Effects of sustained hyperprolactinemia in late gestation on the mammary parenchymal tissue transcriptome of gilts.

BACKGROUND: Gilts experiencing sustained hyperprolactinemia from d 90 to 109 of gestation showed an early onset of lactogenesis coupled with premature mammary involution. To better understand the molecular mechanisms underlying the premature mammary involution observed in these gilts, a transcriptomic analysis was undertaken. Therefore, this study aimed to explore the effect of hyperprolactinemia on the global transcriptome in the mammary tissue of late gestating gilts and identify the molecular pathways involved in triggering premature mammary involution. METHODS: On d 90 of gestation, gilts received daily injections of (1) canola oil until d 109 ± 1 of gestation (CTL, n = 18); (2) domperidone (to induce hyperprolactinemia) until d 96 ± 1 of gestation (T7, n = 17) or; (3) domperidone (until d 109 ± 1 of gestation (T20, n = 17). Mammary tissue was collected on d 110 of gestation and total RNA was isolated from six CTL and six T20 gilts for microarray analysis. The GeneChip® Porcine Gene 1.0 ST Array was used for hybridization. Functional enrichment analyses were performed to explore the biological significance of differentially expressed genes, using the DAVID bioinformatics resource. RESULTS: The expression of 335 genes was up-regulated and that of 505 genes down-regulated in the mammary tissue of T20 vs CTL gilts. Biological process GO terms and KEGG pathways enriched in T20 vs CTL gilts reflected the concurrent premature lactogenesis and mammary involution. When looking at individual genes, it appears that mammary cells from T20 gilts can simultaneously upregulate the transcription of milk proteins such as WAP, CSN1S2 and LALBA, and genes triggering mammary involution such as STAT3, OSMR and IL6R. The down-regulation of PRLR expression and up-regulation of genes known to inactivate the JAK-STAT5 pathway (CISH, PTPN6) suggest the presence of a negative feedback loop trying to counteract the effects of hyperprolactinemia. CONCLUSIONS: Genes and pathways identified in this study suggest that sustained hyperprolactinemia during late-pregnancy, in the absence of suckling piglets, sends conflicting pro-survival and cell death signals to mammary epithelial cells. Reception of these signals results in a mammary gland that can simultaneously synthesize milk proteins and initiate mammary involution.

Effect of muscle fibre types and carnosine levels on the expression of carnosine-related genes in pig skeletal muscle.

It is generally accepted that carnosine (ß-alanyl-L-histidine) content is higher in glycolytic than in oxidative muscle fibres, but the underlying mechanisms responsible for this difference remain to be elucidated. A first study to better understand potential mechanisms involved was undertaken (1) to determine whether differences in the expression of carnosine-related enzymes (CARNS1, CNDP2) and transporters (SLC6A6, SLC15A3, SLC15A4, SLC36A1) exist between oxidative and glycolytic myofibres and (2) to study the effect of carnosine on myoblast proliferative growth and on carnosine-related gene expression in cultured myoblasts isolated from glycolytic and oxidative muscles. Immunohistochemistry analyses were conducted to determine the cellular localization of carnosine-related proteins. Laser-capture microdissection and qPCR analyses were performed to measure the expression of carnosine-related genes in different myofibres isolated from the longissimus dorsi muscle of ten crossbred pigs. Myogenic cells originating from glycolytic and oxidative muscles were cultured to assess the effect of carnosine (0, 10, 25 and 50 mM) on their proliferative growth and on carnosine-related gene expression. The mRNA abundance of CNDP2 and of the studied carnosine transporters was higher in oxidative than in glycolytic myofibres. Since carnosine synthase (CARNS1) mRNA abundance was not affected by either the fibre type or the addition of carnosine to myoblasts, its transcriptional regulation would not be the main process by which carnosine content differences are determined in oxidative and glycolytic muscles. The addition of carnosine to myoblasts leading to a dose-dependent increase in SLC15A3 transcripts, however, suggests a role for this transporter in carnosine uptake and/or efflux to maintain cellular homeostasis.

Linoleic acid, α-linolenic acid and enterolactone affect lipid oxidation and expression of lipid metabolism and antioxidant-related genes in hepatic tissue of dairy cows.

Although beneficial effects have been attributed to PUFA supplementation in high-yielding dairy cows, diets rich in PUFA may also increase oxidative stress in tissues such as the liver. To fully exploit the health benefits of PUFA, we believe that the addition of natural antioxidants could help in preventing oxidative damage. Using an in vitro precision-cut liver slices (PCLS) tissue culture system, we investigated the effects of different linoleic acid (LA, n-6):α-linolenic acid (ALA, n-3) ratios (LA:ALA ratio of 4, LA:ALA ratio of 15 and LA:ALA ratio of 25) in the presence or absence of the antioxidant enterolactone (ENL) on (1) the mRNA abundance of genes with key roles in hepatic lipid metabolism, oxidative stress response and inflammatory processes, (2) oxidative damages to lipids and proteins and (3) superoxide dismutase activity in early-lactating dairy cows. The addition of LA and ALA to PCLS culture media increased oxidative damage to lipids as suggested by higher concentrations of thiobarbituric acid reactive substances and increased the expression of nuclear factor erythroid 2-related factor 2 target genes. The addition of ENL was effective in preventing lipid peroxidation caused by LA and ALA. Transcript abundance of sterol regulatory element-binding transcription factor 1 and its lipogenic target genes acetyl-CoA carboxylase α, fatty acid synthase (FASN) and stearoyl-CoA desaturase (SCD) was decreased with LA and ALA, whereas ENL decreased FASN and SCD gene expression. Our results show that addition of LA and ALA to PCLS culture media lowers hepatic lipogenic gene expression and increases oxidative damages to lipids. On the other hand, addition of ENL prevents oxidative damages provoked by these PUFA.

Effects of different n-6:n-3 fatty acid ratios and of enterolactone on gene expression and PG secretion in bovine endometrial cells.

Feeding flaxseed to dairy cows can modulate gene expression and PG synthesis in the uterus at the time of peri-implantation. The objectives of the present study were to determine which flaxseed components are responsible for these effects and how different endometrial cell types are affected. We evaluated the effects of six different linoleic acid (n-6):α-linolenic acid (n-3) ratios and three concentrations of the lignan enterolactone (ENL) on endometrial stromal cells (SC) and epithelial cells (EC). The mRNA abundance of genes with known or suspected roles in embryo survival or PG synthesis was evaluated, along with PGE2 and PGF2α concentrations in culture media. The mRNA abundance of several genes was modulated by different fatty acid (FA) ratios and/or ENL, and this modulation differed between cell types. The FA4 (FA at an n-6:n-3 ratio of 4) treatment (rich in n-3 FA) increased the mRNA abundance of genes that have positive effects on uterine receptivity and implantation when compared with the FA25 (FA at an n-6:n-3 ratio of 25) treatment (rich in n-6 FA). ENL decreased PGE2 and PGF2α concentrations in both cell types, and this reduction was associated with lower mRNA abundance of the PG synthase genes AKR1B1 and PTGES in SC. The combination of ENL with FA (FA4 treatment) resulted in the greatest reduction in PGF2α concentrations when compared with the addition of FA (FA4) or ENL alone. Because of the known luteolytic properties of PGF2α, a reduction in endometrial PGF2α secretion would favour the establishment and maintenance of pregnancy.

Association of adiponectin and adiponectin receptor genes with sow productivity estimated breeding values.

Our objectives were to estimate frequencies of previously identified single nucleotide polymorphisms (SNPs) in adiponectin (ADIPOQ) and its receptors (ADIPOR1 and ADIPOR2) in a population of Duroc, Landrace and Yorkshire pigs and evaluate the effect of these alleles on sow productivity estimated breeding values (EBVs). Eight SNPs were genotyped on 446 pigs in the ADIPOQ (c.178G>A, c.*300A>G, c.*1094_1095insC and c.*1779A>C), ADIPOR1 (c.*129A>C) and ADIPOR2 (c.*112G>A, c.*295G>C and c.*1455G>A) genes. Association analyses were performed with sow productivity EBVs based on litter records collected in Canadian breeding farms. There were significant associations between ADIPOQ c.178G>A and c.*1094_1095insC SNPs and studied traits. However, none of these associations remained significant after applying a Bonferroni correction. The ADIPOR2 c.*112G>A SNP was associated with the total number of piglets born (TNB, P < 0.001) and litter weight at weaning (LWW, P < 0.001) EBVs. Associations were also observed between the ADIPOR2 [A;C;G] haplotype and TNB and LWW (P < 0.001). Our results demonstrate that a selection in favor of the c.*112G allele or against the [A;C;G] haplotype may have the potential to increase LWW EBVs. However, the c.*112G allele is also associated with lower TNB EBVs. Some of the alleles of the genes studied showed substantial variability and in general, the results corroborated previously reported findings for an independent sow population. However, careful cost-benefits analyses should be performed before using these markers in selection program as an improvement in TNB may translate into lighter LWW, with its associated negative impact on production traits such as growth performances.

Relationship between adiponectin and fertility in the female pig.

Adiponectin isoforms may mediate different aspects of the pleiotropic function of the protein, including the reproductive process. We examined the pattern of circulating adiponectin and adiponectin system expression in fat and ovarian tissues of hyperfertile and subfertile sows. We demonstrated the presence of five different isoforms of adiponectin (90, 158, 180, 250 and >250kDa) in the circulation and identified a subgroup of subfertile females that displayed reduced abundance of all adiponectin isoforms as well as a lack of the 250-kDa adiponectin isoform in both serum and follicular fluid. Subfertility in these animals was associated with fewer large follicles and corpora lutea in the ovaries, as well as lower concentrations of 17ß-oestradiol in the follicular fluid of large follicles. In addition, subfertile females showed higher adiponectin mRNA in fat tissue and altered mRNA and protein expression of adiponectin and its receptors in the ovary. Changes in the abundance and pattern of circulating adiponectin isoforms have been associated with reproductive disorders in animals and humans, including polycystic ovarian syndrome (PCOS). Our findings suggest that the adiponectin system may play an important role in controlling ovarian function and influencing porcine fertility.

mRNA Expression of lipogenic enzymes in mammary tissue and fatty acid profile in milk of dairy cows fed flax hulls and infused with flax oil in the abomasum.

In the present study, the effect of flax hulls with or without flax oil bypassing the rumen on the expression of lipogenic genes in the mammary tissue of dairy cows was investigated. A total of eight dairy cows were used in a replicated 4 × 4 Latin square design. There were four periods of 21 d each and four treatments: control diet with no flax hulls (CONT); diet with 9·88 % flax hulls in the DM (HULL); control diet with 500 g flax oil/d infused in the abomasum (COFO); diet with 9·88 % flax hulls in the DM and 500 g flax oil/d infused in the abomasum (HUFO). A higher mRNA abundance of sterol regulatory element binding transcription factor, fatty acid (FA) synthase, lipoprotein lipase (LPL), PPARγ1, stearoyl-CoA desaturase (SCD) and acetyl-coenzyme A carboxylase-α was observed in cows fed HULL than in those fed CONT, and HUFO had the opposite effect. Compared with CONT, COFO and HUFO lowered the mRNA abundance of SCD, which may explain the lower proportions of MUFA in milk fat with flax oil infusion. The mRNA abundance of LPL in mammary tissue and proportions of long-chain FA in milk fat were higher in cows fed COFO than in those fed CONT. The highest proportions of trans FA were observed when cows were fed HULL. The present study demonstrates that flax hulls with or without flax oil infusion in the abomasum can affect the expression of lipogenic genes in the mammary tissue of dairy cows, which may contribute to the improvement of milk FA profile.

Dual effects of dietary carnosine during in vitro digestion of a Western meal model with added ascorbic acid.

The beneficial role of carnosine during in vitro digestion of meat was previously demonstrated, and it was hypothesized that such benefits could also be obtained in a meal system. The current study, therefore, assessed carnosine effects on markers of lipid and protein oxidation and of advanced glycation end products (AGEs) during gastric and duodenal in vitro digestion of a burger meal model. The model included intrinsic (low) and enhanced (medium and high) carnosine levels in a mix of pork mince and bread, with or without ascorbic acid (AA) and/or fructose as anti- and prooxidants, respectively. In the presence of either AA or fructose, a carnosine prooxidative potential during digestion was observed at the medium carnosine level depending on markers and digestive phases. However, free carnosine found at the high carnosine level exerted a protective effect reducing the formation of 4-hydroxynonenal in the gastric phase and glyoxal in both the gastric and duodenal phases. Dual effects of carnosine are likely concentration related, whereby at the medium level, free radical production increases through carnosine's ferric-reducing capacity, but there is insufficient quantity to reduce the resulting oxidation, while at the higher carnosine level some decreases in oxidation are observed. In order to obtain carnosine benefits during meal digestion, these findings demonstrate that consideration must be given to the amount and nature of other anti- and prooxidants present and any potential interactions. PRACTICAL APPLICATION: Carnosine, a natural compound in meat, is a multifunctional and beneficial molecule for health. However, both pro- and antioxidative effects of carnosine were observed during digestion of a model burger meal when ascorbic acid was included at a supplemental level. Therefore, to obtain benefits of dietary carnosine during digestion of a meal, consideration needs to be given to the amount and nature of all anti- and prooxidants present and any potential interactions.

Mammary gene expression and activity of antioxidant enzymes and oxidative indicators in the blood, milk, mammary tissue and ruminal fluid of dairy cows fed flax meal.

The effects of flax meal (FM) on the activity of antioxidant enzymes (superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT)) in the blood, mammary tissue and ruminal fluid, and oxidative stress indicators (thiobarbituric acid-reactive substances(TBARS) and 1,1-diphenyl-2-picrylhydrazyl-scavenging activity) in the milk, plasma and ruminal fluid of dairy cows were determined.The mRNA abundance of the antioxidant enzymes and oxidative stress-related genes was assessed in mammary tissue. A total of eight Holstein cows were used in a double 4 x 4 Latin square design. There were four treatments in the diet: control with no FM(CON) or 5% FM (5FM), 10% FM (10FM) and 15% FM (15FM). There was an interaction between treatment and time for plasma GPx and CAT activities. Cows supplemented with FM had a linear reduction in TBARS at 2 h after feeding, and there was no treatment effect at 0, 4 and 6 h after feeding. TBARS production decreased in the milk of cows fed the 5FM and 10FM diets. There was a linear increase in nuclear factor (erythroid-derived 2)-like 2 (NFE2L2) mRNA abundance in mammary tissue with FM supplementation.A linear trend for increased mRNA abundance of the CAT gene was observed with higher concentrations of FM. The mRNA abundance of CAT, GPx1, GPx3, SOD1, SOD2, SOD3 and nuclear factor of k light polypeptide gene enhancer in B-cells (NFKB) genes was not affected by the treatment. These findings suggest that FM supplementation can improve the oxidative status of Holstein cows as suggested by decreased TBARS production in ruminal fluid 2 h post-feeding and increased NFE2L2/nuclear factor-E2-related factor 2 (Nrf2) mRNA abundance in mammary tissue.

Identification of single nucleotide polymorphisms in the bovine follicle-stimulating hormone receptor and effects of genotypes on superovulatory response traits.

In dairy cows, there is evidence that failure to respond to superovulation protocols is a heritable trait. In women, genotyping for the p.N680S single nucleotide polymorphism (SNP) in the follicle-stimulating hormone receptor (FSHR) gene may help identify poor responders before ovarian stimulation is initiated. Our objectives were to identify SNPs in the coding region of the bovine FSHR gene and to investigate the effect of FSHR genotypes on superovulatory response in Holstein cattle. Sequencing of FSHR exons 1-10 revealed seven SNPs. Three were non-synonymous mutations (c.337C>G, c.871A>G and c.1973C>G). SNP c.337C>G encodes for a proline-to-alanine (p.Pro113Ala) amino acid replacement in the extracellular ligand-binding domain of the receptor. PCR-RFLP analyses showed that homozygous GG Holstein cows present a higher percentage of viable embryos, whereas GG and CG animals have less unfertilised oocytes. SNP c.871A>G results in an isoleucine-to-valine (p.Ile291Val) modification, and homozygous AA animals present lower embryo yield after superovulatory treatments. SNP c.1973C>G corresponds to a threonine-to-serine (p.The658Ser) modification in the intracellular carboxyl-terminal domain of the FSHR protein, and homozygous GG Holstein cows were associated with a lower embryo yield and a higher percentage of unfertilised oocytes. Our results suggest that specific alleles of the bovine FSHR gene are associated with variations in embryo yield and in the number of unfertilised oocytes.

Effects of leptin and adiponectin on proliferation and protein metabolism of porcine myoblasts.

The aim of this study was to show the abundance of leptin and adiponectin receptors (LEPR, ADIPOR1, ADIPOR2) and to determine the direct effects of leptin and adiponectin on the in vitro growth of porcine skeletal muscle cells. ADIPOR1 and ADIPOR2 were abundant at mRNA and protein level in proliferating and differentiating myoblast cultures derived from semimembranosus and semitendinosus muscles of newborn piglets, whereas LEPR expression was close to the detection limit. Adiponectin (10, 20, 40 µg/ml) attenuated the proliferation of porcine myoblasts, measured as [(3)H]-thymidine incorporation and real-time monitoring of the cells in response to 24- and 48-h exposure, in a dose-dependent manner. This effect resulted from suppressed basic fibroblast growth factor (bFGF)-mediated stimulation of DNA synthesis in serum-free medium (SFM) containing bFGF. No effects of leptin (5, 10, 20, 40, 80 ng/ml) on myoblast proliferation in SFM were detectable. Neither leptin nor adiponectin altered protein synthesis and degradation in differentiating porcine myoblasts cultured in SFM. The results on receptor abundance suggest that porcine skeletal muscle cells may be sensitive to adiponectin and leptin. However, except via inhibitory interaction of adiponectin with bFGF, these adipokines appear not to affect in vitro proliferation and protein metabolism of porcine muscle cells directly under serum-free culture conditions.

Mammary gene expression and activity of antioxidant enzymes and concentration of the mammalian lignan enterolactone in milk and plasma of dairy cows fed flax lignans and infused with flax oil in the abomasum.

The objectives of the study were to investigate the effects of dietary supplementation of flax hulls and/or flax oil on the activity of antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX)) in plasma and the mammary gland and the relative mRNA abundance of antioxidant genes in the mammary gland of dairy cows. A total of eight dairy cows were used in a replicated 4 × 4 Latin square design. There were four treatments: control with no flax hulls (CONT), 9·88% flax hulls in the DM (HULL), control with 500 g flax oil/d infused in the abomasum (COFO), 9·88% flax hulls in the DM and 500 g flax oil/d infused in the abomasum (HUFO). Plasma GPX activity tended to decrease with flax oil supplementation. Cows fed HULL had higher levels of CAT, GPX1 and SOD1 mRNA in the mammary gland and lower mRNA abundance of GPX3, SOD2 and SOD3 compared with those fed CONT. Abundance of CAT, GPX1, GPX3, SOD2 and SOD3 mRNA was down-regulated in the mammary gland of cows fed HUFO compared to those fed CONT. The mRNA abundance of CAT, GPX1, GPX3 and SOD3 was lower in the mammary gland of cows fed COFO than in the mammary gland of cows fed CONT. The present study demonstrates that flax hulls contribute to increasing the abundance of some antioxidant genes, which can contribute to protecting against oxidative stress damage occurring in the mammary gland and other tissues of dairy cows.

Dietary supplementation with lysine (protein) stimulates mammary development in late pregnant gilts.

The goal of this project was to determine if standardized ileal digestible (SID) lysine provided at 40% above estimated requirements, with the concomitant increase in protein intake, from days 90 to 110 of gestation would stimulate mammary development in gilts. From day 90 of gestation, Yorkshire × Landrace gilts were fed 2.65 kg of either a conventional diet (CTL, control, n = 19) providing 18.6 g/d of SID Lys or a diet providing 26.0 g/d of SID Lys via additional soybean meal (HILYS, n = 19). Both diets were isoenergetic. Jugular blood samples obtained on days 90 and 110 of gestation were used to measure concentrations of insulin-like growth factor-1 (IGF-1), metabolites, and amino acids (AA). Gilts were necropsied on day 110 ± 1 of gestation to obtain mammary glands for compositional analyses, immunohistochemistry, and analysis of mRNA abundance for AA transporters and markers of cell proliferation and differentiation. The HILYS gilts gained more body weight (P < 0.01) during the experimental period compared with CTL gilts, and had greater fetal weights (1.29 vs. 1.21 ± 0.03 kg, P < 0.05). There was no difference in circulating IGF-1, glucose, or albumin (P > 0.10) between HILYS and CTL gilts on day 110 of gestation, whereas concentrations of urea and free fatty acids were greater (P < 0.01), and those of Trp and Ala were lower (P < 0.05), in HILYS than CTL gilts. The provision of lysine at 40% above estimated requirements increased total mammary parenchymal mass by 44%, as well as total parenchymal fat, protein, DNA, and RNA (P < 0.01). The mRNA abundance of ACACA was greater (P < 0.05) in HILYS than CTL gilts, while only the AA transporter SLC6A14 tended (P < 0.10) to be greater. Results demonstrate that providing dietary Lys above current National Research Council recommendations in late gestation increases mammary development in gilts. Results also indicate that Lys may have been limiting for protein retention. These data suggest that the use of a two-phase feeding strategy during gestation, whereby dietary Lys is increased from day 90, could benefit potential sow milk yield in the subsequent lactation.

Results indicate that the current National Research Council recommendations for dietary lysine during late pregnancy in pigs, the period when most mammary gland development takes place, are underestimated. From days 90 to 110 of gestation, gilts were fed 2.65 kg of either a conventional diet providing 18.6 g/d of standardized ileal digestible (SID) lysine, or a diet providing 26.0 g/d of SID lysine via the inclusion of additional soybean meal. Diets were isoenergetic. Feeding 26.0 g/d of SID lysine increased the mass of mammary parenchymal tissue (where milk is synthesized) by 44%. Findings suggest that a greater mammary uptake of lysine in supplemented sows supported enhanced accretion of mammary parenchyma. Such information is most pertinent in the actual context where milk yield of hyperprolific sows must be maximized to sustain optimal growth of all their piglets. Furthermore, these data indicate that the use of a two-phase feeding strategy during gestation, whereby dietary lysine is increased from day 90, could benefit potential sow milk yield in the subsequent lactation.

Insulin sensitivity is associated with the observed variation of de novo lipid synthesis and body composition in finishing pigs.

Variations in body composition among pigs can be associated with insulin sensitivity given the insulin anabolic effect. The study objectives were to characterize this association and to compare de novo lipogenesis and the gene expression in the adipose tissue of pigs of the same genetic background. Thirty 30-95 kg of body weight (BW) pigs, catheterized in the jugular vein participated into an oral glucose tolerance test (OGTT; 1.75 g glucose/kg of BW) to calculate insulin-related indexes. The 8 fattest and the 8 leanest pigs were used to determine the relative mRNA abundance of studied genes. The rate of lipogenesis was assessed by incorporation of [U-13C]glucose into lipids. The QUICKI and Matsuda indexes negatively correlated with total body lipids (r = - 0.67 and r = - 0.59; P < 0.01) and de novo lipogenesis (r = - 0.58; P < 0.01). Fat pigs had a higher expression level of lipogenic enzymes (ACACA, ACLY; P < 0.05) than lean pigs. The reduced insulin sensitivity in fat pigs was associated with a higher expression level of glucose-6-phosphate dehydrogenase (G6PD) and a lower expression of peroxisome proliferator-activated receptor-gamma (PPAR-γ). In conclusion, pigs with increased body lipids have lower insulin sensitivity which is associated with increased de novo lipogenesis.

Uterine signaling at the emergence of the embryo from obligate diapause.

Embryonic diapause is the reversible arrest of embryo development prior to implantation under a regime of uterine control that is not well understood. Our objective was to explore uterine modifications associated with the emergence of embryonic diapause in the mink, a species in which embryonic diapause characterizes every gestation. We investigated the uterine transcriptome at reactivation using the suppressive subtractive hybridization technique. A library of 123 differentially expressed genes between uteri with blastocysts in diapause and reactivated blastocysts was generated. Among those genes, 41.5% encode for potential secreted products that are implicated in regulation of cell proliferation (14%), homeostasis (14%), protein folding (11%), electron transport chain (8%), and innate immune response (8%), therefore suggesting that these biological processes are implicated in blastocyst reactivation. Two genes, the high-mobility group nucleosome binding domain 1 (HMGN1), a chromatin remodeling factor, and the secreted protein acidic and cystein-rich (SPARC), which is implicated in extracellular cell-cell interactions, were submitted to more detailed analysis of expression patterns in the mink uterus at blastocyst reactivation. Expression of both HMGN1 and SPARC was increased significantly in the uterus at embryo reactivation compared with diapause, principally in the endometrial epithelium and subepithelial stroma. These results provide new insight into uterine signaling at the emergence of the blastocyst from diapause and highlight the factors HMGN1 and SPARC as potential inductors of uterine environment modifications underlying uterine signaling during emergence of the embryo from embryonic diapause.

Providing domperidone throughout lactation enhances sow lactation performance.

The goal of this project was to determine the effects of domperidone given throughout lactation on hormonal and metabolic status, lactational performance, and gene expression in mammary epithelial cells of sows. Second parity sows were divided in two treatment groups: 1) daily intramuscular injections with canola oil (Control, CTL, n = 24), or 2) daily intramuscular injections with 0.5 mg/kg body weight (BW) of domperidone (DOMP, n = 23). Injections were given at 08h05 starting the day after farrowing until weaning. Over the first 4 d of treatment, DOMP sows also received 0.5 mg/kg BW of domperidone per os twice daily, whereas CTL sows were fed the vehicle. Litter size was standardized to 11 ± 1 within 24 h of birth and piglets were weighed at birth, 24 h postpartum, and on days 7, 22 (weaning on day 23), 35, and 56. Sow feed intake was recorded daily. Representative milk samples were obtained aseptically on day 21 of lactation from 15 sows per treatment for compositional analyses and milk fat globules were used to measure mRNA abundances of various genes. Jugular blood samples were obtained from all sows on days 2, 8, 16, and 23 of lactation to measure concentrations of prolactin, insulin-like growth factor-1 (IGF-1), leptin, adiponectin, insulin, glucose, urea, and free fatty acids (FFA). Concentrations of prolactin (P < 0.001) and FFA (P < 0.01) were increased in DOMP compared with CTL sows, whereas concentrations of insulin were decreased (P < 0.05). Urea concentrations were increased by treatment (P < 0.05) on days 16 and 23 of lactation, and those of IGF-1 were increased (P < 0.01) on day 16. Piglets from DOMP sows were heavier than those from CTL sows on day 22 (P < 0.01). Milk composition was unaffected by treatment. The mRNA abundance in milk fat globules for casein beta and whey acidic protein were lower (P ≤ 0.05) in DOMP than CTL sows. The long form of the prolactin receptor and the signal transducer and activator of transcription 5A mRNA abundances tended to be lower (P < 0.10) in DOMP than CTL sows. In conclusion, hyperprolactinemia induced by domperidone during lactation affected the endocrine and metabolite status of sows and stimulated growth of their suckling piglets.

Protective effects of dietary carnosine during in-vitro digestion of pork differing in fat content and cooking conditions.

Muscle carnosine represents an important health advantage of meat. Ground pork samples with intrinsic or added carnosine; fat content; and cooked under low or high intensity as a 2 × 2 × 2 factorial were digested in-vitro. Changes in free carnosine and in markers of lipid (hexanal, 4-hydroxynonenal (4-HNE), malondialdehyde (MDA) and protein (protein-carbonyls, thiols) oxidation, and of advanced glycation end-products (AGEs) Nε -(carboxymethyl)lysine (CML) were determined in the saliva, gastric, and duodenal digests. During digestion, the different markers overall indicated increased oxidation and decreased free carnosine. Increasing pork carnosine level significantly reduced protein carbonyls, loss of thiols, and 4-HNE during in-vitro gastric digestion, irrespective of fat and cooking level of the meat. Increased carnosine also significantly reduced hexanal, MDA and CML up to the duodenum phase in moderately cooked lean pork. Besides substantiating the formation of AGEs during digestion, these results show a potentially important role of dietary carnosine occurring in the gastrointestinal tract. PRACTICAL APPLICATIONS: The ailments epidemiologically associated with red meat consumption could be counteracted by ingesting carnosine into meat. The health advantages of dietary carnosine, however, have never been demonstrated during digestion, a unique and complex oxidative environment compounded by the composition and cooking of the meat. The results obtained substantiated that AGEs formation occurred in-vitro in the GIT. They also showed that increased carnosine had an immediate health beneficial role during pork digestion in reducing the formation of different harmful molecules, including AGEs, modulated by the composition and cooking of the meat. However, in exerting this protective role in the GIT, the remaining free level of carnosine, gradually decreased during digestion. Carnosine, as an important meat compositional factor may, depending on the fat content and cooking conditions, change the image of meat from representing a health risk to a health benefit. Carnosine level may also explain discrepancies observed in the literature.

Spatial distribution of some microbial trophic groups in a plug-flow-type anaerobic bioreactor treating swine manure.

Anaerobic digestion of swine manure is carried out by a consortium of microbial species, including volatile fatty acid (VFA) producers, VFA-degraders and methanogens. The distribution of five phylogenetic groups within a plug-flow-type anaerobic bioreactor consisting of eight serially-connected tanks was examined through the sequential digestion of swine manure. Quantification was carried out using reverse transcription real-time PCR (RT-Q-PCR) assays targeting the 16S rRNA of Clostridium (cluster XIVa), Peptostreptococcus, Syntrophomonas, Methanosaeta, and Methanosarcina spp. The VFA producers Peptostreptococcus spp. and Clostridium spp. were found predominantly in compartments where hydrolysis/acidogenesis took place. The spatial distribution of the aceticlastic methanogens, Methanosaeta and Methanosarcina, within the bioreactor was not correlated with methanogenic activity. In contrast the VFA-degrading genus Syntrophomonas spp. was more abundant in compartments with elevated methanogenic activity. Multivariate statistical analyses of the RT-Q-PCR data have provided new insights into our understanding of how the various trophic groups were distributed within this bioreactor system. While the distribution of clostridia, peptostreptococci and Syntrophomonas corresponded to their known metabolic functions, aceticlastic methanogens were not apparently linked to the methanogenesis stage occurring in latter compartments, suggesting that hydrogenotrophic methanogens were the primary methane generators in this bioreactor. However, aceticlastic methanogens could be involved in compartments related to the hydrolysis/acidogenesis stage.

Characterisation of intracellular molecular mechanisms modulated by carnosine in porcine myoblasts under basal and oxidative stress conditions.

Carnosine is a naturally occurring histidine-containing dipeptide present at high concentration in mammalian skeletal muscles. Carnosine was shown to affect muscle contraction, prevent the accumulation of oxidative metabolism by-products and act as an intracellular proton buffer maintaining the muscle acid-base balance. The present study was undertaken to gain additional knowledge about the intracellular mechanisms activated by carnosine in porcine myoblast cells under basal and oxidative stress conditions. Satellite cells were isolated from the skeletal muscles of 3 to 4 day-old piglets to study the effect of 0, 10, 25 and 50 mM carnosine pre-treatments in cells that were exposed (0.3 mM H2O2) or not to an H2O2-induced oxidative stress. Study results demonstrated that carnosine acts differently in myoblasts under oxidative stress and in basal conditions, the only exception being with the reduction of reactive oxygen species and protein carbonyls observed in both experimental conditions with carnosine pre-treatment. In oxidative stress conditions, carnosine pre-treatment increased the mRNA abundance of the nuclear factor, erythroid 2 like 2 (NEF2L2) transcription factor and several of its downstream genes known to reduce H2O2. Carnosine prevented the H2O2-mediated activation of p38 MAPK in oxidative stress conditions, whereas it triggered the activation of mTOR under basal conditions. Current results support the protective effect of carnosine against oxidative damage in porcine myoblast cells, an effect that would be mediated through the p38 MAPK intracellular signaling pathway. The activation of the mTOR signaling pathway under basal condition also suggest a role for carnosine in myoblasts proliferation, growth and survival.

Establishment and validation of cell pools using primary muscle cells derived from satellite cells of pig skeletal muscle.

Primary cell cultures derived from satellite cells of skeletal muscle provide an appropriate in vitro model for proliferating myoblasts and differentiating myotubes for muscle biological research. These cell cultures may consist of harvested cells per animal or of a cell pool made of cells from several animals. However, cell pooling reduces the biological variability of the different cell donors. On the other hand, the use of cell pools offers an opportunity to use less donor tissue and to perform long-term projects with a broad spectrum of analysis and replications. In the literature, information about the donors of cell pools, the procedure used for pooling, and the characterization/validation of cell pools is often lacking. In this study, we established three cell pools consisting of M. rhomboideus or M. longissimus from ten or six piglets, each with one gender and medium birth weight. Real-time impedimetric monitoring was used to evaluate the proliferative growth behavior of myoblasts for the cell pools in comparison to their corresponding unpooled cells over a period of 72 h, with a measurement being taken every 30 min. For each of the tested cell pools, cell index, slope, and doubling time did not differ between the cell pool and the unpooled cells of the donor animals. Differentiation capacity and mRNA expression of PAX7, MYOD and MYOG remained unchanged between the cell pool and the unpooled cells. Current results support that the use of cell pools is an appropriate method to reflect the average proliferative growth behavior of unpooled cells.