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Poult Sci
Poult Sci
Poultry Science
0032-5791
1525-3171
Elsevier

S0032-5791(24)00760-0
10.1016/j.psj.2024.104181
104181
MICROBIOLOGY AND FOOD SAFETY
Research Note: Correlation between the reproductive tract microbiota and speckled eggs in laying hens
Cheng Xue *
Wei Yimin *
Liu Yuchen *
Ma Ying *
Zhang Yalan *
Li Wen *
Luo Yuxing *
Yan Wenliang †
Qu Lujiang ⁎
Ning Zhonghua ningzhh@cau.edu.cn
⁎1
⁎ National Engineering Laboratory for Animal Breeding, Department of Animal Genetics and Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China
† Huayu Agricultural Science and Technology Co. Ltd., Handan 057300, China
1 Corresponding author: ningzhh@cau.edu.cn
08 8 2024
11 2024
08 8 2024
103 11 1041816 5 2024
1 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
The microbiomes of the reproductive tract play a crucial role in the egg production and quality and reproductive health of laying hens. Speckled eggs are characterized by shells with brown spots of varying sizes and commonly produced by brown-shelled laying hens. Speckles reduce the economic value of eggs. However, the relationship between oviduct and cloacal microbiomes and the presence of speckled eggs in laying hens remains unclear. In this study, we collected samples from the reproductive tracts (uterus, vagina, and cloaca) of hens laying speckled eggs and those laying normal eggs and compared their microbial structures and relative abundances through 16S rRNA sequencing. We found that the microbial community structure in the reproductive tracts of the hens laying speckled eggs was similar to that in the reproductive tracts of the hens laying normal eggs; however, the relative abundances of Clostridium in the uterus and Turicibacter and Gallibacterium in the vagina of the hens from the speckled group (7.27%, 6.83% and 0.10%, respectively) were significantly higher than those in the normal group (2.00%, 0% and 0%, respectively [P < 0.05]). Additionally, 8, 24, and 11 bacterial taxa in the uterus, vagina, and cloaca were different between the groups of hens laying speckled and normal eggs. At the same time, Clostridium in the uterus may be associated with eggshell speckles. However, further investigations are necessary to understand the functions of these microbiota in the reproductive tracts of laying hens. This study provides novel insights into methods for reducing the occurrence of speckled eggs in laying hens.

Key words

speckled egg
reproductive tract
microbiota
laying hen
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pmcINTRODUCTION

Eggs play a pivotal role in the human diet because of their high protein content and affordability. The shells of speckled chicken eggs are adorned with reddish-brown spots of varying sizes. These distinctive speckles are due to the larger amounts of protoporphyrin IX pigment deposited between the cuticle and vertical crystal layers of the eggshell than that in adjacent areas; they do not affect eggshell strength and thickness but considerably affect eggshell outer aesthetics, thereby reducing the economic value of eggs (Cheng et al., 2023).

The microbiome composition of the reproductive tract is similar to that of the gut because of the physiological structure of chickens (Shterzer et al., 2020). Reproductive tract microbes play a key role in the health and productivity of laying hens (Su et al., 2021; Wen et al., 2021). Eggshell development, cuticle formation, and egg expulsion occur in the uterus, vagina, and cloaca, respectively. However, the correlation between speckled eggs and chicken reproductive tract microbiota is unclear. Therefore, in the present study, we examined the microbial composition of the uterus, vagina, and cloaca of hens laying speckled and normal eggs via 16S rRNA sequencing. The study aimed to determine the influence of reproductive tract microbiota on speckled eggs and provide novel insights into methods for reducing the laying of speckled eggs.

MATERIALS AND METHODS

Ethics Statement

The study was conducted in accordance with the guidelines for experimental animals established by the Animal Care and Use Committee of China Agricultural University, China.

Animals and Sample Collection

A total of 2000 Island White hens (40 wk old) laying brown shell eggs provided by Huayu Agricultural Science and Technology Co., Ltd., China were used in this study. All hens were raised in individual cages placed in an enclosed chicken house with a 16:8 h light: dark cycle. The temperature and relative humidity of the chicken house were maintained at 18 ± 1 °C and 55 ± 5%, respectively. The animals had ad libitum access to water and food. We recorded the eggshell traits (normal or speckle) for 5 consecutive d (Figure 1A) (Cheng et al., 2023). Eight hens that laid normal eggs and 8 hens that laid speckled eggs for 5 consecutive d were selected and assigned to the normal and speckle groups, respectively. Cotton swabs were used to collect microbiota from the cloaca. Subsequently, the hens were euthanized via cervical dislocation, and the mucosa of the uterus and vagina was collected and immediately stored in liquid nitrogen at −80 °C.Figure 1 Results of 16S rRNA sequencing analysis. (A) Normal and speckled eggs. (B) Principal coordinate analysis (PCoA) considering operational taxonomic unit level in the cloaca, vagina, and uterus samples of the hens in the speckle and normal groups. (C) Linear discriminant analysis score in the uterus, vagina, and cloaca between the normal and speckle groups.

Figure 1

DNA Extraction and 16S rRNA Sequencing

Microbial genomic DNA was extracted from the samples using the QIAamp 96 PowerFecal QIAcube HT kit (Cat No.51531 [Qiagen, Hilden, Germany]) following the manufacturer's instructions. Agarose gel electrophoresis was performed to measure DNA concentration and purity. The bacterial 16sV3-V4 region was amplified using qualified DNA as the substrate and 338F (5′-ACTCCTACGGGAGGCAGCA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) primers through polymerase chain reaction (PCR). PCR amplification was performed as follows: denaturation at 98 °C for 5 min, 25 cycles of 98 °C for 30 s, 53 °C for 30 s, 53 °C for 30 s, and elongation at 72 °C for 5 min. The purified amplification products were combined into equal molar concentrations and sequenced on an Illumina MiSeq PE300 platform (Illumina, San Diego, CA) following standard protocols.

Statistical Analysis

Raw reads were filtered using the Trimmomatic software (Github, San Francisco, CA). Qualified reads were assembled using FLASH software (Github). Representative sequences of operational taxonomic units (OTU) were obtained by clustering with 97% similarity using UPARSE. The annotation of representative sequences of OTUs was analyzed using the RDP classifier (v2.2) software (Github), considering the Green-Genes database with a confidence threshold of 0.6. Alpha diversity was calculated using MOTHUR (v1.31.2 [Github]), and the corresponding dilution curve was constructed using R software (v3.1.1 [Rstudio, Boston, MA]). Linear discriminant analysis (LDA) effect size (LEfSe) was used to identify key bacterial taxa. An LDA > 2 and P < 0.05 were considered significant. Differential analysis was performed using the Kruskal–Wallis test. Principal coordinate analysis (PCoA) was conducted using quantitative insights into microbial ecology (v1.80 [University of Colorado, Denver, CO]). Graphical visualization was performed using the R software (Rstudio).

RESULTS AND DISCUSSION

Descriptive Characteristics of Sequencing Data

A total of 48 samples of the uterus, vagina, and cloaca were collected from the hens in the normal and speckle groups. However, vagina samples from 2 chickens in the normal group failed to be collected. Thus, 46 samples were used in subsequent 16S rRNA sequencing. After quality control, the species accumulation curve revealed that as the sample size increased, the number of OTUs detected stabilized, indicating that the sample sequencing depth was reasonable and could be used for subsequent analyses (results not shown).

Comparison and Differences of Microbial Community Diversity

Alpha diversity primarily reflects the richness and evenness of intestinal flora. The ACE, Chao1, Simpson, and Shannon indices had no significant differences in alpha diversity across the different parts of the reproductive tract of layers of the speckle and normal groups (results not shown). Beta diversity compares the differences between samples from different treatment groups using PCoA. The cloaca was distinctly separated from the vagina and uterus; however, the uterus and vagina overlapped to a large extent (Figure 1B), which is consistent with the results of previous studies (Su et al., 2021; Wen et al., 2021). Individuals laying speckled and normal eggs could not be separated by PCoA based on OTU analysis (Figure 1B), indicating that the microbial community structure of the hens in the speckle group was similar to that of the hens in the normal group.

Firmicutes, Proteobacteria, and Actinobacteria were the dominant phyla across the different parts of the reproductive tracts of the hens in both groups. No significant difference in the relative abundance of reproductive tract microbial communities was found between the hens from the 2 groups at the phylum level (results not shown).

The microbial community structure and abundance in the reproductive tracts at the genus level of the hens in the speckle and normal groups are presented in Table 1. The top 15 genera with relative abundance of microorganisms were annotated. Lactobacillus, SMB53, and Peptoniphilus were the dominant bacterial genera. The relative abundance of Clostridium in the uterus of the hens in the speckle group (7.27%) was significantly higher than that of the hens in the normal group (2.00% [P < 0.05]); however, the relative abundances of Turicibacter and Gallibacterium in the vagina of the hens in the speckle group (6.83% and 0.10%, respectively) were significantly higher than that in the normal group (0% and 0%, respectively [P < 0.05]). In the cloaca, no significant differences were found among the top 15 genera, suggesting that the bacterial genera in the cloaca may not be associated with the formation of speckle eggs. Turicibacter is an anaerobic Gram-positive bacterium that participates in overall immune activation, exerts a pro-inflammatory effect, and increases in abundance during enteritis (Lin et al., 2023). However, its function in the reproductive tract and effect on speckled eggs warrant further investigation. Gallibacterium anatis causes respiratory diseases, apoptosis of oviduct epithelial cells, villus shedding, salpingitis, and reduced egg production in chickens (El-Adawy et al., 2018). Thus, Gallibacterium in the vagina may affect the health of the hens. Previous studies have shown that eggshell speckles form in the uterus. Speckled eggs in the uterus are excreted through the vagina and cloaca. Thus, the microorganisms in the uterus possibly affected the microorganisms in the vagina. With artificial insemination, microorganisms can travel from the cloaca to the vagina and uterus, consequently altering the microorganisms in the oviduct. Therefore, the relationship between these microorganisms and speckle eggs requires further exploration.Table 1 Microbial composition of the cloaca, uterus, and vagina at the genus levels (%) in the normal and speckle groups.

Table 1	Cloaca	Uterus	Vagina	
	Normal	Speckle	P-value	Normal	Speckle	P value	Normal	Speckle	P-value	
Lactobacillus	11.83	20.94	0.50	26.90	30.76	0.72	27.45	32.58	0.95	
SMB53	4.01	3.95	1.00	19.05	21.18	0.57	16.66	14.99	0.57	
Peptoniphilus	25.10	20.71	0.65	0.95	1.02	0.34	0.36	0.97	0.49	
Pseudomonas	0.11	0.17	0.44	1.16	0.23	0.38	28.32	0.18	0.28	
Clostridium	2.82	1.89	0.33	2.00b	7.27a	0.01	3.43	8.68	0.14	
Corynebacterium	6.72	5.87	0.96	2.58	3.39	0.96	0.89	4.62	0.06	
Enterococcus	0.96	5.86	0.80	0.80	6.60	0.28	0.71	3.21	0.14	
Ruminococcus	1.89	3.34	0.38	3.59	2.29	0.65	3.96	2.45	0.57	
Turicibacter	3.21	5.75	0.33	0.02	0.03	0.63	0.00b	6.83a	0.01	
Gallibacterium	4.79	8.12	0.28	0.00	0.01	0.40	0.00b	0.10a	0.01	
Faecalibacterium	0.71	1.32	0.28	2.96	0.97	0.19	2.74	1.03	0.34	
Streptococcus	3.30	1.62	0.44	2.62	0.52	0.44	0.20	0.28	0.41	
Actinomyces	2.10	2.86	0.33	0.43	1.55	1.00	0.06	1.23	0.11	
Helcococcus	4.55	2.55	0.28	0.20	0.14	0.87	0.07	0.34	0.61	
a,b Different superscripts within a row means significantly different (P < 0.05).

Linear discriminant analysis effect size and linear discriminant analysis were conducted to identify the bacterial taxa acting as a biomarker between the hens laying normal and speckled eggs. Results revealed that 8, 24, and 11 bacterial taxa in the uterus, vagina, and cloaca, respectively, were significantly different between the normal and speckle groups (Figure 1C). Eggshell speckles were composed of protoporphyrin IX and were deposited in the uterus. Porphyromonas and Prevotella can synthesize protoporphyrin IX (Smalley et al., 2000; Smalley et al., 2003). In the present study, we identified Clostridium as a marker genus in the uterus that was significantly different between the 2 groups. Clostridium can also synthesize uroporphyrinogen III in the uterus, the precursor of protoporphyrin IX (Chen and Friedmann, 1993). Therefore, our results suggest that Clostridium in the uterus may be involved in the formation of eggshell speckles by influencing pigment synthesis.

In summary, our study revealed that the relative abundances of Clostridium in the uterus and Turicibacter and Gallibacterium in the vagina of the hens laying speckled eggs were significantly higher than those in the normal hens. The abundance of Clostridium in the uterus of the speckled group may be involved in the formation of eggshell speckles. However, the effect of these microorganisms on reproductive traits needs further investigation. The findings of this study provide insights into the mechanisms involved in the laying of speckled eggs and provide primary information for reducing speckled eggs in poultry.

DISCLOSURES

No conflict of interest exists in the submission of this manuscript, and manuscript is approved by all authors for publication. On behalf of all the authors, I declare that this paper is original and none of the material in the paper has been published or is under consideration for publication elsewhere. All the authors listed have read the manuscript and approved the submission of the paper to your journal.

ACKNOWLEDGMENTS

We are grateful to Huayu Agricultural Science and Technology Co., Ltd. for providing the laying hens. This work was supported by the China Agriculture Research System (grant number CARS-40 ) and Beijing Agriculture Innovation Consortium (BAIC06-2024 ).
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