Nitrophenols disrupt the expression and activity of biotransformation enzymes (CYP3A and COMT) in chicken ovarian follicles in vivo and in vitro.

Nitrophenols are environmental pollutants and xenobiotics, the main sources of which are diesel exhaust fumes and pesticides. The biotransformation processes that take place in the liver are defence mechanisms against xenobiotics, such as nitrophenols. Our previous study showed that the chicken ovary is an additional xenobiotic detoxification place and that nitrophenols disrupt steroidogenesis in chicken ovarian follicles. Therefore, the present study aimed to determine the in vivo and in vitro effects of 4-nitrophenol (PNP) and 3-methyl-4-nitrophenol (PNMC) on the expression and activity of phase I (CYP3A) and phase II (COMT) biotransformation enzymes in chicken ovary. In an in vivo study, hens were treated with a vehicle or 10 mg PNP or PNMC/kg b.wt. per day for 6 days. In an in vitro study, prehierarchical white and yellowish follicles, as well as the granulosa and theca layers of the three largest preovulatory follicles (F3, F2 and F1), were isolated and then incubated in a control medium or medium supplemented with PNP (10-6 M) or PNMC (10-6 M) for 24 or 48 h. Both in vivo and in vitro studies showed that nitrophenols exert tissue- and compound-dependent (PNP or PNMC) effects on CYP3A and COMT gene (real-time PCR) protein (Western blot) expression and their activity (colorimetric methods). The inhibitory effect of nitrophenols in vivo on the activity of biotransformation enzymes suggest that the ovary has the capacity to metabolise PNP and PNMC.

Combination of emulsifier and xylanase in triticale-based broiler chickens diets.

The current study aimed to investigate the effect of supplementing an emulsifier, xylanase or a combination of both on the growth performance, digestibility of nutrients, microflora activity and intestinal morphology in broiler chickens fed triticale-based diets. A total of 480 one-day-old male Ross 308 broiler chicks were randomly assigned to four dietary treatments: control (CON), control with an added emulsifier (EMU), control with added xylanase (ENZ) and control with emulsifier and xylanase (EMU+ENZ). Xylanase supplemented groups had diminished feed intake (FI) and enhanced body weight gain (BWG) only within the starter period (p ≤ 0.05), while the feed conversion ratio (FCR) in the ENZ and ENZ+EMU groups was lower than CON during the whole experiment period. There was significant ENZ and EMU interaction in apparent metabolisable energy corrected to N equilibrium (AMEN) as well as NDF and DM retention. The viscosity of ileum digesta was the lowest in groups with enzyme addition. Interactions show that caecal galactosidase-α activity was higher in the CON group compared to EMU supplementation, but similar to ENZ and EMU+ENZ (p < 0.05). Activity of glucosidase-α was higher in the CON group related to inclusion of EMU or ENZ alone (p < 0.05) but did not differ from the combined supplementation of EMU+ENZ, whereas the glucosidase-ß activity was higher in the CON group compared to all supplemented diets (p < 0.05). Caecal C2 concentration was greater in the CON group than supplemented diets (p < 0.05). The expression of FATP1, PEPT1 and SGLT1 in the ileum was downregulated after emulsifier addition (p ≤ 0.05). The addition of emulsifier and xylanase indicates a mutual effect on broiler chickens' performance and nutrient digestibility in triticale diets with palm oil during the first nutritional period. Additionally, concomitantly additives usage influenced intestinal microbiome activity, as well.

Effect of emulsifier and multicarbohydrase enzyme supplementation on performance and nutrient digestibility in broiler diets containing rapeseed meal.

The aim of this study was to determine the effect of emulsifier and multicarbohydrase enzyme supplementation on performance, nutrient utilization, and apparent metabolizable energy-nitrogen (AMEN) value of broiler diets containing rapeseed meal (RSM) as well as their influence on the gut morphological structures, excretion of total and free sialic acid, and cecum concentration of short-chain fatty acids (SCFAs) in broiler chickens. A total of 384 male broiler chicks were assigned to four dietary treatments. The diet of the control treatment (CON) consisted of soybean, maize, and RSM (5% in starter, 7% in grower, 15% in finisher) with soybean and palm oils. The diets used for the experimental treatments were the control diet supplemented with an emulsifier (EMU), enzyme (ENZ), or both (EMU + ENZ). The duodenum (n = 10/treatment) and ileum (n = 10/treatment) digesta samples were assessed to determine nutrient digestibility: crude protein (CP), ether extract (EE), starch, Ca. Throughout the experimental period, EMU + ENZ treatment indicated the lowest total average feed intake and feed conversion ratio, with the highest average weight gain among the studied treatments (P < 0.05). The EMU + ENZ treatment also resulted in higher (P < 0.05): apparent prececal digestibility (APD) of CP, total tract neutral detergent fibre (NDF) degradation, apparent total tract digestibility (ATTD) of EE, villus height to crypt depth ratio (P < 0.1). The highest APD of EE was noted in the EMU treatment (P < 0.05). No significant differences were found in the AMEN values of the diets. A greater jejunum villi surface area was found in groups supplemented by enzyme compared to CON (P < 0.05). The EMU + ENZ treatment presented lower sialic acid excretion in the ileum and concentration of cecum SCFAs compared to the CON treatment (P < 0.05). The obtained results indicate that simultaneous usage of additives had beneficial effect on production parameters, nutrient digestibility, NDF degradation, as well as gut mucosa morphology. Based on the SCFAs concentration results, separate or simultaneous addition of emulsifier or/and enzyme did not provoke excessive fermentation activity of cecal bacteria.

Alterations in connexin 43 gene and protein expression in the chicken oviduct following tamoxifen treatment.

Connexins (Cxs) are a group of gap junction proteins involved in the direct exchange of small molecules between neighboring cells. Information concerning the expression and regulation of Cxs in the chicken oviduct is lacking, but likely has potential implications for functioning of the oviduct and the quality of the egg laid by commercially used hens. The present study was designed to examine whether selected Cxs are present in the chicken oviduct and, if so, whether expression of the most abundant Cx changes following tamoxifen (TMX; estrogen receptor modulator) treatment. Hy-Line Brown laying hens were injected (s.c.) daily with a vehicle (n = 6) or with TMX (n = 6), at a dose of 6 mg/kg of body weight for 7 days until complete cessation of egg laying by TMX-treated hens. All oviductal segments (infundibulum, magnum, isthmus, shell gland, and vagina) were collected from hens on day 8 of the experiment. First, the gene expression of GJA1 (i.e. Cx43 protein), GJA4 (Cx39), GJB1 (Cx32), and GJD2 (Cx36) was investigated by real-time PCR in tissues of control birds. The results demonstrated gene- and oviductal segment-dependent expression of GJB1, GJD2, GJA4, and GJA1 mRNA. Since the GJA1 transcript was the most abundant in all oviductal parts, subsequently, the Cx43 expression and localization were examined in the oviduct of all hens. The relative expression of GJA1 mRNA in control hens was highest in the infundibulum and vagina and lowest in the magnum. The pattern of Cx43 protein abundance evaluated by Western blot was similar to that of mRNA. Treatment of hens with TMX decreased the GJA1 mRNA levels in the magnum and isthmus, and Cx43 protein abundances were reduced in the isthmus and vagina. Immunofluorescence demonstrated cell- and segment-dependent localization of Cx43 protein in the oviductal wall; the most intense immunoreactivity was observed in the muscle cells of the shell gland and vagina. In TMX-treated hens, the immunoreactivity for Cx43 in all oviductal segments was slightly reduced and had a different signal pattern compared with control chickens. These results suggest that Cx43 likely takes part in the regulation of oviduct functioning, especially in the coordination of muscle contraction required for egg transport and oviposition. In addition, the results suggest a contribution of estrogen in the regulation of Cx43 expression and/or fates in the chicken oviduct. New insights into the expression and regulation of Cxs in the hen oviduct, indicating their potential involvement in the mechanisms of egg formation and transport that may affect poultry production, were obtained in this study.

In vitro effects of polychlorinated biphenyls and their hydroxylated metabolites on the synthesis and metabolism of iodothyronines in the chicken (Gallus domesticus) thyroid gland.

To assess the effect of polychlorinated biphenyls (PCBs) and their hydroxylated metabolites (OH-PCBs) on thyroid hormone [TH: thyroxine (T4) and triiodothyronine (T3)] secretion, the concentrations of iodothyronine deiodinases (DIO1, DIO2, DIO3), and mRNA expression of genes involved in TH synthesis (TSHR, NIS, TPO, TG), metabolism (DIO1, DIO2, DIO3), and transport (OATP1C1, MCT8, MCT10, LAT1), chicken thyroid explants were incubated in medium supplemented with TSH (250 mU/ml), PCB118, PCB153, 4-OH-PCB107, and 3-OH-PCB153 (0.5 × 10-8 M), and TSH together with each PCB and OH-PCB. The results of the in vitro experiment revealed that, except for 4-OH-PCB107, all applied PCBs and OH-PCBs inhibited basal and TSH-stimulated T4 secretion. Moreover, they increased basal and reduced TSH-stimulated T3 secretion. PCBs and OH-PCBs decreased the TSH-stimulated TSHR expression. Following PCB and OH-PCB exposure, significant changes in mRNA expression of NIS, TPO, and TG were observed. PCBs and OH-PCBs affected DIO1 and DIO3 transcript levels and protein abundances of each DIO. Furthermore, PCB-dependent effects on OATP1C1, MCT8, and MCT10 mRNA expression were found. In conclusion, both PCB118 and PCB153 and their OH-PCBs affect TH synthesis and deiodination processes in the chicken thyroid gland and influence TH transport across the thyrocyte membrane. In addition, the effects of PCBs and OH-PCBs depended mainly on the type of PCB congener and the exposure time. These results indicate that not only parental PCBs but also OH-PCBs are hazardous for the thyroid gland and may disrupt its endocrine function. Further studies are necessary to explain a mechanism of PCB and OH-PCB action in the avian thyroid gland.

Administration of silver nanoparticles affects ovarian steroidogenesis and may influence thyroid hormone metabolism in hens (Gallus domesticus).

This study aimed to determine the in vivo effect of silver nanoparticles (AgNPs) on the concentration of sex steroids (progesterone - P4, estradiol - E2, testosterone - T) and thyroid hormones (thyroxine - T4, triiodothyronine - T3) in the blood plasma as well as the messenger ribonucleic acid (mRNA) and protein expression of HSD3ß, CYP17A1 and CYP19A1 enzymes and steroid hormone concentrations in chicken ovarian follicles. AgNPs did not affect serum steroid hormone levels, but increased T3 levels depending on the size and concentration of AgNPs. At the level of ovarian tissues, AgNPs: (i) affected the levels of E2 and T in prehierachical follicles; (ii) reduced the expression of CYP19A1 mRNA and protein and consequently diminished E2 concentration in small white follicles; and (iii) increased the expression of CYP17A1 mRNA in large white follicles, without changing its protein expression. The results indicate that AgNPs affect chicken ovarian steroidogenesis. The effects of AgNPs depend on exposure time, the type of follicle and the degree of its development and are associated with the modulation of steroidogenic gene expression and E2 and T synthesis. Prehierachical follicles seem to be more susceptible to AgNPs than preovulatory ones. In conclusion, AgNPs by targeting the chicken ovary may indirectly influence the selection processes of prehierarchical follicles to the pre-ovulatory hierarchy and disturb the ovarian steroidogenesis. Furthermore, AgNPs may affect thyroid hormone metabolism in different ways by size which in turn may influence energy homeostasis of the target cells.

Nitrophenols are negative modulators of steroidogenesis in preovulatory follicles of the hen (Gallus domesticus) ovary: An in vitro and in vivo study.

This study assessed the effects of 4-nitrophenol (PNP) and 3-methyl-4-nitrophenol (PNMC) on steroidogenesis in the granulosa layers (GLs) and theca layers (TLs) of chicken preovulatory follicles in vitro and in vivo. In the in vitro experiment, three of the largest yellow preovulatory follicles (F3 < F2 < F1) were exposed to PNP or PNMC (10-8-10-4 M), ovine luteinising hormone (oLH; 10 ng/mL), and combinations of oLH and PNP or PNMC (10-6 M). In the in vivo experiment, laying hens were treated for 6 days with PNP or PNMC (10 mg/kg). In vitro experiments revealed that PNP and PNMC decreased basal and oLH-stimulated P4 secretion from the GL as well as T and E2 secretion from the TLs of F3-F1 follicles. Treatment of laying hens with nitrophenols lowered plasma concentrations of luteinising hormone and all three steroids. The reduction of steroid secretion was associated with decrease in LHR, HSD3B1 and CYP19A1 mRNA expression in the GL and/or TLs of the preovulatory follicles, both in vitro and in vivo. Moreover, PNP decreased HSD3B protein expression in the GL of F2 follicles in vitro and in vivo, while PNMC diminished its expression in the GL of F1 follicles in vivo. In vitro, nitrophenols did not affect CYP19A1 protein expression; however, nitrophenols inhibited its expression in the TLs of F3 and F2 follicles in vivo. The results obtained clearly demonstrate that nitrophenols are negative modulators of steroidogenesis in chicken preovulatory follicles and, in consequence, may not only impair ovulation process, but also affect function of the hypothalamic-pituitary-ovarian axis.

Expression of vitamin D receptor in the porcine uterus and effect of 1,25(OH)2D3 on progesterone and estradiol-17ß secretion by uterine tissues in vitro.

Vitamin D3 acting via its nuclear receptor (VDR) was shown to target many reproductive tissues and regulate their function. Nevertheless, little is known about the role of vitamin D3 and VDR in the uterus. We hypothesized that VDR expression profile varies in the porcine uterus throughout the course of the estrous cycle, and 1,25(OH)2D3 influences uterine steroidogenic activity. The aim of this study was to investigate VDR mRNA expression, VDR protein abundance and immunolocalization in the porcine endometrium and myometrium harvested on Days 2-5, 12-13, 15-16 and 18-20 of the estrous cycle. Additionally, in studied pigs, 25OHD concentration in plasma and uterine flushings was determined by RIA. The effect of 1,25(OH)2D3 (10, 50 and 100 ng/mL) in vitro on progesterone (P4) and estradiol-17ß (E2) release by endometrial and myometrial slices obtained on Days 12-13 of the estrous cycle was also examined. Nuclear VDR immunostaining was found in endometrial (luminal and glandular epithelium, stromal cells) and myometrial cells throughout examined days of the estrous cycle. In the endometrium, the highest VDR mRNA expression was observed on Days 12-13 and 18-20, whereas the greatest VDR protein abundance was noted only on Days 12-13 of the estrous cycle. In the myometrium, either VDR transcript or protein level was the greatest on Days 12-13. Interestingly, the highest 25OHD concentration in plasma and uterine flushings was shown also on Days 12-13 of the estrous cycle. 1,25(OH)2D3 did not affect P4 release by uterine slices while myometrial release of E2 was significantly increased in response to 1,25(OH)2D3 (10 and 50 ng/mL). Overall, obtained results indicate that porcine uterus is a target tissue for vitamin D3 throughout the entire estrous cycle. VDR mRNA expression and protein abundance altered within uterine tissues depending on studied days of the estrous cycle with the greatest protein abundance during mid-luteal phase of the estrous cycle in both uterine tissues. In addition, 1,25(OH)2D3 significantly increased myometrial release of E2 on Days 12-13 of the estrous cycle. These results suggest the role of vitamin D3-VDR system in the uterus, especially as a regulator of myometrial estrogenic activity in pigs during mid-luteal phase of the estrous cycle.

Comparison of the in vitro effects of TCDD, PCB 126 and PCB 153 on thyroid-restricted gene expression and thyroid hormone secretion by the chicken thyroid gland.

The aim of this study was to compare the in vitro effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3,3',4,4',5-pentachlorobiphenyl (PCB 126; a coplanar PCB congener) and 2,2'4,4',5,5'-hexachlorobiphenyl (PCB153; non-coplanar PCB) on mRNA expression of thyroid-restricted genes, i.e. sodium iodide symporter (NIS), thyroid peroxidase (TPO) and thyroglobulin (TG), and thyroid hormone secretion from the thyroid gland of the laying chicken. Relative expression levels of NIS, TG and TPO genes and thyroxine (T4) and triiodothyronine (T3) secretion from the thyroidal explants were quantified by the real-time qPCR and RIA methods, respectively. In comparison with the control group, TCDD and PCB 126 significantly increased mRNA expression of TPO and TG genes. TCDD did not affect NIS mRNA levels, but PCB 126 decreased its expression. No effect of PCB 153 on the expression of these genes was observed. TCDD and PCB 126 significantly decreased T4 and T3 secretion. There was no significant effect of PCB 153 on these hormone secretions. In conclusion, the results obtained show that in comparison with non-coplanar PCB 153, TCDD and coplanar PCB 126 can directly affect thyroid hormone synthesis and secretion, and in consequence, they may disrupt the endocrine function of the thyroid gland of the laying chicken.

[Evaluation of diagnostic usefulness of the cord blood TNF-alpha levels as a marker of early onset neonatal infection]. / Ocena przydatnosci diagnostycznej okreslania stezenia TNF-alpha we krwi pepowinowej jako markera zakazenia wczesnego u noworodka.

OBJECTIVE: The aim of the study was to assess whether cord blood TNF-alpha levels can be useful as a marker of early onset neonatal infection. DESIGN: A prospective survey of a group of 110 newborns performed during the first 3 days of life. METHODS: The study was performed on a group of 110 newborns. In all the examined babies, cord blood TNF-alpha levels was evaluated by RIA. In addition CRP levels, and WBC, RBC, PLT were determined during the first two hours of life. On the basis of clinical symptoms and results of the known laboratory markers of infection the examined babies were divided into two subgroups. 74 of them were a study group and the other 36 -control group. RESULTS: It was found that cord blood TNF-alpha levels were significantly higher (p < 0.05) in the study than in the control group [Me = 31.15 pg/ml (5.0-311.0) versus Me = 21.4 pg/ml (5.0-74.8)]. The differentiation level of the TNF-alpha between study and control group was assessed on the level of 15 pg/ml. It was found that the sensitivity of this test as a marker of early onset infection is 78.0% and specificity is only 41.2%; odds ratio--3.2. Furthermore we did not find a significant correlation between TNF-alpha levels and CRP, as well as WBC, RBC ant PLT count. CONCLUSION: Newborns developing early onset infection are born with higher TNF-alpha levels than healthy subjects are. Low specificity of this test despite high sensitivity is a reason, which can cause a limitation in using this test in clinical practice.