Genotyping by sequencing reveals a new locus for pig teat number

Teat number is an extremely valuable trait for sow reproduction performance and piglet survival. Here, we used genotyping‐by‐sequencing and a general liner model to carry out genome‐wide analysis of sow total teat number in an Erhualian population. The results indicated that eight SNPs on chromosomes 4, 5, 9 and 10 were significant genome wide (Bonferroni method, P < 2.85E‐7) for sow total teat number. Validation analyses were performed in 298 Erhualian and 904 Large White sows using these significant SNPs and the general liner model procedure in sas . Finally, only the SNP on chromosome 5 was found to be significantly associated with sow teat number in both populations. The GG genotype individuals had 2.23 and 0.82 more teat numbers respectively than did the AA genotype individuals in these two populations (P < 0.05). According to the expression and annotation analyses, we inferred the presence of a gene or lincRNA that could affect teat number by regulating other genes and ultimately affecting the mammogenesis of pigs. Further studies using methods such as Cas9 editing and gene silencing analysis are necessary for additional function analysis.     

Identification of new single nucleotide polymorphisms affecting total number born and candidate genes related to ovulation rate in Chinese Erhualian pigs

The Chinese Erhualian pig has the highest record for litter size in the world. However, the genetic mechanism of its high prolificacy remains poorly understood. In our study, large phenotypic variations in litter size were found among Erhualian sows. Significant differences in total number born (TNB) and corpora lutea numbers were observed between sows with high and low estimated breeding values (EBVs) for TNB. To identify single nucleotide polymorphisms (SNPs) associated with TNB, a selective genomic scan was conducted on 18 sows representing the top 10% and 18 sows representing the bottom 10% of EBVs of 177 sows using Illumina Porcine SNP60 genotype data. Genome‐wide fixation coefficient (F_ST) values were calculated for each SNP between the high‐ and low‐EBV groups. A total of 154 SNPs were significantly differentiated loci between the two groups. Of the top 10 highest F_ST SNPs, rs81399474, rs81400131 and rs81405013 on SSC8 and rs81434499 and rs81434489 on SSC 12 corresponded to previously reported QTL for litter size. The other five SNPs, rs81367039 on SSC2, rs80891106 on SSC7, rs81477883 on SSC12 and rs80938898 and rs80971725 on SSC14, appeared to be novel QTL for TNB. Significant associations between rs81399474 on SSC8 and TNB were confirmed in 313 Erhualian sows. Forty genes were identified around the top 10 highest F_ST SNPs, of which UCHL1, adjacent to rs81399474, and RPS6KB1 and CLTC, adjacent to rs81434499, have been reported to affect the ovulation rate in pig. The findings can advance understanding of the genetic variations in litter size of pigs.     

Expression of tissue inhibitor of metalloproteinases-3 messenger RNA and protein in porcine endometrium during implantation

Recent evidence points to a stromal decidualization-like response in the pregnant porcine uterus. The objective of this study was to evaluate expression of tissue inhibitors of metalloproteinase-3 (TIMP-3), a sensitive indicator of endometrial stromal decidualization, in endometrium of pregnant sows and to further investigate this phenomenon. Real-time PCR, Western blot and immunostaining analysis were used to study TIMP-3 expression between/at attachment sites of endometrium of Days 13, 18 and 24 pregnant sows. The results indicate that TIMP-3 protein expression was lowest by Day 13 compared with Day 18 (P < 0.01) and 24 (P < 0.01), and the expression was higher at attachment sites than between attachment sites on Day 13 (P < 0.01) and 18 (P < 0.01). TIMP-3 intensive immunostaining was observed in stroma of endometrium on Days 13, 18 and 24, and the staining at attachment sites was stronger than between attachment sites. Collectively, these results suggest the crucial role of TIMP-3 in successful implantation and embryo survival and indicate the endometrial stromal decidualization-like in pigs.     

Expression of Eph A molecules during swine embryo implantation

Eph–Ephrin system can induce repulsive forces in cell migration and adhesion during embryonic development in various mammals. In this study, the attachment sites of swine endometrium during pregnancy were used and the physiological role of this system in the step of mammalian embryo implantation was estimated to investigate the involvement of the Eph–Ephrin system in swine embryo implantation. Real-time quantitative PCR indicated that mRNA expression of Eph A1 on endometrium increased extremely significantly around the implantation period (P < 0.01), while expression of Eph A2 and A4 decreased significantly during this period (P < 0.05). Immunostaining showed that protein expression of Eph A1, A2 and A4 in the endometrial stroma underlying the luminal epithelium was higher during mid-implantation compared with early or post-implantation. Western blotting examination demonstrated that protein expression of Eph A1, A2 and A4 at the attachment sites of swine endometrium increased from pregnancy day 13 to 18 (P < 0.01), and then decreased from pregnancy day 18 to 24 (P < 0.01). These findings suggest that the Eph–Ephrin A system might play an important role in regulating the swine contact between blastocysts and endometrium during embryo implantation.     

Commensal and pathogenic biofilms differently modulate peri‐implant oral mucosa in an organotypic model

The impact of oral commensal and pathogenic bacteria on peri‐implant mucosa is not well understood, despite the high prevalence of peri‐implant infections. Hence, we investigated responses of the peri‐implant mucosa to Streptococcus oralis or Aggregatibacter actinomycetemcomitans biofilms using a novel in vitro peri‐implant mucosa‐biofilm model. Our 3D model combined three components, organotypic oral mucosa, implant material, and oral biofilm, with structural assembly close to native situation. S. oralis induced a protective stress response in the peri‐implant mucosa through upregulation of heat shock protein (HSP70) genes. Attenuated inflammatory response was indicated by reduced cytokine levels of interleukin‐6 (IL‐6), interleukin‐8 (CXCL8), and monocyte chemoattractant protein‐1 (CCL2). The inflammatory balance was preserved through increased levels of tumor necrosis factor‐alpha (TNF‐α). A. actinomycetemcomitans induced downregulation of genes important for cell survival and host inflammatory response. The reduced cytokine levels of chemokine ligand 1 (CXCL1), CXCL8, and CCL2 also indicated a diminished inflammatory response. The induced immune balance by S. oralis may support oral health, whereas the reduced inflammatory response to A. actinomycetemcomitans may provide colonisation advantage and facilitate later tissue invasion. The comprehensive characterisation of peri‐implant mucosa‐biofilm interactions using our 3D model can provide new knowledge to improve strategies for prevention and therapy of peri‐implant disease.     

Regulation of uterine genes during the peri‐implantation period,and its relationship to the maternal brain in gestating mice

We conducted an integrated analysis of gene expression and chromatin structure of mouse uterus to understand the regulation of uterine‐expressed genes on gestation day 4 (GD4) during the peri‐implantation period. The genes expressed in the uterus showed a significant association (p < .0001) with the presence of the nucleosome‐free region (open chromatin) in the 5′‐untranslated region of the genes. The majority of these upstream open chromatins harbored a common class of regulatory elements known as upstream open reading frames. We also compared the gene expression profiles between the uterus and brain which showed that specific gene pairs were expressed in a correlated manner, either positively or negatively. In addition, specific ligand/receptor genes showed coordinated patterns of expression between the uterus and brain on GD4, and the level of expression of these ligand/receptors altered significantly in the brain during late pregnancy (GD15) compared with the peri‐implantation period (GD4). Collectively, these results suggest that regulation of the uterine genes during the peri‐implantation period is likely to have a functional link with the maternal brain in pregnant mice.     

Genome-wide association analysis reveals genomic regions on Chromosome 13 affecting litter size and candidate genes for uterine horn length in Erhualian pigs

Litter size has a great impact on the profit of swine producers. Uterine development is an important determinant of reproduction efficiency and could hence affect litter size. Chinese Erhualian pig is one of the most prolific breeds in the world, even though large phenotypic variation in litter size was observed within Erhualian sows. To dissect the genetic basis of the phenotypic variation, we herein conducted genome-wide association studies for total number born and number born alive (NBA) of Erhualian sows. In total, one significant single nucleotide polymorphism (SNP) (P<1.78e−06) and 11 suggestive SNPs (P<3.57e−05) were identified on 10 chromosomes, confirming seven previously reported quantitative trait loci (QTL) and uncovering six QTL for litter size or uterus length. One locus on Sus scrofa chromosome (SSC) 13 (79.28 to 90.43 Mb) harbored a cluster of suggestive SNPs associated with multiparous NBA. The SNP (rs81447100) within this region was confirmed to be significantly (P<0.05) associated with litter size in Erhualian (n=313), Sutai (n=173) and Yorkshire (n=488) populations. Retinol binding protein 2 and retinol binding protein 1 functionally related to the development of uterus were located in a region of 2 Mb around rs81447100. Moreover, four genes related to embryo implantation and development were also detected around other significant SNPs. Taken together, our findings provide a potential marker (rs81447100) for the genetic improvement of litter size not only in Chinese Erhualian pigs but also in European commercial pig breeds like Yorkshire, and would facilitate the final identification of causative variant(s) underlying the effect of SSC13 QTL on litter size.     

SNP rs2243828 in MPO associated with myeloperoxidase level and atrial fibrillation risk in Chinese Han population

Previous studies shown that myeloperoxidase (MPO) level is higher in patients with atrial fibrillation (AF); however, no genetic evidence between MPO and AF risk in human population was observed. Therefore, the present study was aimed to investigate the association between rs2243828, a variant in promoter region of MPO and the risk of AF in Chinese GeneID population. The results demonstrated that the minor G allele of rs2243828 showed a significant association with AF in two independent population (GeneID‐north population with 694 AF cases and 710 controls, adjusted P_‐adj = 6.25 × 10^?3 with an odds ratio was 0.77, GeneID‐central population with 1106 cases and 1501 controls, P_‐adj = 9.88 × 10^?5 with an odds ratio was 0.75). The results also showed G allele was significantly associated with lower plasma concentration of myeloperoxidase in general population. We also observed a significant difference of odds ratio between subgroups of hypertension and non‐hypertension. Therefore, our findings identified variant in MPO associated with risk of AF and it may give strong evidence to link the inflammation with the incidence of AF.     

Prognostic value of tumour microenvironment-related genes by TCGA database in rectal cancer

Rectal cancer is a common malignant tumour and the progression is highly affected by the tumour microenvironment (TME). This study intended to assess the relationship between TME and prognosis, and explore prognostic genes of rectal cancer. The gene expression profile of rectal cancer was obtained from TCGA and immune/stromal scores were calculated by Estimation of Stromal and Immune cells in Malignant Tumors using Expression data (ESTIMATE) algorithm. The correlation between immune/stromal scores and survival time as well as clinical characteristics were evaluated. Differentially expressed genes (DEGs) were identified according to the stromal/immune scores, and the functional enrichment analyses were conducted to explore functions and pathways of DEGs. The survival analyses were conducted to clarify the DEGs with prognostic value, and the protein-protein interaction (PPI) network was performed to explore the interrelation of prognostic DEGs. Finally, we validated prognostic DEGs using data from the Gene Expression Omnibus (GEO) database by PrognoScan, and we verified these genes at the protein levels using the Human Protein Atlas (HPA) databases. We downloaded gene expression profiles of 83 rectal cancer patients from The Cancer Genome Atlas (TCGA) database. The Kaplan-Meier plot demonstrated that low-immune score was associated with worse clinical outcome (P = .034), metastasis (M1 vs. M0, P = .031) and lymphatic invasion (+ vs. -, P < .001). A total of 540 genes were screened as DEGs with 539 up-regulated genes and 1 down-regulated gene. In addition, 60 DEGs were identified associated with overall survival. Functional enrichment analyses and PPI networks showed that the DEGs are mainly participated in immune process, and cytokine-cytokine receptor interaction. Finally, 19 prognostic genes were verified by GSE17536 and GSE17537 from GEO, and five genes (ADAM23, ARHGAP20, ICOS, IRF4, MMRN1) were significantly different in tumour tissues compared with normal tissues at the protein level. In summary, our study demonstrated the associations between TME and prognosis as well as clinical characteristics of rectal cancer. Moreover, we explored and verified microenvironment-related genes, which may be the potential key prognostic genes of rectal cancer. Further clinical samples and functional studies are needed to validate this finding.     

Prostaglandin‐endoperoxide synthase 2 is not required for preimplantation ovine conceptus development in sheep

Conceptus development and elongation is required for successful pregnancy establishment in ruminants and is coincident with the production of interferon τ (IFNT) and prostaglandins (PGs). In both the conceptus trophectoderm and endometrium, PGs are primarily synthesized through a prostaglandin‐endoperoxide synthase 2 (PTGS2) pathway and modify endometrial gene expression and thus histotroph composition in the uterine lumen to promote conceptus growth and survival. Chemical inhibition of PG production by both the endometrium and the conceptus prevented elongation in sheep. However, the contributions of conceptus‐derived PGs to preimplantation conceptus development remain unclear. In this study, CRISPR‐Cas9 genome editing was used to inactivate PTGS2 in ovine embryos to determine the role of PTGS2‐derived PGs in conceptus development and elongation. PTGS2 edited conceptuses produced fewer PGs, but secreted similar amounts of IFNT to their Cas9 control counterparts and elongated normally. Expression of PTGS1 was lower in PTGS2 edited conceptuses, but PPARG expression and IFNT secretion were unaffected. Content of PGs in the uterine lumen was similar as was gene expression in the endometrium of ewes who received either Cas9 control or PTGS2 edited conceptuses. These results support the idea that intrinsic PTGS2‐derived PGs are not required for preimplantation embryo or conceptus survival and development in sheep.     

环境丰容对早期发育阶段许氏平鲉趋礁行为的影响

采用实验生态学方法,观测了不同环境丰容水平下培育的许氏平鲉(Sebastes schlegelii)仔、稚、幼鱼的趋礁行为反应,统计分析了两种结构模型礁对实验鱼的诱集效果,初步探究了环境丰容对实验鱼趋礁行为的影响。结果显示:许氏平鲉仔、稚、幼鱼具有明显不同的行为习性与趋礁行为特性,未放入模型礁前,稚鱼在水槽中分布较分散,仔、幼鱼则集中于水槽四周阴影区域,中央区域分布极少;放入模型礁后,仔、稚鱼在礁区的平均分布率无显著变化(P0.05);幼鱼则由1.11%分别增大至47.78%(塔型礁)和54.44%(管型礁)(P0.01),随着个体生长发育,其趋礁性显著增强。环境丰容25 d(即48日龄的稚鱼),对实验鱼群体重心与模型礁的平均距离D_(gr)、个体与模型礁的平均距离D_(fr)、个体间的平均距离D_(ff)及实验鱼在水槽中央区域(VI区)的平均分布率均无显著影响(P0.05)。环境丰容47 d(即70日龄的幼鱼),未放入模型礁前,高水平丰容组(EH组)幼鱼在VI区的平均分布率显著高于空白对照组(C组)(P0.05),低水平丰容组(EL组)与C组、EH组均无显著差异(P0.05),各处理组D_(gr)与D_(fr)大小顺序为:EL组C组EH组;放入模型礁后,各处理组幼鱼在模型礁区域(VI区)的平均分布率:EL组C组EH组,Dfr与Dff大小顺序为:EL组C组EH组;表明低水平环境丰容可有效增强许氏平鲉幼鱼的趋礁行为,减小幼鱼"胆量",这对于提高放流幼鱼在野外的存活率具有重要意义。旨在探讨环境丰容这一行为驯化手段,对于增强早期发育阶段许氏平鲉趋礁行为的可行性,为增殖放流苗种的高效健康培育及增殖型人工鱼礁的选型提供参考。     

Equal nonbreeding period survival in adults and juveniles of a long‐distant migrant bird

In migrant birds, survival estimates for the different life‐history stages between fledging and first breeding are scarce. First‐year survival is shown to be strongly reduced compared with annual survival of adult birds. However, it remains unclear whether the main bottleneck in juvenile long‐distant migrants occurs in the postfledging period within the breeding ranges or en route. Quantifying survival rates during different life‐history stages and during different periods of the migration cycle is crucial to understand forces driving the evolution of optimal life histories in migrant birds. Here, we estimate survival rates of adult and juvenile barn swallows (Hirundo rustica L.) in the breeding and nonbreeding areas using a population model integrating survival estimates in the breeding ranges based on a large radio‐telemetry data set and published estimates of demographic parameters from large‐scale population‐monitoring projects across Switzerland. Input parameters included the country‐wide population trend, annual productivity estimates of the double‐brooded species, and year‐to‐year survival corrected for breeding dispersal. Juvenile survival in the 3‐week postfledging period was low (S = 0.32; SE = 0.05), whereas in the rest of the annual cycle survival estimates of adults and juveniles were similarly high (S > 0.957). Thus, the postfledging period was the main survival bottleneck, revealing the striking result that nonbreeding period mortality (including migration) is not higher for juveniles than for adult birds. Therefore, focusing future research on sources of variation in postfledging mortality can provide new insights into determinants of population dynamics and life‐history evolution of migrant birds.     

Adaptive divergence in embryonic thermal plasticity among Atlantic salmon populations

In the context of global changes, the long‐term viability of populations of endangered ectotherms may depend on their adaptive potential and ability to cope with temperature variations. We measured responses of Atlantic salmon embryos from four populations to temperature variations and used a Q_ST–F_ST approach to study the adaptive divergence among these populations. Embryos were reared under two experimental conditions: a low temperature regime at 4 °C until eyed‐stage and 10 °C until the end of embryonic development and a high temperature regime with a constant temperature of 10 °C throughout embryonic development. Significant variations among populations and population × temperature interactions were observed for embryo survival, incubation time and length. Q_ST was higher than F_ST in all but one comparison suggesting an important effect of divergent selection. Q_ST was also higher under the high‐temperature treatment than at low temperature for length and survival due to a higher variance among populations under the stressful warmer treatment. Interestingly, heritability was lower for survival under high temperature in relation to a lower additive genetic variance under that treatment. Overall, these results reveal an adaptive divergence in thermal plasticity in embryonic life stages of Atlantic salmon suggesting that salmon populations may differentially respond to temperature variations induced by climate change. These results also suggest that changes in temperature may alter not only the adaptive potential of natural populations but also the selection regimes among them.     

Molecular characterization and expression of Kiss2/Kiss2r during embryonic and larval development in (Megalobrama amblycephala Yih, 1955)

In order to test whether Kiss/Kissr systems have potential roles in regulating the embryonic and larval development in teleosts, in this study the Kiss2/Kiss2r full‐length cDNA was cloned from blunt snout bream (Megalobrama amblycephala Yih, 1955) by rapid amplification of cDNA ends and their expression patterns were detected in different tissues of adult and developmental stages for the embryonic and larval periods via quantitative real‐time PCR. Both Kiss and Kissr genes full‐length cDNA sequences of M. amblycephala were obtained and phylogenetic analysis results indicated that these genes belong to the Kiss2/Kiss2r clade. Bioinformatics analyses revealed that there was a conserved decapeptide in M. amblycephala Kiss2 gene putative amino acids, but only two transmembrane domains were predicated in Kiss2r. Tissue distribution analyses showed that both genes were widely expressed in the tissues tested, with high levels in the muscle, gonad and pituitary. At different developmental stages, the mRNA expression of Kiss2/Kiss2r was highest in the blastocyst/15 hpf stage and lowest in the 30 hpf /blastocyst stages for the embryonic period, highest in 7 dph/15 dph and lowest in 30 dph/30 dph for larval period, respectively. These results suggest that the Kiss2/Kiss2r system has varied potential for influencing embryonic and larval development in fish species.