Introduction

Offspring growth, development, and health are influenced by their mothers1,2,3,4. Maternal effects, a subject of extensive long-term research, can impact offspring in the long term and are prevalent in nature5. During biological evolution, maternal effects may have been critical to the adaptation of many organisms to environmental change6,7,8. It has been reported that mothers can influence their offspring’s phenotype through differential resource allocation in response to environmental challenges9,10. In humans, research has demonstrated that leveraging maternal effects can improve the immunity of newborns during early life stages11. In recent years, the potential influence of maternal effects on animal production has gained more recognition12. Our recent research showed that enhancing hen immunity through nutritional interventions can improve gut development and influence the gut microbiota establishment in their offspring embryos13. However, research has indicated that the influence of maternal effects on offspring diminishes as they age14. Therefore, to achieve a more comprehensive understanding of the long-term maternal effects on offspring and to effectively harness maternal effects for improving the health of both human and animal offspring, further research is necessary.

In contrast to mammals, which transfer immune factors from mother to offspring via the placenta and breast milk, hens enrich the egg with all the substances that prepare it for embryonic development, including various immune factors preventing the embryo from pathogens15. Increasing evidence suggests that the immune-mediated maternal effect, such as IgA, IgG, IgM in colostrum as well as IgM, IgG (IgY), and lysozyme (LYZ), and avian beta-defensins (AvBDs) in eggs, is crucial for the health of offspring16,17. However, it has been found that maternal IgG (IgY) induced by myostatin fragment negatively affects the growth and development of chicken offspring18. Thus, it is essential to clarify further which maternal immune factors benefit offspring health, growth, and development, enabling a more practical application of maternal immunity in production management. Our previous findings indicate that enhancing hen immunity with a complex of additives including β-carotene promotes offspring intestinal development by increasing the content of maternal IgA, IgM, IgG, LYZ, and AvBDs in eggs, as well as through microbe-mediated maternal effects13. In addition, egg weight also affects the hatchability as well as body weight and jejunal villous development of birds19,20. Growing evidence indicates that the gut microbiota influences chick growth and development21,22. However, the microbiome establishment and offspring fitness were influenced by immune-mediated maternal effects, such as IgA, and microbe-mediated maternal effects23,24. A substantial microbial population resides in the intestines and oviducts of hens, and research has reported that these microbiomes can influence the establishment of the microbial community in their offspring’s gut25. Most studies on microbe-mediated maternal effects have primarily focused on the individual impacts of the maternal oviduct and gut microbiome on their offspring. However, no research has compared the impact of maternal gut and oviduct microbiome on offspring or examined the influences of microbe-mediated maternal effects on the progression of intestinal microbiota in offspring from the embryonic to post-hatch stages.

Maternal effects are influenced by various factors, with diet being one of the critical determinants26,27. It has been demonstrated that maternal nutritional status influences the vertical transmission of immune factors from mammals to their offspring as well as the initial establishment of the offspring’s gut microbiota28. In poultry, the dietary composition of hens can also influence the immunity system and gut microbiota of their offspring, with these effects potentially being unrelated to the nutritional enhancement of the egg29,30,31. As a pro-vitamin A, β-carotene is widely used in both human diets and animal feeds. Previous study suggested that dietary supplementation with 50 mg/kg β-carotene for 21 days can elevate IgA levels, increase the number of IgA + cells, and upregulate jejunal and ileal gene expression associated with IgA production in weaned mice32. Nishiyama et al. demonstrated that adding 50 mg/kg β-carotene to the diets of mice during pregnancy and lactation not only improves the IgA + cells amount in the ileum and mammary gland but also elevates neonatal acquisition of maternal IgA levels via maternal milk33. Nishijima et al. indicated that β-carotene enhances colostral IgG1 levels in Japanese Black cows34. In poultry, Hui et al. demonstrated that adding 60 mg/kg β-carotene to the diets of layer-type chicks for 28 days promotes growth and development, increases the levels of serum sIgA and mucosal sIgA and IgA in the jejunum, and raises the abundance of Lactobacillus35. Miao et al. found that supplemental β-carotene at 15, 30, 60, or 120 mg/kg decreases the follicular atresia rates and enhances antioxidant activities in the ovaries of hens36. In addition, β-carotene increases the LYZ concentration in the egg albumen of Grey Partridges37, and LYZ plays a crucial protective role in the development of avian embryos15. Nevertheless, it remains unclear whether maternal β-carotene addition affects offspring growth and development through maternal effects. Therefore, our objective was to investigate the impacts and underlying mechanisms of maternal supplemental β-carotene on the growth and development of offspring.

First, we evaluated the effects of dietary inclusion of β-carotene on the immunity of hens. Next, we assessed the impacts of maternal immunity enhanced by β-carotene on the weight, immune factor levels, and incubation performance of breeder eggs, and the body weight, intestinal development parameters, and serum immune factor levels in offspring. We focused on identifying which maternal immune factors contribute to offspring growth and development, while also tracking their origins from the mother. Moreover, we evaluated the role of maternal gut and oviduct microbiome in microbe-mediated maternal effects and investigated the fate of microbial taxa significantly enriched in the gut and magnum of hens with enhanced immunity in the gut of the offspring.

Results

Nutritional supplementation with β-carotene enhances maternal immunity, elevates egg immune factor levels, and improves breeder egg incubation performance

Serum IgG (P < 0.001), LYZ (P = 0.002), and AvBDs (P < 0.001) levels in hens were remarkably elevated in the BC group in comparison with the CON group (Fig. 1a). In contrast, no significant alterations were observed in serum IgA and IgM concentrations between the BC group and the CON group.

Fig. 1
figure 1

Immune-enhanced hens with nutritional intervention can raise maternal immune factor levels in breeding eggs and enhance the hatching performance of these eggs. a Serum immune factor levels in hens as determined by ELISA. n = 5. b Egg quality. Egg weight n = 90; Egg, yolk, and eggshell weight n = 10. c Immune factor levels in eggs as determined by ELISA. n = 10. d Hatching performance of breeding eggs. n = 5, initial weight n = 120. Student’s t-test was conducted. *P < 0.05, **P < 0.01. LYZ, lysozyme; AvBDs, avian beta-defensins. CON, control group with hens fed basic diet; BC, experimental group with their hens receiving a basal diet supplemented with β-carotene.

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Maternal β-carotene nutritional interventions had no significant effects on the weights of breeder eggs and their constituent parts (egg white, yolk, and shell) compared to the CON group (Fig. 1b). However, the BC group exhibited significantly higher levels of yolk IgG (P < 0.001) and albumen LYZ (P < 0.001) and AvBDs (P = 0.015) in comparison with the CON group (Fig. 1c).

Contrasted with the CON group, the hatchability of fertile eggs (P < 0.001) and initial weight (P < 0.001) were significantly increased in the BC group, while the mortality rate was markedly reduced (P < 0.001) (Fig. 1d). The fertility of breeder eggs in the BC group showed no significant changes relative to the CON group.

Enhancing maternal immunity improves offspring growth and intestinal development

As illustrated in Fig. 2a, the main effect of days of age on body weight was significant (P < 0.001). Contrasted with the CON group, the BC group showed significantly greater (P < 0.05) offspring body weights at E21, C7, C14, and C21. There was a remarkable interaction (P < 0.001 ) between maternal β-carotene addition and days of age on offspring body weight.

Fig. 2
figure 2

Enhanced immunity in hens can improve the growth and intestinal development of their offspring. a The weight of offspring embryos and chicks. n = 6. b Villus height in the duodenum, jejunum, and ileum of offspring embryos and chicks. c Crypt depth in the duodenum, jejunum, and ileum of offspring embryos and chicks. Data are shown as the means ± SEMs, n = 6. A two-way ANOVA was performed using GLM, and different letters indicate significant differences (P < 0.05). d Representative morphological images of H&E-stained of the guts of embryos (E15, E19, and E21) and chicks (C7, C14, and C21). Bar = 100 μm, Bar (C7 duodenum) = 200 μm, Bar (C21 jejunum, and ileum) = 250 μm. Bar (C14 and C21 duodenum) = 500 μm. CON, control group with hens fed basic diet; BC, experimental group with their hens receiving a basal diet supplemented with β-carotene.

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There were no significant (P > 0.05) main effects of maternal BC addition or the interaction effects of maternal BC addition and days of age on duodenal villus height and crypt depth (Fig. 2b and c). However, significant (P < 0.05) main effects of maternal BC addition and interaction effects between maternal BC addition and offspring age were observed on villus heights and crypt depths in the jejunum and ileum. Contrasted with the CON group, the villus heights of the jejunum at E21, C7, C14, and C21 and the ileum at E19, E21, C7, C14, and C21 in the BC group were markedly elevated (P < 0.05). Relative to the CON group, the crypt depths of the jejunum (P < 0.05) and ileum (P < 0.05) at E21 were remarkably increased in the BC group, but no significant alterations were noted in duodenal crypt depth. Representative morphological images of HE-stained intestinal tissues are shown in Fig. 2d.

Enhancing maternal immunity improves offspring growth and intestinal development via immune factor-mediated maternal effects

As shown in Fig. 3a, significant interaction effects between maternal BC supplementation and offspring age were noted in the serum concentrations of IgG, LYZ, and AvBDs in offspring. In comparison to the CON group, the serum concentrations of IgG at E19, E21, C7, and C14 and AvBDs at E21, C7, and C14 and C21 and LYZ at E19, E21, C7, C14, and C21 were markedly augmented (P < 0.05) in the BC group.

Fig. 3
figure 3

The growth and developmental benefits of offspring from immune-enhanced hens are correlated with serum maternal immune factors in offspring. a Serum IgG, LYZ, and AvBDs levels in offspring embryos (E15, E19, and E21) and chicks (C7, C14, and C21) as determined by ELISA. n = 6. A two-way ANOVA was performed using GLM, and different letters indicate significant differences (P < 0.05). b Spearman correlation between serum immunity factors and growth and developmental performance in offspring chicks. *P < 0.05, **P < 0.01, n = 6. LYZ, lysozyme; AvBDs, avian beta-defensins; BW, body weight; DVH, duodenum villus height; DCD, duodenum crypt depth; JVH, jejunum villus height; JCD, jejunum crypt depth; IVH, ileum villus height; ICD, ileum crypt depth. CON, control group with their hens fed basic diet; BC, experimental group with their hens receiving a basal diet supplemented with β-carotene.

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Next, we examined the correlations between offspring growth and gut development indices with their serum levels of immune factors. As shown in Fig. 3b, no significant correlations were noted between serum IgG and growth or gut development indicators at any observation window. Notably, serum LYZ levels correlated positively with body weight at E21 (r = 0.678, P = 0.019), C7 (r = 0.602, P = 0.042), C14 (r = 0.657, P = 0.024), and C21 (r = 0.762, P = 0.006); duodenal villus heights at E15 (r = 0.769, P = 0.005) and E19 (r = 0.65, P = 0.026); jejunal villus height at E15 (r = 0.762, P = 0.006), E19 (r = 0.79, P = 0.003), E21 (r = 0.853, P = 0.001), C7 (r = 0.865, P = 0.001), and C14 (r = 0.755, P = 0.006); and ileal villus height at E15 (r = 0.713, P = 0.012), E19 (r = 0.825, P = 0.002), E21 (r = 0.643, P = 0.008), C7 (r = 0.743, P = 0.007), and C14 (r = 0.818, P = 0.002 ). Furthermore, significant negative correlations were observed between serum LYZ levels and the crypt depths in the jejunum (r = -0.741, P = 0.028) and ileum (r = -0.762, P = 0.006) at E21. In addition, serum AvBDs levels exhibited strong positive correlations with body weight at E21 (r = 0.79, P = 0.01), C7 (r = 0.613, P = 0.038), and C21 (r = 0.727, P = 0.01); duodenal villus heights at E15 (r = 0.664, P = 0.022); jejunal villus heights at E15 (r = 0.727, P = 0.01), E19 (r = 0.608, P = 0.04), and C7 (r = 0.676, P = 0.019); and ileal villus heights at E15 (r = 0.678, P = 0.019), C7 (r = 0.676, P = 0.019), and C14 (r = 0.671, P = 0.02).

Maternal nutritional interventions enhance the immunity of the gut and magnum in hens

We conducted RNA-seq of the magnum and the jejunum to trace the maternal sources of the LYZ and AvBDs that improved offspring growth. In the maternal magnum, Principal Component Analysis (PCA) revealed alterations in the transcriptome structures between groups (Fig. 4a). Next, we calculated the differentially expressed genes (DEGs) in the magnum of hens between groups. As shown in Fig. 4b, in the maternal magnum, we identified 128 up-regulated and 51 down-regulated DEGs in the BC group relative to the CON group. The Gene Ontology (GO) enrichment analysis of these DEGs revealed terms that were notably enriched within the BP category, with the top 5 being: response to potassium ion (the number of DEG/ the number of total gene in the term: 2/3), cellular response to potassium ion (2/3), positive regulation of type I interferon-mediated signaling pathway (2/7), iron ion transport (3/26), response to other organism (13/551). Additionally, Fig. 4c highlights the top 30 BP terms exhibiting significant enrichment.

Fig. 4
figure 4

The transcriptome alterations in the gut and magnum of hens are related to substance transport and immunity. PCA of the magnum (a) and gut (d) in the transcriptome. Scatterplot showing DEGs of magnum (b) and gut (e) in the BC group compared with the CON group. Top 30 enriched GO terms with rich factors (the DEG number/total gene number in the term) in the BP category for up-regulated DEGs in the magnum (c) and gut (f) of hen transcriptome. n = 5. CON, control group with their hens fed basic diet; BC, experimental group with their hens receiving a basal diet supplemented with β-carotene.

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In the maternal jejunum, the transcriptome structures exhibited apparent alterations between groups (Fig. 4d). Next, we identified 142 up-regulated DEGs and 144 down-regulated DEGs in the BC group contrasted with the CON group (Fig. 4e). For these DEGs, the top 5 terms that were significantly enriched within the BP category included: anion transport (13/363), organic anion transport (16/271), steroid metabolic process (11/145), sulfate transport (4/13), ion transport (33/995). The top 30 BP terms exhibiting significant enrichment are presented in Fig. 4f.

Notably, in the maternal magnum, the top 30 BP terms were mainly linked to ionic responses and immunity. In the maternal jejunum, the top 30 BP terms were primarily connected to material transport and the immunity-related GO term for defense responses to bacterium was among the top 30 GO terms.

Enhancing maternal immunity affects the microbiota of offspring via microbiota-mediated maternal effects

To explore the impacts of maternal immune enhancement on microbial-mediated maternal effects, we analyzed the microbial composition in the magnum mucosa and cecum of hens, as well as in the cecum of offspring at E19, E21, C7, and C21 through 16 S rRNA sequencing. The microbial communities at the phylum level in the magnum mucosa and cecum of hens and the cecum of offspring embryos and chicks were dominated by Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria (Fig. 5a). As shown in Fig. 5b, the dominant genera in the magnum mucosa and cecum of hens, as well as in the cecum of offspring embryos and chicks, included Faecalibacterium, Ruminococcaceae_Ruminococcus, [Ruminococcus], Oscillospira, Bacteroides, and Lactobacillus (Fig. 5b).

Fig. 5
figure 5

Augmenting hen’s immunity modulates the gut and oviduct microbiota, and increases microbial-mediated maternal effects on offspring gut microbiota. Microbial composition at the phylum (a) and genus (b) levels in the gut and magnum of hens and the gut of their offspring embryos at E19 and E21 and chicks at C7 and C21. c Beta diversity of microbial communities in the gut and magnum of hens, as well as the gut of offspring embryos at E19 and E21 and chicks at C7 and C21 (n = 10). d Shared taxa between the gut (red) and magnum (blue) of hens with the gut of their offspring. CON, control group with their hens fed basic diet; BC, experimental group with their hens receiving a basal diet supplemented with β-carotene.

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To evaluate the diversity of the microbiome in both the magnum and gut of hens, as well as in the gut of their offspring, we assessed the Alpha diversity of the microbiome, including indices such as Chao1, Observed, Shannon, and Simpson. As shown in Fig. S4a, the Chao 1 index was significantly elevated (P = 0.047) in the magnum microbiota of hens of the BC group compared with the CON group. At C7, the Chao 1 (P = 0.009), Observed (P = 0.009), and Shannon (P = 0.009) diversity indices in the offspring gut of the BC group were markedly elevated contrasted with the CON group. Nonmetric Multidimensional scaling (NMDS) analyses revealed that the community composition of the embryonic gut microbiota at E19 and E21 was more closely related to that of the hen magnum than to the hen gut (Fig. 5c). Furthermore, the samples from offspring gut and those from the hen gut and magnum were distinctly separated at C7 and C21. In the gut of offspring, samples from the CON and BC groups exhibited partial separation at E19, C7, and C21 (Fig. S4b). Significantly differentially enriched bacterial taxa between groups, from phylum to genus levels, in the gut and magnum of hens, as well as in the gut of their offspring, were identified using LEfSe. As illustrated in Supplementary Fig. S5, the BC group exhibited significant enrichment of 5/22 (the number of enriched taxa in BC group/total number of enriched taxa), 6/36, 38/52, 10/21, 6/24, and 9/32 in the gut and magnum of hens, as well as in the gut of their offspring at E19, E21, C7, and C21, respectively. Notably, Lactobacillus and Caloramator were markedly enriched in the gut and magnum of the hens, respectively. At E21, offspring from CON group hens exhibited significant enrichment of Desulfovibrio in their gut microbiota, whereas Shigella displayed markedly enrichment in offspring at both C7 and C21 developmental stages.

Next, we quantified shared microbial taxa between the gut and magnum microbiome of hens with their offspring intestinal microbiota, respectively. As shown in Fig. 5d, a total of 1277, 1068, 235, and 539 ASVs were identified as shared between the intestinal microbiome of hens and their offspring at E19, E21, C7, and C21 in the CON group, and 1168, 470, 480, and 487 in the BC group, respectively. Moreover, the number of ASVs shared between the magnum microbiota of hens with the gut microbiota of their offspring at E19, E21, C7, and C21, was 1596, 1287, 286, and 525 in the CON group, and 1701, 719, 1178, and 689 in the BC group, respectively.

Changes in microbiota in the hen’s magnum and gut are associated with immunity and the vertical transfer of maternal microbes is selective

In the gut and magnum of hens, we evaluated the relationship between the relative abundance of taxa that were markedly enriched in the BC group and the immune-related up-regulated DEG expression. In the maternal gut, significant positive correlations were detected between the abundance of Lactobacillus and the expression levels of the following genes: LYZ (r = 0.859, P = 0.001), AvBD10 (r = 0.81, P = 0.005), AvBD11 (r = 0.789, P = 0.007), DDX60 (r = 0.713, P = 0.021), and RAB7B (r = 0.659, P = 0.038) (Fig. 6a). Furthermore, we detected significant positive correlations between the Pediococcus abundance and the expression of EREG (r = 0.718, P = 0.019) and NR1H4 (r = 0.648, P = 0.043) genes. As illustrated in Fig. 6b, in the maternal magnum, the abundance of Caloramtor and Lachnospiraceae_Clostridium exhibited significant positive correlations with the expression levels of the following genes: IKBKE (r = 0.886, P = 0.001 and r = 0.882, P = 0.001), TLR7 (r = 0.677, P = 0.032 and r = 0.863, P = 0.001), ACP5 (r = 0.771, P = 0.009 and r = 0.807, P = 0.005), ISG20 (r = 0.881, P = 0.001 and r = 0.809, P = 0.005), SOCS1 (r = 0.727, P = 0.017 and r = 0.767, P = 0.01), and CTLA4 (r = 0.805, P = 0.005 and r = 0.77, P = 0.009).

Next, we performed monitoring of microbial taxa exhibiting significant enrichment in the intestinal tract and magnum of BC group hens, tracking their relative abundance within the intestinal microbiota of offspring. As illustrated in Fig. 6c, the abundance of Lactobacillus and Pediococcus was markedly elevated (P < 0.05) in the intestinal microbiota of hens in the BC group, whereas it was significantly decreased (P < 0.05) in the intestinal microbiota of their offspring at C21. In addition, their abundance in the intestinal microbiota at C21 was significantly greater (P < 0.05) than at E19, E21, and C7. Significant interaction effects (P < 0.01) between maternal BC supplementation and offspring age were observed for their abundance in the intestinal microbiota. As illustrated in Fig. 6d, the abundance of Synechococcaceae and Lachnospiraceae_Clostridium was markedly raised (P < 0.05) in the magnum microbiota of hens in the BC group, but no significant changes were noted in the intestinal microbiota of their offspring between groups. The Lachnospiraceae_Clostridium abundance was significantly higher (P < 0.05) in the offspring at E21 and C7 than at E19 and C21. The Synechococcaceae abundance at C7 and C21 was notably increased (P < 0.05) compared with E19 and E21, and it was higher (P < 0.05) at C21 than at C7. Notably, Caloramtor, which was significantly enriched (P < 0.05) in the magnum microbiota of BC group hens, also exhibited significant (P < 0.05) enrichment in the intestinal microbiota of their offspring at C21. There was a significant (P < 0.001) interaction effect between maternal BC inclusion and offspring age on the Caloramtor abundance in the progeny intestinal microbiota.

Fig. 6
figure 6

Transcriptome changes in the hen’s gut and magnum are linked to microbial shifts, with the offspring selectively inheriting these microbes. Spearman correlations between up-regulated DEGs and morphological indices in gut (a) and magnum(b). The relative abundance of taxa significantly enriched in the gut (c) and magnum (d) of hens with immune-enhanced in the gut of their offspring. CON, control group with their hens fed basic diet; BC, experimental group with their hens receiving a basal diet supplemented with β-carotene.

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Discussion

Previous studies have shown that maternal antibodies are crucial for protecting offspring from pathogens in early life23. This investment from the mother is known as the maternal effect, which is crucial for the adaptation of animals to their environment and for growth and development38,39. In this study, to enhance the immunity of hens, we supplemented β-carotene into their diet. The results showed that β-carotene nutritional interventions significantly increased serum IgG, LYZ, and AvBDs levels in hens. These results indicated that the immunity of hens was strengthened. Next, we assessed the influences of enhanced maternal immunity through β-carotene addition on offspring. We discovered that the hatchability of fertile eggs from the BC group hens was significantly increased, while the mortality rate of breeder eggs was markedly reduced. Furthermore, the body weight, serum IgG, LYZ, and AvBDs levels, and the villus height in the jejunum and ileum of offspring embryos and chicks were notably enhanced. These results are similar to our previous findings that enhanced maternal immunity with complex additives (β-carotene: 60 mg/kg, curcumin: 250 mg/kg, allicin: 250 mg/kg, and sodium butyrate:500 mg/kg) can reduce the mortality rate of breeder eggs and improve the development of villi and crypts in offspring embryonic gut13. However, there were some differences between this study and our previous study, such as the serum concentrations of IgA and IgM in hens and those of IgG, LYZ, and AvBDs in offspring embryos. We speculate that dosage differences and potential interactions between additives may be the primary reasons for the differing results. Miao et al. reported that 60 and 120 mg/kg β-carotene have different effects on the oxidative stress in hen ovaries36. Furthermore, Shete et al. documented that the bioavailability of β-carotene is influenced by multiple factors, such as other carotenoids, the fat content of the diet, and the type of fat40. However, whether the impacts of β-carotene on avian immunity are influenced by its dose, curcumin, allicin, or sodium butyrate still requires further research. Additionally, our results demonstrate that the immune and growth advantages of the offspring extend from the embryonic stage to 21 days of age after hatching. These findings suggest that maternal β-carotene nutritional interventions can bolster the immunity of offspring by increasing serum IgG, LYZ, and AvBDs levels, as well as improve their growth and development by raising body weight, intestinal villus height, and reducing crypt depth during embryonic and chick stages. However, in poultry, how do maternal immune advantages influence the immunity and growth development of offspring? In addition, significant interaction effects were observed between maternal β-carotene interventions and offspring age on growth development and serum immune parameters in the progeny, indicating that offspring age may modulate the maternal effects induced by nutritional interventions. This discovery is also supported by the study of Gong et al., who reported that the effects of maternal nutritional interventions on offspring body weight and serum immune parameters vary with offspring days of age41.

Birds, like mammals, are higher vertebrates in nature. Avian embryos developing in vitro rely more heavily on maternal effects for protection than mammalian embryos developing in the womb. The successful hatching of healthy chicks is the result of various protective strategies that have evolved over time in oviparous animals42. A variety of anti-microbial proteins, such as LYZ, AvBDs, and IgG, are enriched in eggs from the ovary and oviduct of hens, and these immune proteins provide immune protection to the developing embryo43. Moreover, previous research has shown that the quality of breeder eggs and levels of immune factors both significantly impact the incubation performance of breeder eggs and the early growth and development of offspring chicks44. Therefore, we then investigated breeder egg quality and immune factor levels. Our findings revealed that the quality of breeder eggs did not differ remarkably between the groups. In contrast, the BC group exhibited significantly higher levels of egg white LYZ and AvBDs, as well as yolk IgG, compared with the CON group. In agreement with our findings, prior research has reported that maternal addition of β-carotene 27 mg/kg in Grey Partridge can enhance maternal immunity and elevate the levels of immune-related components, such as LYZ, in eggs without affecting the weight of the yolk and albumen37. In this study, we provide additional information on the impacts of maternal β-carotene inclusion on the albumen AvBDs and yolk IgG content in bird eggs, which has not been documented in prior research. These findings indicate that the improved incubation performance of breeder eggs from the BC group hens, as well as the growth and immune advantages of their offspring, could be linked to raised concentrations of LYZ, AvBDs, and IgG in the breeder eggs.

To further elucidate the reasons for the growth and developmental advantages in offspring, we evaluated the correlations between serum immune factors and growth development indicators in offspring. We found significant positive correlations between serum LYZ and AvBDs levels and both growth and intestinal development indicators in offspring embryos and chicks. Conversely, IgG did not exhibit significant correlations with intestinal development indicators of the offspring embryos and chicks. These findings indicate that the maternal immune-enhanced may have improved offspring growth and development by increasing the vertical transfer of maternal LYZ and AvBDs in this experiment. It has been reported that hens produce IgG in response to antigenic stimulation45, and as a key antibody in the adaptive immune response, maternally derived IgG may benefit the survival prospects of offspring chicks when confronted with specific pathogens46. In our experiment, the potential lack of exposure of offspring to relevant pathogens may be the reason for the lack of a significant correlation between serum IgG levels and their growth and development.

Considering that the egg white in breeder eggs originates from the magnum of the hen’s oviduct47. We carried out further studies on the magnum using RNA-seq and found that the top 30 BP terms significantly enriched are predominantly related to material transport and immunity. However, the expression of AvBDs and LYZ genes did not markedly change between the groups, suggesting that the increased vertical transfer of LYZ and AvBDs from hens with enhanced immunity to their offspring may occur independently of self-synthesis in the magnum. It has been reported that exocrine glands secrete LYZ in humans, which is probably partly derived from local synthesis and partly from blood48. Although there is a lack of reports related to the source of LYZ secreted by the tubular glands of the magnum. Our previous study found that the vertical transfer of LYZ and AvBDs from hens to their offspring is linked to maternal serum levels13. Therefore, we proposed that the increased vertical transmission of LYZ and AVBDs from BC group hens to their offspring may be correlated with elevated serum levels in these hens. In previous studies, the LYZ has been employed as a marker for Pannell cells, which secrete a variety of antimicrobial peptides, including LYZ itself, within the gut49,50. Thus, we performed transcriptome sequencing on the jejunum of the hens and identified that the top 30 BP GO terms significantly enriched were related to material transport and immune function. Notably, maternal β-carotene nutritional intervention significantly enhanced the expression of LYZ, AvBD10, and AvBD11 genes in the hen jejunum. In line with our findings, it has been demonstrated that supplemental β-carotene at 60 mg/kg can enhance the expression of genes associated with intestinal immune and barrier function in chickens via the retinoic acid pathway, including IgA, pIgR, MUC2, and ZO-135. Consequently, these results suggest that β-carotene nutritional supplementation enhances the immune function of the jejunum in hens by increasing the expression of LYZ, AvBD10, and AvBD11 genes. However, whether the mechanism of β-carotene on those gene expressions depends on the retinoic acid pathway requires further investigation. Based on these results, we examined the relationships between the expression levels of jejunal LYZ and AvBDs genes and their corresponding serum concentrations of hens. Our findings indicated that significant positive correlations between the expression levels of jejunal LYZ and AvBDs genes and their respective serum levels (Fig. S3). These results suggest that β-carotene enhances immunity in hens, thereby increasing the vertical transfer of LYZ and AvBDs to their offspring. This effect may be associated with the upregulation of these genes’ expression in the maternal jejunum.

There is evidence that microbial-mediated maternal effects influence the growth and immunity of offspring29,51,52. Does β-carotene modulate the microbiota of hens, thereby affecting chicks via microbial-mediated maternal effects? Previous researches have indicated that β-carotene modulates the gut microbiota in various animal species53, Yang et al. found that supplemental 30 or 90 mg/kg modulates the cecal microbiota of 6-week-old female Kunming mice, such as enhancing the abundance of Akkermansia and reducing the abundance of Alloprevotella54. Li et al. documented that supplemental 40 or 80 mg/kg β-carotene regulates the gut microbiota of 12-day-old piglets, such as raising the abundance of Parabacteroides and diminishing the abundance of Prevotella55. Hui et al. demonstrated that supplementing β-carotene 60 mg/kg to the diet of newborn male chicks can modulate the abundance of specific gut microbes, such as Lactobacillus35. In this study, adding β-carotene 120 mg/kg to diets increased the abundance of five taxa, including Lactobacillus and Pediococcus, and reduced the abundance of 17 taxa, including Desulfovibrio, in the gut microbiota of hens. Additionally, it augmented the abundance of 6 taxa, including Caloramator, in the magnum microbiota of hens and decreased the abundance of 30, including Halomonas. Consistent with the previous findings of Hui et al., we also found that dietary β-carotene significantly enhanced the abundance of Lactobacillus in the chicken gut. However, our study differed in that we performed a more extensive evaluation of the impacts of β-carotene on the intestinal microbiota. Additionally, as far as we know, this is the inaugural documented research examining the impacts of dietary β-carotene on the magnum microbiota in hens. A prior study demonstrated that the microbiome of hens can shape the gut microbiota of their offspring via microbial-mediated maternal effects56. Thus, we speculated that maternal β-carotene nutritional interventions may influence the offspring gut microbiota through modulation of the maternal microbiota. In the offspring, we identified 52, 21, 24, and 32 taxa in the gut microbiota at E19, E21, C7, and C21, respectively, that exhibited significant differences in enrichment between groups. Notably, Desulfovibrio exhibited enrichment in the CON group offspring at E21, while Shigella showed enrichment at C7 and C21.

In birds, during egg laying, the maternal gut microbiota adheres to the eggshell membrane as the egg passes through the cloaca, while the maternal magnum microbes are transferred within the albumen during its formation. Lee et al. have reported that the gut and oviduct microbiome of hens can affect the establishment of the microbiota of their offspring chick gut57. Nevertheless, limited research has directly compared the impacts of maternal gut and reproductive system microbiome on the offspring intestinal microbiota. To contrast the influences of maternal microbes in the gut and oviduct on the intestinal microbiota of offspring, we conducted Beta-diversity analyses, revealing that the offspring embryo’s gut microbiota exhibits a stronger association with the maternal magnum compared to the maternal gut. Next, we analyzed the quantity of ASVs that were common between the magnum of hens and the gut of their offspring, as well as those shared between the gut of hens and their offspring. The findings revealed that the maternal magnum shared a greater number of ASVs with the offspring gut compared to the maternal gut. In agreement with our findings, Li et al. documented that the maternal oviduct microbiota exerts dominant impacts on the offspring’s intestinal microbiota compared to other maternal microbial sources57. Moreover, although the quantity of shared ASVs between the offspring gut and both the maternal gut and magnum gradually reduced during the early life as age increased, the ASVs shared with the maternal gut and oviduct microbiota persisted in the gut microbiota of offspring chicks until C21. In accordance with our results, Gong et al. found that alterations in the maternal magnum microbiota influence the gut microbiota of offspring beginning in the embryonic period13. Savietto et al. demonstrated that there is a gradual decline in the number of microbes derived from the maternal source as age advances58. Our research revealed that the maternal magnum microbiota exerts a stronger influence on offspring gut microbiota relative to the maternal gut, especially during the offspring embryonic period, and the effects of changes in maternal gut and oviduct microbiome on the offspring intestinal microbiota persisted until C21. Furthermore, our results showed that supplemental β-carotene to the maternal diet diminished the influences of the microbes in the intestines of hens on the offspring’s intestinal microbiota at all time points except C7. Conversely, it enhanced the impacts of the microbes in the magnum of hens on the offspring’s intestinal microbiota at all time points except E21. These findings suggest that maternal β-carotene nutritional interventions can regulate the gut and magnum microbiome of hens, such as Lactobacillus, Pediococcus, and Desulfovibrio in the gut, as well as Caloramator and Halomonas in the magnum. This modulation may subsequently influence the offspring’s gut microbiota, such as Desulfovibrio and Shigella, from the embryonic stage through the post-hatch period. However, the mechanisms by which maternal β-carotene nutritional interventions reduce the effects of maternal microbes in the gut and magnum on the offspring intestinal microbiota at E21 still require further investigation.

Host immunity can influence symbiotic microbiota and, conversely, the microbiota significantly contributes to the regulation of host immune responses59,60. Maternal microbiota has been shown to influence the transfer of immune factors from hens to their offspring9. Meanwhile, maternal immunity affects the transfer of microbes from hens to their offspring61. Therefore, we hypothesized that there may be an intrinsic link between the changes in the maternal gut and magnum microbiome and the altered immune status. To investigate the relationship between microbial changes and transcriptomic alterations in the gut and magnum of hens, we assessed the correlations between the expression levels of DEGs that were up-regulated in the maternal jejunum of the BC group and associated with immune function, and the relative abundance of taxa that were markedly enriched within the same group, as well as examined similar correlations in the maternal magnum. Specifically, in the maternal gut, we analyzed the correlations between the expression levels of LYZ, AvBD10, AvBD11, HGF, DDX60, EREG, NR1H4, RAB7B, and SLC26A6 genes and the abundance of Pediococcus and Lactobacillus; in the maternal magnum, we evaluated the correlations between the expression levels of PLAC8, IKBKE, IRF7, TLR7, CMPK2, ACP5, ISG20, SOCS1, and CTLA4 genes and the abundance of Caloramator, Synechococcaceae, and Lachnospiraceae_Clostridium. We found that a total of 5 immune-related DEGs, including LYZ, AvBD10, and AvBD11, were highly correlated with the Lactobacillus in the gut of hens, and a total of 6 immune-related DEGs, including IKBKE, TLR7, and ACP5, were strongly associated with the Caloramator and Lachnospiraceae_Clostridium in the magnum of hens. There is growing evidence that microbes can regulate the host’s immune system62,63, while the host influences microbe colonization through immune pathways64,65. These results suggested that maternal β-carotene nutritional interventions may enhance the immunity of the gut and magnum of hens by modulating the abundance of Lactobacillus in the gut and Caloramator and Lachnospiraceae_Clostridium in the magnum. Thereby enhances the immune-mediated maternal effects and thus improves offspring growth and development.

However, what is the fate of the microbial taxa with significant enrichment in the gut and magnum of BC group hens during transgenerational transfer? We separately investigated the abundance of these taxa in the maternal gut or magnum, as well as in the gut of their offspring. The findings revealed that the abundance of Lactobacillus and Pediococcus, which showed significant enrichment in the gut of hens from the BC group, did not increase in the gut microbiota of their offspring. In contrast, at C21, the relative abundances of these taxa were markedly lower than those in the CON group. Furthermore, among the taxa that were significantly enriched in the magnum microbiota of hens from the BC group, only the abundance of Caloramtor in the offspring’s intestinal microbiota at C21 was markedly raised relative to the CON group. These findings indicate that the transfer of maternal gut and magnum microbes across generations exhibits selectivity. In agreement with our findings, earlier research has indicated that the microbial transfer from the oviduct of hens to the gut of their offspring was selective66. Previous studies have reported that microbial colonization is influenced by multiple factors, with adhesion of microbes as a crucial determinant for their colonization potential, and that the host is also capable of altering the fate of colonized microbes through immune strategies59,67,68. It has been shown that maternal antibodies can enhance the transfer of certain microbial molecules, thereby protecting the offspring from specific microbes23,61. In present research, the gut microbiota of the offspring chicks did not inherit the significantly enriched taxa observed in the hens’ gut and magnum microbiome following maternal β-carotene nutritional intervention. In fact, eggs possess a substantial amount of immune factors originating from hens, which effectively suppress the growth of microbes within the eggs. Therefore, we hypothesized that the selectivity may be associated with the transmission of immune factors from hens to their offspring. However, the mechanisms of the selectivity need to be further investigated with more work. Furthermore, we identified significant interaction effects between maternal β-carotene supplementation and offspring age on the abundance of taxa in the offspring’s intestinal microbiota that are vertically transferred from the maternal gut or magnum. These findings align with previous studies, which demonstrate that maternal nutritional interventions modulate vertical microbial transfer from hens to offspring, with the effects varying across different developmental stages13.

In summary, maternal β-carotene nutritional interventions enhance the immunity of hens and regulate the gut and magnum microbiome, thereby improving breeder egg incubation performance while promoting offspring immunity, growth and development, and intestinal microbiota composition through heightened immune-mediated and microbial-mediated maternal effects. The advantages in the growth and development of offspring may be attributed to an enhanced transfer of LYZ and AvBDs from the hens, while augmented vertical transfer of maternal LYZ and AvBDs may correspond to the upregulated expression of jejunal LYZ and AvBDs genes of hens. The microbiota in the magnum contributes more to microbial-mediated maternal effects than that in the gut of the hens. Additionally, the vertical transmission of microbes from hens to their offspring occurs in a selective manner. According to our findings, we suggest that maternal addition with β-carotene may provide sustained benefits to offspring immunity and growth and development by enhancing maternal effects. Moreover, the influence of maternal effects should be considered in chick management to optimize health strategies for precise management of chick health. Furthermore, microbiota in the maternal magnum may serve as a more effective resource for utilizing microbe-mediated maternal effects to promote offspring health compared to the maternal gut microbiota.

Materials and methods

All procedures were carried out in accordance with the relevant guidelines and regulations.

Hens feeding, experimental design, and samples collection

An experimental design is schematically outlined in supplementary Fig. S1. Eight hundred and ten laying breeder hens (Hy-Line Brown), aged 45 weeks, were randomly allocated to two groups, with each group containing five replicates of eighty-one hens each. The birds were reared in 10 battery cages (3 × 3, 120 × 60 × 60 cm) consisting of 9 units, with 3 birds per unit, featured 6 nipple-type drinkers, and 1 feed trough shared by the three units on each level. Following a 1-week acclimatization period, the control group (CON) received a basal diet, while the experimental group (BC) provided the basal diet and added β-carotene at 120 mg/kg for 6 weeks. The β-carotene (purity: 20%) utilized in this study was extracted from plants and provided by Shaanxi Kingreg Biotech Co., Ltd., Shaanxi, China. The dose of β-carotene was determined based on a previous literature report, which indicated that supplemental β-carotene at 120 mg/kg significantly enhances the antioxidant function in the ovary of hens36. The basal diets of hens were designed based on the guidelines specified in GB/T 5916 − 2020 (supplementary Table S1) and supplied by WELLHOPE Group, Shenyang, China. Throughout the experimental period, all hens had ad libitum access to feed and water. Hens were artificially inseminated every 5 days using semen from healthy breeder roosters (Rhode Island Red) via standard artificial insemination techniques. During the final 3 days of the hen rearing experiment, the breeder eggs of each group were collected separately for subsequent experiments. The hen rearing experiment was carried out at the Changchun Academy of Agricultural Science, Changchun, China.

On the 42nd day of the hen rearing experiment, five birds from each group were randomly selected, with one hen chosen from each replicate. Blood was collected via brachial vein venipuncture, after which the hens were humanely euthanized by cervical dislocation after anesthesia administration. Serum was isolated through centrifugation (3000 rpm, 15 min) of blood, followed by preservation at -20 °C until subsequent use. Samples of magnum mucosa and cecal content were collected, rapidly frozen with liquid nitrogen, and stored at -80 °C for subsequent 16 S rRNA gene sequencing. Tissue samples of the magnum and jejunum were collected and promptly submerged in liquid nitrogen for later RNA-seq analysis.

Breeder egg hatching

In each group, 900 eggs were collected and incubated in five replicates, with each replicate consisting of two trays containing 90 eggs each. Eggs were incubated at 37.8 °C with 50-70% humidity using a fully automatic incubator and turned once every 6 h. Dead embryos and unfertilized eggs were recorded during incubation.

Egg and embryonic sampling

From each group, 90 breeder eggs were randomly collected for egg quality assessment and embryonic development experiment. All eggs were weighed, and ten eggs from each group were randomly chosen for evaluation of the weight of individual components. The egg whites, yolks, and shells were individually separated and weighed. Subsequently, the whites and yolks were stored at -80 °C until utilized for immune factor level assays. The remaining 80 breeder eggs in each group underwent incubation in an automated incubator maintained at 37.8 °C with 50–70% humidity, with the eggs being turned every 6 h. On embryonic days 15, 19, and 21 (referred to as E15, E19, and E21, respectively), six embryos from each group were chosen at random for sampling. Blood was obtained utilizing capillary collection tubes from the yolk sac vein and subsequently centrifuged (4,500 rpm, 15 min) to isolate the serum. The isolated serum was preserved at -20 °C for later analysis of immune factors. The cecum samples were harvested using sterile tubes and promptly preserved in liquid nitrogen for microbial composition analysis. The duodenal, jejunal, and ileal segments were collected and fixed with 4% paraformaldehyde solution until future analysis.

Offspring chick rearing experiment and sampling

Once the chicks had hatched, a total of 90 healthy male chicks were randomly selected from each group and randomly allocated into 6 replicate groups for a 21-day rearing experiment. The offspring chicks were reared in 70 × 60 × 40 cm cages. All chicks were provided with the same basal diet and were allowed ad libitum access to food and water. Detailed information on the basal diet is presented in Supplementary Table S1. The specifics of brooding management are provided in Supplementary Table S2.

On 7, 14, and 21 days post-hatching (referred to as C7, C14, and C21, respectively), six chicks were haphazardly chosen from each group for weighing and sampling. Blood was collected through venipuncture of the brachial vein. Subsequently, the chicks were humanely euthanized by cervical dislocation following anesthesia administration. Serum was achieved by centrifuging blood (3000 rpm, 15 min) and subsequently stored at − 20 °C until further use. Cecal contents were harvested and promptly preserved in liquid nitrogen until used for microbial composition analysis. Approximately 3 cm intestinal segments (duodenum, jejunum, and ileum) were harvested and fixed with 4% paraformaldehyde solution for morphological examination of the offspring chicks.

ELISA

Following the protocol outlined by Hamal et al.69, proteins were isolated from egg yolk and egg white samples for subsequent use in ELISAs. The concentrations were quantified as mass per gram of egg yolk or white. Following the manufacturer’s protocol, the concentrations of IgA (MM-0913O1), IgG (MM-0505O1), IgM (MM-0912O1), lysozyme (LYZ) (MM-34261O1), and avian β-defensins (AvBDs) (MM-34120O1) were quantified in the serum of hens, egg yolks, egg whites, and the serum of offspring embryos and chicks utilizing commercial ELISA kits supplied by MeiMian Industrial Co., Ltd. (Yancheng, China). During the measurements, samples were 5-fold diluted, and 3 technical replicates were performed.

Intestinal morphological observations of offspring embryos and chicks

The duodenal, jejunal, and ileal segments from offspring embryos and chicks were fixed with 4% paraformaldehyde. After fixation, the tissues were processed through trimming, dehydration, clearing, and embedding in paraffin wax. The embedded samples were cut into sections with 5 μm and stained with hematoxylin and eosin (H&E). Subsequently, the stained sections were examined and imaged under an Olympus IX74 microscope at appropriate magnifications. Finally, according to the methodology described by Liu et al.70, we used cellSens software ( v 3.1) at the appropriate field of view to quantify intestinal villus height and crypt depth. For each sample, three slides were generated with five intact villi were measured per slide, and the mean was then calculated.

RNA extraction and RNA-Seq

The gene expression in the jejunum and magnum of hens was comprehensively investigated using RNA-seq. RNA samples were obtained from the magnum and jejunum tissues utilizing TRIzol Reagent (Invitrogen, Carlsbad, CA# 15 596 − 026) following the manufacturer’s guidelines. The sequencing libraries were constructed for each sample utilizing a commercial kit (NEBNext Ultra II RNA Library Prep Kit for Illumina). After the extraction, purification, and library construction of sample RNA, second-generation sequencing technology (Next-Generation Sequencing, NGS) was employed utilizing the NovaSeq 6000 platform (Illumina) to conduct paired-end (Paired-end, PE) sequencing on these libraries.

Bioinformatics analyses of RNA-seq data

To ensure data quality, the cutadapt R package was used to filter the sequencing reads. The quality control outcomes are detailed in Supplementary Table S3. The filtered reads underwent alignment against Gallus gallus reference genomes sourced from the Ensembl database (http://asia.ensembl.org/Gallus_gallus/Info/Index) using HISAT2 software under default parameters. The mapping quality control was conducted using mapping summary (Supplementary Table S4) and gene coverage data (Supplementary Fig. S2). The FPKM method (fragments per kilobase of transcript per million mapped reads) was employed for normalization of gene expression quantification. The DESeq R package (v 1.30.0) was utilized to perform Principal Component Analysis (PCA) for each sample based on expression levels, and to identify differentially expressed genes (DEGs) with criteria of |log2FoldChange|>1 and P-value < 0.05. Subsequently, we utilized the ggplot2 R package (v 3.3.3) to generate volcano plots for visualizing differentially expressed genes (DEGs). Next, we annotated the identified DEGs based on the Gene Ontology (GO) database (http://geneontology.org). GO term enrichment was subsequently analyzed utilizing the topGO R package (v 3.14), highlighting terms with significant enrichment (P-value < 0.05).

DNA extraction and 16 S rRNA gene sequencing

Total microbial DNA was isolated from maternal cecal content and magnum scrape samples, as well as offspring cecal content collected at E19, E21, C7, and C21, utilizing the FastDNA®SPIN Kit (MP Biomedical, 11654600). The hypervariable V3-V4 domains of the 16S rRNA gene were PCR-amplified with primers 338F (5’-ACTCCTACGGGAGGCAGCA-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’). Purification of amplification products was achieved utilizing the Agencourt AMPure XP Kit (Beckman Coulter, A63881) and their concentrations were determined with the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, P7589). Quantitative measurements of purified amplicons are documented in Supplementary Table S5. Subsequently, paired-end 2 × 250 bp sequencing of the purified amplicons was carried out on the Illumina NovaSeq PE250 platform.

Microbial bioinformatics analysis

Following the previously described methods13, the 16 S rRNA sequencing datasets were analyzed through the QIIME2 platform (v 2019.4). Briefly, the demux plugin was used to perform demultiplexing on the raw sequencing reads, and the cutadapt plugin was utilized to trim the primers. The DADA2 plugin was utilized for quality control of the sequencing data. The non-singleton amplicon sequence variants (ASVs) were subjected to multiple sequence alignment using MAFFT, followed by phylogenetic reconstruction employing the FastTree2. ASVs were classified into taxa utilizing the feature-classifier plugin, with reference to the SILVA database (v 138).

Alpha diversity (Chao 1, observed species, Shannon, and Simpson) was analyzed utilizing the normalized ASV matrix through the vegan (version 2.4.0) and phyloseq (version 1.14.0) packages in R71. To evaluate the dissimilarity in community structure (Beta-diversity), we utilized the diversity plugin with the Bray-Curtis dissimilarity matrix to conduct nonmetric multidimensional scaling (NMDS). The results were visualized using the vegan R package (v 2.5-7). Taxa exhibiting significant enrichment across phylogenetic hierarchies (phylum to genus) were identified through Linear discriminant analysis (LDA) effect size (LEfSe) implemented on the Galaxy computational platform, employing Kruskal-Wallis/Wilcoxon tests with one-against-all comparisons (selection criteria: log10(LDA score) > 2, P < 0.05) on normalized ASV table. The number of shared ASVs between the maternal gut and magnum microbiome with the offspring gut microbiota was calculated using the VennDiagram R package (v 1.7.0).

Statistical analysis

We performed the Spearman correlation analyses to examine the correlations between serum immune factor levels and growth and development parameters (body weight, villus height, and crypt depth) in offspring chciks, as well as between the expression levels (FPKM values) of up-regulated genes and the relative abundance of LEfSe-identified clade termini taxa in the gut and magnum of hens. These analyses were performed using IBM SPSS Statistics 26 (v 26.0.0.0 ), and the resulting data were subsequently presented as heatmaps utilizing GraphPad Prism 9 (v 9.5.1). Longitudinal profiling of LEfSe-identified differentially abundant taxa (originally enriched in breeder hen intestinal/magnum microbiome) across offspring intestinal microbiota developmental trajectories was visualized using GraphPad Prism 9, and the differences between groups in maternal gut and magnum were determined based on the results of LEfSe. Except for the sequencing data, the normality of the data was evaluated utilizing the one-sample Kolmogorov-Smirnov (K-S) test, and the homogeneity of variance was assessed utilizing Levene’s test in SPSS software. For hens and breeder eggs data, intergroup statistical differences were evaluated utilizing Student’s t-test. When the assumption of homogeneity of variance between groups was violated, Welch’s correction was applied to adjust the t-test results. For the data on body weight, intestinal development, serum immune parameters, and the relative abundance of taxa in offspring chicks, a two-way ANOVA was conducted via the general linear model (GLM) to analyze the main effects (maternal β-carotene supplementation and age) and the interaction effect. If the interaction was significant and the data followed a normal distribution and exhibited homogeneous variance, they were analyzed applying one-way ANOVA. Conversely, data that deviated from normality or exhibited heterogeneous variance were analyzed applying Kruskal-Wallis tests. The findings were graphically represented using Prism 9 software and presented as measured values ± SEM or means ± SEM. Statistical significance was defined as P-values < 0.05 and is represented in the figures as: ns for P ≥ 0.05, * for P < 0.05, and ** for P < 0.01.