The chitooligosaccharide ( COS ) and chlorella polysaccharide ( CPS ) have been used as feed supplements in the poultry industry for improving growth performance and immunity. However, the benefits of these prebiotics on the gut health of chickens when used in early nutrition are unknown. This study evaluated the effects of in ovo feeding of COS and CPS on the cecal microbiome, metabolic pathways, and fermentation metabolites of chickens. A total of 240 fertile eggs were divided into 6 groups (n = 4; 10 eggs/replicate): 1) no-injection control, 2) normal saline control, 3) COS 5 mg, 4) COS 20 mg, 5) CPS 5 mg, and 6) CPS 20 mg injection. On day 12.5 of egg incubation, test substrate was injected into the amniotic sac of eggs in respective treatments. The hatched chicks were raised for 21 D under standard husbandry practices. On day 3 and 21, cecal digesta were collected to determine microbiota by shotgun metagenomic sequencing and short-chain fatty acids by gas chromatography. The cecal microbial composition was not different ( P > 0.05) among the treatment groups on day 3 but was different ( P < 0.05) on day 21. At the species level, the polysaccharide-utilizing bacteria including Lactobacillus johnsonii, Bacteroides coprocola , and Bacteroides salanitronis were higher in the COS group, whereas the relative abundance of some opportunistic pathogenic bacteria were lower than those in the CPS and control groups. At the functional level, the pathways of gluconeogenesis, L-isoleucine degradation, L-histidine biosynthesis, and fatty acid biosynthesis were enriched in the COS group. In addition, propionic acid content was higher ( P < 0.05) in the COS group. A network based on the correlation between the COS and other factors was constructed to illuminate the potential action mechanism of the COS in chicken early nutrition. In conclusion, in ovo inoculation of COS 5 mg showed positive effects on the cecal microbiota, metabolic pathways, and propionic acid, thus can be used as in ovo feeding to modulate the gut health of chickens.

The stable gut microbiome plays a key role in sustaining host health, while the instability of gut microbiome also has been found to be a risk factor of various metabolic diseases. At the ecological and evolutionary scales, the inevitable competition between the ingested probiotic and indigenous gut microbiome can lead to an increase in the instability. It remains largely unclear if and how exogenous prebiotic can improve the overall gut microbiome stability in probiotic consumption. In this study, we used Lactobacillus plantarum HNU082 (Lp082) as a model probiotic to examine the impact of the continuous or pulsed supplementation of galactooligosaccharide (GOS) on the gut microbiome stability in mice using shotgun metagenomic sequencing. Only continuous GOS supplement promoted the growth of probiotic and decreased its single-nucleotide polymorphisms (SNPs) mutation under competitive conditions. Besides, persistent GOS supplementation increased the overall stability, reshaped the probiotic competitive interactions with Bacteroides species in the indigenous microbiome, which was also evident by over-abundance of carbohydrate-active enzymes (CAZymes) accordingly. Also, we identified a total of 793 SNPs arisen in probiotic administration in the indigenous microbiome. Over 90% of them derived from Bacteroides species, which involved genes encoding transposase, CAZymes, and membrane proteins. However, neither GOS supplementation here de-escalated the overall adaptive mutations within the indigenous microbes during probiotic intake. Collectively, our study demonstrated the beneficial effect of continuous prebiotic supplementation on the ecological and genetic stability of gut microbiomes.

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Myostatin (MSTN) is a negative regulator of skeletal muscle growth, thus it was hypothesized that immunization of hens against MSTN would enhance post-hatch growth and muscle mass via suppression of MSTN activity by anti-MSTN IgY in fertilized eggs. This study investigated the effects of immunization of hens against chicken MSTN (chMSTN) or a MSTN fragment (Myo2) on the growth and muscle mass of offspring. In Experiment 1, hens mixed with roosters were divided into two groups and hens in the Control and chMSTN groups were immunized with 0 and 0.5 mg of chMSTN, respectively. In Experiment 2, hens in the chMSTN group were divided into chMSTN and Myo2 groups while the Control group remained the same. The Control and chMSTN groups were immunized in the same way as Experiment 1. The Myo2 group was immunized against MSTN peptide fragment (Myo2) conjugated to KLH. Eggs collected from each group were incubated, and chicks were reared to examine growth and carcass parameters. ELISA showed the production of IgYs against chMSTN and Myo2 and the presence of these antibodies in egg yolk. IgY from the chMSTN and Myo2 groups showed binding affinity to chMSTN, Myo2, and commercial MSTN in Western blot analysis but did not show MSTN-inhibitory capacity in a reporter gene assay. In Experiment 1, no difference was observed in the body weight and carcass parameters of offspring between the Control and chMSTN groups. In Experiment 2, the body weight of chicks from the Myo2 group was significantly lower than that of the Control or chMSTN groups. The dressing percentage and breast muscle mass of the chMSTN and Myo2 groups were significantly lower than those of the Control group, and the breast muscle mass of Myo2 was significantly lower than that of the chMSTN. In summary, in contrast to our hypothesis, maternal immunization of hens did not increase but decreased the body weight and muscle mass of offspring.