[4] Yu M.W., Robinson F.E., Etches R.J. Effect of feed allowance during rearing and breeding on female broiler breeders. 3. Ovarian steroidogenesis. Poult. Sci. 1992;71:1762–1767. doi: 10.3382/ps.0711762. - DOI - PubMed

[5] Chen C.Y., Lin H.Y., Chen Y.W., Ko Y.J., Liu Y.J., Chen Y.H., Walzem R.L., Chen S.E. Obesity-associated cardiac pathogenesis in broiler breeder hens: Pathological adaption of cardiac hypertrophy. Poult. Sci. 2017;96:2428–2437. doi: 10.3382/ps/pex015. - DOI - PubMed

[6] Chen C.Y., Lin H.Y., Chen Y.W., Ko Y.J., Liu Y.J., Chen Y.H., Walzem R.L., Chen S.E. Obesity-associated cardiac pathogenesis in broiler breeder hens: Pathological adaption of cardiac hypertrophy. Poult. Sci. 2017;96:2428–2437. doi: 10.3382/ps/pex015. - DOI - PubMed

[7] Chen C.Y., Huang Y.F., Ko Y.J., Liu Y.J., Chen Y.H., Walzem R.L., Chen S.E. Obesity-associated cardiac pathogenesis in broiler breeder hens: Development of metabolic cardiomyopathy. Poult. Sci. 2017;96:2438–2446. doi: 10.3382/ps/pex016. - DOI - PubMed

[8] Chen C.Y., Huang Y.F., Ko Y.J., Liu Y.J., Chen Y.H., Walzem R.L., Chen S.E. Obesity-associated cardiac pathogenesis in broiler breeder hens: Development of metabolic cardiomyopathy. Poult. Sci. 2017;96:2438–2446. doi: 10.3382/ps/pex016. - DOI - PubMed

[9] Zou A., Nadeau K., Wang P.W., Lee J.Y., Guttman D.S., Sharif S., Korver D.R., Brumell J.H., Parkinson J. Accumulation of genetic variants associated with immunity in the selective breeding of broilers. BMC Genet. 2020;21:5. doi: 10.1186/s12863-020-0807-z. - DOI - PMC - PubMed

[10] Zou A., Nadeau K., Wang P.W., Lee J.Y., Guttman D.S., Sharif S., Korver D.R., Brumell J.H., Parkinson J. Accumulation of genetic variants associated with immunity in the selective breeding of broilers. BMC Genet. 2020;21:5. doi: 10.1186/s12863-020-0807-z. - DOI - PMC - PubMed

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Differential Modulation of 25-hydroxycholecalciferol on Innate Immunity of Broiler Breeder Hens

Affiliations

Differential Modulation of 25-hydroxycholecalciferol on Innate Immunity of Broiler Breeder Hens

Authors

Affiliations

Abstract

Conflict of interest statement

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References

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