Effect of porcine glucagon‐like peptides‐2 on tight junction in GLP‐2R + IPEC‐J2 cell through the PI3k/Akt/mTOR/p70S6K signalling pathway

Because of rare glucagon‐like peptide‐2 (GLP‐2) receptor (+) cells within the gut mucosa, the molecular mechanisms transducing the diverse actions of GLP‐2 remain largely obscure. This research identified the naturally occurring intestinal cell lines that endogenously express GLP‐2R and determined the molecular mechanisms of the protective effects of GLP‐2‐mediated tight junctions (TJ) in GLP‐2R (+) cell line. (i) Immunohistochemistry results showed that GLP‐2R is localised to the epithelia, laminae propriae and muscle layers of the small and large bowels of newborn piglets. (ii) GLP‐2R expression was apparent in the cytoplasm of endocrine cells in IPEC‐J2 cell lines. (iii) The protein expressions of ZO‐1, claudin‐1, occludin, p‐PI₃K, p‐Akt, p‐mTOR and p‐p70^S6K significantly (p < 0.05) increased in GLP‐2‐treated IPEC‐J2 cells, and all of them significantly (p < 0.05) decreased when LY‐294002 or rapamycin was added. GLP‐2 improves intestinal TJ expression of GLP‐2R (+) cells through the PI₃k/Akt/mTOR/p70^S6K signalling pathway.     

Effects of age and zinc supplementation on transport properties in the jejunum of piglets

Zinc is effective in the prevention and treatment of post‐weaning diarrhoea and in promoting piglet growth. Its effects on the absorption of nutrients and the secretory capacity of the intestinal epithelium are controversial. We investigated the effects of age, dietary pharmacological zinc supplementation and acute zinc exposure in vitro on small‐intestinal transport properties of weaned piglets. We further examined whether the effect of zinc on secretory responses depended on the pathway by which chloride secretion is activated. A total of 96 piglets were weaned at 26 days of age and allocated to diets containing three different levels of zinc oxide (50, 150 and 2500 ppm). At the age of 32, 39, 46 and 53 days, piglets were killed, and isolated epithelia from the mid‐jejunum were used for intestinal transport studies in conventional Ussing chambers, with 23 μm ZnSO₄ being added to the serosal side for testing acute effects. Absorptive transport was stimulated by mucosal addition of d ‐glucose or l ‐glutamine. Secretion was activated by serosal addition of prostaglandin E₂, carbachol or by mucosal application of Escherichia coli heat‐stable enterotoxin (St_p). Jejunal transport properties showed significant age‐dependent alterations (p < 0.03). Both absorptive and secretory responses were highest in the youngest piglets (32 d). The dietary zinc supplementation had no significant influence on jejunal absorptive and secretory responses. However, the pre‐treatment of epithelia with ZnSO₄ in vitro led to a small but significant decrease in both absorptive and secretory capacities (p < 0.05), with an exception for carbachol (p = 0.07). The results showed that, in piglets, chronic supplementation with zinc did not sustainably influence the jejunal transport properties in the post‐weaning phase. Because transport properties are influenced by the addition of zinc in vitro, we suggest that possible epithelial effects of zinc depend on the acute presence of this ion.     

Aspartame supplementation in starter accelerates small intestinal epithelial cell cycle and stimulates secretion of glucagon‐like peptide‐2 in pre‐weaned lambs

The objective of this study was to test the hypothesis that aspartame supplementation in starter diet accelerates small intestinal cell cycle by stimulating secretion and expression of glucagon‐like peptide ?2 (GLP‐2) in pre‐weaned lambs using animal and cell culture experiments. In vivo, twelve 14‐day‐old lambs were selected and allocated randomly to two groups; one was treated with plain starter diet (Con, n = 6) and the other was treated with starter supplemented with 200 mg of aspartame/kg starter (APM, n = 6). Results showed that the lambs received APM treatment for 35 d had higher (p < .05) GLP‐2 concentration in the plasma and greater jejunum weight/live body weight (BW) and jejunal crypt depth. Furthermore, APM treatment significantly upregulated (p < .05) the mRNA expression of cyclin D1 in duodenum; and cyclin A2, cyclin D1, cyclin‐dependent kinases 6 (CDK6) in jejunum; and cyclin A2, cyclin D1, CDK4 in ileum. Moreover, APM treatment increased (p < .05) the mRNA expression of glucagon (GCG), insulin‐like growth factor 1 (IGF‐1) in the jejunum and ileum and mRNA expression of GLP‐2 receptor (GLP‐2R) in the jejunum. In vitro, when jejunal cells were treated with GLP‐2 for 2 hr, the 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2‐H‐tetrazolium bromide (MTT) OD, IGF‐1 concentration, and the mRNA expression of IGF‐1, cyclin D1 and CDK6 were increased (p < .05). Furthermore, IGF‐1 receptor (IGF‐1R) inhibitor decreased (p < .05) the mRNA expression of IGF‐1, cyclin A2, cyclin D1 and CDK6 in GLP‐2 treatment jejunal cells. These results suggest that aspartame supplementation in starter accelerates small intestinal cell cycle that may, in part, be related to stimulate secretion and expression of GLP‐2 in pre‐weaning lambs. Furthermore, GLP‐2 can indirectly promote the proliferation of jejunal cells mainly through the IGF‐1 pathway. These findings provide new insights into nutritional interventions that promote the development of small intestines in young ruminants.     

Both butyrate incubation and hypoxia upregulate genes involved in the ruminal transport of SCFA and their metabolites

Butyrate modulates the differentiation, proliferation and gene expression profiles of various cell types. Ruminal epithelium is exposed to a high intraluminal concentration and inflow of n‐butyrate. We aimed to investigate the influence of n‐butyrate on the mRNA expression of proteins involved in the transmembranal transfer of n‐butyrate metabolites and short‐chain fatty acids in ruminal epithelium. N‐butyrate‐induced changes were compared with the effects of hypoxia because metabolite accumulation after O₂ depletion is at least partly comparable to the accumulation of metabolites after n‐butyrate exposure. Furthermore, in various tissues, O₂ depletion modulates the expression of transport proteins that are also involved in the extrusion of metabolites derived from n‐butyrate breakdown in ruminal epithelium. Sheep ruminal epithelia mounted in Ussing chambers were exposed to 50 mM n‐butyrate or incubated under hypoxic conditions for 6 h. Electrophysiological measurements showed hypoxia‐induced damage in the epithelia. The mRNA expression levels of monocarboxylate transporters (MCT) 1 and 4, anion exchanger (AE) 2, downregulated in adenoma (DRA), putative anion transporter (PAT) 1 and glucose transporter (GLUT) 1 were assessed by RT‐qPCR. We also examined the mRNA expression of nuclear factor (NF) κB, cyclooxygenase (COX) 2, hypoxia‐inducible factor (HIF) 1α and acyl‐CoA oxidase (ACO) to elucidate the possible signalling pathways involved in the modulation of gene expression. The mRNA expression levels of MCT 1, MCT 4, GLUT 1, HIF 1α and COX 2 were upregulated after both n‐butyrate exposure and hypoxia. ACO and PAT 1 were upregulated only after n‐butyrate incubation. Upregulation of both MCT isoforms and NFκB after n‐butyrate incubation could be detected on protein level as well. Our study suggests key roles for MCT 1 and 4 in the adaptation to an increased intracellular load of metabolites, whereas an involvement of PAT 1 in the transport of n‐butyrate also seems possible.     

Gene expression and morphological changes in the intestinal mucosa associated with increased permeability induced by short‐term fasting in chickens

Short‐term fasting for 4.5 and 9 hr has been demonstrated to increase intestinal permeability (IP) in chickens. This study aimed to investigate the effects of 0, 4.5, 9 and 19.5 hr fasting on intestinal gene expression and villus‐crypt architecture of enterocytes in jejunal and ileal samples. On day 38, Ross‐308 male birds were fasted according to their group and then euthanised. Two separate intestinal sections (each 2 cm long, jejunum and ileum) were collected. One section was utilised for villus height and crypt depth measurements. The second section was snap‐frozen in liquid nitrogen for quantitative polymerase chain reaction (qPCR) analysis of tight junction proteins (TJP) including claudin‐1, claudin‐3, occludin, zonula occludens (ZO‐1, ZO‐2), junctional adhesion molecules (JAM) and E‐cadherin. Additionally genes involved in enterocyte protection including glucagon‐like peptide (GLP‐2), heat‐shock protein (HSP‐70), intestinal alkaline phosphatase (IAP), mammalian target of rapamycin (mTOR), toll‐like receptors (TLR‐4), mucin (MUC‐2), cluster differentiation (CD‐36) and fatty acid‐binding protein (FABP‐6) were also analysed. Normally distributed data were analysed using one‐way analysis of variance ANOVA. Other data were analysed by non‐parametric one‐way ANOVA. Villus height and crypt depth were increased (p < .05) only in the ileum after fasting for 4.5 and 9 hr compared with non‐fasting group. mRNA expression of claudin‐3 was significantly reduced in the ileum of birds fasted for 9 and 19.5 hr, suggesting a role in IP modulation. However, all other TJP genes examined were not statistically different from control. Nevertheless, ileal FABP‐6 of all fasted groups was significantly reduced, which could possibly be due to reduced bile acid production during fasting.     

Exogenous Estradiol Benzoate Induces Spermatogenesis Disorder through Influencing Apoptosis and Oestrogen Receptor Signalling Pathway

As the exact role for exogenous oestrogen in spermatogenesis is not fully understood, the aim of this study was to investigate the effect of estradiol benzoate (EB) exposure to male mice on their spermatogenesis and fertility. Sixty male mice aged 4 weeks were randomly divided into three groups, including a control group and two treatment groups. The mice of the control group were injected with 250 μl paraffin oil only by every other day subcutaneous injection for 4 weeks. Meantime, the mice of the treatment groups were injected with EB at the concentration of 5 or 10 mg/kg, respectively. Results showed that EB slowed down the body weight gains and generated testicular atrophy with spermatogenesis disorder compared with that of the control mice, and consequently induced their infertility. Moreover, the number of TUNEL‐positive cells in the testis of EB‐treated mice was significantly increased with the EB concentration rise. In comparison with controls, the mRNA expression level of pro‐apoptosis factors (Fas, TNF, Cytochrome C, Apaf1, Chop, Caspase‐3, Caspase‐8, Caspase‐9 and Caspase‐12) and key genes in oestrogen receptor (ER) signalling pathway (ER α, ER β, Erk1/2, Hsp90 and DAX‐1) were upregulated in the testes of the treatment groups. Furthermore, Western blotting results proved the protein expression level of Fas, TNF, Cytochrome C, Chop, Caspase‐3, cleaved Caspase‐3, Caspase‐9, Erk1/2 and Hsp90 were upregulated, and the phosphorylation level of Erk1/2 was also increased. These results indicate that EB may impair spermatogenesis through influencing the apoptosis and ER signalling pathway.     

Gamma‐aminobutyric acid (GABA) permeates ovine ruminal and jejunal epithelia,mainly by passive diffusion

Gamma‐aminobutyric acid (GABA) represents the most abundant inhibitory neurotransmitter in the mammalian brain. GABA is also produced in plants and/or by the microbial conversion of amino acids. Thus, ruminants may be forced to take up significant amounts of GABA from their diet. However, it is not known whether exogenously acquired GABA might permeate the gastrointestinal barrier in such quantities as to induce systemic alterations. Thus, this study pursues the question of where within the ruminant's GI tract and by which pathways GABA may be taken up from the ingesta. The jejunal and ruminal epithelia of sheep were mounted in Ussing chambers under short‐circuit conditions. The flux rates of radiolabelled GABA from the mucosal to the serosal side (J_ms) and vice versa (J_sm) were measured. GABA was applied in various concentrations with adjustment of the mucosal pH to 6.1 or 7.4. Furthermore, beta‐alanine or glycine was used as a competitive inhibitor for GABA transport. In both the jejunal and ruminal epithelium, the J_ms of GABA was linearly correlated to the mucosal GABA concentration. However, J_ms across the jejunal epithelium was approximately 10‐fold higher than J_ms across the ruminal epithelium. When 0.5 mmol/l GABA was applied on both sides of the epithelium, no net flux could be observed in the jejunal epithelia. Additionally, there was no effect of decreased mucosal pH or the application of glycine or beta‐alanine under these conditions. The J_ms and J_sm of GABA were linearly correlated to the transepithelial conductance. Our results suggest that GABA is taken up from the small intestine rather than from the rumen. Due to the lack of influence of pH and competitive inhibitors, this uptake seems to occur primarily via passive diffusion.     

Expression of Heparin‐Binding EGF‐Like Growth Factor (HB‐EGF) in Bovine Endometrium: Effects of HB‐EGF and Interferon‐τ on Prostaglandin Production

Heparin‐binding EGF‐like growth factor (HB‐EGF) regulates several cell functions by binding to its membrane receptor (ErbB1 and ErbB4). Experimental evidences suggest that HB‐EGF, prostaglandins (PGs) and interferon‐τ (IFN‐τ) regulate uterine function for pregnancy establishment in ruminants. In this study, the mRNA expressions of HB‐EGF, ErbB1 and ErbB4 in bovine endometrium and the effects of HB‐EGF and IFN‐τ on PGE2 and PGF2‐α production by endometrial cells were investigated. RT‐PCR analysis revealed that HB‐EGF mRNA was greater at the mid‐luteal stage than at the early and regressed luteal stages (p < 0.05). ErbB1 mRNA expression was greater at the mid‐ and late luteal stages than at the other luteal stages (p < 0.05). IFN‐τ increased the expression of HB‐EGF, ErbB1 and ErbB4 mRNA in epithelial cells (p < 0.05). HB‐EGF did not affect PGF2‐α or PGE2 production by bovine endometrial epithelial cells, but increased PGF2‐α and PGE2 production by bovine endometrial stromal cells (p < 0.05). IFN‐τ significantly decreased HB‐EGF‐stimulated PGF2‐α (p < 0.05), but not PGE2 (p > 0.05) production by stromal cells. These results indicate that HB‐EGF and its receptors expression changed in bovine endometrium throughout the oestrous cycle. IFN‐τ increased their expression in cultured endometrial cells. HB‐EGF and IFN‐τ have the ability to regulate PGs production by stromal cells and therefore may play a role in the local regulation of uterine function at the time of implantation in cattle.     

Effects of dietary supplementation with epidermal growth factor on nutrient digestibility,intestinal development and expression of nutrient transporters in early‐weaned piglets

The abnormalities in intestinal morphology and digestive function during weaning are associated with the loss of milk‐borne growth factors. Epidermal growth factor (EGF) has been shown to stimulate the growth of animals. This study was to determine the effect of dietary EGF on nutrient digestibility, intestinal development and the expression of genes encoding nutrient transporters in weaned piglets. Forty‐two piglets were weaned at 21 days and assigned to one of three treatment groups: (1) basal diet (control), (2) basal diet + 200 µg/kg EGF or (3) basal diet + 400 µg/kg EGF. Each treatment consisted of 14 replicates, and seven piglets from each treatment were sampled on day 7 and 14. The EGF supplementation significantly elevated (p < 0.05) the coefficients of total tract apparent digestibility of crude protein, calcium and phosphorus, but tended to decrease sucrase activity (p < 0.10) than the control group. At day 7 post‐weaning, animals receiving EGF diets showed a tendency (p < 0.10) towards greater ileal villus height (VH), jejunal crypt depth (CD) and duodenal VH:CD when compared with the control group. Moreover, the mRNA levels of glucose transporter 2 (Slc2a2), neutral amino acid transporter (Slc6a19) and calbindin D9k (S100G) tended to be higher (p < 0.10) for EGF groups than the control group. By day 14, EGF supplementation markedly enhanced (p < 0.05) the VH, CD and VH:CD in the jejunum compared to the control group. This addition also up‐regulated (p < 0.05) the mRNA level and the protein abundance of peptide transporter 1 than the control group. These findings demonstrated that dietary EGF beneficially enhanced nutrient digestibility, improved intestinal development and increased the mRNA expression of nutrient transporters in weaned piglets.     

Comparative study of stress response,growth and development of uteri in post‐weaning gilts challenged with zearalenone and estradiol benzoate

The objective of this study was to evaluate the effects of zearalenone (ZEA) and estradiol benzoate (EB) on stress injury and uterine development in post‐weaning gilts. Thirty healthy post‐weaning female gilts (Duroc × Landrace × Large White) aged 28–32 days were randomly allocated to three treatments as follows: (a) basal diet (Control), (b) basal diet plus 1.0 mg/kg purified ZEA (ZEA) and (c) basal diet plus 0.75 ml (1.5 mg) EB per pig at 3‐days intervals by intramuscular injection (EB). The serum estradiol (E₂), the final and the increased vulvar area, uterine index, thickness of the myometrium and endometrium, and protein expression of heat shock protein 70 (HSP70) in ZEA group were higher than those in the control group (p < .05), but lower than those in the EB group (p < .05). The serum luteinizing hormone in ZEA group was lower than that of the control group (p < .05), but higher than that in the EB group (p < .05). Higher serum follicle‐stimulating hormone and progesterone were observed in the ZEA and control groups than those in the EB group (p < .05). The serum glutathione peroxidase activity in the ZEA group was lower than that in the control and EB groups (p < .001), and the malondialdehyde in the ZEA group was higher than that in the control and EB groups (p < .001). Moreover, the relative mRNA and protein expression of growth hormone receptor (GHR) and relative mRNA expression of HSP70 in the ZEA and EB groups were higher than those in the control group (p < .05). In conclusion, both ZEA (1.0 mg/kg) and EB (1.5 mg at 3 days intervals by intramuscular injection) stimulated vulvar swelling and uterine hypertrophy by disordering serum hormones and up‐regulating GHR expression, and induced stress by different mechanisms in this study. Furthermore, the observed up‐regulating HSP70 expression challenged by ZEA or EB may be part of the mechanism to resist stress injury.     

Effects of dissolved carbon dioxide on the integrity of the rumen epithelium: An agent in the development of ruminal acidosis

The carbon dioxide released and dissolved in rumen fluid may easily permeate across the epithelial cell membrane. Thus, we hypothesized that CO₂ may act as proton carrier and induce epithelial damage under acidotic conditions. Ovine ruminal epithelia were mounted in Ussing chambers under short‐circuit conditions. The serosal buffer solution had a constant pH of 7.4 and was gassed either with 100% oxygen or with carbogen (95% O₂/5% CO₂). The mucosal solution was gassed with either 100% oxygen or 100% carbon dioxide. The mucosal pH was lowered stepwise from 6.6 to 5.0 in the presence or absence of short‐chain fatty acids (SCFA). The transepithelial conductance (G_t) as an indicator of epithelial integrity and the short‐circuit current (I_sc) as an indicator of active electrogenic ion transfer were continuously monitored. At an initial mucosal pH of 6.6, there was no significant difference in G_t between the treatment groups. In the absence of both SCFA and CO₂, G_t remained constant when the mucosal solution was acidified to pH 5.0. In the presence of SCFA, mucosal acidification induced a significant rise in G_t when the solutions were gassed with oxygen. A small increase in G_t was observed in the mucosal presence of CO₂. However, no difference in final G_t was observed between SCFA‐containing and SCFA‐free conditions under carbon dioxide gassing during stepwise mucosal acidification. The SCFA+proton‐induced increase in G_t could also be minimized by serosal gassing with carbogen. Because of the SCFA+proton‐induced changes in G_t and their attenuation by CO₂, a protective role for mucosally available carbon dioxide may be assumed. We suggest that this effect may be due to the intraepithelial conversion of carbon dioxide to bicarbonate. However, the serosal presence of CO₂ at a physiological concentration may be sufficient to protect the epithelia from SCFA+proton‐induced damage for a certain period of time.     

Effect of melatonin on regulation of apoptosis and steroidogenesis in cultured buffalo granulosa cells

This study was aimed to address melatonin receptor expression, mRNA level of hypothalamus and hypophysis hormone receptors (GnRHR, FSHR, and LHR), steroidogenesis, cell cycle, apoptosis, and their regulatory factors after addition of melatonin for 24 hr in cultured buffalo granulosa cells (GCs). The results revealed that direct addition of different concentrations of melatonin (100 pM, 1 nM, and 100 nM) resulted in significant upregulation (p < 0.05) of mRNA level of melatonin receptor 1a (MT1) without affecting melatonin receptor 1b (MT2). Melatonin treatment significantly downregulated (p < 0.05) mRNA level of FSH and GnRH receptors, whereas 100 nM dose of melatonin significantly increased mRNA level of LH receptor. Treatment with 100 nM of melatonin significantly decreased the basal progesterone production with significant decrease (p < 0.05) in mRNA levels of StAR and p450ssc, and lower mRNA level of genes (Insig1, Lipe, and Scrab1) that affect cholesterol availability. Melatonin supplementation suppressed apoptosis (100 nM, p < 0.05) and enhanced G2/M phase (1 nM, 100 nM, p < 0.05) of cell cycle progression which was further corroborated by decrease in protein expression of caspase‐3, p21, and p27 and increase in bcl2. Our results demonstrate that melatonin regulates gonadotrophin receptors and ovarian steroidogenesis through MT1. Furthermore, the notion of its incorporation in apoptosis and proliferation of buffalo GCs extends its role in buffalo ovaries.     

Montmorillonite improved the intestinal mucosal barrier functions of laying hens in late production

This study was conducted to investigate the effects of dietary supplementation with montmorillonite (MMT) on performance, intestinal endotoxin concentration, gut mucosal oxidation status, intestinal morphology and permeability, and immunological barrier function of laying hens during late production. Four hundred and eighty 75‐week‐old laying hens (Lohmann Brown) were randomly assigned to five treatments with eight replicates per treatment and 12 hens in each replicate. The hens were fed the basal diet supplemented with 0 (control), 0.3, 0.6, 0.9, or 1.2 g MMT/kg for 70 days. Compared with the control, supplemented with 0.9 g MMT/kg increased egg mass significantly (p < 0.05) during weeks 1–5 of the experiment. Supplemented with 0.6 and 0.9 g MMT/kg also increased the endotoxin concentration in the ileal digesta (p < 0.05), but decreased the MDA concentration in the ileum significantly (p < 0.05). The T‐AOC in the jejunum of the group fed 0.3 g MMT/kg was significantly increased (p < 0.05). Compared with the control, the villus height:crypt depth of ileum from the groups fed 0.6, 0.9, and 1.2 g MMT/kg increased significantly (p < 0.05). The sIgA concentration of jejunum in the groups fed 0.6 and 0.9 g MMT/kg was higher (p < 0.05) than the control. The MMT supplementation linearly increased (p < 0.05) the mRNA expression of claudin‐1 and claudin‐5 in the jejunum. Dietary MMT supplementation down‐regulated the mRNA expression of NF‐κB P65 and TNF‐α in the jejunum in a linear and quadratic manner (p < 0.05). The IL‐1β mRNA expression of jejunum in the group fed 0.6 g MMT/kg was lower (p < 0.05) than the control. In conclusion, dietary supplementation with MMT may improve the gut barrier functions and suggests that 0.9 g/kg of MMT in diets may be the optimal supplemental level for laying hens in late production.     

The effects of phospholipase C on oestradiol and progesterone secretion in porcine granulosa cells cultured in vitro

Granulosa cells play important roles in the regulation of ovarian functions. Phospholipase C is crucial in several signalling pathways and could participate in the molecular mechanisms of cell proliferation, differentiation and ageing. The objective of this study was to identify the effects of phospholipase C on the steroidogenesis of oestradiol and progesterone in porcine granulosa cells cultured in vitro. Inhibitor U73122 or activator m‐3M3FBS of phospholipase C was added to the in vitro medium of porcine granulosa cells, respectively. The secretion of oestradiol decreased after 2 hr, 8 hr, 12 hr, 24 hr and 48 hr of treatment with 500 nM U73122 (p < .05) and decreased after 2 hr of treatment in the 500 nM m‐3M3FBS addition group (p < .05). The secretion of progesterone increased after 4 hr of treatment with 500 nM U73122 (p < .05) and increased after 2 hr and 8 hr of treatment in the 500 nM m‐3M3FBS addition group (p < .05). The ratio of oestradiol to progesterone decreased at each time point, except 8 hr after the addition of 500 nM U73122 (p < .05). The ratio of oestradiol to progesterone decreased after 2 hr (p < .05) of treatment with 500 nM m‐3M3FBS. In genes that regulate the synthesis of oestradiol or progesterone, the mRNA expression of CYP11A1 was markedly increased (p < .05), and the mRNA expression of other genes did not change significantly in the U73122 treatment group, while the addition of m‐3M3FBS did not change those genes significantly despite the contrary trend. Our results demonstrated that phospholipase C can be a potential target to stimulate the secretion of oestradiol and suppress progesterone secretion in porcine granulosa cells cultured in vitro, which shed light on a novel biological function of phospholipase C in porcine granulosa cells.     

Protein Localization of Epidermal Growth Factor in Sheep Ovaries and Improvement of Follicle Survival and Antrum Formation In Vitro

The aims of this study were to characterize EGF protein expression in ovine ovaries and to verify the effect of EGF on the in vitro development of isolated pre‐antral follicles. After collection, ovarian tissue was fixed for immunohistochemical analysis. Additional pairs of ovaries were collected, and secondary follicles were cultured for 18 days in α‐MEM⁺ (control) alone or supplemented with EGF (1, 10 or 50 ng/ml). The immunostaining for EGF was observed in oocytes from pre‐antral and antral follicles, in granulosa cells of primary and secondary follicles, as well as in cumulus and mural cells of antral follicles. After 18 days, the results showed that treatment with 50 ng/ml EGF significantly increased the percentage of morphologically normal follicles compared with the control group (α‐MEM⁺) and significantly reduced the precocious extrusion of oocytes and increased the percentage of antral follicles compared with the control and 1 ng/ml EGF. All the treatments induced a progressive and significant increase of the follicular diameter throughout the period of culture. However, there were no significant differences in follicular diameter or in the daily growth rate among treatments. In conclusion, this study demonstrated the presence of EGF in ovine ovaries. Moreover, 50 ng/ml EGF increased the percentage of normal follicles and improved antrum formation in isolated ovine follicles after 18 days of in vitro culture.     

Ruminal epithelial insulin‐like growth factor‐binding proteins 2, 3, and 6 are associated with epithelial cell proliferation

The aim of this study was to identify factors that regulate ruminal epithelial insulin‐like growth factor‐binding protein (IGFBP) expression and determine its role in rumen epithelial cell proliferation. Primary bovine rumen epithelial cells (BREC) were incubated with short‐chain fatty acids (SCFAs) at pH 7.4 or 5.6, lactate, lipopolysaccharide (LPS), insulin‐like growth factor‐I (IGF‐I), ‐II (IGF‐II), or recombinant bovine IGFBP2 (rbIGFBP2). The mRNA expression levels of IGFBP in BREC were analyzed using quantitative real‐time polymerase chain reaction (qRT‐PCR). The proliferation rate of BREC was analyzed using a WST‐1 assay. IGFBP2 gene expression tended to be lower with SCFA treatment (p < .1), and IGFBP6 gene expression was significantly lower with SCFA treatment (p < .05). IGFBP3 and IGFBP6 gene expression tended to be higher with d ‐Lactate treatment (p < .1). IGFBP3 gene expression was significantly higher (p < .05) with LPS treatment. BREC treated with IGF‐I grew more rapidly than vehicle control‐treated cells (p < .01); however, recombinant bovine rbIGFBP2 inhibited IGF‐I‐induced proliferation. IGF‐II and/or rbIGFBP2 did not affect BREC proliferation. Taken together, SCFA treatment decreased IGFBP2 and IGFBP6 expression in rumen epithelial cells, and lower expression of these IGFBP might promote rumen epithelial cell proliferation by facilitating IGF‐I.     

Effect of estradiol cypionate and GnRH treatment on plasma estradiol‐17β concentrations,synchronization of ovulation and on pregnancy rates in suckled beef cows treated with FTAI‐based protocols

Two experiments were conducted to evaluate the effect of different ovulation inducers on E‐17β plasma concentrations, synchronized ovulations and pregnancy rates. In Experiment 1, cows received a progesterone intravaginal device (PID) with 1 g of progesterone (P4) plus 2 mg of estradiol benzoate (EB) (day 0). At PID removal (day 8), cows received 0.150 mg of D‐cloprostenol and were randomly assigned to four treatment groups (n = 10/treatment): Group ECP: 1 mg of estradiol cypionate at PID removal, Group EB: 1 mg of EB 24 hr after PID removal, Group GnRH: 10 μg of GnRH 48 hr after PID removal, Group ECP‐GnRH: 1 mg of ECP at PID removal plus 10 μg of GnRH 48 hr later. Ultrasonographic examinations were performed to detect the dominant follicle and ovulation. GnRH‐treated cows ovulated later (p < .05) compared to ECP‐ and ECP+GnRH‐treated cows. There were effects of treatment, time and their interaction on E‐17β concentrations (p < .05). ECP treatment affected plasma E‐17β concentration, which increased earlier and decreased later compared to treatments without ECP. In Experiment 2, cows received (i) ECP: n = 126; (ii) EB: n = 126; (iii) GnRH: n = 136; (iv) ECP+GnRH: n = 139; FTAI was performed 48–50 hr after PID removal. Pregnancy rates did not differ among ovulation inducers (p > .05; ECP: 54.0%, 68/126; EB: 49.2%, 62/126; GnRH: 40.4%, 55/136; ECP+GnRH: 43.9%, 61/139). In conclusion, ECP administration (ECP and ECP+GnRH treatments) affected E‐17β concentrations, determining its earlier increase and later decrease compared to treatments without ECP (EB and GnRH treatments). ECP+GnRH‐treated cows achieved the best distribution of ovulations without affecting pregnancy rates.     

Metabolic parameters and their relationship to energy balance in multiparous Simmental,Brown Swiss and Holstein cows in the periparturient period as influenced by energy supply pre‐ and post‐calving

A study was conducted to evaluate the effects of three energy supply (E) levels [low (L), medium (M), high (H)], both pre‐partum (PRE) and post‐partum (POST), and their interactions on metabolic parameters and energy balance (EB) in dairy cows of three breeds. In both phases, E levels applied to a total of 81 multiparous cows of breeds Simmental (SI), Brown Swiss (BS) and Holstein–Friesian (HF; n = 27 for each breed) were 75%, 100% and 125% of recommendations of the German Society of Nutrition Physiology, using a 3 × 3 factorial arrangement of treatments. During the pre‐calving period, serum concentrations of non‐esterified fatty acids (NEFA) were higher for L_PRE cows, and glucose concentrations were elevated for H_PRE cows. During the lactation period, NEFA concentrations were greatest for treatment L_POST. Mean concentrations of β‐hydroxybutyrate (BHB) were highest for cows of the L_POST treatment, intermediate for M_POST and lowest for H_POST. Glucose concentrations were lower for L_POST cows. SI cows had lower BHB concentrations both pre‐ and post‐calving and higher glucose concentrations during early lactation than the other breeds. BHB concentration POST was highest for BS cows. Restricted feeding PRE resulted in a better energy status of cows fed above energy requirements POST (E_PRE × E_POST interaction). HF cows had a higher EB pre‐calving, whereas SI cows had a less negative EB during early lactation, compared with the other breeds respectively. Correlations of serum NEFA, BHB and glucose concentrations with EB were strongest during the transition period. Results suggest that controlling energy intake during the dry period might be advantageous for the energy status of dairy cows after calving, whereas energy restriction in early lactation leads to metabolic stress. Evidence is provided of a clear relationship between EB and the blood metabolites NEFA and BHB, especially in the transition period.     

Eleutheroside B increase tight junction proteins and anti‐inflammatory cytokines expression in intestinal porcine jejunum epithelial cells (IPEC‐J2)

Eleutheroside B (EB) is a phenylpropanoid glycoside with anti‐inflammatory properties, neuroprotective abilities, immunomodulatory effects, antinociceptive effects, and regulation of blood glucose. The aim of this study was to investigate the effects of EB on the barrier function in the intestinal porcine epithelial cells J2 (IPEC‐J2). The IPEC‐J2 cells were inoculated into 96‐well plates at a density of 5 × 10³ cells per well for 100% confluence. The cells were cultured in the presence of EB at concentrations of 0, 0.05, 0.10, and 0.20 mg/ml for 48 hr. Then, 0.10 mg/ml was selected as the suitable concentration for the estimation of transepithelial electric resistance (TEER) value, alkaline phosphatase activity, proinflammatory cytokines mRNA expression, tight junction mRNA and protein expression. The results of this study indicated that the supplementation of EB in IPEC‐J2 cells decreased cellular membrane permeability and mRNA expression of proinflammatory cytokines, including interleukin‐6 (IL‐6), interferon‐γ (INF‐γ), and tumour necrosis factor‐α (TNF‐α). The supplementation of EB in IPEC‐J2 cells increased tight junction protein expression and anti‐inflammatory cytokines, interleukin 10 (IL‐10) and transforming growth factor beta (TGF‐β). In addition, the western blotting and real‐time quantitative polymerase chain reaction (RT‐qPCR) results indicated that EB significantly (p < 0.05) increased the mRNA and protein expression of intestinal tight junction proteins, Claudin‐3, Occludin, and Zonula Occludins protein‐1 (ZO‐1). Therefore, dietary supplementation of EB may increase intestinal barrier function, tight junction protein expression, anti‐inflammatory cytokines, and decrease proinflammatory cytokines synthesis in IPEC‐J2 cells.     

Hepatic biotransformation pathways and ruminal metabolic stability of the novel anthelmintic monepantel in sheep and cattle

Monepantel (MNP) is a new amino‐acetonitrile derivative anthelmintic drug used for the treatment of gastrointestinal (GI) nematodes in sheep. The present work investigated the main enzymatic pathways involved in the hepatic biotransformation of MNP in sheep and cattle. The metabolic stability in ruminal fluid of both the parent drug and its main metabolite (monepantel sulphone, MNPSO₂) was characterized as well. Additionally, the relative distribution of both anthelmintic molecules between the fluid and particulate phases of the ruminal content was studied. Liver microsomal fractions from six (6) rams and five (5) steers were incubated with a 40 μm of MNP. Heat pretreatment (50 °C for 2 min) of liver microsomes was performed for inactivation of the flavin‐monooxygenase (FMO) system. Additionally, MNP was incubated in the presence of 4, 40, and 80 μm of methimazole (MTZ), a FMO inhibitor, or equimolar concentrations of piperonyl butoxide (PBx), a well‐known general cytochrome P450 (CYP) inhibitor. In both ruminant species, MNPSO₂ was the main metabolite detected after MNP incubation with liver microsomes. The conversion rate of MNP into MNPSO₂ was fivefold higher (P < 0.05) in sheep (0.15 ± 0.08 nmol/min·mg) compared to cattle. In sheep, the relative involvement of both FMO and CYP systems (FMO/CYP) was 36/64. Virtually, only the CYP system appeared to be involved in the production of MNPSO₂ in cattle liver. Methimazole significantly reduced (41 to 79%) the rate of MNPSO₂ production in sheep liver microsomes whereas it did not inhibit MNP oxidation in cattle liver microsomes. On the other hand, PBx inhibited the production of MNPSO₂ in liver microsomes of both sheep (58 to 98%, in a dose‐dependent manner) and cattle (almost 100%, independently of the PBx concentration added). The incubation of MNP and MNPSO₂ with ruminal contents of both species showed a high chemical stability without evident metabolism and/or degradation as well as an extensive degree of adsorption (83% to 90%) to the solid phase of the ruminal content. Overall, these results are a further contribution to the understanding of the metabolic fate of this anthelmintic drug in ruminants.