Engormix/Poultry Industry/Technical articles

Effect of Cordyceps militaris Hot Water Extract on Immunomodulation-associated Gene Expression in Broilers, Gallus gallus (Extract)

Published on: 11/8/2022
Author/s : Life Rainbow
Studies in the past decade have shown that Cordyceps militaris has multiple pharmacological functions, including anti-oxidant, anti-inflammatory, antimicrobial, anti-tumor, and anti-angiogenic activities and immunopotentiation ability. The chemical structure of cordycepin is similar to that of adenosine. Cordycepin can be used as a nucleotide during RNA synthesis, which leads to premature termination of chain elongation because of the absence of oxygen in the 3′ position of its ribose ring. In addition to cordycepin, the polysaccharides of Cordyceps militaris also possess antiviral and anti-tumor activities.
The purpose of this study was to investigate the effects of cordycepin from Cordyceps militaris hot water extract (CMHW) on growth performance and immunomodulation in response to vaccination and LPS challenge. 
 
Materials and Methods
Animal Experiment I
  1. 48 one-day-old male broilers (Ross 308) were purchased from a local commercial hatchery. 
  2. All the broilers were randomly divided into four groups with three replicates. CMHW powder was supplied in drinking water for 5 days after vaccination on days 4 and 14, respectively.
  3. The four groups (n=12 per group) were: (1) basal diet (control), (2) basal diet plus 1 g/L (9.69 mg cordycepin) CMHW in drinking water, (3) basal diet plus 2 g/L (19.38 mg cordycepin) CMHW in drinking water (4) basal diet plus 4 g/L (38.76 mg cordycepin) CMHW in drinking water.
  4. Broilers were vaccinated by nose-drop administration with combined Newcastle disease (ND)-infectious bronchitis (IB) live vaccines at 4 and 14 days of age.
Animal Experiment II
  1. 48 one-day-old male broilers (Ross 308) were purchased from a local commercial hatchery. 
  2. All the broilers were randomly divided into four groups with three replicates.
  3. The four groups (n=12 per group) were: 
(1) control group, 
(2) supplementation of 2 g/L CMHW in drinking water for 3 days before vaccination (A), 
(3) supplementation of 2 g/L CMHW in drinking water for 3 days before and after vaccination (AP), (4) supplementation of 2 g/L CMHW in drinking water for 3 days after vaccination (P).
  1. Broilers were vaccinated by nose-drop administration with combined ND-IB live vaccines when they were 4, 14, and 28 days old.
Animal Experiment III
  1. 36 one-day-old male broilers (Ross 308) were purchased from a local commercial hatchery.
  2. All the broilers were randomly divided into three groups with three replicates each.
  3. The three groups (n=12 per group) were: 
(1) basal diet (control), 
(2) basal diet plus intraperitoneal administration of LPS (1 mg/kg), 
(3) basal diet plus intraperitoneal administration of LPS (1 mg/kg) in combination with 2 g/L CMHW in drinking water.
Quantitative Reverse Transcription-polymerase Chain Reaction (qRT-PCR)
  1. Six birds from each group were randomly selected, sacrificed, and used for gene expression analysis.
  2. Gene include iNOS, COX-2, IFN-γ, IL-4 and β-actin.
 
Results
Effect of CMHW Supplementation on Growth Performance and Vaccine Adjuvant Activity in Broilers
To examine the effect of CMHW supplementation on growth performance, broilers were fed with 1, 2, and 4 g/L CMHW for four weeks. However, there were no statistically confirmed differences between body weight and CMHW supplementation during the entire feeding period (Table 1). Broilers fed 1 g/L CMHW had lower feed intake from day 14 to 28 compared to other treatments (Table 1, P< 0.05). Supplementation of high concentration of CMHW had the highest potential to improve FCR in broilers from day 1 to 14, whereas this beneficial effect was reversed from day 14 to 28 (Table 1, P< 0.05). Supplementation of CMHW in drinking water 3 days before vaccination increased the body weight at 21 days of age compared to other treatments (Table 2, P< 0.05). Similar results were also observed in daily gain and feed intake from day 1 to 21 (Table 2, P< 0.05). However, broilers fed CMHW for 3 d after vaccination showed higher FCR from day 1 to 21 (Table 2, P< 0.05). CMHW supplementation did not improve the response of antibody titer to ND and IB viruses (Fig. 1a and b). In addition, different CMHW supplementation periods did not affect the antibody titer of ND and IB viruses (Fig. 1c and and1d1d).
Effect of CMHW Supplementation on Regulation of Immunomodulation-related Gene Expression in Broilers
To examine the effect of different concentrations of CMHW on inflammation-related gene expression in the spleen and bursa of Fabricius, broilers were fed 1, 2, and 4 g/L CMHW. Results showed that CMHW supplementation in drinking water for 14 d had no significant effect on iNOS mRNA level in the spleen (Fig. 2a), whereas it was significantly reduced after 28 day-treatment with 2 g/L of CMHW (Fig. 2b, P< 0.05). Similarly, CMHW supplementation did not inhibit COX-2 expression in the spleen of 14-day-old broilers (Fig. 2c). However, COX-2 mRNA level was significantly reduced in the spleen in a dose-dependent manner in 28-day-old broilers (Fig. 2d, P< 0.05). CMHW treatment did not affect iNOS mRNA level in the bursa of Fabricius of 14-day-old broilers (Fig. 2e), whereas it efficiently inhibited iNOS expression in 28-day-old broilers (Fig. 2f, P< 0.05). The high concentration of CMHW (4 g/L) attenuated COX-2 expression in the bursa of Fabricius of 14-day-old broilers (Fig. 2g, P< 0.05). Similar inhibitory effects on COX-2 mRNA levels after CMHW treatment were also observed in 28-day-old broilers (Fig. 2h, P< 0.05).
To investigate whether CMHW has an immunomodulatory role in the spleen and bursa of Fabricius, broilers were fed 2 g/L CMHW before or after vaccination. Results showed that compared to other treatments, posterior supplementation of CMHW in drinking water reduced iNOS mRNA level in the spleen of 21-day-old broilers (Fig. 3a, P< 0.05). In contrast, iNOS mRNA level in the spleen was induced after anterior and posterior supplementation of CMHW in 35-day-old broilers (Fig. 3b, P< 0.05). Compared to other treatments, anterior supplementation of CMHW reduced COX-2 mRNA levels in the spleen of 21- and 35-day-old broilers (Fig. 3c and d, P< 0. 05). iNOS expression was attenuated in the bursa of Fabricius of 21-day-old broilers at all CMHW supplementation periods (Fig. 3e, P< 0.05). However, CMHW supplementation during vaccination did not significantly affect iNOS expression in the bursa of Fabricius of 35-day-old broilers (Fig. 3f). Anterior only and anterior in combination with posterior supplementation of CMHW inhibited COX-2 expression in the bursa of Fabricius of 21 day-old broilers (Fig. 3g, P< 0.05). Similar to iNOS expression in the bursa of Fabricius, CMHW supplementation during vaccination did not significantly affect COX-2 expression in the bursa of Fabricius of 35-day-old broilers (Fig. 3h).
Effect of CMHW Supplementation on Regulation of Immunomodulation-related Gene Expression in LPS-challenged Broilers
CMHW supplementation significantly alleviated LPS-induced iNOS expression in the spleen of broilers 3 h post-injection of LPS (Fig. 4a, P< 0.05). No significant difference was observed in iNOS mRNA level in the spleen of broilers 24 h post-injection of LPS in combination with CMHW treatment (Fig. 4b). LPS-induced COX-2 expression in the spleen of broilers was efficiently alleviated by CMHW treatment at 3 and 24 h post-injection (Fig. 4c and d, P< 0.05). Interestingly, iNOS mRNA level in the bursa of Fabricius 3 h post-injection of LPS was further reduced in chickens challenged with LPS compared to the control group (Fig. 4e, P< 0.05). No significant difference was observed regarding iNOS mRNA level in the bursa of Fabricius of broilers 24 h post-injection of LPS in combination with CMHW treatment (Fig. 4f). Similar to the spleen, LPS-induced COX-2 expression in the bursa of Fabricius of broilers was efficiently alleviated by CMHW treatment 3 and 24 h post-injection (Fig. 4g and h, P< 0.05). CMHW supplementation significantly reduced LPS-induced IFN-γ mRNA level in the spleen of broilers 3 and 24 h postinjection of LPS (Fig. 5a and b, P< 0.05). IL-4 mRNA levels were increased in the spleen 3 and 24 h post-injection of LPS in combination with CMHW treatment (Fig. 5c and d, P< 0.05). LPS-induced IFN-γ expression in the bursa of Fabricius of broilers were efficiently attenuated by CMHW treatment 3 and 24 h post-injection (Fig. 5e and f, P< 0.05). Similar to the spleen, IL-4 mRNA levels were significantly increased in the bursa of Fabricius 3 and 24 h post-injection of LPS in combination with CMHW treatment (Fig. 5g and h, P< 0.05).
Table 1. Effects of different concentration of CMHW on growth performance in broilers
Table 1. Effects of different concentration of CMHW on growth performance in broilers
Table 2. Effects of CMHW on growth performance in broilers during vaccination
Table 2. Effects of CMHW on growth performance in broilers during vaccination
Fig. 1. Effects of CMHW supplementation on antibody titer in broilers. (a) Effect of different concentrations of CMHW (1, 2, and 4 g/L in drinking water) on antibody titers against Newcastle disease (ND) in 14-day-old broilers. (b) Effect of different concentrations of CMHW (1, 2, and 4 g/L in drinking water) on antibody titers against infectious bronchitis (IB) in 14-day-old broilers. (c) Effect of different supplementation period (A, anterior only; AP: anterior and posterior; P, posterior only) of CMHW (2 g/L in drinking water) on antibody titers against ND in 35-day-old broilers. (d) Effect of different supplementation period (A, anterior only; AP: anterior and posterior; P, posterior only) of CMHW (2 g/L in drinking water) on antibody titers against IB in 35-day-old broilers. Values represent mean±SD (n=3).
Fig. 1. Effects of CMHW supplementation on antibody titer in broilers. (a) Effect of different concentrations of CMHW (1, 2, and 4 g/L in drinking water) on antibody titers against Newcastle disease (ND) in 14-day-old broilers. (b) Effect of different concentrations of CMHW (1, 2, and 4 g/L in drinking water) on antibody titers against infectious bronchitis (IB) in 14-day-old broilers. (c) Effect of different supplementation period (A, anterior only; AP: anterior and posterior; P, posterior only) of CMHW (2 g/L in drinking water) on antibody titers against ND in 35-day-old broilers. (d) Effect of different supplementation period (A, anterior only; AP: anterior and posterior; P, posterior only) of CMHW (2 g/L in drinking water) on antibody titers against IB in 35-day-old broilers. Values represent mean±SD (n=3).
Fig. 2. Effects of different concentrations of CMHW on inflammation-related gene expression in broilers. Effect of different concentrations of CMHW (1, 2, and 4 g/L in drinking water) on iNOS mRNA level in the spleen of 14-day-old (a) and 28-day-old broilers (b). COX-2 mRNA level in the spleen of 14-day-old (c) and 28-day-old broilers (d). Effect of different concentrations of CMHW (1, 2, and 4 g/L in drinking water) on iNOS mRNA level in the bursa of Fabricius of 14-day-old (e) and 28-day-old broilers (f). COX-2 mRNA level in the bursa of Fabricius of 14-day-old (g) and 28-day-old broilers (h). Values represent mean±SD (n=6). Means with different letter superscripts are significantly different (P< 0.05)
Fig. 2. Effects of different concentrations of CMHW on inflammation-related gene expression in broilers. Effect of different concentrations of CMHW (1, 2, and 4 g/L in drinking water) on iNOS mRNA level in the spleen of 14-day-old (a) and 28-day-old broilers (b). COX-2 mRNA level in the spleen of 14-day-old (c) and 28-day-old broilers (d). Effect of different concentrations of CMHW (1, 2, and 4 g/L in drinking water) on iNOS mRNA level in the bursa of Fabricius of 14-day-old (e) and 28-day-old broilers (f). COX-2 mRNA level in the bursa of Fabricius of 14-day-old (g) and 28-day-old broilers (h). Values represent mean±SD (n=6). Means with different letter superscripts are significantly different (P< 0.05)
Fig. 3. Effects of CMHW supplementation on inflammation-associated gene expression in broilers during vaccination. Effect of different supplementation period (A, anterior only; AP, anterior and posterior; P, posterior only) of CMHW (2 g/L in drinking water) on iNOS mRNA level in the spleen of 21-day-old (a) and 35-day-old broilers (b). COX-2 mRNA level in the spleen of 21-day-old (c) and 35-day-old broilers (d). Effect of different supplementation period (A, anterior only; AP, anterior and posterior; P, posterior only) of CMHW (2 g/L in drinking water) on iNOS mRNA expression in the bursa of Fabricius of 21-day-old (e) and 35-day-old broilers (f). COX-2 mRNA level in the bursa of Fabricius of 21-day-old (g) and 35-day-old broilers (h). Values represent mean±SD (n=6). Means with different letter superscripts are significantly different (P< 0.05)
Fig. 3. Effects of CMHW supplementation on inflammation-associated gene expression in broilers during vaccination. Effect of different supplementation period (A, anterior only; AP, anterior and posterior; P, posterior only) of CMHW (2 g/L in drinking water) on iNOS mRNA level in the spleen of 21-day-old (a) and 35-day-old broilers (b). COX-2 mRNA level in the spleen of 21-day-old (c) and 35-day-old broilers (d). Effect of different supplementation period (A, anterior only; AP, anterior and posterior; P, posterior only) of CMHW (2 g/L in drinking water) on iNOS mRNA expression in the bursa of Fabricius of 21-day-old (e) and 35-day-old broilers (f). COX-2 mRNA level in the bursa of Fabricius of 21-day-old (g) and 35-day-old broilers (h). Values represent mean±SD (n=6). Means with different letter superscripts are significantly different (P< 0.05)
Fig. 4. Effects of CMHW supplementation on inflammation-related gene expression in LPS-challenged broilers. Effect of CMHW supplementation on iNOS mRNA level in the spleen of broilers 3 h (a) and 24 h (b) post-injection of LPS. COX-2 mRNA level in the spleen of broilers 3 h (c) and 24 h (d) post-injection of LPS. Effect of CMHW supplementation on iNOS mRNA expression in the bursa of Fabricius of broilers 3 h (e) and 24 h (f) post-injection of LPS. COX-2 mRNA level in the bursa of Fabricius of broilers 3 h (g) and 24 h (h) post-injection of LPS. Values are expressed as mean±SD (n=6). Means with different letter superscripts are significantly different (P< 0.05)
Fig. 4. Effects of CMHW supplementation on inflammation-related gene expression in LPS-challenged broilers. Effect of CMHW supplementation on iNOS mRNA level in the spleen of broilers 3 h (a) and 24 h (b) post-injection of LPS. COX-2 mRNA level in the spleen of broilers 3 h (c) and 24 h (d) post-injection of LPS. Effect of CMHW supplementation on iNOS mRNA expression in the bursa of Fabricius of broilers 3 h (e) and 24 h (f) post-injection of LPS. COX-2 mRNA level in the bursa of Fabricius of broilers 3 h (g) and 24 h (h) post-injection of LPS. Values are expressed as mean±SD (n=6). Means with different letter superscripts are significantly different (P< 0.05)
Fig. 5. Effects of CMHW supplementation on type 1 helper T-lymphocytes (Th1) /type 2 helper T-lymphocytes (Th2) cytokine expression in LPS-challenged broilers. Effect of CMHW supplementation on IFN-γ mRNA level in the spleen of broilers 3 h (a) and 24 h (b) post-injection of LPS, and IL-4 mRNA level in the spleen of broilers 3 h (c) and 24 h (d) post-injection of LPS. Effect of CMHW supplementation on IFN-γ mRNA level in the bursa of Fabricius of broilers 3 h (e) and 24 h (f) post-injection of LPS. IL-4 mRNA level in the bursa of Fabricius of broilers 3 h (g) and 24 h (h) post-injection of LPS. Values represent mean±SD (n=6). Means with different letter superscripts are significantly different (P< 0.05).
Fig. 5. Effects of CMHW supplementation on type 1 helper T-lymphocytes (Th1) /type 2 helper T-lymphocytes (Th2) cytokine expression in LPS-challenged broilers. Effect of CMHW supplementation on IFN-γ mRNA level in the spleen of broilers 3 h (a) and 24 h (b) post-injection of LPS, and IL-4 mRNA level in the spleen of broilers 3 h (c) and 24 h (d) post-injection of LPS. Effect of CMHW supplementation on IFN-γ mRNA level in the bursa of Fabricius of broilers 3 h (e) and 24 h (f) post-injection of LPS. IL-4 mRNA level in the bursa of Fabricius of broilers 3 h (g) and 24 h (h) post-injection of LPS. Values represent mean±SD (n=6). Means with different letter superscripts are significantly different (P< 0.05).
In conclusion, CMHW exerts an immunomodulatory role and inhibits inflammation-related gene expression in response to vaccination and LPS stimulation in broilers. Therefore, CMHW containing cordycepin has high potential for development as a feed additive that may act as an alternative source for immunomodulation in farm animals.

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