PKM2 induces mitophagy through the AMPK-mTOR pathway promoting CSFV proliferation.

Viruses exploit the host cell's energy metabolism system to support their replication. Mitochondria, known as the powerhouse of the cell, play a critical role in regulating cell survival and virus replication. Our prior research indicated that the classical swine fever virus (CSFV) alters mitochondrial dynamics and triggers glycolytic metabolic reprogramming. However, the role and mechanism of PKM2, a key regulatory enzyme of glycolytic metabolism, in CSFV replication remain unclear. In this study, we discovered that CSFV enhances PKM2 expression and utilizes PKM2 to inhibit pyruvate production. Using an affinity purification coupled mass spectrometry system, we successfully identified PKM as a novel interaction partner of the CSFV non-structural protein NS4A. Furthermore, we validated the interaction between PKM2 and both CSFV NS4A and NS5A through co-immunoprecipitation and confocal analysis. PKM2 was found to promote the expression of both NS4A and NS5A. Moreover, we observed that PKM2 induces mitophagy by activating the AMPK-mTOR signaling pathway, thereby facilitating CSFV proliferation. In summary, our data reveal a novel mechanism whereby PKM2, a metabolic enzyme, promotes CSFV proliferation by inducing mitophagy. These findings offer a new avenue for developing antiviral strategies. IMPORTANCE: Viruses rely on the host cell's material-energy metabolic system for replication, inducing host metabolic disorders and subsequent immunosuppression-a major contributor to persistent viral infections. Classical swine fever virus (CSFV) is no exception. Classical swine fever is a severe acute infectious disease caused by CSFV, resulting in significant economic losses to the global pig industry. While the role of the metabolic enzyme PKM2 (pyruvate dehydrogenase) in the glycolytic pathway of tumor cells has been extensively studied, its involvement in viral infection remains relatively unknown. Our data unveil a new mechanism by which the metabolic enzyme PKM2 mediates CSFV infection, offering novel avenues for the development of antiviral strategies.

Toxoplasma gondii infection regulates apoptosis of host cells via miR-185/ARAF axis.

BACKGROUND: Toxoplasmosis is a zoonosis with a worldwide presence that is caused by the intracellular parasite Toxoplasma gondii. Active regulation of apoptosis is an important immune mechanism by which host cells resist the growth of T. gondii or avoid excessive pathological damage induced by this parasite. Previous studies found that upregulated expression of microRNA-185 (miR-185) during T. gondii infection has a potential role in regulating the expression of the ARAF gene, which is reported to be associated with cell proliferation and apoptosis. METHODS: The expression levels of miR-185 and the ARAF gene were evaluated by qPCR and Western blot, respectively, in mice tissues, porcine kidney epithelial cells (PK-15) and porcine alveolar macrophages (3D4/21) following infection with the T. gondii ToxoDB#9 and RH strains. The dual luciferase reporter assay was then used to verify the relationship between miR-185 and ARAF targets in PK-15 cells. PK-15 and 3D4/21 cell lines with stable knockout of the ARAF gene were established by CRISPR, and then the apoptosis rates of the cells following T. gondii infection were detected using cell flow cytometry assays. Simultaneously, the activities of cleaved caspase-3, as a key apoptosis executive protein, were detected by Western blot to evaluate the apoptosis levels of cells. RESULTS: Infection with both the T. gondii ToxoDB#9 and RH strains induced an increased expression of miR-185 and a decreased expression of ARAF in mice tissues, PK-15 and 3D4/21 cells. MiR-185 mimic transfections showed a significantly negative correlation in expression levels between miR-185 and the ARAF gene. The dual luciferase reporter assay confirmed that ARAF was a target of miR-185. Functional investigation revealed that T. gondii infection induced the apoptosis of PK-15 and 3D4/21 cells, which could be inhibited by ARAF knockout or overexpression of miR-185. The expression levels of cleaved caspase-3 protein were significantly lower in cells with ARAF knockout than in normal cells, which were consistent with the results of the cell flow cytometry assays. CONCLUSIONS: Toxoplasma gondii infection could lead to the upregulation of miR-185 and the downregulation of ARAF, which was not related to the strain of T. gondii and the host cells. Toxoplasma gondii infection could regulate the apoptosis of host cells via the miR-185/ARAF axis, which represents an additional strategy used by T. gondii to counteract host-cell apoptosis in order to maintain survival and reproduce in the host cells.

Phenotypic link between protein efficiency and pig welfare suggests no apparent trade-offs for mitigating nitrogen pollution.

Pig manure contributes significantly to environmental pollution through nitrogen compounds. Reducing protein in feed can help, but it may lead to damaging behaviors if pigs' nutritional needs are not met. Breeding pigs for higher protein efficiency (PE) is a long-term solution to reduce nitrogen pollution, but concerns about pig welfare remain. We studied 95 pigs involved in a project on the genetic basis of PE on a 20% protein restricted diet to investigate the phenotypic connection between PE and welfare. These pigs represented natural PE variations in the population. At around 100 days, before their PE was known, we observed their behaviors. Only three pigs engaged in tail biting and manipulation of vulnerable regions, but this was not associated with PE. There was no clear link between PE and manipulating pen mates' less vulnerable regions. Such behaviors are normal but can cause stress and injury if carried out excessively due to boredom or stress. Overall, pigs with higher PE showed no major behavioral abnormalities in this study. Considering the lack of genetic knowledge, the risk of increased harmful behaviors when selecting for higher PE appears low when inferred from this purely phenotypic association.

The effects of standardized ileal digestible His to Lys ratio on growth performance, intestinal health, and mobilization of histidine-containing proteins in pigs at 7 to 11 kg body weight.

The objectives were to evaluate the effects of standardized ileal digestible (SID) His:Lys ratio above the current NRC requirement on growth performance, intestinal health, and mobilization of His-containing proteins, including hemoglobin, carnosine, and trypsinogen, in nursery pigs from 7 to 11 kg body weight (BW). Forty pigs (26 d of age; initial BW of 7.1 ±â€…0.5 kg) were allotted to 5 dietary treatments based on a randomized complete block design with sex and initial BW as blocks. Dietary treatments were supplemented with varying SID His to Lys ratios of 26%, 32%, 38%, 43%, and 49% and fed to pigs for 14 d (SID Lys = 1.22%). Feed intake and BW were recorded at d 0, 7, and 14 to measure growth performance. Blood samples were collected on d 12. Pigs were euthanized on d 14 to collect pancreas, longissimus dorsi muscles, mid-jejunum, and jejunal mucosa. Data were analyzed using the Proc Mixed of SAS. Growth performance was not affected, whereas varying SID His to Lys ratio affected hemoglobin (P < 0.05, max: 12 g/dL at 36%), immunoglobulin A (IgA, P < 0.05, min: 1.25 µg/mg at 35%) in jejunal mucosa, villus height (P = 0.065, max: 536 µm at 40%) in jejunum, trypsinogen (P = 0.083, max: 242 pg/mg at 41%) in pancreas, and carnosine (P = 0.051, max: 4.7 ng/mg at 38%) in muscles. Varying SID His to Lys ratios linearly increased (P < 0.05, from 1.95 to 2.80 nmol/mg) protein carbonyl in muscles and decreased (P < 0.05, from 29.1% to 26.9%) enterocyte proliferation. In conclusion, SID His to Lys ratio between 35% and 41% in diets fed to nursery pigs at 7 to 11 kg enhanced intestinal health and maximized concentrations of His-containing proteins, indicating that His-containing proteins are effective response criteria when determining His requirement.

Histidine is an essential amino acid for protein synthesis, but it also plays a vital role in the metabolic system of pigs. An accurate assessment of His requirement provides pivotal information for efficient growth and health of pigs. Growth performance and plasma His concentration have been used to assess His requirement, but they may not be the effective parameters due to the contribution of His from mobilization of His-containing proteins, such as hemoglobin, carnosine, and pancreatic enzymes. Hemoglobin is a transport protein and the main component in red blood cells, enabling oxygen transport throughout the body. Most carnosine is stored in muscles at 3 to 4 g/kg wet weight and has antioxidative effects to prevent cells from oxidative damages. In addition, His has a critical role in serine peptidases as a part of the catalytic triad. In this study, growth performance did not respond to His deficiency due to the compensation of His from His-containing proteins and potentially due to a short experimental period. Standardized ileal digestible His to Lys ratio between 35% and 41% maximized concentrations of His-containing proteins and enhanced intestinal health in pigs at 7 to 11 kg body weight. This study indicated that hemoglobin, carnosine, and trypsinogen are effective response criteria when determining His requirement.

Effects of the feeding level in early gestation on body reserves and the productive and reproductive performance of primiparous and multiparous sows.

Early gestation may be the best period for sows to recover body reserve losses from previous lactation. The aim of this study was to investigate the effect of different levels of restricted feeding in early gestation on the body status, productive and reproductive performance, and hormonal-metabolic status of primiparous and multiparous sows. A total of 130 sows were randomly assigned to one of three feeding levels: Treatment I, which sows were fed at the level commonly used from day 3 to 28 of gestation (2.5 kg·d-1 of a diet with 2.18 Mcal NE·kg-1 and 13.72 g CP·kg-1), and Treatments II and III, where feed was increased by 25% and 50%, respectively. Sow body status, litter size and weight, early mortalities, reproductive rates, weaning-to-estrus interval, and hormones linked to metabolism were recorded. The highest weight gain, body condition score, and backfat thickness were found in sows fed Treatment III compared to those fed the usual feeding level (Treatment I). No differences among treatment groups were found in litter size or litter weight, although a tendency for more live born piglets and fewer stillbirths was found in sows fed Treatment III. In contrast, litters from sows fed at higher feeding levels had a higher mortality at 72 h compared to those fed at the lowest feeding level (I), which was partly linked to a higher percentage of piglets culled at birth and piglets weighing less than 800 g. There were no differences in conception and farrowing rates, leptin, progesterone, insulin, or cortisol among treatment groups applied in early gestation. In conclusion, increasing the feeding level in sows during early gestation to improve their short-term productive and reproductive performance remains controversial. Further studies are needed to focus on how the restricted feeding level applied could affect the viability and proportion of low-weight piglets.

Construction of circRNA-related ceRNA networks in longissimus dorsi muscle of Queshan Black and Large White pigs.

Circular RNA (circRNA) is a class of non-coding RNA (ncRNA) that plays an important regulatory role in various biological processes of the organisms and has a major function in muscle growth and development. However, its molecular mechanisms of how it regulates pork quality remain unclear at present. In this study, we compared the longissimus dorsi (LD) muscle expression profiles of Queshan Black (QS) and Large White (LW) pigs to explore the role of circRNAs in meat quality using transcriptome sequencing. A total of 62 differentially expressed circRNAs (DECs), including 46 up- and 16 down-regulated, 39 differentially expressed miRNAs (DEmiRNAs), including 21 up- and 18 down-regulated and 404 differentially expressed mRNAs (DEMs), including 174 up- and 230 down-regulated were identified, and most circRNAs were composed of exons. Our results indicated that the DEC parent genes and DEMs were enriched in the positive regulation of fast-twitch skeletal muscle fiber contraction, relaxation of skeletal muscle, regulation of myoblast proliferation, AMPK signaling pathway, Wnt and Jak-STAT signaling pathway. Furthermore, circSETBP1/ssc-miR-149/PIK3CD and circGUCY2C/ssc-miR-425-3p/CFL1 were selected by constructing the competitive endogenous RNA (ceRNA) regulatory network, circSETBP1, circGUCY2C, PIK3CD and CFL1 had low expression level in QS, while ssc-miR-149 and ssc-miR-425-3p had higher expression level than LW, our analysis revealed that circSETBP1, circGUCY2C, ssc-miR-149, ssc-miR-425-3p, PIK3CD and CFL1 were associated with lipid regulation, cell proliferation and differentiation, so the two ceRNAs regulatory networks may play an important role in regulating intramuscular fat (IMF) deposition, thereby affecting pork quality. In conclusion, we described the gene regulation by the circRNA-miRNA-mRNA ceRNA networks by comparing QS and LW pigs LD muscle transcriptome, and the two new circRNA-associated ceRNA regulatory networks that could help to elucidate the formation mechanism of pork quality. The results provide a theoretical basis for further understanding the genetic mechanism of meat quality formation.

Selenium deficiency induced apoptosis via mitochondrial pathway caused by Oxidative Stress in porcine gastric tissues.

Selenium (Se) is an essential nutrient for the body, which can ensure GSH-Px activity and has antioxidant effect. Se deficiency may lead to apoptosis in various tissues and organs in animals. Pigs as major livestock in the farming industry, Se deficiency can cause various types of diseases such as white muscle disease, and mulberry heart disease.The aim of this experiment was to investigate the effect and mechanism of Se deficiency on apoptosis in porcine gastric tissue. Forty weaned piglets were randomly divided into Se deficiency group and control group, and fed with low Se diet and normal diet for six weeks respectively. The histochemical characteristics, antioxidant indexes, apoptotic genes and apoptotic protein expression of gastric cells in Se-deficient piglets were detected. The results of antioxidant index, TUNEL, RT-PCR and Western blot showed that Se deficiency decreased the activities of CAT, SOD and GSH-Px, increased the apoptotic rate of porcine gastric tissue, increased the expression of Bax and Caspase-3, and decreased the expression of Bcl-2. The results demonstrated that Se deficiency could induce apoptosis in porcine gastric tissue cells through oxidative stress-induced mitochondrial pathway. The stomach was a key target of Se deficiency and may play a key role in the response to Se deficiency. Our study may provide new ideas for the prevention and treatment of swine gastric diseases caused by Se deficiency and is beneficial to the development of pig farming industry.

Effects of phytase and 25-hydroxyvitamin D3 supplementation on growth performance and bone development in weaned piglets in Ca- and P-deficient dietary.

BACKGROUND: The beneficial function of phytase and 25-hydroxyvitamin D3 (HyD) on the feed utilization rate has been widely investigated. However, studies concerning its influence on weaned piglets largely lag behind. The aim of this study was to investigate the effects of phytase and HyD supplementation on the growth performance and bone development in weaned piglets under dietary Ca and P deficiency. RESULTS: The results showed that dietary Ca and P deficiency decreased (P < 0.05) the content of serum P in 6-10 kg piglets, as well as reducing (P < 0.05) the contents of serum Ca and P, average daily gain (ADG), bone mineral density (BMD), breaking force (BF), bone ash and femur Ca in 10-20 kg piglets. Compared with the control group, the feed-to-gain ratio (F/G) of 6-10 kg piglets in the Phy group was decreased (P < 0.05), whereas the ADG, blood Ca and P, BMD, BF, bone ash, P apparent digestibility, Ca and P retention rate of 10-20 kg piglets were increased (P < 0.05). The contents of serum osteocalcin and HyD in 6-10 kg piglets and ADG were higher than in the control group (P < 0.05), as well as the contents of serum Ca and HyD in 10-20 kg piglets in the HyD treatment group. Supplementation with both Phy and HyD decreased the F/D (P < 0.05) and increased the contents of serum Ca, P and HyD in 6-10 kg piglets as well as enhancing the ADG, BMD, BF, bone ash, femur Ca and P, serum Ca and P, HyD, and the apparent digestibility and retention of Ca and P (P < 0.05) in 10-20 kg piglets. Supplementation with Phy and HyD in Ca- and P-deficient dietary decreased bone resorption, and improved tight arrangement of collagen fibers and oblique fibers in weaned piglets. CONCLUSION: These data indicated that supplementation with both 1500 U kg-1 Phy and 50 µg kg-1 HyD could enhance dietary Ca and P utilization and promote bone development in low Ca and P dietary, and supplementation with both Phy and HyD had a significant synergy effect compared to single supplement. © 2021 Society of Chemical Industry.

Effects of 25-hydroxyvitamin D3 on growth performance, serum parameters, fecal microbiota, and metabolites in weaned piglets fed diets with low calcium and phosphorus.

BACKGROUND: With the development of intensive farming, long-term exposure of pigs to poor light conditions is not conducive to the production of vitamin D3 , and vitamin D3 deficiency could affect absorption and metabolism of calcium (Ca) and phosphorus (P). 25-Hydroxyvitamin D3 (25OHD3 ) has higher bioactivity than regular vitamin D3 . This study investigated the effects of 25OHD3 on performance, serum parameters, fecal microbiota, and metabolites in weaned piglets fed with low Ca-P diet. RESULTS: It was found that a low Ca-P diet supplemented with 50 µg/kg 25OHD3 (NC + 25-D) improved (P < 0.05) average daily gain (ADG) in phase 2 and in the overall period of the experiment, and increased (P < 0.05) the immunoglobulin G (IgG), immunoglobulin A (IgA), catalase (CAT), bone-specific alkaline phosphatase (BALP), and osteocalcin (OC) serum content on day 28 compared with a low Ca-P diet (NC), but no differences were observed between a normal Ca-P diet (PC) and the NC + 25-D diet. Compared with NC, the abundance of Firmicutes was higher (P < 0.05) in PC and NC + 25-D. NC + 25-D decreased (P < 0.05) the abundance of Streptococcaceae compared with PC and NC, and increased (P < 0.05) the abundance of Lachnospiraceae compared with NC. Serum 25OHD3 was negatively correlated with the abundance of fecal Streptococcaceae (P < 0.05), and positively correlated with the abundance of fecal Lachnospiraceae (P < 0.05). CONCLUSION: Supplementation of 25OHD3 in a low Ca-P diet improved serum immunity, bone biochemical parameters, and fecal microbiota such as decreased Streptococcaceae abundance and increased Lachnospiraceae abundance, which could subsequently promote growth of piglets. The effects were similar to that of a normal Ca-P diet. © 2021 Society of Chemical Industry.

Soybean meal particle size for pigs during the nursery phase / Tamanho da partícula do farelo de soja em suínos no período de creche

This study evaluated the effect of soybean meal (SBM) particle size on nutrient digestibility and the growth performance of nursery piglets. Sixty-three piglets (BW = 6.86 kg ± 0.56; 23 d of age) were distributed in a randomized block design (by initial weight and sex) with 3 dietary treatments: diets with 1,017 µm (unground); 585 µm; and 411µm SBM, with 7 replicates of 3 piglets each. All diets were offered ad libitum in mash form, formulated differently according to three growing phases: (1) with 20% of SBM, from 23 to 32 d of age; (2) with 25% of SBM, from 32 to 44 d of age, and (3) with 30% of SBM, from 44 to 63 d of age. For the first 21 d, pigs fed diets with a medium particle size of SBM (585mm) had better average weight gain and feed/gain ratio (P<0.05). The average feed intake, average body weight gain, and feed/gain ratio from 44 to 63 d improved (P<0.05) with increasing SBM particle sizes, and the average live weight for the overall period increased with coarser SBM (P<0.05). There was a marginally improvement (P < 0.1) on digestible energy as particle size of SBM decreased; although, no differences (P > 0.05) in the coefficients of apparent digestibility of dry matter and crude protein for the assessed SBM particle sizes were observed. It was concluded that the grinding of dietary SBM is not required for piglets during the nursery phase.

O objetivo deste estudo foi avaliar o efeito do tamanho da partícula do farelo de soja (FS) sobre o desempenho e digestibilidade de leitões no período de creche. 63 leitões (6,86 kg ± 0,56; 23 dias de idade) foram distribuídos aleatoriamente em um delineamento de blocos casualizados (peso inicial e sexo) entre os tratamentos. As dietas experimentais foram produzidas a partir de diferentes tamanhos médios de partículas do FS moídos ou não: 1,017 µm (sem moer), 585 µm (moído em peneira de 10 mm), e 411µm (moído em peneira de 3 mm), totalizando três tratamentos com sete repetições de três animais cada. Todas as dietas foram fornecidas na forma farelada e ad libitum, sendo divididas em três fases: 1) com 20% de FS, dos 23 a 32 dias de idade; 2) com 25% de FS, dos 32 a 44 dias de idade; e 3) com 30% de FS, dos 44 a 62 dias de idade. Nos primeiros 21 dias de experimento, os animais consumindo a dieta com o tamanho médio (585 µm) das partículas do FS apresentaram melhores resultados para o ganho de peso médio e conversão alimentar. No período seguinte (44 a 63 d), houve (P<0.05) aumento do consumo de ração médio, do ganho do peso médio e melhor conversão alimentar dos leitões conforme o aumento do tamanho da partícula de FS incluída na ração. Consequentemente, ao final do experimento houve melhora linear do peso vivo médio dos animais com o aumento do tamanho do FS consumido. Foi observado melhora marginalmente significativa (P < 0.1) da energia digestível conforme a redução no tamanho do FS, entretanto, não houve diferença (P > 0.05) no coeficiente de digestibilidade aparente da matéria seca e da proteína bruta dos animais entre os diferentes tamanhos do FS. Em conclusão, de acordo com as condições deste estudo, não se faz necessária a moagem do farelo de soja para leitões no período de creche.

Fruit and Vegetable Supplemented Diet Modulates the Pig Transcriptome and Microbiome after a Two-Week Feeding Intervention.

A study was conducted to determine the effects of a diet supplemented with fruits and vegetables (FV) on the host whole blood cell (WBC) transcriptome and the composition and function of the intestinal microbiome. Nine six-week-old pigs were fed a pig grower diet alone or supplemented with lyophilized FV equivalent to half the daily recommended amount prescribed for humans by the Dietary Guideline for Americans (DGA) for two weeks. Host transcriptome changes in the WBC were evaluated by RNA sequencing. Isolated DNA from the fecal microbiome was used for 16S rDNA taxonomic analysis and prediction of metabolomic function. Feeding an FV-supplemented diet to pigs induced differential expression of several genes associated with an increase in B-cell development and differentiation and the regulation of cellular movement, inflammatory response, and cell-to-cell signaling. Linear discriminant analysis effect size (LEfSe) in fecal microbiome samples showed differential increases in genera from Lachnospiraceae and Ruminococcaceae families within the order Clostridiales and Erysipelotrichaceae family with a predicted reduction in rgpE-glucosyltransferase protein associated with lipopolysaccharide biosynthesis in pigs fed the FV-supplemented diet. These results suggest that feeding an FV-supplemented diet for two weeks modulated markers of cellular inflammatory and immune function in the WBC transcriptome and the composition of the intestinal microbiome by increasing the abundance of bacterial taxa that have been associated with improved intestinal health.

Tripeptidyl peptidase I activity in porcine lumbar spinal ganglia - a histochemical study.

Distribution of tripeptidyl peptidase I (TPPI) activity in the structures of porcine lumbar spinal ganglia (LSG) was studied by enzyme histochemistry on cryostat sections from all the ganglia using the substrate glycyl-L-prolyl-L-methionyl-5-chloro-1-anthraquinonyl hydrazide (GPM-CAH) and 4-nitrobenzaldehyde (NBA) as visualization factor. Light microscopic observations showed TPPI activity in almost all the LSG structures. The enzyme reaction in different cell types was compared semi-quantitatively. Strong reaction was observed in the small neurons, satellite ganglia cells and some nerve fibers. Weak reactivity was found in the large sensory somatic neurons, whereas moderate reaction for TPPI was determined in the middle sensory somatic neurons and some nerve fibers. Statistical analysis by one-way ANOVA showed no significance of difference (when p⟨0.05) for the number of TPPI positive neurons per mm2. The original data obtained by the enzyme histochemistry method give us a reason to presume that TPPI actively participates in the functions of all the neuronal structures in porcine LSG. According to our results, it could be suggested that TPPI activity is important for the functions of autonomic and somatic sensory neurons.

The genome variation and developmental transcriptome maps reveal genetic differentiation of skeletal muscle in pigs.

Natural and artificial directional selections have resulted in significantly genetic and phenotypic differences across breeds in domestic animals. However, the molecular regulation of skeletal muscle diversity remains largely unknown. Here, we conducted transcriptome profiling of skeletal muscle across 27 time points, and performed whole-genome re-sequencing in Landrace (lean-type) and Tongcheng (obese-type) pigs. The transcription activity decreased with development, and the high-resolution transcriptome precisely captured the characterizations of skeletal muscle with distinct biological events in four developmental phases: Embryonic, Fetal, Neonatal, and Adult. A divergence in the developmental timing and asynchronous development between the two breeds was observed; Landrace showed a developmental lag and stronger abilities of myoblast proliferation and cell migration, whereas Tongcheng had higher ATP synthase activity in postnatal periods. The miR-24-3p driven network targeting insulin signaling pathway regulated glucose metabolism. Notably, integrated analysis suggested SATB2 and XLOC_036765 contributed to skeletal muscle diversity via regulating the myoblast migration and proliferation, respectively. Overall, our results provide insights into the molecular regulation of skeletal muscle development and diversity in mammals.

Total Aseptization of Boar Semen, to Increase the Biosecurity of Reproduction in Swine.

The aim of the study was to establish the complete microbiological profile of boar semen (Sus scrofa domesticus) and to choose the most effective antiseptic measures in order to control and optimize AI reproduction in pig farms. One hundred and one semen samples were collected and analyzed from several pig farms. The microbiological profile of ejaculates was determined by evaluating the degree of contamination of fresh semen and after dilution with specific extenders. The bacterial and fungal load of fresh boar semen recorded an average value of 82.41/0.149 × 103 CFU/mL, while after diluting the ejaculates the contamination value was 0.354/0.140 × 103 CFU/mL. Twenty-four germs (15 bacterial and 9 fungal species) were isolated, the most common being Candida parapsilosis/sake (92%) and Escherichia coli (81.2%). Modification of the sperm collection protocol (HPBC) reduced contamination in raw sperm by 49.85% in bacteria (significant (p < 0.00001) and by 9.67% in fungi (non-significant (p < 0.111491). The load in bacteria and filamentous fungi can be controllable, but not in levuras fungi. Some fluconazole-added extenders (12.5 mg%), ensure fungal aseptization, and even an increase in sperm progressivity (8.39%) for at least a 12 h shelf life after dilution. Validation of sperm aseptization was done by maintaining sow fecundity unchanged after AI (insignificant p > 0.05).

Heritability and genetic correlations of plasma metabolites of pigs with production, resilience and carcass traits under natural polymicrobial disease challenge.

Metabolites in plasma of healthy nursery pigs were quantified using nuclear magnetic resonance. Heritabilities of metabolite concentration were estimated along with their phenotypic and genetic correlations with performance, resilience, and carcass traits in growing pigs exposed to a natural polymicrobial disease challenge. Variance components were estimated by GBLUP. Heritability estimates were low to moderate (0.11 ± 0.08 to 0.19 ± 0.08) for 14 metabolites, moderate to high (0.22 ± 0.09 to 0.39 ± 0.08) for 17 metabolites, and highest for L-glutamic acid (0.41 ± 0.09) and hypoxanthine (0.42 ± 0.08). Phenotypic correlation estimates of plasma metabolites with performance and carcass traits were generally very low. Significant genetic correlation estimates with performance and carcass traits were found for several measures of growth and feed intake. Interestingly the plasma concentration of oxoglutarate was genetically negatively correlated with treatments received across the challenge nursery and finisher (- 0.49 ± 0.28; P < 0.05) and creatinine was positively correlated with mortality in the challenge nursery (0.85 ± 0.76; P < 0.05). These results suggest that some plasma metabolite phenotypes collected from healthy nursery pigs are moderately heritable and genetic correlations with measures of performance and resilience after disease challenge suggest they may be potential genetic indicators of disease resilience.

Nutritional restriction during the peri-conceptional period alters the myometrial transcriptome during the peri-implantation period.

This study hypothesized that female peri-conceptional undernutrition evokes transcriptomic alterations in the pig myometrium during the peri-implantation period. Myometrium was collected on days 15-16 of pregnancy from pigs fed a normal- (n = 4) or restricted-diet (n = 4) from conception until day 9th of pregnancy, and the transcriptomic profiles of the tissue were compared using Porcine (V2) Expression Microarrays 4 × 44 K. In restricted diet-fed pigs, 1021 differentially expressed genes (DEGs) with fold change ≥ 1.5, P ≤ 0.05 were revealed, and 708 of them were up-regulated. Based on the count score, the top within GOs was GO cellular components "extracellular exosome", and the top KEGG pathway was the metabolic pathway. Ten selected DEGs, i.e. hydroxysteroid (17ß) dehydrogenase 8, cyclooxygenase 2, prostaglandin F receptor, progesterone receptor membrane component 1, progesterone receptor membrane component 2, annexin A2, homeobox A10, S-phase cyclin A-associated protein in the ER, SRC proto-oncogene, non-receptor tyrosine kinase, and proliferating cell nuclear antigen were conducted through qPCR to validate microarray data. In conclusion, dietary restriction during the peri-conceptional period causes alterations in the expression of genes encoding proteins involved i.a. in the endocrine activity of the myometrium, embryo-maternal interactions, and mechanisms regulating cell cycle and proliferation.

The Effect of Dietary Protein Imbalance during Pregnancy on the Growth, Metabolism and Circulatory Metabolome of Neonatal and Weaned Juvenile Porcine Offspring.

Protein imbalance during pregnancy affects women in underdeveloped and developing countries and is associated with compromised offspring growth and an increased risk of metabolic diseases in later life. We studied in a porcine model the glucose and urea metabolism, and circulatory hormone and metabolite profile of offspring exposed during gestation, to maternal isoenergetic low-high (LP-HC), high-low (HP-LC) or adequate (AP) protein-carbohydrate ratio diets. At birth, LP-HC were lighter and the plasma acetylcarnitine to free carnitine ratios at 1 day of life was lower compared to AP offspring. Plasma urea concentrations were lower in 1 day old LP-HC offspring than HP-LC. In the juvenile period, increased insulin concentrations were observed in LP-HC and HP-LC offspring compared to AP, as was body weight from HP-LC compared to LP-HC. Plasma triglyceride concentrations were lower in 80 than 1 day old HP-LC offspring, and glucagon concentrations lower in 80 than 1 day old AP and HP-LC offspring. Plasma urea and the ratio of glucagon to insulin were lower in all 80 than 1 day old offspring. Aminoacyl-tRNA, arginine and phenylalanine, tyrosine and tryptophan metabolism, histidine and beta-alanine metabolism differed between 1 and 80 day old AP and HP-LC offspring. Maternal protein imbalance throughout pregnancy did not result in significant consequences in offspring metabolism compared to AP, indicating enormous plasticity by the placenta and developing offspring.

Intestinal organoid-based 2D monolayers mimic physiological and pathophysiological properties of the pig intestine.

Gastrointestinal infectious diseases remain an important issue for human and animal health. Investigations on gastrointestinal infectious diseases are classically performed in laboratory animals leading to the problem that species-specific models are scarcely available, especially when it comes to farm animals. The 3R principles of Russel and Burch were achieved using intestinal organoids of porcine jejunum. These organoids seem to be a promising tool to generate species-specific in vitro models of intestinal epithelium. 3D Organoids were grown in an extracellular matrix and characterized by qPCR. Organoids were also seeded on permeable filter supports in order to generate 2D epithelial monolayers. The organoid-based 2D monolayers were characterized morphologically and were investigated regarding their potential to study physiological transport properties and pathophysiological processes. They showed a monolayer structure containing different cell types. Moreover, their functional activity was demonstrated by their increasing transepithelial electrical resistance over 18 days and by an active glucose transport and chloride secretion. Furthermore, the organoid-based 2D monolayers were also confronted with cholera toxin derived from Vibrio cholerae as a proof of concept. Incubation with cholera toxin led to an increase of short-circuit current indicating an enhanced epithelial chloride secretion, which is a typical characteristic of cholera infections. Taken this together, our model allows the investigation of physiological and pathophysiological mechanisms focusing on the small intestine of pigs. This is in line with the 3R principle and allows the reduction of classical animal experiments.

Hyaluronan and Collagen Are Prominent Extracellular Matrix Components in Bovine and Porcine Ovaries.

The extracellular matrix (ECM) is a major component of the ovarian stroma. Collagen and hyaluronan (HA) are critical ovarian stromal ECM molecules that undergo age-dependent changes in the mouse and human. How these matrix components are regulated and organized in other mammalian species with reproductive characteristics similar to women such as cows and pigs, has not been systematically investigated. Therefore, we performed histological, molecular, and biochemical analyses to characterize collagen and HA in these animals. Bovine ovaries had more collagen than porcine ovaries when assessed biochemically, and this was associated with species-specific differences in collagen gene transcripts: Col3a1 was predominant in cow ovaries while Col1a1 was predominant in pig ovaries. We also observed more HA in the porcine vs. bovine ovary. HA was distributed across three molecular weight ranges (<100 kDa, 100-300 kDa, and >300 kDa) in ovarian tissue and follicular fluid, with tissue having more >300 kDa HA than the other two ranges. Transcripts for HA synthesis and degradation enzymes, Has3 and Hyal2, respectively, were predominant in cow ovaries, whereas Has2, Kiaa1199, and Tmem2 tended to be predominant in pig ovaries. Together, our findings have implications for the composition, organization, and regulation of the ovarian ECM in large mammalian species, including humans.