Background: The selection of stably expressed reference genes is a prerequisite when evaluating gene expression, via real-time PCR, in cells in response to viral infections. The objective of our study was to identify suitable reference genes for mRNA expression analysis in chicken embryonic fibroblasts (CEF) after infection with avian leukosis virus subgroup J (ALV-J). Findings: The expression levels of 11 potential reference genes in CEF infected with ALV-J were determined by real-time PCR. The expression stability of these genes were analyzed and ranked using the geNorm tool. Analysis indicated that the genes RPL30 (ribosomal protein L30) and SDHA (succinate dehydrogenase complex, subunit A) were the most stably expressed genes in the ALV-J infected CEF. Conclusions: The RPL30 and SDHA were deemed suitable for use as reference genes for real-time PCR analysis of mRNA gene expression during ALV-J infection, whereas commonly used ACTB and GAPDH are unsuitable to be reference genes.

Earthworm has a variety of molecular biological characteristic, for example, growth promo-tion, antioxidant, and anti-bacteria. Thus, we decom-posed earthworm by earthworm's own protease for preparing of earthworm hydrolysate. Muscovy ducks were fed with basal diet that formulated to contain 1.5% and 2.5% earthworm hydrolysate. Then, we investigated the influences of earthworm hydrolysate on growth per-formance in Muscovy ducks by performance terminology and measurement for poultry (NY/T 823-2020). The morphology of duodenum and number of intraepithelial lymphocytes were tested by HE staining and immuno-histochemical method. Serum biochemical parameters and antioxidant capacity were also determined. High -throughput sequencing technology can sequence 16S rDNA of cecal contents from experimental Muscovy ducks. Results showed that 1.5% earthworm hydrolysate increased ADG (16-70 days old), ALB, HDL-C, T - AOC, CAT, SOD, GSH-PX, villi length, intestine thick- ness and surface area of villi (P < 0.05 or P < 0.01), and reduced FCR (16-70 days old), UREA, CRE, LDL-C, MDA (P < 0.05 or P < 0.01). Meanwhile, 2.5% improved ADG (16-70 days old), abdominal fat yield, breast mus- cle yield, heart index, spleen index, ALP, UA, T-AOC, CAT, SOD, GSH-PX, villi length, crypt depth, intestine thickness, surface area of villi, the percentage of intraepi- thelial lymphocytes (P < 0.05 or P < 0.01), and decreased FCR (42-70 days old and 16-70 days old), UREA, UA, MDA (P < 0.05 or P < 0.01). The sequenc- ing results of gut flora demonstrated that earthworm hydrolysate improved variety of the gut flora in the V4 area of ducks immensely. In a word, our results provide the foundation for preliminary researching the potential principles of earthworm hydrolysate in promoting pro- duction performance, adjusting antioxidant function and intestinal functions in the Muscovy duck industry.

The study aimed to examine the effects of unfermented and fermented Andrographis paniculata on growth performance, carcass traits, immune function, and intestinal health in Muscovy ducks. A total of 450 (16-day-old) Muscovy ducks weighing 271.44 8.25 g were randomly assigned to 5 dietary treatments (6 repli-cate pens of 15 ducks per treatment), consisting of one control treatment (basal diet without A. paniculata), one unfermented A. paniculata treatment (basal diet plus 30 g/kg unfermented A. paniculata) and 3 fer-mented A. paniculata treatments (basal diet plus 10, 30, and 50 g/kg). 30 g/kg unfermented A. paniculata increased the ADG, thymus index, peripheral blood lym-phocyte conversion rate, villi height, intestinal thick-ness, villi surface area, intraepithelial lymphocytes rate, while decreased the FCR. 10 g/kg fermented A. panicu-lata markedly boosted ADG, bursa of fabricius index, thymus index, serum lysozyme, lymphocyte conversion rate, villi height, vilii width, intestinal thickness, villi surface area, while decreased the FCR. 30 g/kg fer-mented A. paniculata clearly improved ADG, bursa of fabricius index, thymus index, serum lysozyme, lympho-cyte conversion rate, villi height, vilii width, intestinal thickness, villi surface area, intraepithelial lymphocytes, while decreased FCR. 50 g/kg fermented A. paniculata significantly increased villi height, vilii width, and villi surface area, while clearly reduced BW. Additionally, compared to 30 g/kg unfermented A. paniculata, 30 g/kg fermented A. paniculata obviously increased bursa of fabricius indices, lymphocyte conversion rate, vilii width, villi surface area. On top of that, supplemen-tation with unfermented and fermented A. paniculata (30 g/kg each) decreased the relative abundance of harmful bacteria (Succinivibrio, Succinatimonas, Sphaerochaeta, and Mucispirillum) and increase the abundance of beneficial bacteria (Rikenellaceae, Metha-nocorpusculum, Fournierella, Ruminococcaceae) in the ceca of the ducks. However, fermented A. paniculata had considerable better effects than unfermented A. paniculate on all above measured indices. Overall, these results revealed that supplementation with unfermented and fermented A. paniculata across different treatments improved growth, immune status, intestinal morphol-ogy, and intestinal microbiota composition and struc-ture in Muscovy ducks, making it a potential alternative to antibiotics in poultry production.

Rare-earth mines worldwide urgently need sustainable, low-cost methods to restore soil quality and productivity. The application of remediation plants, such as Morus alba, combined with organic fertilisers (vermicompost), is promising for enhancing soil quality around rare-earth tailings and improving crop yields; however, the effects of phytoremediation combined with organic fertiliser are not fully understood. We conducted a 4-year study to assess the impact of four fertilisation regimes combined with M. alba on soil properties, enzyme activities, microbial diversity, and crop yield in rare-earth tailings. Organic amendments markedly improved soil quality. The treatment combining vermicompost and biogas slurry increased soil pH from 4.37 in the chemical fertiliser control to 6.67, soil organic matter from 4.87 to 30.58 g·kg^-1, and available phosphorus from 8.31 to 264.52 mg·kg^-1, while significantly enhancing soil enzyme activity. In soil bacterial communities, relative abundances of environmentally adapted taxa (e.g., Acidobacteria and Verrucomicrobia) increased, accompanied by a tendency toward greater stochastic assembly. The combination of earthworm casts and biogas slurry increased crop yield from 6.72 t·ha^-1 in the control to 42.18 t·ha^-1 in 2023. Functional prediction suggested enhanced microbial potential for carbon and nitrogen cycling. Random forest and structural equation modelling revealed that the dominant driver of crop yield shifted from available nitrogen in the early stage to organic matter in the later stage. Collectively, these results suggest that organic fertilisation combined with phytoremediation is a viable strategy for mine tailings, supporting global efforts toward sustainable ecological restoration and improved soil health.