Mapping short tandem repeats for liver gene expression traits helps prioritize potential causal variants for complex traits in pigs

Background Short tandem repeats (STRs) were recently found to have significant impacts on gene expression and diseases in humans, but their roles on gene expression and complex traits in pigs remain unexplored. This study investigates the effects of STRs on gene expression in liver tissues based on the whole-genome sequences and RNA-Seq data of a discovery cohort of 260 F6 individuals and a validation population of 296 F7 individuals from a heterogeneous population generated from crosses among eight pig breeds. Results We identified 5203 and 5868 significantly expression STRs (eSTRs, FDR < 1%) in the F6 and F7 populations, respectively, most of which could be reciprocally validated (π1 = 0.92). The eSTRs explained 27.5% of the cis-heritability of gene expression traits on average. We further identified 235 and 298 fine-mapped STRs through the Bayesian fine-mapping approach in the F6 and F7 pigs, respectively, which were significantly enriched in intron, ATAC peak, compartment A and H3K4me3 regions. We identified 20 fine-mapped STRs located in 100 kb windows upstream and downstream of published complex trait-associated SNPs, which colocalized with epigenetic markers such as H3K27ac and ATAC peaks. These included eSTR of the CLPB, PGLS, PSMD6 and DHDH genes, which are linked with genome-wide association study (GWAS) SNPs for blood-related traits, leg conformation, growth-related traits, and meat quality traits, respectively. Conclusions This study provides insights into the effects of STRs on gene expression traits. The identified eSTRs are valuable resources for prioritizing causal STRs for complex traits in pigs.

Dietary Coleus amboinicus Lour. decreases ruminal methanogenesis and biohydrogenation, and improves meat quality and fatty acid composition in longissimus thoracis muscle of lambs

Background Methane production and fatty acids (FA) biohydrogenation in the rumen are two main constraints in ruminant production causing environmental burden and reducing food product quality. Rumen functions can be modulated by the biologically active compounds (BACs) of plant origins as shown in several studies e.g. reduction in methane emission, modulation of FA composition with positive impact on the ruminant products. Coleus amboinicus Lour. (CAL) contains high concentration of polyphenols that may potentially reduce methane production and modulate ruminal biohydrogenation of unsaturated FA. This study aimed to investigate the effect of BAC of Coleus amboinicus Lour. (CAL) fed to growing lambs on ruminal methane production, biohydrogenation of unsaturated FA and meat characteristics. In this study, the in vitro experiment aiming at determining the most effective CAL dose for in vivo experiments was followed by two in vivo experiments in rumen-cannulated rams and growing lambs. Experiment 1 (RUSITEC) comprised of control and three experimental diets differing in CAL content (10%, 15%, and 20% of the total diet). The two in vivo experiments were conducted on six growing, rumen-cannulated lambs (Exp. 2) and 16 growing lambs (Exp. 3). Animals were assigned into the control (CON) and experimental (20% of CAL) groups. Several parameters were examined in vitro (pH, ammonia and VFA concentrations, protozoa, methanogens and select bacteria populations) and in vivo (methane production, digestibility, ruminal microorganism populations, meat quality, fatty acids profiles in rumen fluid and meat, transcript expression of 5 genes in meat). Results CAL lowered in vitro methane production by 51%. In the in vivo Exp. 3, CAL decreased methane production by 20% compared with the CON group, which corresponded to reduction of total methanogen counts by up to 28% in all experiments, notably Methanobacteriales. In Exp. 3, CAL increased or tended to increase populations of some rumen bacteria (Ruminococcus albus, Megasphaera elsdenii, Butyrivibrio proteoclasticus, and Butyrivibrio fibrisolvens). Dietary CAL suppressed the Holotricha population, but increased or tended to increase Entodiniomorpha population in vivo. An increase in the polyunsaturated fatty acid (PUFA) proportion in the rumen of lambs was noted in response to the CAL diet, which was mainly attributable to the increase in C18:3 cis-9 cis-12 cis-15 (LNA) proportion. CAL reduced the mRNA expression of four out of five genes investigated in meat (fatty acid synthase, stearoyl-CoA desaturase, lipoprotein lipase, and fatty acid desaturase 1). Conclusions Summarizing, polyphenols of CAL origin (20% in diet) mitigated ruminal methane production by inhibiting the methanogen communities. CAL supplementation also improved ruminal environment by modulating ruminal bacteria involved in fermentation and biohydrogenation of FA. Besides, CAL elevated the LNA concentration, which improved meat quality through increased deposition of n-3 PUFA.

Microbial and metabolomic mechanisms mediating the effects of dietary inulin and cellulose supplementation on porcine oocyte and uterine development

Background Dietary fiber (DF) is often eschewed in swine diet due to its anti-nutritional effects, but DF is attracting growing attention for its reproductive benefits. The objective of this study was to investigate the effects of DF intake level on oocyte maturation and uterine development, to determine the optimal DF intake for gilts, and gain microbial and metabolomic insight into the underlying mechanisms involved. Methods Seventy-six Landrace × Yorkshire (LY) crossbred replacement gilts of similar age (92.6 ± 0.6 d; mean ± standard deviation [SD]) and body weight (BW, 33.8 ± 3.9 kg; mean ± SD) were randomly allocated to 4 dietary treatment groups (n = 19); a basal diet without extra DF intake (DF 1.0), and 3 dietary groups ingesting an extra 50% (DF 1.5), 75% (DF 1.75), and 100% (DF 2.0) dietary fiber mixture consisting of inulin and cellulose (1:4). Oocyte maturation and uterine development were assessed on 19 d of the 2nd oestrous cycle. Microbial diversity of faecal samples was analysed by high-throughput pyrosequencing (16S rRNA) and blood samples were subjected to untargeted metabolomics. Results The rates of oocytes showing first polar bodies after in vitro maturation for 44 h and uterine development increased linearly with increasing DF intake; DF 1.75 gilts had a 19.8% faster oocyte maturation rate and a 48.9 cm longer uterus than DF 1.0 gilts (P < 0.05). Among the top 10 microbiota components at the phylum level, 8 increased linearly with increasing DF level, and the relative abundance of 30 of 53 microbiota components at the genus level (> 0.1%) increased linearly or quadratically with increasing DF intake. Untargeted metabolic analysis revealed significant changes in serum metabolites that were closely associated with microbiota, including serotonin, a gut-derived signal that stimulates oocyte maturation. Conclusions The findings provide evidence of the benefits of increased DF intake by supplementing inulin and cellulose on oocyte maturation and uterine development in gilts, and new microbial and metabolomic insight into the mechanisms mediating the effects of DF on reproductive performance of replacement gilts.

Supplementation of oligosaccharide-based polymer enhanced growth and disease resistance of weaned pigs by modulating intestinal integrity and systemic immunity

Background There is a great demand for antibiotic alternatives to maintain animal health and productivity. The objective of this experiment was to determine the efficacy of dietary supplementation of a blood group A6 type 1 antigen oligosaccharides-based polymer (Coligo) on growth performance, diarrhea severity, intestinal health, and systemic immunity of weaned pigs experimentally infected with an enterotoxigenic Escherichia coli (ETEC), when compared with antibiotics. Results Pigs in antibiotic carbadox or Coligo treatment groups had greater (P < 0.05) body weight on d 5 or d 11 post-inoculation (PI) than pigs in the control group, respectively. Supplementation of antibiotics or Coligo enhanced (P < 0.05) feed efficiency from d 0 to 5 PI and reduced (P < 0.05) frequency of diarrhea throughout the experiment, compared with pigs in the control group. Supplementation of antibiotics reduced (P < 0.05) fecal β-hemolytic coliforms on d 2, 5, and 8 PI. Pigs in antibiotics or Coligo groups had reduced (P < 0.05) neutrophil counts and serum haptoglobin concentration compared to pigs in the control group on d 2 and 5 PI. Pigs in Coligo had reduced (P < 0.05) total coliforms in mesenteric lymph nodes on d 5 and 11 PI, whereas pigs in antibiotics or Coligo groups had reduced (P < 0.05) total coliforms in spleen on d 11 PI compared with pigs in the control group. On d 5 PI, pigs in the Coligo group had greater (P < 0.05) gene expression of ZO1 in jejunal mucosa, but less (P < 0.05) mRNA expression of IL1B, IL6, and TNF in ileal mucosa, in comparison with pigs in the control group. Supplementation of antibiotics enhanced (P < 0.05) the gene expression of OCLN in jejunal mucosa but decreased (P < 0.05) IL1B and IL6 gene expression in ileal mucosa, compared with the control. On d 11 PI, supplementation of antibiotics or Coligo up-regulated (P < 0.05) gene expression of CLDN1 in jejunal mucosa, but Coligo reduced (P < 0.05) IL6 gene expression in ileal mucosa compared to pigs in the control group. Conclusions Supplementation of Coligo improved growth performance, alleviated diarrhea severity, and enhanced gut health in weaned pigs infected with ETEC F18 in a manner similar to in-feed antibiotics.

Effect of Bacillus amyloliquefaciens and Bacillus subtilis on fermentation, dynamics of bacterial community and their functional shifts of whole-plant corn silage

Background Bacillus amyloliquefaciens (BA) and Bacillus subtilis (BS) are usually used as feed supplements directly or bacterial inoculants in biological feeds for animals. However, few research have reported the effects of BA and BS on fermentation characteristics and bacterial community successions of whole-plant corn silage during ensiling. If the BA and BS inoculants have positive effects on silages, then they could not only improve fermentation characteristics, but also deliver BA or BS viable cells to ruminants, which would play its probiotic effect. Therefore, the objectives of this study were to investigate the effects of BA and BS on the fermentation, chemical characteristics, bacterial community and their metabolic pathway of whole-plant corn silage. Results Freshly chopped whole-plant corn was inoculated without or with BA and BS, respectively, and ensiled for 1, 3, 7, 14 and 60 d. Results showed that BA and BS inoculations increased lactic acid concentrations of whole-plant corn silages compared with control, and BA inoculation decreased acetic acid concentrations, whereas BS inoculation decreased fiber contents and increased crude protein (CP) content. Higher water-soluble carbohydrate contents and lower starch contents were observed in BA- and BS-inoculated silages compared with that in control. The decreased CP content and increased non-protein nitrogen content were observed in BA-inoculated silage, which was consistent with the higher amino acid metabolism abundances observed in BA-inoculated silage. In addition, it was noteworthy that BA and BS inoculations increased the metabolism of cofactors and vitamins, and decreased the relative abundances of drug resistance: antimicrobial pathways. We also found that the bacterial metabolism pathways were clearly separated into three clusters based on the ensiling times of whole-plant corn silage in the present study. There were no significant differences in bacterial community compositions among the three groups during ensiling. However, BA and BS inoculations decreased the relative abundances of undesirable bacteria such as Acetobacter and Acinetobacter. Conclusion Our findings suggested that the BS strain was more suitable as silage inoculants than the BA strain in whole-plant corn silage in this study.

Role of milk carbohydrates in intestinal health of nursery pigs: a review

Intestinal health is essential for the resistance to enteric diseases and for nutrient digestion and absorption to support growth. The intestine of nursery pigs are immature and vulnerable to external challenges, which cause negative impacts on the structure and function of the intestine. Among nutritional interventions, the benefits of milk are significant for the intestinal health of pigs. Milk coproducts have traditionally been used in starter feeds to improve the growth of nursery pigs, but their use is somewhat limited due to the high costs and potential risks of excessive lactose on the intestine. Thus, understanding a proper feeding level of milk carbohydrates is an important start of the feeding strategy. For nursery pigs, lactose is considered a highly digestible energy source compared with plant-based starch, whereas milk oligosaccharides are considered bioactive compounds modulating intestinal immunity and microbiota. Therefore, milk carbohydrates, mainly composed of lactose and oligosaccharides, have essential roles in the intestinal development and functions of nursery pigs. The proper feeding levels of lactose in starter feeds could be variable by weaning age, body weight, or genetic lines. Effects of lactose and milk oligosaccharides have been broadly studied in human health and animal production. Therefore, this review focuses on the mechanisms of lactose and milk oligosaccharides affecting intestinal maturation and functions through modulation of enterocyte proliferation, intestinal immunity, and intestinal microbiota of nursery pigs.

Alterations in intestinal microbiota composition coincide with impaired intestinal morphology and dysfunctional ileal immune response in growing-finishing pigs under constant chronic heat stress

Background Previous studies had shown that short-term acute heat stress (HS) affected the host’s metabolism and intestinal microbiota independent of feed intake (FI) reduction, and long-term calorie restriction caused intestinal morphological injuries and gut microbial alterations. However, research on the effects of constant chronic HS on intestinal microbial composition and the roles of FI reduction played in is limited. This study aimed to investigate the effects of 7-day constant chronic HS on the composition of intestinal microbes in growing-finishing pigs, and its relationship with pigs’ performance, intestinal morphology, and ileal immune response. Twenty-four growing-finishing pigs (Duroc × Large White × Landrace, 30 ± 1 kg body weight) were randomly assigned to three treatments (n = 8), 1) thermal neutral (TN) conditions (25 ± 1 °C) with ad libitum FI, 2) HS conditions (35 ± 1 °C) with ad libitum FI, 3) pair-fed (PF) with HS under TN conditions to discriminate the confounding effects of dissimilar FI, and the FI was the previous day’s average FI of HS. The small intestinal segments (duodenum, jejunum, and ileum) and feces were collected on d 8. Results Results indicated that HS drastically declined (P < 0.05) average daily gain (ADG) and average daily feed intake (ADFI) (about 61%) in comparison with TN, and caused hyperpyrexia, meanwhile PF caused hypothermia. Morphological observation by light and electron microscopes showed that both HS and PF treatment decreased (P < 0.05) the villus and microvillus height compared with TN. Additionally, HS increased (P < 0.05) protein expression of heat shock protein 70 in the duodenum, jejunum, and ileum. Furthermore, the expression of tight junction protein zonula occluden-1 (ZO-1) in the duodenum and ileum, and Occludin in the ileum were enhanced (P < 0.05) compared with TN and PF. Moreover, HS significantly enhanced (P < 0.05) the mRNA relative expression of inflammatory cytokines (TLR-2, TLR-4, and tumor necrosis factor-α (TNF-α), IL-6, IL-8, PG1–5, β-defensin 2 (pBD-2)), mucins (mucin-1 and mucin-2) and P65 protein level in the ileal mucosa tissue. Intestinal microbiota analysis by 16S rRNA sequencing showed lower (P < 0.10) α diversity in both HS and PF, and a separated cluster of β diversity among groups. Compared with TN, HS but not PF mainly reduced (FDR < 0.05) Bacteroidetes (phylum), Bacteroidia (class) and elevated the proportions of Proteobacteria (phylum, FDR < 0.05), Bacillales (order, FDR < 0.05), Planococcaceae (family, FDR < 0.05), Kurthia (genus, FDR < 0.05), Streptococcaceae (family, FDR < 0.10) and Streptococcus (genus, FDR < 0.10). Notably, Lactobacillales (order) was decreased (FDR < 0.05) by PF alone. Furthermore, the Spearman correlation analysis indicated that the microbes prevalent in HS were positively (P < 0.05) associated with intestinal morphological injuries indicators and ileal immune response parameters, and the microbes reduced in HS were negatively (P < 0.05) with the performance data. Conclusions Intestinal morphological injuries and ileal immune response caused by constant chronic HS independent of FI showed close connections with alterations in intestinal microbiota in growing-finishing pigs.

Single-cell RNA-sequencing reveals the dynamic process and novel markers in porcine spermatogenesis

Background Spermatogenesis is the process by which male gametes are formed from spermatogonial stem cells and it is essential for the reliable transmission of genetic information between generations. To date, the dynamic transcriptional changes of defined populations of male germ cells in pigs have not been reported. Results To characterize the atlas of porcine spermatogenesis, we profiled the transcriptomes of ~ 16,966 testicular cells from a 150-day-old pig testis through single-cell RNA-sequencing (scRNA-seq). The scRNA-seq analysis identified spermatogonia, spermatocytes, spermatids and three somatic cell types in porcine testes. The functional enrichment analysis demonstrated that these cell types played diverse roles in porcine spermatogenesis. The accuracy of the defined porcine germ cell types was further validated by comparing the data from scRNA-seq with those from bulk RNA-seq. Since we delineated four distinct spermatogonial subsets, we further identified CD99 and PODXL2 as novel cell surface markers for undifferentiated and differentiating spermatogonia, respectively. Conclusions The present study has for the first time analyzed the transcriptome of male germ cells and somatic cells in porcine testes through scRNA-seq. Four subsets of spermatogonia were identified and two novel cell surface markers were discovered, which would be helpful for studies on spermatogonial differentiation in pigs. The datasets offer valuable information on porcine spermatogenesis, and pave the way for identification of key molecular markers involved in development of male germ cells.

Soy saponin improves egg-laying performance and immune function of laying hens

Background Soy saponin (SS), an active compound in soybean meals, has been widely studied in the medical field. However, it was considered as an anti-nutritional factor in poultry diets. The objective of this experiment was to measure the effects of dietary SS using three dietary treatments on egg-laying performance and immune function of laying hens. Birds were fed a low soybean meal basal diet (CON), a low-SS diet (50 SS) containing 50 mg/kg SS, or a high-SS diet (500 SS) containing 500 mg/kg SS for 10 weeks. At the end of the 5th and 10th week of the trial, samples were collected for analysis. Results Results showed that with 50 mg/kg SS supplementation, the egg production rate, feed conversion ratio (FCR), and eggshell quality tended to be improved. Serum follicle stimulating hormone (FSH) and Interleukin-4 (IL-4) levels were also elevated as well as the peripheral blood LPS stimulation index, the proportion of B lymphocytes, and antibody titer of bovine serum albumin (BSA). We also found that mRNA levels of follicle stimulating hormone receptor (FSHR) in ovarian, nuclear transcription factor kappa B (NF-κB), Transforming growth factor (TGF-β) and interferon γ (IFN-γ) in spleen were up-regulated at the end of the trial. Additionally, dietary 50 mg/kg SS improved the ileal flora via up-regulating the relative abundance of Lactobacillus, Romboutsia and Lactobacillus delbrueckii. Although the immune related indicators were improved with 500 mg/kg SS supplemented, it seemed to have a negative influence on the laying-performance. Specifically, serum alanine aminotransferase (ALT), alkaline phosphatase (ALP), and the ratio of IFN-γ to IL-4 were increased in the 500 SS group at the end of the trial. The mRNA levels of gonadotropin releasing hormone 1 (GnRH1) in Hypothalamus, the estrogen related receptor (ERR) in ovaries were downregulated as well as the egg production rate during the trial with 500 mg/kg SS supplemented. Conclusions The egg production performance was improved by dietary supplemented with 50 mg/kg SS via increasing ovarian FSHR transcription level and serum estrogen level. A beneficial shift in intestinal microflora was recorded, and the immune function of laying hens was also improved with 50 mg/kg SS supplementation. Surprisingly, the long-term supplementation of 500 mg/kg SS exerted a negative impact on the laying performance and physiological functions of the liver of laying hens.

Lovastatin as a supplement to mitigate rumen methanogenesis: an overview

Methane from enteric fermentation is the gas with the greatest environmental impact emitted by ruminants. Lovastatin (Lv) addition to feedstocks could be a strategy to mitigate rumen methane emissions via decreasing the population of methanogenic archaea (MA). Thus, this paper provides the first overview of the effects of Lv supplementation, focusing on the inhibition of methane production, rumen microbiota, and ruminal fermentation. Results indicated that Lv treatment had a strong anti-methanogenic effect on pure strains of MA. However, there are uncertainties from in vitro rumen fermentation trials with complex substrates and rumen inoculum.Solid-state fermentation (SSF) has emerged as a cost-effective option to produce Lv. In this way, SSF of agricultural residues as an Lv-carrier supplement in sheep and goats demonstrated a consistent decrease in ruminal methane emissions. The experimental evidence for in vitro conditions showed that Lv did not affect the volatile fatty acids (VFA). However, in vivo experiments demonstrated that the production of VFA was decreased. Lv did not negatively affect the digestibility of dry matter during in vitro and in vivo methods, and there is even evidence that it can induce an increase in digestibility. Regarding the rumen microbiota, populations of MA were reduced, and no differences were detected in alpha and beta diversity associated with Lv treatment. However, some changes in the relative abundance of the microbiota were induced. Further studies are recommended on: (i) Lv biodegradation products and stability, as well as its adsorption onto the solid matter in the rumen, to gain more insight on the “available” or effective Lv concentration; and (ii) to determine whether the effect of Lv on ruminal fermentation also depends on the feed composition and different ruminants.