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

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. 

 

Animal Experiment I

Animal Experiment II

(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).

Animal Experiment III

(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)

 

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).

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).

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).

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.