Asinine milk mitigates stress-mediated immune, cortisol and behavioral responses of piglets to weaning: A study to foster future interventions in humans.

Introduction: The present study assessed whether asinine milk supplementation improved the immune and behavioral responses of piglets during an early life weaning stress event as a model for its future use in humans. Methods: For this, 48 piglets from 4 different litters were used. At 20 days of age, piglets were weighed and allocated with their litter and dam into group pens until 28 days of age. Four piglets from each litter were then randomly assigned to either (1) asinine milk supplementation (n = 16) (2), skimmed cow milk supplementation (n = 16) or (3) no supplementation (n = 16; control group). The supplementations were voluntarily administered for 3 days preweaning and 3 days postweaning using a baby bottle. The effects on the weaning stress response were assessed through salivary cortisol measurements; behavioral tests such as the open field, novel object end elevated plus maze tests; and gene expression of HSD11B1, NR3C1 and IL1B in PBMCs, which was determined by RT-qPCR and normalized to GAPDH and UBB. To test the effect of the supplementations on weight, milk intake, gene expression, and behavior, a randomized block design was used with repeated measurements over time by the PROC MIXED procedure. Results and discussion: The effects on salivary cortisol were determined using the ratio between the morning and afternoon concentrations, considering the time before and after the weaning event. Principal component analysis (PCA) and Fisher's test were performed to evaluate the behavior test data. When comparing salivary cortisol concentrations between the pre- and postweaning periods, there was a difference (p < 0.05) between the supplementation groups in the afternoon period, suggesting that piglets fed asinine milk had lower afternoon cortisol concentrations postweaning than their counterparts. For the behavioral tests, the supplementations had no measurable effects. No difference was between groups pre- and postweaning for the expression of HSD11B2, which codes for an enzyme that breaks down cortisol. However, the expression of NR3C1, which encodes the glucocorticoid receptor, was significantly upregulated in piglets supplemented with cow milk (mean 1.245; p < 0.05). Conclusion: Asinine milk downregulated 1L1B gene expression, which codes for an inflammatory cytokine. In conclusion, these results suggest that supplementation with asinine milk may represent a strategy to diminish the damage associated with an early life event by modulating IL1B expression and reducing salivary cortisol levels in piglets undergoing weaning stress. Further transcriptomic and metabolomic studies may improve our understanding of the molecular pathways that mediate this systemic immune-mediated response.

Streptococcus canis prevalence on the normal and abnormal ocular surface of dogs referred for ophthalmic disease in Canada.

Streptococcus canis is a beta-haemolytic, Gram-positive cocci commonly identified on the canine ocular surface under both healthy and diseased conditions. The objective of the study was to determine the prevalence of S. canis on the normal and abnormal ocular surface of a canine ophthalmology referral population in Canada, and to investigate potential clinical aspects that may be associated with its presence. Included were 59 dogs (118 eyes) with unilateral or bilateral ocular disease diagnosed at the time of conjunctival sampling. A real-time PCR specific for S. canis was standardized for use with conjunctival swabs. Total DNA was extracted from 118 samples and used as template for the diagnostic assay. Samples were considered positive if amplification was detected and dissociation temperature matched a positive control. Signalment and other clinical data were also collected at the time of sampling. Of the 118 eyes sampled, 8 tested positive for S. canis (6.8%). No association between the detection of S. canis and breed, cephalic conformation, sex, age, use of ophthalmic antibiotics or other topical medications, ophthalmic diagnosis, use of systemic antibiotics or other systemic medications, or systemic diagnosis was identified. In conclusion, S. canis may be present on the ocular surface of dogs at a higher rate than previously reported. It is suggested that this may be linked to the use of PCR for pathogen detection instead of culture.

Whole-Exome Sequencing Indicated New Candidate Genes Associated with Unilateral Cryptorchidism in Pigs.

INTRODUCTION: 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. METHODS: 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 (Sus scrofa 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. RESULTS: A total of 63 SNPs were identified across the genes PIGB, CCPG1, COMMD9, LDLRAD3, TRIM44, MYLPF, SEPTIN, ZNF48, TIA1, FAIM2, KRT18, FBP1, FBP2, CTSL, DAPK1, DHX8, GPR179, DEPDC1B, ENSSSCG00000049573, ENSSSCG00000016384, ENSSSCG00000022657, ENSSSCG00000038825, and ENSSSCG00000001229. Using pathway enrichment analyses and network prospection, we have identified the following significant adjusted p value threshold of 0.001 involved with the biological function pathways of estrogen signaling, cytoskeleton organization, and the pentose phosphate pathway. CONCLUSION: 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 GPR179 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.

Vaginal Microbiota Diversity in Response to Lipopolysaccharide in Gilts Housed Under Three Housing Systems.

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.

From Mendel laws to whole genetic association study to decipher the swine mulefoot phenotype.

The swine mulefoot (SM) is a rare condition characterized by a non-cloven hoof due to the partial or total fusion of the phalanges. No comprehensive study has been conducted to identify associated markers with this phenotype until now. We aimed to characterize the association between SNP and the mulefoot phenotype using a Genome-Wide Association Study (GWAS). An experimental population was produced using a half-sib mating where the male had the mulefoot phenotype and the females (n = 6) had cloven hoofs. The cross resulted in 27 (47%) animals with the mulefoot characteristic and 30 (53%) normal animals, indicating the possible dominant gene action. Animals were further genotyped using the Illumina PorcineSNP50k BeadChip, and SNPs were tested for associations. Twenty-nine SNPs located on the SSC15, SSC4, and SSCX were associated with the mulefoot phenotype (p-value <5 × 10-5). Six markers were found in the intronic regions of VWC2L, CATIP, PDK3, PCYT1B, and POLA1 genes. The marker rs81277626, on SSC15:116,886,110 bp, is located in the Von Willebrand Factor C Domain (VWC2L), a possible functional candidate gene. The VWC2L is part of a biological process involved with the bone morphogenetic protein (BMP) signaling pathway, previously associated with syndactyly in other species. In conclusion, the identified markers suggest the involvement of the VWC2L gene in the SM phenotype in this population.

Sperm Methylome Profiling Can Discern Fertility Levels in the Porcine Biomedical Model.

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.

Pesticide and trace element bioaccumulation in wild owls in Brazil.

Owls are outstanding environmental quality bioindicators due to their position at the top of the food chain and susceptibility to pollutant accumulation. Exposure to chemical contaminants is often a risk for these animals. Moreover, studies addressing the bioaccumulation of trace elements and pesticide residues in tropical nocturnal raptor species are scarce. We analyzed the 26 organs (heart, liver, and kidney) of Tyto furcata (n=3), Megascops spp. (n=5), Pulsatrix koeniswaldiana (n=1), and Asio stygius (n=1) carcasses, collected from June 2018 to May 2019 in the Southern region of Brazil. The original vegetation consisted of areas of Araucaria forests and grassy-woody steppes with gallery forests, which were greatly modified by the introduction of agriculture. In four animals and eight organs, the pesticides abamectin, atrazine, chlorpyrifos-ethyl, and diurom were analyzed through high-performance liquid chromatography coupled with a mass detector. In six animals and eighteen organs, the trace elements cadmium, lead, chromium, and nickel were identified via atomic absorption spectrophotometry. Chlorpyrifos-ethyl was detected in the livers of the genus Megascops. Chromium was found at high concentrations in all matrices analyzed for this trace element. Moreover, P. koeniswaldiana presented lead levels indicative of high exposure. The bioaccumulation of these toxics in owls described here can impact the population levels of these species, impact on its ecological function, and consequently unbalance the ecosystem. Moreover, owls are considered bioindicators; therefore, the occurrence of bioaccumulation indirectly gives us information about the quality of the environment.

Study of vaginal microbiota of Holstein cows submitted to an estrus synchronization protocol with the use of intravaginal progesterone device.

The characterization of vaginal microbiota will help to understand some of the reproductive problems and mechanisms to improve cattle reproduction. The objective of this study was to characterize the vaginal microbiota of cyclical Holstein cows with different parturition orders using 16S rDNA sequencing. Animals were submitted to an estrus synchronization protocol with the use of intravaginal progesterone (P4) implants and were treated or not with ceftiofur hydrochloride. DNA samples were extracted from vaginal swabs on day 0 and 10 of the synchronization, and sequenced with the Illumina MiSeq platform with an average coverage rate of 10.000 reads per samples using a Single-End library for fragments of 300 bp. The main bacterial phyla found in the vaginal tract of Holstein cows, were Firmicutes (37.61%), Tenericutes (29.45%), Proteobacteria (17.47%) and Bacteriodetes (13.73%), followed by Actinobacteria (0.82%) and Spirochaetae (0.45%). The use of intravaginal P4 devices has increased the relative abundance of the genera Family XIII AD3011 and Family XIII unclassified (p < .049). We have also observed an effect of the number of calving on the vaginal microbiota composition, showing that multiparous cows have a greater bacterial diversity than primiparous animals (p < .05). The use of ceftiofur hydrochloride was effective to reduce the vaginal bacteria proliferation. This study describes for the first time the vaginal microbiota of cows synchronized with intravaginal progesterone devices, different from the traditional methods such as microbiological culture and biochemical tests. We have identified a large number of microorganisms commonly found in the gastrointestinal tract of cows, colonizing the vaginal microbiota.