Environmental levels of cadmium, lead and mercury in brown hares and their relation to blood metabolic parameters.

The purpose of this study was to examine concentrations of selected heavy metals in the liver and kidney of brown hares (Lepus europaeus). In addition, correlations between heavy metals and biochemical parameters in blood plasma were determined. The average concentrations of heavy metals (mmol/L) +/- SD were as follows: liver: Pb 0.221 +/- 0.189, Cd 0.160 +/- 0.140, Hg 0.021 +/- 0.030, kidney: Pb 0.115 +/- 0.125, Cd 1.570 +/- 1.103, Hg 0.030 +/- 0.053. The average concentrations of biochemical parameters in the blood plasma were as follows: Ca 3.16 mmol/L, P 2.19 mmol/L, Mg 1.40 mmol/L, Na 148.71 mmol/L, K 8.12 mmol/L, glucose 6.56 mmol/L, total proteins 56.49 g/L, urea 5.00 mmol/L, total lipids 1.40 g/L, bilirubin 3.97 micro mol/L, cholesterol 1.53 mmol/L, aspartate aminotransferase (AST) 6.06 micro kat/L and alanine aminotransferase (ALT) 1.94 micro kat/L. Average levels of hormones (ng/mL) were as follows: testosterone 2.94, androstendiol 0.13, estradiol 501.59, progesterone 6.63, oxytocin 328.60. Tissue analysis showed an accumulation of lead, cadmium and mercury in the liver and kidney of brown hares. There were no significant correlations between levels of heavy metals in liver, kidney, and biochemical parameters.

Evidence that gamma-aminobutyric acid is part of the neural circuit mediating estradiol negative feedback in anestrous ewes.

Seasonal anestrus in ewes is driven by an increase in response to estradiol (E2) negative feedback. Compelling evidence indicates that inhibitory A15 dopaminergic (DA) neurons mediate the increased inhibitory actions of E2 in anestrus, but these neurons do not contain estrogen receptors. Therefore, we have proposed that estrogen-responsive afferents to A15 neurons are part of the neural circuit mediating E2 negative feedback in anestrus. This study examined the possible role of afferents containing gamma-aminobutyric acid (GABA) and nitric oxide (NO) in modulating the activity of A15 neurons. Local administration of NO synthase inhibitors to the A15 had no effect on LH, but GABA receptor ligands produced dramatic changes. Administration of either a GABA A or GABA B receptor agonist to the A15 increased LH secretion in ovary-intact ewes, suggesting that GABA inhibits A15 neural activity. In ovariectomized anestrous ewes, the same doses of GABA receptor agonist had no effect, but combined administration of a GABA A and GABA B receptor antagonist to the A15 inhibited LH secretion. These data are consistent with the hypothesis that endogenous GABA release within the A15 is low in ovary-intact anestrous ewes and elevated after ovariectomy. Using dual immunocytochemistry, we observed that GABAergic varicosities make close contacts on to A15 neurons and that A15 neurons contain both the GABA A-alpha1 and the GABA B-R1 receptor subunits. Based on these data, we propose that in anestrous ewes, E2 inhibits release of GABA from afferents to A15 DA neurons, increasing the activity of these DA neurons and thus suppressing episodic secretion of GnRH and LH.

Does nitric oxide act in the ventromedial preoptic area to mediate oestrogen negative feedback in the seasonally anoestrous ewe?

Seasonal anoestrus in the ewe results from enhanced oestrogen negative feedback. Recent data have implicated the ventromedial preoptic area (vmPOA) as an important site of oestrogen action. This study addressed whether NO acts within the vmPOA to inhibit LH during seasonal anoestrus. In Experiment 1, microimplants containing Nomega-nitro-l-arginine methyl ester (l-NAME, NOS inhibitor), S-methyl thiocitrulline (SMTC, neural NOS (nNOS) inhibitor) or empty implants (control) were administered during mid-anoestrus to the vmPOA. l-NAME, but not SMTC, significantly increased LH pulse frequency. For Experiment 2, ewes in late anoestrus were administered 7-nitroindazole (7NI; nNOS inhibitor), l-NAME, SMTC, or empty implants. 7NI, but not l-NAME or SMTC, increased LH pulse frequency. In Experiment 3, the effects of microimplants and microinjections of l-NAME were compared in mid-anoestrus. Microinjections of l-NAME (300 nl at 10 microg/microl) increased LH pulse frequency, but microimplants did not. In late anoestrus, similar microinjections were ineffective. Taken together, the results of Experiments 1-3 suggested that NO inhibition may be stronger during the middle than at the end of seasonal anoestrus. To test this hypothesis, ewes in Experiment 4 received microinjection of l-NAME or vehicle thrice during the non-breeding season; none of the treatments increased LH pulse frequency. These results indicate that NO plays a role in the vmPOA in suppressing LH secretion during seasonal anoestrus because NOS inhibitors were consistently stimulatory when LH pulse frequency was low. However, the inconsistent and modest effects of these inhibitors suggest that NO actions in this area cannot completely account for the effects of inhibitory photoperiod.

Accumulation of some metals in muscles of five fish species from lower Nitra river.

This paper reports the results of accumulated selected metals concentrations (Fe, Mn, Zn, Cu, Ni, Co, Cr, Pb, Cd, Hg and meHg) in the muscle of five common Slovak fish species (Chub-Leuciscus cephalus, Common carp-Cyprinus carpio, Prussian carp-Carassius gibelio, Roach-Rutilus rutilus, and Wels catfish-Silurus glanis). Furthermore, correlations among the selected metals and order of metal accumulation in the fish muscle were determined. The concentrations of metals (mg/kg wet weight basis) ranged as follows: Fe 3.70-21.10; Mn 0.27-1.50; Zn 3.72-42.82; Cu 0.26-1.82; Ni 0.02-0.29; Co 0.06-0.28; Cr 0.09-0.28; Pb 0.08-34.59; Cd 0.06-2.76, Hg 0.34-3.64 and meHg 0.08-1.20. The level of lead and mercury exceeded the maximum allowed concentration in Slovakia by the Codex Alimentarius for safe human consumption (0.2 and 0.5 mg/kg, respectively) in the majority of samples (94.6 and 82.1%, respectively). Content of Cd (0.88 +/- 0.76 mg/kg wet weight) in the fish muscle exceeded maximum allowed levels (0.05 mg/kg) in all samples. An average, the order of metal concentrations in the fish muscle was: Fe > Zn > Pb > Cd > Hg > Cu > Mn > meHg > Ni > Cr > Co.

Heavy metals content and microbiological quality of carp (Cyprinus carpio, L.) muscle from two Southwestern Slovak fish farms.

The purpose of this study was to assess concentration and correlation of selected heavy metals and level of some microbiological indicators (total bacteria count-TBC, mesophilic anaerobic sporulating bacteria-MASB) in the muscle of common carp (Cyprinus carpio). Fish were collected by seine net within the frame of pondfishing, in October (pond Horné Obdokovce--pond A) and December (pond Budmerice--pond B) 2003. Concentrations of selected metals were measured using an atomic absorption spectrophotometer Pye Unicam SP9. The concentrations of metals (mg/kg wet weight basis) ranged as follows: Fe 3.47-15.15; Mn 0.14-0.42; Zn 3.47-9.52; Cu 0.24-1.32; Co 0.05-0.17; Ni 0.07-0.42; Cr 0.08-0.19; Pb 0.11-0.30; and Cd 0.01-0.05. The count of MASB and TCB (in CFU/g) varied as follow: 1.12-7.76 x 10(3) and 0.03-7.59 x 10(6), respectively. Significant differences (P < 0.05) for Cu, Ni, and Cr bioaccumulation, as well as for count of MASB between ponds were recorded. An opposite trend of bioaccumulation was found in only 12 of 36 cases. Positive correlations between counts of MASB and accumulated heavy metals, except Ni and negative correlations between TBC and accumulated heavy metals, except Cr, were recorded, (P < 0.05) for TBC-Mn and TBC-Cd relationship. Lead concentrations exceeded the maximum values allowed concentration in Slovakia by Codex Alimentarius (0.2 mg/kg) by 60% for pond-A and 40% for pond-B. The level of MASB count exceeded the maximum allowed concentration in 100% by both ponds. On average, the order of metal concentrations in the fish muscle was: (Pond-A) Fe > Zn > Cu > Mn > Pb > Cr > Ni > Co > Cd; (Pond-B) Fe > Zn > Cu > Mn > Pb > Ni > Cr > Co > Cd.

Inhibition of luteinizing hormone secretion by localized administration of estrogen, but not dihydrotestosterone, is enhanced in the ventromedial hypothalamus during feed restriction in the young wether.

The ability of steroids to inhibit LH secretion is enhanced during undernutrition. To identify potential hypothalamic sites at which this enhancement may occur, we examined LH secretion in feed-restricted or fed young wethers treated with locally administered metabolites of testosterone. In experiment 1, microimplants containing crystalline estradiol-17beta (E) or cholesterol were administered via chronic guide tubes directed to the preoptic area (POA) or ventromedial hypothalamus (VMH) in fed or feed-restricted wethers. E treatment in the VMH decreased LH pulse frequency, pulse amplitude, and mean LH concentration in feed-restricted, but not fed, wethers. E may act in the POA to suppress LH under feed restriction, but definite conclusions cannot be drawn because of steroid-independent effects of feed restriction on LH pulse frequency. In experiment 2, the effect of dihydrotestosterone (DHT) in the VMH was determined. DHT administration to the VMH did not alter LH secretion in either feed-restricted or fed wethers. Thus the VMH is one site wherein E negative feedback is enhanced during feed restriction in the wether. In contrast, we found no evidence for enhanced responsiveness to androgen negative feedback within the VMH of feed-restricted wethers. We suggest that increased sensitivity within the VMH to E, but not to DHT, is important for suppressing LH secretion in undernourished male sheep.

Concentration of selected metals in muscle of various fish species.

The purpose of this study was to assess concentration of selected metals (Fe, Mn, Zn, Cu, Ni, Co, Cr, Pb, Cd, and Hg) in the muscle of four common Slovak fish species (chub--Leuciscus cephalus, barbel--Barbus barbus, roach--Rutilus rutilus, and perch--Perca fluviatilis). Furthermore, correlations among the selected metals and order of metal accumulation in the fish muscle were determined. An electrofishing technique was used for collecting the fish from the Nitra River (Slovakia) in September 2003. Concentrations of selected metals were measured using an atomic absorption spectrophotometer Pye Unicam SP9. The concentrations of metals (mg/kg wet weight basis) ranged as follows: Fe 3.41-15.14; Mn 0.20-0.81; Zn 3.51-15.64; Cu 0.25-0.78; Ni 0.07-0.25; Co 0.05-0.19; Cr 0.11-0.42; Pb 0.20-5.81; Cd 0.06-0.56, and Hg 1.35-6.52. Significant correlations (P < 0.05) between Fe-Cu, Fe-Ni, Fe-Cr, Mn-Ni, Mn-Cr, Mn-Cr, Cu-Ni, and Ni-Cr were observed. The level of lead exceeded the maximum allowable concentration in Slovakia by Codex Alimentarius for safe human consumption (0.2 mg/kg) in the majority of samples (97.2%). Content of Cd (0.23 +/- 0.13 mg/kg wet weight) and Hg (2.85 +/- 1.22 mg/kg wet weight) in the fish muscle exceeded maximum allowed levels in all samples. On average, the order of metal concentrations in the fish muscle was: Fe > Zn > Hg > Pb > Cu > Mn > Cd > Cr > Ni > Co.

Progesterone increases dynorphin a concentrations in cerebrospinal fluid and preprodynorphin messenger ribonucleic Acid levels in a subset of dynorphin neurons in the sheep.

Recent studies suggest that the endogenous opioid peptide, dynorphin, is an important mediator of progesterone negative feedback on GnRH pulse frequency in the ewe. These experiments tested this hypothesis by examining the effects of progesterone on dynorphin A concentrations in cerebrospinal fluid (CSF) collected from the third ventricle and expression of preprodynorphin (PPD) mRNA in hypothalamic nuclei. CSF was collected every 10 min for 5 h in three groups of ewes: 1) ovary-intact ewes during the luteal phase (d 6-7 of estrous cycle); 2) ewes 6-7 d after ovariectomy (OVX); and 3) OVX ewes treated for 6-7 d with implants that produced luteal-phase progesterone levels. Diencephalic tissue from these ewes was then collected and processed for in situ hybridization using an ovine cDNA probe against PPD. Progesterone treatment increased dynorphin A concentrations in CSF over that observed in untreated OVX ewes; CSF dynorphin A concentrations in ovary-intact ewes were midway between the other groups. OVX significantly decreased the number of PPD mRNA-expressing cells in the preoptic area (POA), anterior hypothalamic area (AHA), and arcuate nucleus (ARC), with no change seen in any other PPD-expressing nuclei. Progesterone treatment of OVX ewes restored PPD expression in the POA and AHA to levels seen in luteal-phase animals but had no effect on PPD expression in the ARC. These results are consistent with the hypothesis that progesterone acts via dynorphin neurons to inhibit pulsatile GnRH secretion and point to dynorphin neurons in the POA, AHA, and ARC as potential mediators of this action during the luteal phase.

Evidence that thyroid hormones act in the ventromedial preoptic area and the premammillary region of the brain to allow the termination of the breeding season in the ewe.

Thyroid hormones are permissive for various species to enter seasonal anestrus. In the ewe they act centrally to permit the onset of potent estradiol-negative feedback responsible for anestrus, but the specific sites of action are unknown. Therefore, we tested whether T(4) replacement via chronic microimplants in any of five brain areas could reverse the reproductive effects of thyroidectomy. Diffusion of (125)I-T(4) from the microimplant was largely (>98%) limited to a 1.2-mm radius. A marked decline in LH concentration in ovariectomized, estradiol-treated ewes was used as an index for anestrus. In experiment 1, all thyroidectomized (THX) ewes with microimplants in the medial preoptic area, A15 area, and medial basal hypothalamus failed to enter anestrus; instead, LH levels remained elevated, similar to those in untreated THX controls. In ventromedial preoptic area (vmPOA)-microimplanted ewes, only the two animals with the most caudal microimplants entered anestrus, as did thyroid-intact controls and THX ewes receiving icv or sc T(4) replacement. In experiment 2, all vmPOA-treated ewes with similar placements to those effective in experiment 1 along with all ewes microimplanted in the premammillary region entered neuroendocrine anestrus. Thus, the premammillary region and vmPOA are brain sites in which thyroid hormones act to permit the onset of seasonal anestrus.

Thyroid hormones mediate steroid-independent seasonal changes in luteinizing hormone pulsatility in the ewe.

Thyroid hormones permit the increase in response to estradiol negative feedback in ewes at the transition to anestrus. In this study, we tested whether the thyroid hormones are also required for steroid-independent seasonal changes in pulsatile LH secretion. In experiment 1, Suffolk ewes were ovariectomized and thyroidectomized (THX) or ovariectomized only (controls) in late November. LH pulse frequency and amplitude were measured for 4 h in December, April, May, June, and August. Pulse frequency was also measured in the presence of estradiol-containing implants during the breeding (December) and early anestrus (March) seasons. As expected, in the presence of estradiol, pulse frequency declined between December and March in control but not THX ewes. In the absence of estradiol, a seasonal decline in frequency and an increase in amplitude occurred in control ewes, concurrent with lengthening photoperiod. A similar trend was seen in THX ewes, but the seasonal changes were lower in magnitude and not significant. In experiment 2, the same protocol was used (pulse measurements in December, May, and June) with a larger THX group size (n = 7). Results were similar to those of experiment 1 for controls. In THX ewes, pulse frequency did not change over time and was significantly elevated relative to that of controls during the summer. Pulse amplitude in THX ewes tended to increase during summer and did not differ from pulse amplitudes in control ewes. These results demonstrate that thyroid hormones are required for steroid-independent cycles in LH pulse frequency; however, some seasonal changes in amplitude still occur in the absence of thyroid hormones. This finding contrasts with the changes in estradiol negative feedback at the transition to anestrus, which are entirely thyroid hormone dependent.