A combined Genotyping By Sequencing (GBS) and methylated DNA immunoprecipitation (MeDIP) protocol was used to identify—in parallel—genetic variation (Genomic-Wide Association Studies (GWAS) and epigenetic differences of Differentially Methylated Regions (DMR) in the genome of spermatozoa from the porcine animal model. Breeding boars with good semen quality (n = 11) and specific and well-documented differences in fertility (farrowing rate, FR) and prolificacy (litter size, LS) (n = 7) in artificial insemination programs, using combined FR and LS, were categorized as High Fertile (HF, n = 4) or Low Fertile (LF, n = 3), and boars with Unknown Fertility (UF, n = 4) were tested for eventual epigenetical similarity with those fertility-proven. We identified 165,944 Single Nucleotide Polymorphisms (SNPs) that explained 14–15% of variance among selection lines. Between HF and LF individuals (n = 7, 4 HF and 3 LF), we identified 169 SNPs with p ≤ 0.00015, which explained 58% of the variance. For the epigenetic analyses, we considered fertility and period of ejaculate collection (late-summer and mid-autumn). Approximately three times more DMRs were observed in HF than in LF boars across these periods. Interestingly, UF boars were clearly clustered with one of the other HF or LF groups. The highest differences in DMRs between HF and LF experimental groups across the pig genome were located in the chr 3, 9, 13, and 16, with most DMRs being hypermethylated in LF boars. In both HF and LF boars, DMRs were mostly hypermethylated in late-summer compared to mid-autumn. Three overlaps were detected between SNPs (p ≤ 0.0005, n = 1318) and CpG sites within DMRs. In conclusion, fertility levels in breeding males including FR and LS can be discerned using methylome analyses. The findings in this biomedical animal model ought to be applied besides sire selection for andrological diagnosis of idiopathic sub/infertility.
In this paper, we have used two approaches to detect genetic associations with scrotal hernias in commercial pigs. Firstly, we have investigated the effects of runs of homozygosity (ROH) with the appearance of scrotal hernias, followed by a Genome Wide Association Study (GWAS). The phenotype classification was based on visual appearance of scrotal hernias. Each affected animal was matched to a healthy control from the same pen. In the total, 68 animals were genotyped using the Porcine SNP60 Beadchip, out of those, 41 animals had the presence of hernias and 27 were healthy animals. Fifteen animals were removed from the analysis due to differences in genetic background, leaving 18 healthy animals and 35 piglets with scrotal hernia. Further, the detection of extended haplotypes shared ROH were conducted for health (control) and affected (case) animals and a permutation test was used to test whether the ROH segments were more frequent in case/case pairs than non-case/case pairs. Using the ROH, we have identified an association (p = 0.019) on chromosome 2(SSC2) being segregated on animals with the presence of scrotal hernias. Using a GWAS, a region composed by 3 SNPs on the sexual chromosome X (SSCX) were associated with scrotal hernias (p < 1.6 × 10−5), this region harbors the Androgen Receptor Gene (AR).
Epigenetics works as an interface between the individual and its environment to provide phenotypic plasticity to increase individual adaptation capabilities. Recently, a wide variety of epi-genetic findings have indicated evidence for its application in the development of putative epi-biomarkers of stress in farm animals. The purpose of this study was to evaluate previously reported stress epi-biomarkers in swine and encourage researchers to investigate potential paths for the development of a robust molecular tool for animal welfare certification. In this literature review, we report on the scientific concerns in the swine production chain, the management carried out on the farms, and the potential implications of these practices for the animals’ welfare and their epigenome. To assess reported epi-biomarkers, we identified, from previous studies, potentially stress-related genes surrounding epi-biomarkers. With those genes, we carried out a functional enrichment analysis of differentially methylated regions (DMRs) of the DNA of swine subjected to different stress-related conditions (e.g., heat stress, intrauterine insult, and sanitary challenges). We identified potential epi-biomarkers for target analysis, which could be added to the current guidelines and certification schemes to guarantee and certify animal welfare on farms. We believe that this technology may have the power to increase consumers’ trust in animal welfare.
The United Kingdom and European Union have banned crates for pregnant sows. However, animals are kept in a restrictive environment for up to four weeks after mating, leading to stress and different responses of the animals’ immune system. Here, we used vaginal flushing of gilts to investigate whether housing systems or an experimental inflammatory challenge with lipopolysaccharide (LPS) can modify the gilt vaginal microbiome. Alpha-diversity indices showed differences in the microbiota of gilts housed under different systems (q = 0.04). Shannon alpha-diversity richness was higher in gilts group-housed in pens than in gilts housed in crates (q = 0.035), but not higher than in other groups. The relative abundance of the operational taxonomic unit (OTU) (q < 0.05) revealed specific differences in housing systems before a LPS or saline (SAL control) challenge. We found different abundances in taxa of Actinobacteria, Bacteroidetes, Cyanobacteria, Firmicutes, and Proteobacteria in gilts housed in the different systems before challenge. After the LPS challenge, significant differences were detected in the relative abundance of OTUs (q < 0.05) for the LPS-challenged group compared with SAL animals for each housing system. The phylum Staphylococcus showed higher abundance among the LPS-challenged gilts than in SAL-challenged animals. Furthermore, Enterobacter was more abundant in the LPS-challenged gilts housed in crates than in SAL-challenged gilts housed in crates. Streptococcus suis, Conchiformibius, Globicatella and Actinobacillus were more abundant in LPS-challenged gilts in indoor group housing than in SAL gilts in the same housing system. Gilts kept outdoors did not show changes in vaginal microbiota after an LPS challenge. Gilts housed in crates showed clinical signs of urogenital infection, whereas gilts housed outdoors and in indoor group housing did not. The relationship between environment, immune response, and microbiota suggested that animals in a poor environments experience difficulties responding to a challenge and their vaginal microbiota is altered as a consequence, with decreased richness of normal vaginal microbiota, and increased opportunistic bacteria. Welfare indicators measured by gilts’ responses to housing systems however, do not fully explain mechanisms associated with the unique signature in vaginal microbiota encountered in the different housing systems.
<b><i>Introduction:</i></b> Cryptorchidism is a hereditary anomaly characterized by the incomplete descent of one or both testicles to the scrotum. One of the challenges of this anomaly is that the retained testicle maintains its endocrine function. As a consequence, cryptorchid animals produce hormone-tainted meat in comparison to castrated animals and are likely to be more aggressive. Cryptorchidism can lead to reduced animal welfare outcomes and cause economic losses. Identifying genetic markers for cryptorchidism is an essential step toward mitigating these negative outcomes and may facilitate genome manipulation to reduce the occurrence of cryptorchidism. Attempts to identify such markers have used genome-wide association studies. Using whole-exome sequencing, we aimed to identify single nucleotide polymorphisms (SNPs) in the coding regions of cryptorchid pigs and to characterize functional pathways concerning these SNPs. <b><i>Methods:</i></b> DNA was extracted and sequenced from 5 healthy and 5 cryptorchid animals from the Landrace breed, using the Illumina HiSeq 2500 platform. Data were pre-processed using the SeqyClean tool and further mapped against the swine reference genome (<i>Sus scrofa</i> 11.1) using BWA software. GATK was used to identify polymorphisms (SNPs and InDels), which were annotated using the VEP tool. Network prediction and gene ontology enrichment analysis were conducted using the Cytoscape platform, and STRING software was used for visualization. <b><i>Results:</i></b> A total of 63 SNPs were identified across the genes <i>PIGB</i>, <i>CCPG1</i>, <i>COMMD9</i>, <i>LDLRAD3</i>, <i>TRIM44</i>, <i>MYLPF</i>, <i>SEPTIN</i>, <i>ZNF48</i>, <i>TIA1</i>, <i>FAIM2</i>, <i>KRT18</i>, <i>FBP1</i>, <i>FBP2</i>, <i>CTSL</i>, <i>DAPK1</i>, <i>DHX8</i>, <i>GPR179</i>, <i>DEPDC1B</i>, ENSSSCG00000049573, ENSSSCG00000016384, ENSSSCG00000022657, ENSSSCG00000038825, and ENSSSCG00000001229. Using pathway enrichment analyses and network prospection, we have identified the following significant adjusted <i>p</i> value threshold of 0.001 involved with the biological function pathways of estrogen signaling, cytoskeleton organization, and the pentose phosphate pathway. <b><i>Conclusion:</i></b> Our data suggest the involvement of new SNPs and genes in developing cryptorchidism in pigs. However, further studies are needed to validate our results in a larger cohort population. Variations in the <i>GPR179</i> gene, with implications at the protein level, may be associated with the appearance of this anomaly in the swine. Finally, we are showing that the estrogen signaling pathway may be involved in the pathophysiological mechanisms of this congenital anomaly as previously reported in GWAS.
The corpus luteum (CL) is a temporary endocrine gland that plays a decisive role in the reproductive physiology of gilts. Recently, it has been suggested that exogenous factors may compromise the normal functioning of the CL. In the present study, we aimed to understand to what extent an acute and systemic challenge with lipopolysaccharide (LPS) on the day of estrus could compromise gene expression of gilts’ CLs housed in different welfare conditions. For this, we housed 42 gilts in three different housing systems: crates, indoor group pens, and outdoor housing. Then, we challenged six females from each group with LPS and eight with saline (SAL) on the day of estrus. After slaughtering the gilts on the fifth day after the challenge, ovaries were collected for gene expression analysis by RT-qPCR. Housing system and LPS challenge did not have a significant interaction for any genes evaluated; thus, their effects were studied separately. We identified significant (p < 0.05) downregulation of the angiogenic genes VEGF and FTL1 among LPS-challenged animals. Meanwhile, we also observed upregulation of HSD3B1 gene among LPS-challenged animals. We found that STAR and LHCGR genes were differentially expressed depending on the housing system, which indicates that the environment may affect adaptation capabilities. Our results indicate that an acute health challenge on the estrus day alters CL gene expression; however, the role of the housing system remains uncertain.
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