Characteristics of the Jejunal Microbiota in 35-Day-Old Saba and Landrace Piglets
Article Category: original-paper
Data publikacji: 08 wrz 2020
Zakres stron: 367 - 378
Otrzymano: 18 cze 2020
Przyjęty: 19 sie 2020
DOI: https://doi.org/10.33073/pjm-2020-041
Słowa kluczowe
© 2020 Huan Gao et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
The healthy creature intestine is home to microorganisms (Eckburg et al. 2005; Ley et al. 2006; Lozupone et al. 2012). Although microbiota resides in the intestines, it plays a critical role in the digestion and absorption of nutrients, maturation of immune system, anti-colonization, and stimulation of diverse host functions (Turnbaugh et al. 2006; Levy et al. 2017). The jejunum is a significant site for nutrient absorption (Martinez-Guryn et al. 2019). The jejunal microbiota is closely related to amino acid metabolism (Dai et al. 2010) and lipid deposition (Li et al. 2019).
Previous studies indicated that the host’s genetics shapes the microbial repertoire (Goodrich et al. 2014; Goodrich et al. 2016). It was discovered that the intestinal microbiota in exotic pig breeds varies from Chinese indigenous pig breeds (Yang et al. 2014). To further explore this observation, two pig breeds with different host genetics (Saba and Landrace) were selected as the subjects in this study. The Saba pig is an indigenous breed in Chuxiong of Yunnan Province, China, and it is on the list of National Conservation Program for Chinese Indigenous Livestock Germplasm. Saba pigs grow slow, but this breed is characteristic of a high propensity for meat quality, ability to adapt to the environment, and disease resistance (Jeong et al. 2014; Diao et al. 2019). In contrast, the Landrace breed was commercially selected over generations for rapid growth and enhanced carcass yield (Briggs 1983).
Before birth the intestine of newborns is believed to be free of microbes (Turnbaugh and Turnbaugh 2008). Due to contact with sows and exposure to the surrounding environment, a complex microbial community rapidly colonizes the newborn mammal (Frese et al. 2015). The balanced microbiological system (diverse intestinal microbes) is a significant hallmark of piglet health (Patil et al. 2019). Suckling piglets are an essential stage in the life of pigs, and thus more attention should be paid to the intestinal microbiota of piglets. The 35-day-old piglets easy to cause any diseases or dramatic internal environmental changes, they are about to wean; therefore, we selected 35-day-old Saba and Landrace suckling piglets as the subjects of this study. A comparison of their jejunal microbiota diversity will help comprehend the composition and functionality of gut microbiota in Chinese indigenous pigs.
The OTU profiling table and alpha/beta diversity were also achieved by Python scripts of QIIME. Alpha diversity was the species diversity in each sample, including community abundance (Chao1 index), the diversity (Shannon and Simpson index), the phylogenetic diversity index (PD whole tree), and coverage (Good’s coverage values). QIIME software was used to calculate the samples’ alpha diversity index based on the OTU results and to generate the corresponding dilution curve. The Bray-Curtis distance was calculated to estimate the dissimilarity in the community structure, which was visualized using principal coordinates analysis (PCoA). Analysis of similarities (ANOSIM) was performed in the Mothur v1.380. We determined the strength of these groups using multiresponse permutation procedures (MRPP). Both analyses were performed in the PC-ORD. In addition to
The linear discriminant analysis (LDA) effect size (LEfSe) method (
We used Spearman’s test to estimate the correlation between KEGG pathway and jejunal microbial composition and host growth performance.
Growth performance of Saba and Landrace piglets.
| SB | LA | |
|---|---|---|
| Birth weight (kg) | 0.76 ± 0.20B | 1.99 ± 0.14A |
| Body weight (kg) | 4.69 ± 1.14B | 10.22 ± 0.57A |
| Average daily gain (kg) | 0.11 ± 0.03B | 0.24 ± 0.02A |
| Stem length (cm) | 37.00 ± 4.84B | 50.00 ± 2.27A |
| Height at withers (cm) | 22.75 ± 1.83B | 29.00 ± 1.6A |
| Chest measurement (cm) | 37.88 ± 3.48B | 48.38 ± 1.41A |
| Chest depth (cm) | 10.75 ± 0.71B | 14.38 ± 2.07A |
| Abdominal girth (cm) | 38.75 ± 3.85B | 49.50 ± 1.60A |
| Cannon circumference (cm) | 8.06 ± 0.56B | 10.13 ± 0.35A |
Different superscripts in the same column indicate significant difference (
SB – Saba piglets, LA – Landrace piglets
Description of the assembly results of jejunum microbiota from piglets.
| Sample name | Clean Reads | Bases (bp) | Q20 (%) | Q30 (%) | GC (%) | Average length (bp) |
|---|---|---|---|---|---|---|
| LA-1 | 58278 | 24288798 | 0.9602 | 0.8873 | 0.5229 | 416 |
| LA-2 | 55975 | 23383577 | 0.9591 | 0.8855 | 0.5188 | 417 |
| LA-3 | 62396 | 25557801 | 0.9696 | 0.9081 | 0.527 | 409 |
| LA-4 | 56565 | 23778906 | 0.9614 | 0.8922 | 0.5365 | 420 |
| LA-5 | 55547 | 22929087 | 0.9671 | 0.9054 | 0.5221 | 412 |
| LA-6 | 56336 | 23469885 | 0.9612 | 0.8928 | 0.5234 | 416 |
| LA-7 | 57139 | 23771848 | 0.96 | 0.8902 | 0.5332 | 416 |
| SB-1 | 63393 | 26990140 | 0.9609 | 0.8917 | 0.5154 | 425 |
| SB-2 | 55959 | 22667528 | 0.9697 | 0.9119 | 0.5271 | 405 |
| SB-3 | 62484 | 25749078 | 0.9661 | 0.9035 | 0.5185 | 412 |
| SB-4 | 61527 | 26100871 | 0.9594 | 0.8867 | 0.5117 | 424 |
| SB-5 | 56907 | 24136231 | 0.9602 | 0.8897 | 0.5123 | 424 |
| SB-6 | 63409 | 26890638 | 0.958 | 0.8848 | 0.5496 | 424 |
| SB-7 | 58920 | 23815087 | 0.9656 | 0.9029 | 0.5306 | 404 |
| SB-8 | 60147 | 24637940 | 0.9626 | 0.8966 | 0.5253 | 409 |
SB – Saba piglets, LA – Landrace piglets
Fig. 1.
Venn diagram of OTUs clustered at 97% sequence identity of microbiotas from Saba and Landrace piglets. The number of overlapping parts is the total number of OTUs between the groups, while the numbers in non-overlapping parts indicate the number of unique OTUs for each group. SB – Saba piglets, LA – Landrace piglets.
Alpha diversity in jejunal microbiota between Saba and Landrace piglets.
| LA | SB | ||
|---|---|---|---|
| Chao1 index | 229.04 ± 38.23 | 242.85 ± 10.92 | 0.531 |
| The observed_species index | 124.75 ± 24.27 | 139.75 ± 36.57 | 0.520 |
| PD_whole_tree | 13.31 ± 1.98 | 17.76 ± 2.17 | 0.023 |
| Shannon index | 2.84 ± 0.42 | 3.06 ± 0.89 | 0.671 |
| Simpson index | 0.76 ± 0.077 | 0.79 ± 0.096 | 0.665 |
| Goods_coverage | 0.998 ± 0.00015 | 0.999 ± 0.00024 | 0.149 |
SB – Saba piglets, LA – Landrace piglets
MRPP of the 16S rRNA gene between Saba and Landrace piglets.
| A | Observe Delta | Expect Delta | Significance | |
|---|---|---|---|---|
| The weighted_unifrac | 0.0312699850241734 | 0.276682987004487 | 0.285614136784429 | 0.102 |
| The unweighted_unifrac | 0.0338353054352021 | 0.551581668259676 | 0.570898182641762 | 0.00 |
SB – Saba piglets, LA – Landrace piglets
Fig. 2.
Principal coordinate analysis (PCoA) illustrated bacterial community structures based on Bray-Curtis distances. On the PCoA plot, each color represents one group. Unweighted and weighted PCoA of β-diversity measures of all samples. PCOA1 (19.67%) and PCOA2 (13.63%).
Fig. 3.
Community composition of the jejunum microbial of Saba and Landrace piglets at the phylum (A) and genus (B) levels, respectively. Data are expressed as means + MSE, *
The abundance of bacterial species within jejunal taxa is shown in Fig. 3B. The two most abundant genera were
Fig. 4.
Alteration of the relative abundance of bacteria in the Saba and Landrace piglets using linear discriminant analysis effect size (LEfSe). Each bar represents the log 10 effect size (LDA score) for a specific taxon. A longer bar represents a higher LDA score. Only taxa meeting an LDA significant threshold of 2 are shown. These taxa showed a statistically significant difference between the Saba and Landrace piglets (
p – phylum, c – class, o – order, f – family, and g – genus.
Fig. 5.
A cladogram showed a comparison of the bacterial microbial profiles from Saba and Landrace piglets.
p – phylum, c – class, o – order, f – family, and g – genus.
Fig. 6.
Heatmap analysis of the correlation between microbiota and growth performance.
Fig. 7.
KEGG enrichment analysis of the difference within groups at the L3 hierarchy.
We used Spearman’s correlation heatmap (Fig. 8) to study the correlation between the jejunal microbiota and the KEGG pathway. The “d-glutamine and d-glutamate metabolism” pathway was positively correlated with the presence of Firmicutes (family,
Fig. 8.
Pearson’s correlation analysis of microorganisms and signal pathways in Saba and Landrace piglets. Heatmap analysis of the correlation between microorganisms and signal pathways. Correlations with
The Landrace pig from Denmark is a typical commercial pig breed of fast growth and high carcass yield (Briggs 1983). The previous research reported that the body weight of Landrace piglets was 1.68 kg and 6.52 kg on day 1 and day 27, respectively (Li et al. 2013). In contrast, the Saba pig is an indigenous breed from China, with a relatively slow growth rate. In our study, the birth weight, body weight (day 35), and average daily gain of Landrace piglets (1.99 kg, 10.22 kg, and 0.24 kg/d, respectively) were higher than those of Saba piglets (0.76 kg, 4.69 kg, and 0.11 kg/d, respectively). The data indicated that the growth performance of Landrace piglets was higher than Saba piglets. Previous studies have shown that growth performance and intestinal microbes were different in Jinhua pigs and Landrace pigs of the same age (Xiao et al. 2018). In addition, our results show that the
It is generally believed that intestinal microorganisms have abundant metabolic profiles to maintain their basic life and have a considerable impact on host growth and health (Turnbaugh et al. 2006). The accumulating evidence suggested that diet (Pluske 2013), environment (Thompson et al. 2008), and host’s genetics (Büsing and Zeyner 2015; Hancox et al. 2015) can affect the composition of intestinal microbiota. The previous research (Yang et al. 2014) revealed that the percentages of Firmicutes and Bacteroidetes in the Chinese indigenous pig breeds (Xiaomeishan, Meishan, and Bama sows) were higher than those of exotic breeds (Landrace, Yorkshire, and Duroc sows). It is consistent with our finding that the Firmicutes, Proteobacteria, and Bacteroidetes dominated in the jejunum of both pig breeds. Furthermore, the percentage of Bacteroidetes in Saba piglets was significantly higher than in Landrace piglets. Saba piglets are obese, and Landrace piglets are lean. A previous study has shown that fat deposition is positively correlated with the presence of Bacteroidetes and Firmicutes within the intestinal microbiota (Turnbaugh et al. 2006). Nevertheless, the mechanisms between intestinal microbiota and fat deposition are still unclear, and further study is needed.
At the genus level, the two most numerous genera in the pig’s small intestine were
Furthermore, some taxa are recognized as negatively correlated with host health.
The gut microbiome is beneficial for pigs, contributing to improved vitamin K production, cellulose fermentation, and increased resistance to pathogens (Kim and Isaacson 2015; Stokes 2017; Yang et al. 2017). PICRUSt analysis of jejunal microbiotas’ metabolic potential showed that metabolic pathways were significantly different in Saba and Landrace piglets. It is noteworthy that the different amino acid metabolism pathways were enriched in Saba and Landrace piglets. The “d-glutamine and d-glutamate metabolism” pathway was enriched in Saba piglets, while “tryptophan metabolism” pathway was enriched in Landrace piglets. The “d-glutamine and d-glutamate metabolism” pathway participates in the inhibition of lipid peroxidation and quenches free radicals during oxidative stress (Qu et al. 2020). It has been reported that tryptophan is related to the immune response regulation, inflammation, and oxidative stress (Anesi et al. 2019; Liu et al. 2019). Therefore, different pig breeds might have regulated their health through different metabolic pathways.
The central part of amino acids absorption is the small intestine (Wu 1998). The catabolism of arginine and lysine in the jejunum could exceed their transport into intestinal cells (Dai et al. 2010). This phenomenon may be due to the role of intestinal microorganisms. Besides, owing to the deficiency of several key enzymes, the threonine, tryptophan, histidine, lysine, and methionine cannot be metabolized by porcine intestinal cells in the presence of amino acids at physiological concentrations (Chen et al. 2007). However, the histidine, glutamate, threonine, and lysine were utilized by microbiota of the porcine small intestine (Dai et al. 2010). The above results indicated that jejunal microorganisms could participate in amino acid metabolism. In our study, the Firmicutes enriched in the jejunal microbiome of Saba piglets were positively associated with the “d-glutamine and d-glutamate metabolism” pathway. In contrast Fusobacteriaceae and
In summary, the growth performance was higher for Landrace piglets compared to Saba piglets due to their different genetic characteristics. The rich diversity and fewer infection-associated taxa were observed in Saba piglets, partially accounting for their high adaptability to environmental perturbations compared to Landrace piglets. Several taxa in the jejunum of Saba and Landrace piglets were associated with “d-glutamine and d-glutamate metabolism” and “tryptophan metabolism” respectively, suggesting that pig breeds may regulate their health through different metabolic pathways. Although the interaction between pig and microbiota needs further extensive investigations, our study would shed more light on the functional exploration and resource development of local pig intestinal microbiota in China.