Influence of Phytogenic Supplement on Layer Production During the Peak of Heat Stress

Phytogenic products are used as an antibiotic alternative, with other alternatives like prebiotics, probiotics, organic acids and other products with antimicrobial properties. The ability of phytogens to reduce the load of significant pathogens such as Clostridium (2020), Escherichia (Zou et al., 2016), and Salmonella (Abudabos et al., 2016), and improve the health and performance of chickens makes them increasingly popular. Phytogens also improve critical layer production parameters such as the quality and quantity of eggs and feed conversion ratio (Abou-Elkhair et al., 2018). Other effects reported include phytogen interaction with levels of cholesterol (Abou-Elkhair et al., 2018), gut-brain axis (Bajagai et al., 2021), levels of sex hormones and immune systems readiness (Wang et al., 2021). Despite the reported benefits of phytogenic products, running open-range production systems is still a major challenge for the poultry industry. The phytogenic blend comprised of a mixture of essential oil from the Myrtaceae and Asteraceae plant families and saponins (Phy) was used in this experiment. The effects of this product have been examined to investigate the health, performance, egg quality and intestinal microbiota composition, and changes in microbiota functional capability via shotgun metagenomics and gene expression via RNAseq under a free-range system at the time of the year corresponding to the highest heat stress and disease outbreak peak. The effects on performance, microbiota and metagenomics functional alterations have been published in the Applied and Environmental Microbiology (Yu et al., 2022b) and the detailed RNAseq analyses on the effects of Phy on ileum gene expression in layers was published in Antibiotics (Yu et al., 2022a). Here we will present an overview of the most relevant findings from this study.

The study was performed on a commercial layer farm in Queensland, Australia using Lohman Brown breed and was approved by the Animal Ethics Committee of Central Queensland University under approval number 0000022879. When the pullets reached 16 weeks of age, they were moved to production sheds with ad libitum access to feed and water and with 20,000 birds each in the treatment and control group.

The birds were fed a proprietary commercial layer diet as an unsupplemented (Ctr) or with phytogen-supplemented feed (Phy). The two groups were grown in special research sheds with groups housed on separate sides of the shed, divided by the utility room through the middle of the shed and by the fence in the range. There was no physical contact between the Ctr and Phy birds indoors or in the range. The supplementation lasted from week 16 to week 40 of bird age. The aim was to evaluate the efficacy of Phy against Spotty Liver Disease (SLD) reoccurring during the hottest summer period. The supplementation started just before the summer’s height, and the birds’ sampling was at 30 weeks of age. The birds were sampled at the end of the peak of the lay and precisely at the peak of summer heat, with the maximum daily temperature of 38 ◦ C on the sampling day. Birds used in this study were randomly selected from different parts of the shed, rejecting the outlier birds.

At 30 weeks of age, 50 cloacal swabs were collected for microbiota analysis from both control and treatment sheds. Ten birds from each treatment were euthanised, and ileum tissue was collected for RNAseq analysis and histology. Ileum tissue for RNAseq was snap-frozen in liquid nitrogen and stored at −80 ◦ C, and for histology, it was stored in 10% neutral buffered formalin. The V3-V4 region of 16S rRNA gene was amplified using forward primer 338F, and the reverse primer 806R. All samples were sequenced in Azenta Live Sciences (China). The alignment of the sequences with the chicken genome and transcriptome analysis was done using CLC Genomic Workbench 21.0.3 (Qiagen, Germany). The differential genes were selected using the DESeq2 R package, and further pathway investigation was done using Ingenuity Pathway Analysis (QIAGEN IPA, version 76765844). Genes included in IPA analysis were DESeq2 P < 0.05, with an absolute fold change of 1.2.

The SLD outbreak was confirmed at week 37 of bird age when the mortality climbed and birds were diagnosed with the disease. Figure 1 shows some of the most affected performance parameters, and details on performance are available in Yu et al., (2022b). The phytogen consistently reduced mortality and increased the rate of lay and average egg weight even during SLD outbreak.

Cumulative hen-housed eggs were higher throughout the phytogen application resulting in approximately 8,333 additional cartons of dozen eggs or expressed as an egg mass improvement, approximately surplus of 5.8 tons over the supplementation period (Yu et al., 2022b). The phytogen showed protective effects during the SLD outbreak via considerably lower mortality and larger eggs than control birds (Yu et al., 2022b).

Our data indicate that microbiota change targeted the specific pathogenic and growth-related functions across microbial community membership, affecting complete community functional capabilities. Figure 2 shows the functional diversity of the microbial community (left) and RDA plot (right), representing the relationship between control and treatment functional profiles. Phy-supplemented birds show a more reproducible number of functions compared to variable functional diversity in Ctr.

Phytogen supplementation had a significant effect on the ileum gene expression. Phy significantly inhibited major cholesterol pathways and altered disease prediction analysis towards improved cardiovascular health, reduced disease susceptibility for 26 cancer categories and improved intestinal health (Yu et al., 2022a). Major metabolic shifts were identified in lipid and carbohydrate metabolism, resulting in a decrease in the Obesity category, higher bird weight, and altering the ratio of fat and carbohydrate metabolism toward lower fat storage (Figure 3). RNAseq functional analysis identified genes involved in increased goblet cell function, which was confirmed using ileum histology (Yu et al., 2022a).

Performance data established vital welfare and production-related improvements in a large-scale commercial layer system. Phytogen could not prevent the outbreak of SLD, but cumulative mortalities were constantly lower, and egg production was improved during the disease outbreak. Similarly, performance parameters, including the cumulative hen-housed eggs, and weight and number of individual eggs produced, were better in the phytogen-supplemented flocks, representing enhancements in general healthiness and performance. This is of high implication for the layer industry, where re-emerging of SLD can have serious consequences for bird welfare. Campylobacter hepaticus is frequently found in range soil, insects, rodents and wild birds.

Shotgun sequencing of cecal metagenomes shows that a range of essential vitamin, energy, and amino acid production functions was devastated in Phy-supplemented birds’ bacterial community. Reducing the diversity of functional abilities have promoted hosts’ productivity in heat and disease outbreak. This is well reflected in RNAseq data, especially in disease and function analysis, where the evidence pointed out better cardiovascular and intestinal health (significantly inhibited diseases categories) and, less relevant to poultry, reduced cancer predisposition due to supplementation. Histology confirmed an increase in the number of goblet cells identified via RNAseq disease and function algorithm, which is also highly relevant to overall intestinal health. Interesting rearrangements occurred in carbohydrate and lipid metabolism leading to slightly heavier yet less fat birds. This outcome is relevant for use in poultry and other livestock where the fat percentage has a role in meat quality scoring.

Our data displays a wide range of the benefits of the plant phytogen antimicrobials supplementation in poultry and suggests that identifying the precise functional alterations in the microbiota community (loss of pathogenic functions, toxin production or motility in bacteria, for example) via shotgun metagenomics functional analysis, in combination with disease and vital metabolic functions analysis via RNAseq, can be the first step in personalised nutritional supplementation. This can be custom tailored for each farm, or even more precise, specific shed, by choosing a particular phytogenic supplement capable of best addressing particular issues, including shed disease outbreak history or performance issues.