Wei Guo,Jinghui Zhou,Yanyan Liu,Junxia Bai,Yufei Zhu,Xin Yang,Xiaojun Yang
Embryonic injection of Lactobacillus plantarum PA01 alters the microbial diversity in the gastrointestinal tract of the broilers before and after hatching
broiler,embryonic stage,Lactobacillus,in ovo injection,gastrointestinal microbes
The total number of intestinal microbiotas is low, and the intestinal tract develops rapidly and imperfectly at the embryonic stage. Embryonic period as a particular physiological stage is an important time window to explore how to regulate organismal health by probiotics. Therefore, this experiment was conducted to investigate the effect of embryonic injection of Lactobacillus plantarum PA01 at embryonic d 14 (E14) on the microbiome of the contents of the gizzard, cecum at embryonic d 20 (E20) and cecum at d 1 posthatch (D1) by 16S rRNA sequencing. Results showed that PA01 had no significant effect on broiler body weight and yolk sac weight at E20 and D1 (P > 0.05). PA-01 altered the Shannon index and β diversity of the gizzard at E20 (P < 0.05), increased the abundance of Firmicutes (P < 0.05), and decreased the relative abundance of Proteobacteria, Bacteroidota, and Actinobacteriota (P < 0.05). At the genus level of the microbiota, PA01 significantly increased the relative abundance of Lactiplantibacillus (P < 0.05). At 20 embryos, PA01 altered the α and β diversity indices (P < 0.05) and decreased the relative abundance of Salmonella (P < 0.05) of the cecal microbiota. The biomarkers of PA01 group were Lactobacillales, Blautia, Lachnospiraceae, and Asinibacterium. Embryonic injection of PA01 altered the E20 intestinal microbes. PA01 altered the β-diversity index of the 1-day-old cecum (P < 0.05), and there was no significant effect on microbial composition at the phylum and genus level (P > 0.05). LefSe analysis revealed that the biomarkers of the PA01 group were Lactobacillaceae, Lactiplantibacillus, Moraxellaceae, and Acinetobacter. Biomarkers in the Con group were Devosia, Bacillus, Nordella, Mesorhizobium, and Pseudolabrys. PA01 increased acetic acid in the gastrointestinal tract at E20 along with acetic and butyric acid in cecum of 1-day-old. In conclusion, embryo-injected L. plantarum PA01 altered the structure and metabolites of the microbial flora before and after hatching, in particular promoting the colonization of Lactobacillus.
Embryonic injection of Lactobacillus plantarum PA01 alters the microbial diversity in the gastrointestinal tract of the broilers before and after hatching

The total number of intestinal microbiotas is low, and the intestinal tract develops rapidly and imperfectly at the embryonic stage. Embryonic period as a particular physiological stage is an important time window to explore how to regulate organismal health by probiotics. Therefore, this experiment was conducted to investigate the effect of embryonic injection of Lactobacillus plantarum PA01 at embryonic d 14 (E14) on the microbiome of the contents of the gizzard, cecum at embryonic d 20 (E20) and cecum at d 1 posthatch (D1) by 16S rRNA sequencing. Results showed that PA01 had no significant effect on broiler body weight and yolk sac weight at E20 and D1 (P > 0.05). PA-01 altered the Shannon index and β diversity of the gizzard at E20 (P < 0.05), increased the abundance of Firmicutes (P < 0.05), and decreased the relative abundance of Proteobacteria, Bacteroidota, and Actinobacteriota (P < 0.05). At the genus level of the microbiota, PA01 significantly increased the relative abundance of Lactiplantibacillus (P < 0.05). At 20 embryos, PA01 altered the α and β diversity indices (P < 0.05) and decreased the relative abundance of Salmonella (P < 0.05) of the cecal microbiota. The biomarkers of PA01 group were Lactobacillales, Blautia, Lachnospiraceae, and Asinibacterium. Embryonic injection of PA01 altered the E20 intestinal microbes. PA01 altered the β-diversity index of the 1-day-old cecum (P < 0.05), and there was no significant effect on microbial composition at the phylum and genus level (P > 0.05). LefSe analysis revealed that the biomarkers of the PA01 group were Lactobacillaceae, Lactiplantibacillus, Moraxellaceae, and Acinetobacter. Biomarkers in the Con group were Devosia, Bacillus, Nordella, Mesorhizobium, and Pseudolabrys. PA01 increased acetic acid in the gastrointestinal tract at E20 along with acetic and butyric acid in cecum of 1-day-old. In conclusion, embryo-injected L. plantarum PA01 altered the structure and metabolites of the microbial flora before and after hatching, in particular promoting the colonization of Lactobacillus.

Researches on microbiota in broiler chickens have been very broad, but those have mainly focused on the posthatching period. There are few reports on whether microbes are present in broiler embryo eggs during incubation, or whether gastrointestinal microbes play a key role in broiler embryo incubation (Ding et al., 2022). The establishment of the early gut microbiota in neonates directly affects the intestinal mucosal ecosystem (Roto et al., 2016). The gut microbiota during the early stage is unstable in composition and very sensitive to invasion by external pathogenic microorganisms (Al Nabhani and Eberl, 2020; Renz and Skevaki, 2021), which is the reason why young animals are sensitive to disease problems (Prescott et al., 2016; Roubaud-Baudron et al., 2019). Neonatal microbial colonization is important for establishment and has a long-term impact on the host immune system (Bergström et al., 2014; Al Nabhani and Eberl, 2020). Studies of the colonization of the gut microbiota during the embryonic period of broilers are still at an early stage, and its influencing factors and modulation have not been systematically investigated. The exploration in this area will play a key role in the health and growth of young animals. Kogut (2019) revealed that there are 4 window periods for regulating broiler gut microbes by studying the changes in gut microbes throughout the broiler period, which are the early shelling incipient stage; the middle broiler feed switch stage (usually around 14 or 28 d of age); the later stage is the feed break before broiler slaughter; and another window period is the period when broiler disease or gut dysbiosis problems occur. Broiler gut microbes are most volatile from hatching to about 1 wk after shelling, and the composition and diversity of microorganisms are extremely unstable, which is the best time for manual intervention (Jurburg et al., 2019). Early regulatory intervention in the embryonic stage of chickens has received widespread attention. With the continuous progress of broiler breeding technology and feeding management, the cycle time of chicken production has been shortened, and the hatching period of chicken embryo accounts for about 1/3 of the whole life cycle of commercial broiler chicken. So, here is great research value in the early microbiota regulatory intervention at the embryonic stage. It was found that providing chick embryos with probiotic substances such as Lactobacillus (Wilson et al., 2019), galactooligosaccharides (Slawinska et al., 2019) or combined probiotic and oligosaccharide preparations (Dunislawska et al., 2017) by in ovo feeding can significantly increase the relative abundance of probiotic bacteria in the intestine of broiler chickens. Therefore, the effect of in ovo feeding of Lactobacillus plantarum PA01 on the microbial diversity in the gizzard, cecum at embryonic d 20 and cecum at d 1 posthatch of broilers.

All the birds and experimental protocols in this study were approved by the Institution Animal Care and Use Committee of the Northwest A&F University.

The breeder eggs (Arbor Acres, 38 wk old) were purchased from the Xi'an Dacheng Poultry Industry Co. Ltd. (Shaanxi, China). After disinfection, all eggs were placed in an automatic incubator (Beijing LanTianJiao Electronic Technology Co., Ltd, Beijing, China). After egg candling at 14 embryonic age (E14), the unfertilized breeding eggs were carried out. Then, 350 fourteen embryonic age AA broiler eggs of near normal size were selected and randomly divided into 2 groups with 7 replicates of 25 eggs each. The treatment groups were the control group (no treatment) and the L. plantarum injection group (100 µL of L. plantarum PA01 physiological saline resuspension, 9 × 107 CFU/egg) (Guo et al., 2023). L. plantarum was isolated from pickles by our team and had good probiotic characteristics (Feng et al., 2016). Four embryos or 1 chick in each repeat group were randomly selected for slaughter and sample collection at embryonic d 20 (E20) and d 1 posthatch (D1). Weighing and recording analysis of body weight and yolk sac weight at 20-embryo (n = 28) and 1 d (n = 7) of age. Twenty-embryonic ages embryonic gizzard (n = 7), cecum (n = 7), and 1-day-old chicks cecum (n = 7), contents were collected for 16S rRNA sequencing. Use the gas chromatography to measure short chain fatty acids (SCFAs) in the contents of the gizzard and cecum.

16S rRNA sequencing was used to determine the diversity and structural comparisons of the bacterial species in gizzard and cecal samples using the Illumina MiSeq platform (Shanghai BIOZERON Co., Ltd.) according to the standard protocols. The V4–V5 regions of the bacteria 16S ribosomal RNA gene were amplified by PCR (95°C for 2 min, followed by 25 cycles at 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s and a final extension at 72°C for 5 min) using primers 515F 5′-barcode-GTGCCAGCMGCCGCGG)-3′ and 907R 5′-CCGTCAATTCMTTTRAGTTT-3′, where the barcode is an 8-base sequence unique to each sample. The PE reads obtained by Illumina PE250 sequencing are first spliced according to the overlap relationship, and the sequence quality is controlled and filtered at the same time. Operational Taxonomic Unit (OTU) clustering analysis and species taxonomic analysis were performed after distinguishing samples. Based on the results of OTU clustering, a variety of diversity index analyses and sequencing depth detection can be performed on OTU. Based on taxonomic information, statistical analysis of community structure can be performed at various taxonomic levels. Based on the above analysis, a series of in-depth statistical and visual analyses such as community structure and phylogeny can be carried out. The rarefaction analysis based on Mothur v.1.21.1 (Schloss et al., 2009) was conducted to reveal the Shannon diversity indices. The beta diversity analysis was performed using UniFrac (Lozupone et al., 2011) to compare the results of the principal component analysis using the community ecology package, R-forge (Vegan 2.0 package was used to generate a principal component analysis figure). Taxa abundance was statistically compared between samples or groups at the phylum, order, family, and genus levels by Metastats. For identification of biomarkers for highly dimensional colonic bacteria, linear discriminant analysis effect size (LEfSe) analysis was done (Segata et al., 2011). Kruskal–Wallis sum-rank test was performed to examine the changes and dissimilarities among classes followed by Linear Discriminant Analysis to determine the size effect of each distinctively abundant taxa (Ijaz et al., 2018).

All statistical analyses of 16S rRNA were performed by R stats package and cloud platform (http://www.cloud.biomicroclass.com/CloudPlatform). The SPSS 21.0 software was used for all statistical analyses in this study. The t test of the data on the Organ index and the contents of SCFAs were used. All the analysis results were considered to be statistically significant at P < 0.05.

At E20 and D1, embryo injection of L. plantarum PA01 at embryonic d 14 had no effect on broiler body weight and yolk sac weight at E20 and D1 (Figure 1; P > 0.05). L. plantarum PA01 has no influence on the development of the embryo and the uptake of the yolk sac.

Microorganisms of the chicken embryo gizzard were examined in control (ES-C) and L. plantarum PA01 injection (ES-T) groups at E20. L. plantarum PA01 significantly reduced the microbial α diversity index (Shannon index) (P = 0.0058) and altered the β-diversity (P < 0.05; Figure 2). Analysis of the structural components of the microbiota showed that ES-T significantly changed the structure of the flora, changing the Proteobacteria to Firmicutes (P < 0.05), and also reducing the relative abundance of Bacteroidota and Actinobacteriota (Figure 3A; P < 0.05). The change in microbial composition at the genus level was also significant, with nearly 70% of Lactiplantibacillus in the microbiome of gizzard after injection of L. plantarum PA01 (P < 0.05). The TOP5 genus of the ES-C group were Ralstonia, Enterococcus, Phyllobacterium, Mesorhizobium, and others. ES-T group were Lactiplantibacillus, Enterococcus, Phyllobacterium, Mesorhizobium, and Vibrionimonas (Figure 3B). The LefSe analysis of the ES-T and ES-C groups showed that the marker microorganisms in the ES-T group were Firmicutes at the phylum level and Lactiplantibacillus and Enterococcus at the genus level, whereas the ES-C group had 40 genera of Proteobacteria, Actinobacteriota, and Firmicutes were found as differential markers (Figure 4). Microbiota composition and LefSe analysis data show that embryonic injection of L. plantarum PA01 allowed the Lactobacillus to reach the stomach and cause a dramatic change in the microbiota.

The microbial results of the cecal contents of the control (EC-C) and Lactobacillus injection (EC-T) groups showed that L. plantarum PA01 significantly altered the α (P = 0.021; Figure 5A) and β diversity (PLS-DA) of the microbiota (Figure 5B; P < 0.05). No significant effects on the relative abundance of Proteobacteria, Actinobacteriota, Firmicutes, and Bacteroides were found in the cecum of broiler chickens at 20 embryonic days (Figure 5C; P > 0.05). At the genus level, L. plantarum PA01 significantly reduced the relative abundance of Salmonella (P < 0.05). LefSe analysis (Figure 6) revealed that the marker microorganisms in the EC-C group were Salmonella. The marker microorganisms in EC-T group were Lactobacillales, Blautia, Lachnospiraceae, and Asinibacterium. Embryo injection of L. plantarum PA01 was able to modify the microflora of the cecum of broiler at 20 embryonic ages.

The microflora of the cecum of 1-day-old broiler chickens was analyzed to determine whether embryonic injection of L. plantarum PA01 could break the physiological behavior of hatching and continue through the subsequent growth period. A total of 314 bacteria OTUs were identified in the injection group (DC-T) and 120 in the control group (DC-C), respectively, giving a total of 177 in both groups (Figure 7A). DC-T altered the α and β diversity (PLS-DA analysis) index of cecum microbiotas of 1-day-old broiler (Figure 7B and 7C; P < 0.05). No significant differences were seen in the relative abundance of microorganisms at the phylum and genus levels (P > 0.05; Figure S1). LefSe analysis revealed that the typical microorganisms of the DC-T group were Lactobacillaceae, Lactiplantibacillus, Moraxellaceae, and Acinetobacter. The DC-C group were Devosia, Bacillus, Nordella, Mesorhizobium, and Pseudolabrys (Figure 7D). The microorganisms in the cecum of 1-day-old broilers, especially the Lactobacillus-like genera, could be modified by the injection of L. plantarum PA01 at 14 embryonic ages.

Short-chain fatty acids such as acetic acid, propionic acid, butyric acid, valeric acid, and isovaleric acid were measured in the contents of the gastrointestinal tract of broiler chickens (Figure 8). At 20 d of embryonic age, only acetic acid was found in the gastrointestinal tract of chicken embryos. L. plantarum PA01 significantly increased the levels of acetic acid in the gizzard and cecum of broilers (Figure 8A; P < 0.05). L. plantarum PA01 increased the levels of acetic acid and butyric acid in the cecum of 1-day-old broilers (P < 0.05), but had no significant effect on the levels of propionic acid (Figure 8B; P > 0.05).

This experiment was designed to study the effect of L. plantarum PA01 on the microbiota of the digestive tract at the embryonic and hatching stages. Embryonic and hatching stages are the early stage of microbiota establishment which is a specific physiological stage with a relatively low absolute number of microbes. In this experiment, the microbiota in the gizzard and cecum of 20-embroyo-day broilers and cecum of 1-day-old broilers were examined by 16S rRNA sequencing. Kizerwetter-Świda and Binek (2008) examined the bacterial counts of E18, E20, and 1 d broiler cecum contents, liver and yolk sac by the aid of the plate count method and found that with the embryo age increased, the intestinal flora became more differentiated and the number of specific flora was higher, with levels of Enterobacter, Staphylococcus, and Micrococcus detected at 102 CFU/g in the embryonic stage. So, there exits low levels of total bacteria in the embryonic stage compared to the growth stage. Different methods were applied to study the colonization of the gastrointestinal tract of chicks with probiotics before hatching, both probiotic feeding to hens and prehatch eggshell spraying can detect probiotic colonization in the gastrointestinal tract of chicks after incubation (Thofner et al., 2021). Oviparous embryos develop in closed eggs, and the embryonic microbiota is associated with the maternal reproductive tract, feathers, nest environment (Barrow, 1994). Also, salivary and physical contact from parental feeding behavior after hatching can also promote the spread of gut microbiota and subsequent colonization of the chick's gut (Li et al., 2022a,b). Almost embryonic development of all bird is influenced by external environmental and incubation conditions (Reed and Clark, 2011). Similarly, the incubation environment can influence microbial colonization and evolution in the digestive tract of broiler embryos. Ding et al. (2017) tested the gut microbiomes of broiler embryos, chicks and breeding hens and found that the core flora of embryos and breeding hens were moderately correlated and some of the microorganisms in the early embryos may have been inherited from the hens. Therefore, it is feasible to change the microflora structure by embryonic injection of L. plantarum PA01. Southwell (2006) studied and illustrated the status of gut development in chick embryos from 2 to 10 embryonic ages in detail, where the contour of the intestine appeared at 4 embryonic ages, the cecum began to sprout and extend at 5 embryonic ages, the duodenal loop formed at 7 embryonic ages, and by 10 d of age, the basic proportions of the small intestine, large intestine and cecum are formed. Changes in mucosal morphology and enterocyte dynamics were studied in broiler chickens from 1- to 12-embryonic days of incubation. Results shows that all epithelial cells proliferated in the duodenum, jejunum and ileum in different patterns, the cell density of the crypt and villi reached a plateau at 100h of incubation, and the total absorptive surface in the different intestines increased continuously with time (Geyra et al., 2001). By microscopic observation and analysis of intestinal samples of turkeys from 15 embryonic age to 12 d posthatching, found that there was a substantial increase in the height of the villi (nearly 5-fold changes), the apical part of the epithelium was gradually tightened by the attachment complex and mature goblet cells could be observed (Bohorquez et al., 2011). The maturation of the intestinal epithelium was influenced by the swallowing of amniotic fluid at embryonic stage, the embryo injection of nutrients and the supplementation of intestinal regulators after hatching. The chicken embryo develops to d 12 and begins to swallow proteins when the intestine becomes functional (Hamburger and Hamilton, 1951). The ratio of intestine to body weight in chickens increases rapidly from 1% at 17 embryonic ages to 3.5% at hatching (Uni et al., 2003). The rapid growth of the intestine coincided with the swallowing of amniotic fluid, which promoted the continuous proliferation of the small intestine (Trahair and Sangild, 2002; De Oliveira, 2007). So, we chose to inject L. plantarum PA01 into the egg protein at the tip of the egg at 14-embryonic days to make L. plantarum PA01 better colonize in the gastrointestinal tract and regulate the microbiota through the embryo's swallowing of amniotic fluid. Fluorescently labeled Escherichia coli were injected into the air chambers and yolk sac of broiler chickens at 18 embryonic ages and found that the E. coli were detected in the jejunum, ileum, and cecum of broiler embryos at 21 embryonic ages (Castaneda et al., 2019). This is a visual method to show that external environmental bacteria can migrate to and colonize the embryonic digestive tract (Siwek et al., 2018). PCR techniques can identify the embryo-injected Lactobacillus strains in the digestive tract contents of 3-day-old broilers (Aleksandrzak-Piekarczyk et al., 2019). Injections of Lactobacillus reuteri (Edens et al., 1997), Enterococcus (Skjoet-Rasmussen et al., 2017), and Lactobacillus salivarius (Andreatti et al., 2006) increased their intestinal colonization through competitive exclusion effects and reduced Salmonella and E. coli colonization in the gut of embryos and chicks. Similar findings were obtained in embryonic injection or egg soak with Lactobacillus acidophilus, Lactobacillus fermentum, and L. salivarius combinations (Yamawaki et al., 2013). Probiotic injections in broiler embryos can alter gut microbes during the embryonic period and throughout the rearing cycle, injections of a combination of inulin and Enterococcus in the amniotic sac can increase the number of probiotic bacteria in the cecum contents of broilers (Calik et al., 2017). Fluorescence in situ hybridization analysis of microbial communities in ileal and cecum contents of broilers at 21 d of age injected with combinations of prebiotics and probiotics in the air chamber at 12 embryonic age revealed that galactooligosaccharides and raffinose family oligosaccharides reduced the number of Lactobacillus spp. and Enterococcus spp. in ileal contents; L. salivarius reduced the total bacterial count, such as Bacteroides–Prevotella cluster, Clostridium leptum subgroup, Eubacterium rectale cluster, whereas L. plantarum had opposite results. In addition, the L. salivarius group had higher abundance of Lactobacillus and Enterococcus in cecum (Dunislawska et al., 2017). Amniotic fluid injection of Bacillus subtilis, Enterococcus, and Lactobacillus lactis at 18 embryonic ages reduced the intestinal E. coli and increased the Lactobacillus in 7-day-old broilers (Majidi-Mosleh et al., 2017). Injections of Lactobacillus, Citrobacter freundii, and Citrobacter species at 18 embryonic ages altered the microbial composition of the intestinal of broilers at 1, 3, and 10 d of age, especially the abundance of Lactobacillus, Enterococcaceae, and Enterococcus, but the effect varied by ages (Wilson et al., 2019). The Lactobacillus injection at 14 embryonic ages significantly increased the content of acetic, propionic, butyric, and total acid in the cecum contents of 14-day-old broilers (Guo et al., 2023). Probiotic mixture of L. plantarum strains significant increase in acetic acid and butyric acid (Li et al., 2020). Oligosaccharides and Pediococcus acidilactici BBC1 induced high abundance of butyric acid bacteria of broiler chickens (Wu et al., 2021). Embryonic injections can alter late embryonic broiler microbiota, exposing broiler embryos to beneficial bacteria can influence intestinal microbiota and improve broiler intestinal health throughout the life cycle. It is possible to regulate the gut microbiota during the embryonic stage of broiler chickens, and this modification can be transmitted to posthatching. We examined microbes in the gizzard and cecum at 20 embryonic age and in the cecum of broiler chickens at 1 d of postincubation. L. plantarum significantly reduced the Shannon index of microbial α diversity and altered β diversity in the gizzard at 20 embryonic ages, achieving a reversal from Proteobacteria to Firmicutes, indicating that our embryonic injection model was very successful. The results of the cecum microbiota at 20 embryonic age showed that L. plantarum PA01 significantly altered the α and β diversity indices, reduced the relative abundance of the Salmonella and increased Lactobacillales and so on. At 1-day-old, the PA01 altered the β diversity index and enriched Lactobacillus and Lactiplantibacillus. Simply, embryonic injection of L. plantarum PA01 altered the microflora structure of the embryonic gastrointestinal tract, increasing the colonization abundance of Lactobacillus and affecting the content of SCFAs, and these changes could persist until after hatching.