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Background In recent decades, the incidence of metabolic disorders has increased internationally. This increase has been linked to exposure to persistent organic pollutants (POPs) but little is known about the metabolic effects of realistic human exposure mixtures at relevant concentrations. Objectives In this study we tested if POPs, representing real life exposure profiles and concentrations, were able to disrupt development and functions of adipose tissue in a direct way. Methods The lipogenic potency of a POP mixture modelled on levels found in human blood as detected in the Scandinavian population was assessed. The Total mixture comprises 29 compounds divided over three groups: chlorinated (Cl), brominated (Br), and perfluorinated compounds (PFAA). Individual PFAA chemicals, the Total mixture, and sub-mixtures (Cl, Br, PFAA, Cl + Br, Cl + PFAA, and Br + PFAA) at five (× 1/10, × 1, × 50, × 100, and × 500) human blood levels were tested in an optimised high content analysis (HCA) 3T3-L1 adipogenesis assay. Results Individual PFAAs; perfluorohexanesulfonic acid (PFHxS), perfluorooctanesulfonic acid (PFOS), perfluorononanoic acid (PFNA), and perfluoroundecanoic acid (PFUnDA) promoted lipid accumulation in 3T3-L1 cells. The Total mixture, and the Cl, PFAA, Cl + Br, and Cl + PFAA sub-mixtures, promoted adipogenic differentiation and lipid accumulation. Increased lipid accumulation promoted adipose tissue expansion. Conclusions To the authors knowledge, this is the first in vitro bioassay study assessing the adipogenic effects of POP mixtures modelled on real-life human exposure levels. The findings highlight that such exposures may alter adipose tissue development and function, thus potentially playing a role in the globally increasing escalation of metabolic disorders.

Persistent organic pollutants (POPs) can reach the fetal brain and contribute to developmental neurotoxicity. To explore the distribution of POPs to the fetal brain, we exposed chicken embryos to a POP mixture, containing 29 different compounds with concentrations based on blood levels measured in the Scandinavian human population. The mixture was injected into the allantois at embryonic day 13 (E13), aiming at a theoretical concentration of 10 times human blood levels. POPs concentrations in the brain were measured at 0.5, 1, 2, 4, 6, 24, 48, and 72 h after administration. Twenty-seven of the individual compounds were detected during at least one of the time-points analyzed. Generally, the concentrations of most of the measured compounds were within the order of magnitude of those reported in human brain samples. Differences in the speed of distribution to the brain were observed between the per- and polyfluoroalkyl substances (PFASs), which have protein binding potential, and the lipophilic polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs) and brominated flame retardants (BFRs). Based on pharmacokinetic modeling, PFASs were best described by a one compartment model. PFASs displayed relatively slow elimination (Kel) and persisted at high levels in the brain. Lipophilic OCPs and PCBs could be fitted to a 2-compartment model. These showed high levels in the brain relative to the dose administrated as calculated by area under the curve (AUC)/Dose. Altogether, our study showed that chicken is a relevant model to explore the distribution of POPs into the developing brain at concentrations which are relevant for humans.

Persistent organic pollutants (POPs) are widespread throughout the environment and some are suspected to induce reproductive toxicity. As animals and humans are exposed to complex mixtures of POPs, it is reasonable to assess how such mixtures could interact with the reproductive system. Our aim is to investigate how maternal exposure to a mixture of 29 different persistent organic pollutants, formulated to mimic the relative POP levels in the food basket of the Scandinavian population, could alter reproductive endpoints. Female mice were exposed via feed from weaning, during pregnancy and lactation in 3 exposure groups (control (C), low (L) and high (H)). Testicular morphometric endpoints, epididymal sperm concentration and sperm DNA integrity were assessed in adult male offspring. We found that the number of tubules, proportion of tubule compartments and epididymal sperm concentration significantly decreased in both POP exposed groups. Epididymal sperm from both POP exposed groups showed increased DNA fragmentation. It is concluded that maternal exposure to a defined POP mixture relevant to human exposure can affect testicular development, sperm production and sperm chromatin integrity.

An extended lifespan of spermatozoa following artificial insemination (AI) can make the timing of insemination less critical, as previously demonstrated with immobilized spermatozoa that are gradually released from an alginate gel. The purpose was to examine the in vivo dissolution of SpermVital (SV) alginate gel over time by endoscopy and secondly to assess spermatozoa quality after incubation of the gel. In vivo endoscopy showed SV gel in the uterus 3, 6, 20 and 24 hr after AI, demonstrating the potential release of spermatozoa to the uterus during this period. In utero ex vivo incubation of the semen demonstrated that high motility and viability of sperm cells was sustained following overnight incubation.

Environmental exposure to persistent organic pollutants (POPs) has been suggested as a contributing factor for the increased rate of type 2 diabetes and obesity. A complex mixture of 29 POPs (Total mixture), based on human blood concentrations, was used to expose a glucagon-like peptide 1 (GLP-1) secreting enteroendocrine cell line (pGIP/neo: STC-1) in vitro for 3 and 24 h. Significant increases of GLP-1 occurred when cells were exposed to the Total mixture at ×500 blood levels. Six sub-mixtures representing chlorinated (Cl), brominated (Br), and perfluorinated chemicals (PFAA), and their combinations (Cl + Br, Cl + PFAA, Br + PFAA) were also tested at ×500. Secretion levels seen for these remained lower than the Total mixture, and the Br mixture had no effect. After 24 h, increased secretion was seen with all mixtures at ×1 blood levels. Cytotoxicity was present for ×100 and ×500 blood levels. When tested in a GLP-1 receptor translocation assay (U2OS-GLP1R-EGFP), neither agonistic nor antagonist effects on receptor internalization were seen for any of the mixtures. We conclude individual classes of POPs, alone or in combination, can affect GLP-1 secretion and may contribute as a molecular mechanism linking environmental toxicants and diabetes.

Male and female mice pups were exposed to a low and high dose of a human relevant mixture of persistent organic pollutants (POPs) during pregnancy and lactation. Most compounds detected in the dams were found in offspring brains. The mice offspring exhibited changed expression of hippocampal genes involved in cognitive function (Adora2a, Auts2, Crlf1, Chrnb2, Gdnf, Gnal, Kcnh3), neuroinflammation (Cd47, Il1a), circadian rhythm (Per1, Clock), redox signalling (Hmox2) and aryl hydrocarbon receptor activation (Cyp1b1). A few genes were differentially expressed in males versus females. Mostly, similar patterns of gene expression changes were observed between the low and high dose groups. Effects on learning and memory function measured in the Barnes maze (not moving, escape latency) were found in the high dose group when combined with moderate stress exposure (air flow from a fan). Mediation analysis indicated adaptation to the effects of exposure since gene expression compensated for learning disabilities (escape latency, walking distance and time spent not moving in the maze). Additionally, random forest analysis indicated that Kcnh3, Gnal, and Crlf1 were the most important genes for escape latency, while Hip1, Gnal and the low exposure level were the most important explanatory factors for passive behaviour (not moving). Altogether, this study showed transfer of POPs to the offspring brains after maternal exposure, modulating the expression level of genes involved in brain function.

Perfluoroalkyl acids (PFAAs) are detectable in human blood. PFAA exposure may contribute to androgen receptor (AR)-related health effects such as prostate cancer (PCa). In Norway and Sweden, exposures to PFAAs and PCa are very real concerns. In vitro studies conventionally do not investigate PFAA-induced AR disruption at human blood-based concentrations, thus limiting application to human health. We aim to determine the endocrine disrupting activity of PFAAs based upon human exposure levels, on AR transactivation and translocation. PFAAs (PFOS, PFOA, PFNA, PFDA, PFHxS, and PFUnDA) were tested at concentrations ranging from 1/10 × to 500 × relative to human blood based upon the exposure levels observed in a Scandinavian population. Translocation was measured by high content analysis (HCA) and transactivation was measured by reporter gene assay (RGA). No agonist activity (translocation or transactivation) was detected for any PFAAs. In the presence of testosterone, AR translocation increased following exposure to PFOS 1/10 × and 100 ×, PFOA 1/10 ×, and PFNA 1 × and 500 × (P < 0.05). In the presence of testosterone, PFOS 500 × antagonised AR transactivation, whereas PFDA 500 × increased AR transactivation (P < 0.05). PFAAs may contribute to AR-related adverse health effects such as PCa. PFAAs can disrupt AR signalling via two major components: translocation and transactivation. PFAAs which disrupt one signalling component do not necessarily disrupt both. Therefore, to fully investigate the disruptive effect of human exposure-based contaminants on AR signalling, it is imperative to analyse multiple molecular components as not all compounds induce a disruptive effect at the same level of receptor signalling.

This study describes the utilization of an LC-MS/MS based H295R assay to assess an environmentally relevant mixture of persistent organic pollutants (POPs). H295R cells were exposed to the POP mixture in two conditions stimulated with 10 μM forskolin and unstimulated. Most importantly, the unstimulated cells responded to the low concentration of the mixture with a significant down-regulation of dehydroepiandrosterone (DHEA). This response was not observed in forskolin-stimulated cells. In stimulated H295R cells, exposure to the highest concentration showed a trend towards induced production of mineralocorticoids and glucocorticoids, although this was not significant. On the other hand, in the same exposure concentration and condition, estrogen and androgen production tended to be down-regulated. In addition to these patterns of responses being different in the stimulated vs unstimulated condition, four steroids were not detectable in the unstimulated condition.

Seafood products, including fish and fish oils, are major sources of persistent organic pollutants (POPs) which may cause endocrine disruption related to reproductive dysfunction in males. Primary porcine neonatal Leydig cells were exposed to three extracts of POPs obtained from different stages in production of cod liver oil dietary supplement, in the absence and presence of luteinizing hormone (LH). No reduced viability was observed and all POP extracts showed increased testosterone and estradiol levels in unstimulated cells and decreased testosterone and estradiol secretion in LH-stimulated cells. A decrease in central steriodogenic genes including STAR, CYP11A1, HSD3B and CYP17A1 was obtained in both culture conditions with all POP extracts. We implicate both small differences in composition and concentration of compounds as well as "old" POPs to be important for the observed steroidogenic effects.

The ability to evaluate the extent of poor welfare during clinical disease is of great importance for animal welfare. Chronic exposure to stressful situations has a negative impact on animal welfare, and there is a need for valid and reliable biomarkers of chronic stress. The aim of this study was (1) to evaluate hair cortisol (HC) and hair cortisone (HCn) as measures of chronic stress in sheep and (2) to assess stress and pain associated with ovine footrot using different physiological [fecal cortisol metabolites (FCM), HC and HCn] and behavioral measures. The study was performed as a single foot inoculation using a boot. The right hind foot of 24 lambs was inoculated with different strains of Dichelobacter nodosus, and the animals were subsequently monitored daily for two weeks to assess lameness and pain. Hair was collected from both hind limbs at the start of the study and before treatment with gamithromycin subsequent to the trial period, and fecal samples were collected weekly for four weeks. Clinical signs of footrot developed in all experimental groups. There was an increase in FCM from week 0 to 2 (p

Larval zebrafish (Danio rerio) are a tool for assessing endocrine disruption during early development. Here, we investigated the extent to which a simple light/dark behavioral test at five days post fertilization could compliment current methods within the field. We exposed fertilized embryos to hormones (17β-estradiol, testosterone, dihydrotestosterone, 11-ketotestosterone, thyroxine, triiodothyronine, progesterone, and hydrocortisone) and other relevant compounds (17α ethinylestradiol, bisphenol A, bisphenol S, nonylphenol, flutamide, nilutamide, linuron, drospirenone, potassium perchlorate, mifepristone, and fadrozole) to screen for behavioral effects between 96 and 118h post fertilization (hpf). With the exception of progesterone, all the hormones tested resulted in altered behaviors. However, some inconsistencies were observed regarding the age of the larvae at testing. For example, the xenoestrogens 17α- ethinylestradiol and nonylphenol had behavioral effects at 96hpf, but not at 118hpf. Furthermore, although thyroxine exposure had pronounced effects on behavior, the thyroid disruptor potassium perchlorate did not. Finally, we were unable to demonstrate a role of nuclear receptors following testosterone and 17α- ethinylestradiol exposure, as neither the androgen receptor antagonist flutamide nor the general estrogen receptor inhibitor fulvestrant (ICI) could rescue the observed behavioral effects, respectively. Similarly, molecular markers for androgen and estrogen disruption were upregulated at concentrations below which behavioral effects were observed. These results demonstrate hormones and endocrine disruptors can alter the behavior of larval zebrafish, but the mechanistic pathways remain unclear.

Both autoimmune disease prevalence and exposure to immunotoxic chemicals have increased the last decades. As a first screening of immunotoxic chemicals possibly affecting development of autoimmunity through attenuated macrophage function, we demonstrate a promising model measuring macrophage function in isolated peritoneal macrophages (PCM) from Wistar rats and C57Bl/6 mice. Immunotoxic effects of bisphenol A (BPA) and a selection of perfluoroalkyl acids (PFAAs) were analysed in vitro assessing phagocytic function of macrophages from different sources. Phagocytosis was reduced in PCM of C57Bl/6 mice and Wistar rats after BPA and perfluoroundecanoic acid (PFUnDA) exposure, but not in macrophages derived from human and rat monocyte derived macrophages (MDM). On the other hand, in vitro exposure to mixtures of persistent organic pollutants (POPs) showed similar reductions in rat PCM and rat and human MDM phagocytosis. Reduced phagocytosis was partly due to cytotoxicity. PCM isolated from non-obese diabetic (NOD) mice, interleukin 1α/β knockout (IL-1KO) mice and new-born rats were less sensitive to the xenobiotics than PCM from adult wild type rodents. Finally, in vivo studies with NOD mice verified that POP exposure also decreased the number of pancreatic macrophages in pancreatic islets, reflecting early signs of autoimmunity development, similarly as previously described for BPA.

Polychlorinated biphenyls (PCB) are environmental pollutants linked to adverse health effects including endocrine disruption and disturbance of reproductive development. This study aimed to determine whether exposure of pregnant sheep to three different mixtures of PCB 153 and PCB 118 affected fetal testis development. Ewes were treated by oral gavage from mating until euthanasia (d 134), producing three groups of fetuses with distinct adipose tissue PCB levels: high PCB 153/low PCB 118 (n = 13), high PCB 118/low PCB 153 (n = 14), and low PCB 153/low PCB 118 (n = 14). Fetal testes and blood samples were collected for investigation of testosterone, testis morphology, and testis proteome. The body weight of the offspring was lower in the high PCB compared to the low PCB group, but there were no significant differences in testis weight between groups when corrected for body weight. PCB exposure did not markedly affect circulating testosterone. There were no significant differences between groups in number of seminiferous tubules, Sertoli cell only tubules, and ratio between relative areas of seminiferous tubules and interstitium. Two-dimensional (2D) gel-based proteomics was used to screen for proteomic alterations in the high exposed groups relative to low PCB 153/low PCB 118 group. Twenty-six significantly altered spots were identified by liquid chromatography (LC)-mass spectroscopy (MS)/MS. Changes in protein regulation affected cellular processes as stress response, protein synthesis, and cytoskeleton regulation. The study demonstrates that in utero exposure to different environmental relevant PCB mixtures exerted subtle effects on developing fetal testis proteome but did not significantly disturb testis morphology and testosterone production.

Polychlorinated biphenyls (PCB), synthetic, persistent organic pollutants (POP), are detected ubiquitously, in water, soil, air, and sediments, as well as in animals and humans. PCB are associated with range of adverse health effects, such as interference with the immune system and nervous system, reproductive abnormalities, fetotoxicity, carcinogenicity, and endocrine disruption. Our objective was to determine the effects of three structurally different PCB congeners, PCB118, PCB 126, and PCB 153, each at two concentrations, on the steroidogenic capacity and proteome of human adrenocortical carcinoma cell line cultures (H295R) . After 48 h of exposure, cell viability was monitored and estradiol, testosterone, cortisol and progesterone secretion measured to quantify steroidogenic capacity of the cells. Two-dimensional (2D) gel-based proteomics was used to screen for proteome alterations in H295R cells in response to the PCB. Exposure to PCB 118 increased estradiol and cortisol secretion, while exposure to PCB 153 elevated estradiol secretion. PCB 126 was the most potent congener, increasing estradiol, cortisol, and progesterone secretion in exposed H295R cells. Seventy-three of the 711 spots analyzed showed a significant difference in normalized spot volumes between controls (vehicle only) and at least one exposure group. Fourteen of these protein spots were identified by liquid chromatography with mass spectroscopy (LC-MS/MS). Exposure to three PCB congeners with different chemical structure perturbed steroidogenesis and protein expression in the H295R in vitro model. This study represents an initial analysis of the effects on proteins and hormones in the H295R cell model, and additional studies are required in order to obtain a more complete understanding of the pathways disturbed by PCB congeners in H295R cells. Overall, alterations in protein regulation and steroid hormone synthesis suggest that exposure to PCB disturbs several cellular processes, including protein synthesis, stress response, and apoptosis.

Alternariol (AOH) is a mycotoxin commonly produced by Alternaria alternata on a wide range of foods. Few studies to date have been performed to evaluate the effects of AOH on endocrine activity. The present study makes use of in vitro mammalian cellular based assays and gene expression to investigate the ability of AOH to act as an endocrine disruptor by various modes of action. Reporter gene assays (RGAs), incorporating natural steroid hormone receptors for oestrogens, androgens, progestagens and glucocorticoids were used to identify endocrine disruption at the level of nuclear receptor transcriptional activity, and the H295R steroidogenesis assay was used to assess endocrine disruption at the level of gene expression and steroid hormone production. AOH exhibited a weak oestrogenic response when tested in the oestrogen responsive RGA and binding of progesterone to the progestagen receptor was shown to be synergistically increased in the presence of AOH. H295R cells when exposed to 0.1–1000 ng/ml AOH, did not cause a significant change in testosterone and cortisol hormones but exposure to 1000 ng/ml (3.87 μM) AOH resulted in a significant increase in estradiol and progesterone production. In the gene expression study following exposure to 1000 ng/ml (3.87 μM) AOH, only one gene NR0B1 was down-regulated, whereas expression of mRNA for CYP1A1, MC2R, HSD3B2, CYP17, CYP21, CYP11B2 and CYP19 was up-regulated. Expression of the other genes investigated did not change significantly. In conclusion AOH is a weak oestrogenic mycotoxin that also has the ability to interfere with the steroidogenesis pathway.

Background Normal brain maturation is the result of molecular changes that can be modulated by endocrine variables associated with brain plasticity and results in sex- and age specific changes in cognitive performance. Using a sheep model, we have previously shown that peri-pubertal pharmacological blockade of gonadotropin releasing hormone (GnRH) receptors results in increased sex-differences in cognitive executive function and emotional control. In this study we explore effects of this treatment regime on hippocampal gene expression and spatial orientation. Methods The study was conducted with 30 same-sex twin lambs, half of which were treated with the GnRH analog (GnRHa) goserelin acetate every 4th week, beginning before puberty, until 50 weeks of age. Animals were tested in their spatial orientation ability at 48 weeks of age. Quantitative real time PCR analysis was conducted to examine effects of treatment on the expression of genes associated with synaptic plasticity and endocrine signaling. Results GnRHa treatment was associated with significant sex- and hemisphere specific changes in mRNA expression for some of the genes studied. The treatment had no significant effect on spatial orientation. However, there was a tendency that females performed better than males in spatial orientation. Conclusion Our results indicate that GnRH directly and/or indirectly, is involved in the regulation of sex- and side-specific expression patterns of genes. Hence, these results should be considered when long-term peri-pubertal GnRHa treatment is used in children.

Persistent organic pollutants such as polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs) and dichlorodiphenyltrichloroethane (DDT) are present in high concentrations in livers of burbot (Lota lota) in Lake Mjøsa, Norway. In order to assess effects of such pollutants on fish gonadal morphology, female zebrafish were exposed in two generations by food to mixtures of pollutants extracted from livers of burbot from Lake Mjøsa (high and low dose) and Lake Losna, which represents background pollution, and compared to a control group. Ovarian follicle counts detected a significant decrease in late vitellogenic follicle stages in fish exposed to the Losna and the high concentrations of Mjøsa mixtures in fish from the first generation. In addition, proliferation of granulosa cells, visualized by immunohistochemistry against proliferating cell nuclear antigen (PCNA), was decreased in all exposure groups in either early or late vitellogenic follicle stages compared to control. This was accompanied by increased apoptosis of granulosa cells. There was a decrease in proliferation of liver hepatocytes with exposure to both Mjøsa mixtures. In addition, immunopositivity for vitellogenin in the liver was significantly lower in the Mjøsa high group than in the control group. When analysing effects of parental exposure, fish with parents exposed to Mjøsa high mixture had significantly higher numbers of perinucleolar follicles than fish with control parents. We conclude that long-term exposure of a real-life mixture of pollutants containing high- and background levels of chemicals supress ovarian follicle development, liver vitellogenin immunostaining intensity and hepatocyte proliferation in the zebrafish model.

The aim of this study was to screen the inhibitory potential of several testicular steroids on cytochrome P450 3A (CYP3A) and 2C (CYP2C) activities in porcine liver microsomes. The microsomes used in this study were obtained from pubertal male pigs of two breeds, Landrace and Duroc. For the in vitro inhibition study, porcine microsomes were incubated in the presence of 17β-estradiol, 17α-estradiol, androstenone, dehydroepiandrosterone and dihydrotestosterone. Both reversible and mechanism-based inhibitions were examined. 7-benzyloxyresorufin (BR) and 7-benzyloxy-4-trifluoromethylcoumarin (BFC) were used as substrates for CYP3A, and diclofenac and tolbutamide (TB) as substrates for CYP2C. 7-benzyloxyresorufin O-dealkylase (BROD) activity was inhibited by all tested steroids in the microsomes from Landrace pigs via mechanism-based mode, but in the microsomes from Duroc pigs, BROD activities were inhibited only in the presence of 17β-oestradiol. Mechanism-based inhibition of BFC metabolism by the tested steroids was observed in the microsomes from both breeds, but this inhibition was weak and did not exceed 20%. TB hydroxylase (TBOH) activity in the microsomes from Duroc pigs was inhibited by 17α-oestradiol through the mechanism-based mode of inhibition. None of the investigated steroids inhibited TBOH activity in Landrace pigs. For the in vivo study, male pigs were injected with a single dose of human chorionic gonadotropin (hCG) to stimulate testicular steroid production by the Leydig cells. In vivo stimulation with hGC did not alter BROD activity either in Landrace or in Duroc pigs. BFC metabolism was significantly induced by hCG stimulation in both breeds and TBOH activity only in Duroc pigs. Activity of diclofenac hydroxylase was not detected in either Landrace or Duroc pigs. Breed significantly affected BROD and TBOH activity with BROD being higher in Landrace and TBOH in Duroc pigs. This study improved our understanding of the role of testicular steroids in the regulation of porcine CYP450 activity.

Perfluorinated compounds (PFCs) comprise a large class of man-made chemicals of which some are persistent and present throughout the ecosystem. This raises concerns about potential harmful effects of such PFCs on humans and the environment. In order to investigate the effects of potentially harmful PFCs on steroid hormone production, human adrenocortical H295R cells were exposed to three persistent PFCs including perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA) and perfluorononanoic acid (PFNA) at six different concentrations (6nM to 600μM) for 48h. Exposure to 600μM PFOS resulted in a dose-responsive increase in oestradiol as well as a smaller dose-responsive increase in progesterone and testosterone secretion measured using radioimmunoassay. The aromatase activity was not significantly altered by PFOS. Only small changes in hormone secretion were detected following exposure to PFOA and PFNA. Gene expression of CYP11A, quantified using qRT-PCR was decreased by all exposure doses of PFOA, whereas HMGR expression was decreased by 60nM PFNA. The viability markedly decreased by exposure to 600μM of PFOA or PFNA, but not PFOS. Flow cytometric analysis demonstrated a significant increase in apoptosis following exposure to PFNA at the highest concentration. We conclude that PFOS is capable of altering steroidogenesis in the H295R in vitro model by a mechanism other than changes in gene expression or activity of aromatase. Additionally, PFCs appear to differentially affect cell viability with induction of cell death via apoptosis at high doses of PFNA.

Proper management of threatened species requires knowledge of population sizes and struc- tures, however current techniques to gather this information are generally impractical and costly and can be stressful on the animals. Non-invasive methods that can produce high quality and accurate results are better alternatives. In winter 2010, we collected blood and fecal samples from 2 reindeer (Rangifer tarandus) populations (Kaamanen, Finland and Svalbard, Norway) to investigate the feasibility of using fecal proges- terone metabolites to help estimate the reproductive status, the sex, and the age structures of the populations. We first examined the relationship between plasma progesterone and fecal progesterone metabolite con- centrations. We further assessed whether fecal progesterone metabolite levels would clearly differ among calf, yearling, and adult and between pregnant and non-pregnant females. We quantified fecal progesterone metabolites (using enzyme immunoassay) and plasma progesterone (using radio immunoassay) of females and males of different ages from the 2 herds. We found in both populations that fecal progesterone metabolite levels reflected plasma progesterone concentrations. However, the range of fecal progesterone metabolite concentration was much wider in Finland than in Svalbard, possibly due to differences in diet or body condition. We determined a threshold value of 1.31 ng/ml plasma progesterone and 2025.93 ng/g dried fecal progesterone metabolites to identify pregnant reindeer from non-pregnant animals with 100% accuracy. We found a significant difference in fecal progesterone metabolite concentrations only between calves and yearlings/adults in Finland. We could not differentiate among males, non-pregnant adults, or calves of either sex; therefore identification of sex may have to rely on the use of DNA techniques. Our results suggest that hormone concentration, in combination with fecal DNA and pellet morphometry techniques, may provide important population parameters and is a valuable tool for the monitoring of reindeer and may have an application for threatened populations of woodland caribou throughout the winter and early spring. 2011 The Wildlife Society.

Obesity is reaching epidemic proportions worldwide, and is associated with chronic illnesses such as diabetes, cardiovascular disease, hypertension and dyslipidemias (metabolic syndrome). Commonly held causes of obesity are overeating coupled with a sedentary lifestyle. However, it has also been postulated that exposure to endocrine disrupting chemicals (EDCs) may be related to the significant increase in the prevalence of obesity and associated diseases. In the present study, developmental and reproductive effects of lifelong exposure to environmentally relevant concentrations of two natural mixtures of persistent organic pollutants (POPs) were investigated using classical and molecular methods in a controlled zebrafish model. The mixtures used were extracted from burbot ( Lota lota ) liver originating from freshwater systems in Norway (Lake Mjosa and Lake Losna). The concentration of POPs in the zebrafish ranged from levels detected in wild fish (Lake Mjosa and Lake Losna), to concentrations reported in human and wildlife populations. Phenotypic effects observed in both exposure groups included (1) earlier onset of puberty, (2) elevated male/female sex ratio, and (3) increased body weight at 5 months of age. Interestingly, genome-wide transcription profiling identified functional networks of genes, in which key regulators of weight homeostasis (PPARs, glucocoricoids, CEBPs, estradiol), steroid hormone functions (glucocoricoids, estradiol, NCOA3) and insulin signaling (HNF4A, CEBPs, PPARG) occupied central positions. The increased weight and the regulation of genes associated with weight homeostasis and insulin signaling observed in the present study suggest that environmental pollution may affect the endocrine regulation of the metabolism, possibly leading to increased weight gain and obesity.

The main objective of the present study was to gain knowledge about the disposition of the PCB congeners 118 and 153 in a sheep model where ewes were given oral doses of PCB in two consecutive gestations. The study aimed to investigate how maternal exposure with PCBs affected tissue concentrations in lambs and fetuses and to carry out time trend studies in ewes between gestations, as well as to increase the knowledge on metabolic processes by studying OH-metabolites. The results showed that the fetuses accumulated PCBs in levels and composition comparable to the ewes. The mean fetus/mother ratios of adipose tissue levels ranged from 0.71 to 0.82 for both PCB 118 and PCB 153 in the two exposure groups. Furthermore, the fetal brain/fat ratios ranged from 0.15 to 0.20 for both congeners. Body burden calculations (BB) showed that 16% and 43% of the total PCB 118 and PCB 153, respectively, still remained in ewes, lambs and fetuses after exposure during two gestations. The different accumulation of the two congeners is assumed to be a result of differences in metabolism. The present study demonstrate that high levels of PCB 118 and PCB 153 cross the placenta and accumulate in the fetal adipose tissue and brain thus exposing the fetus during vulnerable periods of extensive development.

In the present study, developmental and reproductive effects of lifelong exposure to environmental relevant concentrations of two natural mixtures of persistent organic pollutants (POP) were investigated using classical and molecular methods in a controlled zebrafish model. The mixtures used were extracted from burbot (Lota lota) liver originating from freshwater systems in Norway: one mixture with high levels and one mixture with background levels of polybrominated diphenyl ethers (PBDE), polychlorinated biphenyls (PCB), and dichlorodiphenyltrichloroethane metabolites (DDT). The concentration of POP measured in the zebrafish ranged from levels detected in wild fish from Lake Mjøsa to concentrations reported in human and wildlife populations, indicating that the experimental fish were exposed to concentrations comparable with wild fish. Phenotypic effects observed in both exposure groups included earlier onset of puberty, increased male/female sex ratio, and differences in body weight at 5 mo of age. Interestingly, genome-wide transcription profiling showed changes in regulation of genes involved in endocrine signaling and growth. The transcriptomics changes include key regulator genes for steroid hormone functions (ncoa3), and growth (c/ebp, ncoa3). The effects observed in the experimental zebrafish model raise the question whether chemical pollution represents a risk to reproductive health of wild fish inhabitating the freshwater system.

The aim of the present study was to investigate whether low levels of mono-ortho PCB 118 and di-ortho PCB 153, affect bone composition and strength in ewes (Dala breed) and their foetuses following exposure starting at conception and ending a week before expected delivery. In male foetuses, trabecular bone mineral content at the metaphysis was almost 30% lower in the PCB 118 (49 microg/kg body wt/day) group compared to the control group (corn oil) (ANCOVA, P

Contents The effect of human chorionic gonadotropin (hCG) stimulation on the activities of ethoxyresorufin O-deethylase (EROD), methoxyresorufin O-demethylase (MROD) and pentoxyresorufin O-depentylase (PROD) was studied in intact male pigs of purebred Landrace and Duroc breeds. Pigs were divided into four groups: two control groups of each breed, without hCG stimulation (n = 20 for each breed), and two experimental groups (n = 18 for each breed), with hCG stimulation (Pregnyl®; N.V. Organon, Oss, The Netherlands, 30 IU/kg live weight). Pigs were slaughtered 3 days after hCG stimulation and enzyme activities were measured in hepatic microsomes using two approaches. First, only one substrate concentration was used for the analysis of each enzyme activity. We found that EROD activity was suppressed by hCG-stimulation in Landrace (p = 0.004), but not Duroc pigs (p > 0.05). Generally, EROD activity was higher in Duroc pigs compared with Landrace (p = 0.017). Methoxyresorufin O-demethylase and PROD activities did not differ between groups (p > 0.05). To further characterize EROD, MROD and PROD, enzyme kinetic studies were performed. Vmax values for EROD and MROD in both breeds were lower after hCG stimuation (p

Short-term stress exposure is associated with activation of the hypothalamic-pituitary-adrenal (HPA) axis and a consequent rise in blood glucocorticoids and catecholamines, from the adrenal cortex and medulla, respectively. The HPA axis is a potential target for some persistent organic pollutants, among which polychlorinated biphenyls (PCB) were found to be modulators of the mammalian endocrine system. PCB are distributed globally in the environment, in food chains, and are transferred to the fetuses of pregnant animals and via mother's milk to suckling offspring. In the present study it was postulated that intrauterine and lactational exposure to either of two single congeners of PCB (PCB 153 and PCB 126, respectively) might affect basal cortisol concentrations, and also the cortisol response to short-term stress in adulthood. Thus, pregnant goats were orally exposed to one of these PCB congeners from d 60 of gestation until delivery, and their offspring studied. Low-dose exposure to PCB 153 and PCB 126 resulted in significantly lower mean basal cortisol concentrations in goat offspring during certain periods of pubertal development and their first breeding season. Male goat kids exposed to either PCB congener showed a greater and more prolonged rise in plasma cortisol levels than controls when animals were subjected to mild stress at 9 mo of age using frequent blood sampling. Neither the basal maternal cortisol plasma level nor goat kid adrenal masses were affected by PCB exposure.

We recently reported on a stimulatory effect of a mixture of persistent organic pollutants (POPs) extracted from the liver oil of Atlantic cod (Gadus morhua) on estradiol (E2) secretion by porcine ovarian follicular cells [Gregoraszczuk EL, Milczarek K, Wójtowicz A, Berg V, Skaare JU, Ropstad E. Steroid secretion following exposure of ovarian follicular cells to three different natural mixtures of persistent organic pollutants (POPs). Reproductive Toxicol 2007;25:58-66]. The objective of the present study was to identify what compound or compounds within this natural mixture could be responsible for the stimulatory action on E2 secretion. Co-cultures of porcine ovarian theca and granulosa cells were exposed for 48h to individual congeners of polychlorinated biphenyls (PCBs) alone or in combination, at environmentally relevant concentrations (PCB153 at 8microg/ml; PCB118 at 3microg/ml; PCB138 at 8microg/ml; PCB180 at 3microg/ml; PCB126 at 8ng/ml; DDT at 5microg/ml; p,p'-DDE at 10microg/ml). At the end of culture media samples were collected and used for cell viability and steroid determination, and cells were used for the measurement of caspase-3 activity. A threefold decrease in E2 secretion was noted when PCB126 was added in combination with PCB153. Only DDT and DDE could reverse the action of PCB126. Stimulatory secretion of E2 occurred in parallel with a decrease in testosterone (T) secretion. Among the single congeners used in the present study, DDE had the strongest stimulatory action on E2 secretion. In conclusion, the present findings indicate that the overall effect on steroid secretion by ovarian follicular cells could be dependent on the concentration of DDE in environmental POPs and that the predicted effect of a mixture based on a sum of independent effects of individual congeners is higher than that noted under the influence of defined mixture.

Valproate (VPA) is a major antiepileptic drug with a broad spectrum of antiepileptic activity. There is, however, increasing concern about the possible effects of VPA on reproductive endocrine function. This study investigated the effects of valproate, on the endocrine and reproductive system of adolescent, non-epileptic, goat bucks. Nine goat bucks were orally treated with 62.5mg/kg valproate twice daily from 2 to 10 months of age in order to sustain therapeutic plasma concentrations of between 300 and 600 micromol/l. Seven bucks served as controls. Body weights and testicular diameters were recorded. Blood samples were collected for measurement of luteinising hormone (LH), follicle stimulating hormone (FSH) and testosterone three times weekly until sacrifice at approximately 40 weeks of age. Conventional reproductive endpoints were recorded and flow cytometric (FCM) analyses of spermatogenesis, including the sperm chromatin structure were conducted. Valproate-treated bucks had on average a higher body weight, but a lower testis diameter than controls. No significant differences were found for plasma FSH in comparison to controls. Valproate-treated bucks differed significantly from the control group by showing lower plasma concentrations of LH and testosterone and a later onset of puberty. A significantly higher proportion of sperm from valproate-treated bucks showed abnormal chromatin, demonstrating a harmful effect on DNA from valproate treatment. These results demonstrate that valproate was able to induce reproductive effects in goat bucks related to the hypothalamic-pituitary-axis, as well as to the testes.

The main objective of the present study was to describe in vitro effects of individual congeners of polybrominateddibenzoethers (PBDEs) (PBDE-47, 99, 100, 209), p,p� -dichlorodiphenyltrichloroethane (DDT) and p,p � -dichlorodiphenyldichloroethylene (DDE) present at the highest amount in real-life mixtures of POPs extracted from liver oil of burbot from Lake Mjosa in Norway, and defined combinations of these compounds on steroidogenesis in porcine ovarian follicular cells. Co-culture of theca and granulosa cells (Gc) was exposed by individual congeners of PBDEs or mixture. Media samples were collected and used for cell viability and steroid determination, while cells were used for the measurement of caspase-3 activity. All investigated PBDEs congeners increased both testosterone (T) and estradiol (E2) secretion. We noted additive stimulatory action of BDE-47 and BDE-100 on both T and E2 secretion, and then inhibitory action on both of these hormones production when additionally BDE-99 and then BDE-209 were included in this mixture. The situation was different when DDT and DDE were added to the defined mixture. Stimulatory action on E2 secretion with parallel decrease of T secretion was noted. In conclusion, the data indicated: (1) stimulatory action of individual congeners of PBDEs on both E2 and T secretion by ovarian follicular cells points to stimulatory action on enzymes responsible for testosterone secretion; (2) the overall effect of a real-life mixture on the steroidogenic response in ovarian follicular cells could be dependent on the concentration of DDTs in the mixture; (3) the predicted effect of mixture, calculated based on a sum of independent action of individual congeners, was higher than that noted under the influence of defined mixture.

This study investigated in vitro endocrine disrupting effects of three mixtures of POPs: 'Marine mix' extracted from Atlantic cod liver, and two mixtures extracted from burbot liver, 'Mjosa mix' and 'Losna mix'. The POP mixtures were chemically characterized. Co-culture of theca and granulosa cells, were exposed for 48h with different doses of 'Marine mix', 'Mjosa mix' or 'Losna mix'. As an end point cell viability was determinated by LDH test, steroid analysis by EIA and caspase-3 by colorimetric substrate. Chemical characterization of the mixtures demonstrated that the 'Marine mix' contained high levels of DDTs and PCBs. In the 'Mjosa mix', the dominant pollutants were BDEs and HBCD. The concentrations of POPs measured in the 'Losna mix' were considerably lower. All mixtures used in the present study had a stimulatory effect on testosterone and estradiol secretion with 'Marine mix'>'Mjosa mix'>'Losna mix'. These results show that even a mixture containing background concentrations of POPs significantly affected steroid secretion. A higher steroidogenic response in low dose ranges, compared with high dose ranges indicated xenobiotic-conditioning hormesis. This could complicate predictions of effects in risk assessments.

Exposure to organohalogens in endotherms has been suggested to impose chemically induced stress by affecting functions related to maintenance energy requirements. Effects on basal metabolic rate (BMR) have been suggested to be, in part, mediated through interactions with the thyroid hormones (THs). We investigated the relationships between plasma concentrations of major organochlorines, PBDEs, hydroxylated (OH)- and methoxylated (MeO)-PBDEs and OH-PCBs, circulating TH levels and BMR in breeding glaucous gulls (Larus hyperboreus) from the Norwegian Arctic. Negative associations were found between BMR and concentrations of sigma PCB, Sigma DDT and particularly Sigma chlordane, which combined made up 91% of the total contaminant burden. Levels of THs (thyroxine and triiodothyronine) were not associated significantly with variation of BMR or concentrations of any of the compounds determined. The present study suggests that BMR may be altered in glaucous gulls exposed to high loadings of persistent contaminants in the Norwegian Arctic environment.

The present study was based on data obtained from artificial inseminations (AIs) performed with cryopreserved semen from elite bulls used in the Norwegian breeding program. Semen was diluted to standardize the number of spermatozoa to 18 million per AI dose. The aim of the study was to investigate whether the net sperm concentration at semen collection and the storage period in liquid nitrogen have any effect on probability of conception in dairy cattle. We demonstrated that the natural range of sperm concentration at semen collection within some of the bulls was associated with the probability of conception. However, no primary trend among bulls was found on the effect of sperm concentration at semen collection. This appears to be due to differences among bulls in their response to the dilution ratio of seminal plasma to extender. The effect of storage time was investigated in semen that had been stored between 1000 days and 2400 days in AI straws in liquid nitrogen at the AI center. Our findings showed that use of semen with the longest storage period, i.e. 1951-2400 days, resulted in a more than one percentage point lower probability of conception than semen with a shorter storage period. In conclusion, the net sperm concentration at semen collection, which affects the dilution ratio of seminal plasma to extender, should be considered individually among bulls to achieve optimal reproductive performance. Furthermore, this study gives support to the idea that a measurable degree of damage to the spermatozoa could occur during the preservation time in liquid nitrogen.

The main objective of this study was to describe the growth patterns of 4 large dog breeds [Newfoundland (NF), Labrador retriever (LR), Leonberger (LEO), and Irish wolfhound (IW)] raised in domestic environments and concomitant changes in 2 growth-related clinical variables: total serum alkaline phosphatase (ALP) and the circumference of the distal radius and ulna (CDRU). The second objective was to investigate whether these measurements were affected by a range of independent variables like age, sex, litter number, and birth weight. Seven hundred dogs were included in the study, and BW data, separated by breed and sex, were fitted to the Gompertz function. Birth weight, adjusted for litter number, differed significantly between sexes for 3 breeds (LEO, P = 0.004; NF, P = 0.02; LR, P = 0.009) and approached significance for IW (P = 0.07). Estimated mean BW increased rapidly during the first 100 d after birth in all 4 breeds, then plateaued, with maturity being reached between 351 (female LR) and 413 d (male NF). Estimated mature BW ranged from 30.8 kg for the female LR up to 65.7 kg for the male IW. Weight gain, as expressed by the derivative of the Gompertz function, reached its peak in the smallest breed (LR) at the youngest age, 89 d for the females and 95 d for males. Log-transformed BW was significantly related to age, breed, and sex, and the age x sex and age x breed interactions. Within breeds, age, birth weight, and litter number had a significant effect on log-transformed BW. The estimated average CDRU increased from 90 d of age toward a peak at 180 d. Thereafter, CDRU declined and stabilized at about 1 yr of age. The estimated total ALP concentrations decreased from 90 to 360 d of age, after which they stabilized, at mean concentrations varying among breeds from 98 to 131 IU/L. Maximum least squares mean total ALP concentrations were found at 3 mo of age in all breeds, with the greatest least squares mean concentration in the IW breed (713 IU/L). In a mixed model analysis of the complete data set, total ALP was affected (P < 0.001) by age, breed, and the interaction of age x breed. This study described the main factors influencing growth and provided reference data for other studies, including those related to nutrition and disorders of growth.

Persistent organic pollutants (POPs) are toxic substances, highly resistant to environmental degradation, which can bio-accumulate and have long-range atmospheric transport potential (UNEP 2001). The majority of studies on endocrine disruption have focused on interferences on the sexual steroid hormones and so have overlooked disruption to glucocorticoid hormones. Here the endocrine disrupting potential of individual POPs and their mixtures has been investigated in vitro to identify any disruption to glucocorticoid nuclear receptor transcriptional activity. POP mixtures were screened for glucocorticoid receptor (GR) translocation using a GR redistribution assay (RA) on a CellInsight(TM) NXT High Content Screening (HCS) platform. A mammalian reporter gene assay (RGA) was then used to assess the individual POPs, and their mixtures, for effects on glucocorticoid nuclear receptor transactivation. POP mixtures did not induce GR translocation in the GR RA or produce an agonist response in the GR RGA. However, in the antagonist test, in the presence of cortisol, an individual POP, p,p'-dichlorodiphenyldichloroethylene (DDE), was found to decrease glucocorticoid nuclear receptor transcriptional activity to 72.5% (in comparison to the positive cortisol control). Enhanced nuclear transcriptional activity, in the presence of cortisol, was evident for the two lowest concentrations of perfluorodecanoic acid (PFOS) potassium salt (0.0147mg/ml and 0.0294mg/ml), the two highest concentrations of perfluorodecanoic acid (PFDA) (0.0025mg/ml and 0.005mg/ml) and the highest concentration of 2,2',4,4'-tetrabromodiphenyl ether (BDE 47) (0.0000858mg/ml). It is important to gain a better understanding of how POPs can interact with GRs as the disruption of glucocorticoid action is thought to contribute to complex diseases.

Summary: Purpose: Long-term valproate (VPA) treatment has been associated with reproductive endocrine disorders characterized by hyperandrogenism and polycystic changes in the ovaries in women with epilepsy. Levetiracetam (LEV) is a promising, new antiepileptic drug that may represent an alternative to VPA for many patients. Here the effect of LEV and VPA on basal and gonadotropin-stimulated steroid secretion from prepubertal porcine ovarian follicular cells was compared and the conversion of testosterone to estradiol is measured. Methods: Ovarian follicles were obtained from prepubertal pigs. Follicular theca and granulosa cells were cocultured and different concentrations of LEV or VPA added to the control or gonadotropin-stimulated cultures. Results: VPA, but not LEV, caused a significant increase of LH-stimulated testosterone secretion and decreased FSHstimulated estradiol secretion. VPA decreased conversion of testosterone to estradiol in both basal and FSH-stimulated cultures, while LEV only decreased testosterone to estradiol conversion after FSH stimulation and only at the highest, nontherapeutic drug concentration. Both drugs increased basal testosterone secretion at therapeutic drug levels. VPA also reduced basal estradiol secretion, while LEV decreased basal estradiol secretion only at nontherapeutic drug levels. Conclusion: Both LEV and VPA affect endocrine function in the prepubertal ovary. But while VPA alters both basal and gonadotropin-stimulated testosterone and estradiol secretion at therapeutic drug concentrations, LEV only affects basal hormone secretion at this concentration level. The possibility that LEV could be an alternative treatment to VPA if reproductive endocrine problems emerge in adult women, is discussed. However, extrapolation to the clinical situation is problematic and particular emphasis is placed on the need for further studies. Key Words: Levetiracetam—Valproate—Steroidogenesis—Ovary.

Plasma cortisol concentrations were determined from the blood of eight mature female reindeer (Rangifer tarandus tarandus) after an intravenous injection of either saline (control) or 100, 250 or 500 μg of synthetic ACTH. Blood samples were collected at 0, 2, 4, 6, 8, 10, 15, 20, 25, 30, 45, 60, 75, 90, 120, 150, and 180 min after the injections. The aims were to determine the appropriate dose of ACTH for adrenal stimulation tests, to define the dose level of ACTH which elicited a maximal cortisol response and to describe the range of blood cortisol concentrations for reference when evaluating the stress responses of reindeer.The mean plasma cortisol concentrations (s.e.) at the zero sample times (t0) of the control and the ACTH treatments varied between 93·4 (11·8) and 132·5 (18·1) nmol/l. The total plasma cortisol response (area under curve, AUC, nmol/l × min) increased with increasing dose of ACTH (P < 0·001). The AUC of the control treatment was significantly smaller than of the ACTH treatments (P < 0·001). The highest dose of ACTH (500 μg) gave a significantly bigger AUC than the lowest dose (100 μg) (P = 0·008). The maximal plasma cortisol concentrations (CMAX) were achieved within 60 min of the ACTH injections. The ranges of individual CMAX were 59·0 to 136·8 nmol/l for the control treatment, and 110·0 to 252·0, 152·0 to 247·5 and 135·1 to 257·1 nmol/l for 100, 250 and 500 μg ACTH, respectively. The difference in CMAX was significant between the control treatment and the ACTH treatments (P < 0·001) but not between the different doses of ACTH. The plasma cortisol concentrations at the end of the observation period at t180 were not significantly affected by the ACTH treatment (P > 0·05).In conclusion, the 100-μg dose of ACTH was sufficient to produce a significant cortisol response compared with the control treatment. Increasing the dose did not increase the maximal response, but tended to elongate the response profile. The blood sampling frequency used in the study was found suitable for detection of the cortisol response in reindeer.

In this study, female goats were orally exposed to PCB126 or PCB153, at 49 ng/kg body weight per day and 98 μg/kg body weight per day respectively, from gestational day 60 until delivery at approximately day 150. Exposure of the offspring continued via lactation until postnatal day 40. Reproductive toxicity in the male offspring was studied by the evaluation of conventional reproductive endpoints as well as flow cytometric analyses of spermatogenesis and sperm chromatin structure. PCB153-treated animals showed a significant smaller testis diameter in comparison to the control group. Neither of the treated groups showed differences for plasma FSH in comparison to controls. PCB153-treated animals differed significantly from the control group with respect to plasma LH and testosterone levels, whereas PCB126-treated animals only differed from the controls in plasma testosterone concentrations. Neither the PCB126 nor the PCB153 group differed from the controls with respect to the conventional sperm parameters or testis histology. A significant lower ratio of interstitium area to seminiferous tubules area and proportion of diploid testis cells were observed for the PCB153 group. Sperm from PCB153-treated animals showed a significantly higher percentage of sperm with damaged DNA. From the results of the present study it was concluded that PCB153 was able to induce alterations in reproductive endpoints related to the hypothalamic-pituitary-axis as well as to the testis. The effects observed in male kids after a long-term maternal exposure to PCB153 support the concept that exposure to endocrine-disrupting compounds during foetal development may lead to adverse reproductive effects in adult life.

The effects of four seasons of semen collection and of artificial insemination on conception in dairy cows were studied. The solstices and equinoxes (December, March, June and September) defined the beginning and/or end of each season. Semen was collected from 973 progeny-test bulls over 8 years at the two Norwegian AI stations at 60.8 degrees N and 63.4 degrees N where artificial light was used to provide a minimum photoperiod of 10 h/day. The effect of using semen of elite bulls during progeny testing and after selection as elite sires also was investigated. Norwegian Red (NRF) cows were inseminated over a 7-year period using progeny test semen and over the last 4 years of the same period using the semen of the elite sires. The probability of conception to only first inseminations for cows up to, and including, the fifth lactation was assessed by 56-day non-return rate (56d NRR) and calving rate. Two data sets were analysed which excluded cows culled within 270 days of AI or included such cows as non-calving. The reasons for culling were categorised as those for fertility problems or all other reasons. Semen was used for AI irrespective of the season in which it had been collected. Season of semen collection did not affect 56d NRR but calving rate was significantly higher (by 0.5-0.8%, approximately; P < 0.01) for semen collected in the December-March period, when photoperiod was increasing, than at other times of the year. The season in which AI was performed showed a peak of 56d NRR in spring for heifers (P < 0.01) and in summer for parous animals (P < 0.01). For calving rate, however, no seasonal peak was found in heifers, whereas pluriparous cows had much higher calving rates in summer and autumn/early winter than late winter and spring (P < 0.01). Semen of elite sires resulted in higher calving rates by 0.5 (NS) to 1.9% (P < 0.01) when used after selection than when used during progeny testing. The difference between the calving rate achieved when the semen from elite sires was used during progeny testing and after selection indicates that farmers select different classes of cows for submission to AI by progeny test bulls and sires. The 56d NRR was not as good as calving rate for assessing seasonal and other effects on conception rates.

The endocrinology was studied throughout pregnancy in reindeer (Rangifer tarandus tarandus) located in Oulu, Finland (65 degrees N, 25 degrees E) with 13 captive, semi domestic adult females. Blood samples were analyzed for plasma progesterone (P4), estradiol (E2) and estrone sulphate (E1SO4), 15-ketodihydro-PGF2alpha (PG-metabolite) and pregnancy associated glycoproteins (PAG). The mean plasma P4 concentration peaked twice during gestation: at around 24 and three weeks prior to calving. In pregnant females the plasma PAG concentration increased over basal concentrations 21-30 days after the estimated day of conception and peaked at the time of calving. The concentrations of E2 and E1SO4 remained low until 60 days before calving when a rapid increase was found for both hormones. The mean plasma concentration of PG-metabolite increased throughout pregnancy to a maximum at parturition. The estimated mean (range) gestation length was 216 (212-220) days. Judged from measures on reproductive organs collected from 86 free-ranging, semi-domestic female reindeer of unknown age presented for slaughter at Roros, Norway (63 degrees N, 11 degrees E) in the second week of December 1999, it was concluded that the breeding season lasted from early September until the end of November. The results also showed that plasma PAG concentration could provide a tool for detection of pregnancy in reindeer.

This study examined the effects of acute exposure to PCB99 (2,2′,4,4′,5-pentachlorobiphenyl), and PCB153 (2,2′,4,4′5,5′-hexachlorobiphenyl), on spermatogenesis in 8-week-old C57BL6 mice. The mice were randomly allocated to PCB99 and PCB153 and a single dose of respectively 10 and 100 mg/kg was given by oral gavage. During the 6-week experiment, six mice per treatment group were sacrificed weekly, body weights were recorded and samples with respect to the male reproductive system were collected until further analysis. None of the treatments, showed changes in body weight or reproductive endpoints. Flow cytometric analysis revealed spermatogenesis to be unaffected. However, PCB99 and PCB153 showed a significant increase in Leydig cell apoptosis. The results from the present study indicate that the male reproductive system is relatively refractory to PCB99 and PCB153 at levels exceeding those of wildlife and humans, when exposed during adult life. However, the finding of apoptotic Leydig cells merits further investigation.

The effects of manual blood sampling and remote blood sampling using automatic blood sampling equipment (ABSE) on plasma cortisol and catecholamine concentrations were studied on eight adult female reindeer (Rangifer tarandus tarandus). Contemporary body temperatures and heart rates were also recorded to determine their utility as other possible stress indicators. The animals were blood sampled once every hour with ABSE on 9-10 May and then by manual blood sampling on 13-14 May. Animals were also fitted with equipment to record heart rate and body temperature. Heart rate and body temperature were also recorded continuously without blood sampling on 17-18 May in undisturbed control conditions. Plasma cortisol concentrations were five-to-six fold greater during manual blood sampling compared to sampling with ABSE (F(1,3) = 13.34, P < 0.05). Plasma noradrenaline concentrations were significantly higher (F(1,3) = 22.98, P < 0.05) during manual blood sampling compared to sampling with ABSE, whereas plasma adrenaline concentrations did not differ. Heart rate was higher during manual blood sampling compared to control values. Body temperature was significantly higher during manual sampling compared to values recorded without blood sampling (F(1,4)= 31.65, P < 0.01). In conclusion, plasma cortisol concentration provides an excellent indicator of handling stress in reindeer. The use of ABSE for blood sampling enables measurements of plasma cortisol levels close to basal concentrations that may be used for reference values in studies where indicators of physiological stress are required.

Summary: Purpose: Long-term valproate (VPA) treatment has been associated with hyperandrogenism and polycystic ovaries in women with epilepsy. The exact mechanisms of action of the drug on sex steroid hormone function are still unsettled. The aim of the present study was to investigate the action of VPA on basal and gonadotropin-stimulated steroid secretion in porcine ovarian follicular cells and to measure the conversion of testosterone to estradiol. Second, the action of VPA on proliferation and apoptosis of follicular cells was investigated. Methods: Small and medium follicles were obtained from pig ovaries on days 8‐10 and 14‐16 of the estrus cycle. Both follicular compartments, theca and granulosa cells, were cultured as a coculture resembling follicles in vivo. VPA in concentrations of 100 and 250 µg/ml was added to the control or gonadotropinstimulated cultures. Results: VPA caused a significant increase in basal and luteinizing hormone (LH)-stimulated testosterone secretion from small follicles, whereas in medium follicles, an increased basal but decreased LH-stimulated testosterone secretion was found. VPA caused decreased basal and follicle-stimulating hormone (FSH)-stimulated estradiol secretion by small follicles, whereas only the higher concentration decreased estradiol secretion in medium follicles. The conversion of testosterone to estradiol by small follicles was decreased under the influence of VPA in testosterone-alone and in testosterone-plus-FSH‐stimulated cultures, whereas this was seen at only the higher VPA concentration in medium follicles. VPA had no effect on cell proliferation and viability, whereas in a dose-dependent manner, VPA increased caspase-3 activity. Conclusions: VPA affected steroidogenesis in both unstimulated and gonadotropin-stimulated porcine ovarian follicular cells and inhibited the conversion of testosterone to estradiol. In addition, VPA may act as an apoptotic agent in both small and medium-sized follicles. Key Words: Valproate—Testosterone—Estrogen—LH—FSH—Polycystic ovaries—Apoptosis—Caspase-3.

High concentrations of polychlorinated biphenyls (PCBs) in polar bears from Svalbard have increased concern for that population's reproductive health. We examined whether there were associations between the plasma concentrations of PCBs and reproductive hormones [progesterone (P4)] and 17 beta-estradiol (E2)] in free-living female polar bears from Svalbard. Concentrations of P4 depended on reproductive status, and concentrations were lowest in females with offspring--females with cubs and females with yearlings. In these females, the P4 concentrations were positively correlated with plasma sigma PCBs (sum of all analyzed polychlorinated biphenyl congeners) concentrations. The sigma PCBs concentrations explained 27% of the variation in the P4 concentrations. There were no correlations between sigma PCBs and E2 and cortisol in any of the groups of polar bears, or between sigma PCBs and P4 in single polar bears. Although the sigma PCBs-P4 relationship in female polar bears with offspring is not evidence per se of a direct cause-effect association, the results indicate that PCBs may affect levels of P4 in polar bear females. There is a clear need to further assess the hormone balance and population health of polar bears at Svalbard.

Normal sexual development and subsequent reproductive function are dependent on appropriate testosterone production and action. The regulation of steroid hormones, including androgens, can be influenced by both biological and environmental factors, including environmental chemicals. Concentrations of organochlorines are considerably greater in Svalbard polar bears than in polar bears from other regions. Between 1995 and 1998, samples were collected from 121 male polar bears (Ursus maritimus) from the Svalbard area. In this study, testosterone concentration variations were described for male polar bears during different seasons and for all age groups. To study possible relationships between plasma testosterone concentrations and biological factors, such as age, axial girth, and extractable plasma fat, and organochlorine contaminants including hexachlorocyclohexanes, hexachlorobenzene, chlordanes, p,p'-DDE, and 16 individual polychlorinated biphenyl (PCB) congeners, identical statistical analyses were performed on the total population and a subsample of reproductively active adults. Of the biological factors, axial girth showed a significant positive relationship and percentage extractable fat and a significant negative relationship with the testosterone concentrations. Both the epsilon pesticides and epsilon PCBs made significant negative contributions to the variation of the plasma testosterone concentration. The continuous presence of high concentrations of organochlorines in male polar bears throughout their life could possibly aggravate any reproductive toxicity that may have occurred during fetal and early postnatal development.

This study addressed the effect of breed on estrus length and estrous behavior by observing 20 Holstein-Friesian (HF) and 20 Norwegian Red (NRF) cows on an outdoor wood- chip pad through 1 estrous cycle (22 d). Detailed behavioral data were collected by continuous (24 h) video monitoring of all cows. Accurate estimation of duration of estrous periods, behavioral signs (sum per period and counts per hour), and duration and number of sexually active groups were reported through all stages of mount estrus (prestand, standing estrus, and poststand). These dependent variables were analyzed with a basic statistical model that included fixed effects for breed and lactation group. Other independent variables (milk yield, body condition score, and number of cows in standing estrus) were added to the basic model one by one and included in an expanded model if they had an effect on the respective dependent variables. Estrus duration was considerably shorter in HF compared with NRF cows for all the major periods: mount estrus (11.2 +/- 3.0 vs. 21.3 +/- 2.7 h), standing estrus (7.1 +/- 1.4 vs. 11.7 +/- 1.3 h), mounting period (6.9 +/- 2.7 vs. 18.2 +/- 2.4 h), and mounted period (9.2 +/- 2.8 vs. 17.5 +/- 2.6 h). Additionally, the NRF cows spent more time in sexually active groups (36.1 +/- 4.0 vs. 17.6 +/- 4.8%) during standing estrus compared with HF cows. The NRF cows participated in a greater number of sexually active groups (9.6 +/- 1.3 vs. 5.5 +/- 1.3) with longer average duration (0.42 +/- 0.04 vs. 0.20 +/- 0.04 h) and continued to be more active in these groups through late stages of estrus (poststand) compared with the HF breed. Mounting activity differed between breeds as NRF mounted more times in total (46.3 +/- 6.2 vs. 18.1 +/- 6.3) and per hour (2.6 +/- 0.4 vs. 1.5 +/- 0.5) during mount estrus. In addition, NRF tended to express the primary estrous sign, standing when mounted, more often during standing estrus (32.4 +/- 5.0 vs. 18.5 +/- 5.2). The HF initiated more unsuccessful mounts (1.6 +/- 0.3 vs. 0.6 +/- 0.3) per hour than did NRF during mount estrus. A significant effect of milk yield was demonstrated only on this behavior. For other estrous signs, HF cows initiated chase-up (2.0 +/- 0.5 vs. 0.5 +/- 0.4) and anogenital sniff (3.7 +/- 0.6 vs. 2.0 +/- 0.5) more frequently (counts per hour), whereas NRF expressed more total head butt behavior (32.3 +/- 4.7 vs. 14.2 +/- 4.8) during mount estrus. Body condition score had a significant effect on receptive behavior. Measures of estrus duration, sexually active group activity, and behavior related to estrus should be subjected to larger studies for improved heat detection and possible implementation in breeding programs.

Relationships among milk progesterone, concentrate allocation, energy balance (EB), milk yield and conception rate were studied in 146 lactations in 94 moderate yielding cows. All animals were of the dual purpose breed Norwegian cattle, and were monitored through their first and second lactations. The cows were assigned three different concentrate allocations and had free access to grass silage. Energy balance was estimated by subtracting energy required for maintenance and lactation from energy intake. Milk progesterone concentration was determined three times weekly from calving until pregnancy. The cumulative progesterone concentration was calculated as area under the progesterone curve for the first three luteal phases postpartum. The conception rate increased linearly by rising milk progesterone for values of cumulative progesterone in the lowest third of the range, whereas the likelihood of conception did not differ between milk progesterone concentrations within the upper two-thirds. This implies that the progesterone values were below a threshold value for optimal reproductive success in one-third of the services performed in this study. Milk progesterone concentrations during the third luteal phase postpartum were low when the high-energy diet was fed. Negative EB was associated with reduced values for milk progesterone during the third luteal phase in second parity cows. Likewise, milk yield was inversely related to progesterone levels during both the first and third luteal phases postpartum in second parity cows. Energy balance was higher and milk yield lower during peak lactation among second parity cows that conceived compared to cows that remained open after the first artificial insemination. The present study have demonstrated an association between likelihood of conception and the energy coverage in Norwegian cattle. This relationship is possibly mediated through progesterone deficiency.

Evidence that some of the fungal metabolites present in food and feed may act as potential endocrine disruptors is increasing. Enniatin B (ENN B) is among the emerging Fusarium mycotoxins known to contaminate cereals. In this study, the H295R and neonatal porcine Leydig cell (LC) models, and reporter gene assays (RGAs) have been used to investigate the endocrine disrupting activity of ENN B. Aspects of cell viability, cell cycle distribution, hormone production as well as the expression of key steroidogenic genes were assessed using the H295R cell model. Cell viability and hormone production levels were determined in the LC model, while cell viability and steroid hormone nuclear receptor transcriptional activity were measured using the RGAs. ENN B (0.01-100μM) was cytotoxic in the H295R and LC models used; following 48h incubation with 100μM. Flow cytometry analysis showed that ENN B exposure (0.1-25μM) led to an increased proportion of cells in the S phase at higher ENN B doses (10μM) while cells at G0/G1 phase were reduced. At the receptor level, ENN B (0.00156-15.6μM) did not appear to induce any specific (ant) agonistic responses in reporter gene assays (RGAs), however cell viability was affected at 15.6μM. Measurement of hormone levels in H295R cells revealed that the production of progesterone, testosterone and cortisol in exposed cells were reduced, but the level of estradiol was not significantly affected. There was a general reduction of estradiol and testosterone levels in exposed LC. Only the highest dose (100μM) used had a significant effect, suggesting the observed inhibitory effect is more likely associated with the cytotoxic effect observed at this dose. Gene transcription analysis in H295R cells showed that twelve of the sixteen genes were significantly modulated (p0.05) by ENN B (10μM) compared to the control. Genes HMGR, StAR, CYP11A, 3βHSD2 and CYP17 were downregulated, whereas the expression of CYP1A1, NR0B1, MC2R, CYP21, CYP11B1, CYP11B2 and CYP19 were upregulated. The reduction of hormones and modulation of genes at the lower dose (10μM) in the H295R cells suggests that adrenal endocrine toxicity is an important potential hazard.

The relationship between milk fat progesterone concentration at first artificial insemination (AI) and reproductive performance of Norwegian Red Cattle dairy cows was investigated in a field study. Fifteen AI technicians collected milk samples from 2250 cows from 458 herds and progesterone was analysed in the milk fat portion of the samples. Logistic regression revealed decreased probability of non-return, and increased likelihood of repeat-breeding, with increasing progesterone concentrations at insemination. The odds ratio for non-return and for repeat-breeding between the minimum and maximum value for milk fat concentrations between 4 and 32.2ng/ml was 4.5 (P0.01) and 3.2 (P = 0.01), respectively. The variable 'Technician' did not significantly influence non-return rate or repeat-breeding. Progesterone concentration at AI was positively related to calving to last insemination interval, and to number of inseminations per cow (P0.05). The association between the progesterone concentration at insemination and calving interval was only marginally significant. Technician was significantly (P0.01) associated with interval from calving to first AI, interval from calving to last AI (P = 0.05), and number of AI per cow (P = 0.01). Technician was not significantly related to the calving interval. We conclude that increased suprabasal progesterone concentrations at the time of first insemination, and higher return rate at AI may, at least partly, be due to endocrinological asynchrony at AI, a condition which may lead to decreased fertility.

The aim of the present study was to investigate whether long-term exposure to valproate (VPA) alters follicular steroidogenesis and whether or not this effect is dependent on the degree of follicular development. Small- and medium-sized follicles were obtained from pig ovaries collected, respectively, at days 8–10 and 14–16 of oestrus cycle. Theca interna and granulosa cells were isolated from follicles and placed in the same well in the ratio 1 : 3 with or without the VPA in doses of 100, 300 and 500 μ g ml−1. The culture medium was changed after 2, 4, 6 and 8 days. In both types of follicles, VPA caused a significant and dose-dependent reduction in both testosterone and estradiol secretion from follicular cells. In small-sized follicles, the testosterone to oestrogen ratio increased at all doses used and after all lengths of time in culture. In medium-sized follicles, a significant increase in the testosterone to oestrogen ratio was only observed at the highest dose level. All doses of VPA caused a marked inhibition of progesterone secretion after 48 hours while during long-term VPA exposure progesterone gradually increased demonstrating luteinization of cells. In conclusion, the present study demonstrates a direct effect of VPA on steroidogenesis. The effect seems to differ to some extent depending on the follicular stage of development. The elevated ratio of testosterone to estradiol suggests that VPA inhibits the conversion of testosterone to estradiol.