Developmental programming: Impact of preconceptional and gestational exposure to a real-life environmental chemical mixture on maternal steroid, cytokine and oxidative stress milieus in sheep.

BACKGROUND: Gestational exposure to environmental chemicals (ECs) is associated with adverse, sex-specific offspring health effects of global concern. As the maternal steroid, cytokine and oxidative stress milieus can have critical effects on pregnancy outcomes and the programming of diseases in offspring, it is important to study the impact of real-life EC exposure, i.e., chronic low levels of mixtures of ECs on these milieus. Sheep exposed to biosolids, derived from human waste, is an impactful model representing the ECs humans are exposed to in real-life. Offspring of sheep grazed on biosolids-treated pasture are characterized by reproductive and metabolic disruptions. OBJECTIVE: To determine if biosolids exposure disrupts the maternal steroid, cytokine and oxidative stress milieus, in a fetal sex-specific manner. METHODS: Ewes were maintained before mating and through gestation on pastures fertilized with biosolids (BTP), or inorganic fertilizer (Control). From maternal plasma collected mid-gestation, 19 steroids, 14 cytokines, 6 oxidative stress markers were quantified. Unpaired t-test and ANOVA were used to test for differences between control and BTP groups (n = 15/group) and between groups based on fetal sex, respectively. Correlation between the different markers was assessed by Spearman correlation. RESULTS: Concentrations of the mineralocorticoids - deoxycorticosterone, corticosterone, the glucocorticoids - deoxycortisol, cortisol, cortisone, the sex steroids - androstenedione, dehydroepiandrosterone, 16-OH-progesterone and reactive oxygen metabolites were higher in the BTP ewes compared to Controls, while the proinflammatory cytokines IL-1ß and IL-17A and anti-inflammatory IL-36RA were decreased in the BTP group. BTP ewes with a female fetus had lower levels of IP-10. DISCUSSION: These findings suggest that pre-conceptional and gestational exposure to ECs in biosolids increases steroids, reactive oxygen metabolites and disrupts cytokines in maternal circulation, likely contributors to the aberrant phenotypic outcomes seen in offspring of BTP sheep - a translationally relevant precocial model.

Developmental programming: Preconceptional and gestational exposure of sheep to a real-life environmental chemical mixture alters maternal metabolome in a fetal sex-specific manner.

BACKGROUND: Everyday, humans are exposed to a mixture of environmental chemicals some of which have endocrine and/or metabolism disrupting actions which may contribute to non-communicable diseases. The adverse health impacts of real-world chemical exposure, characterized by chronic low doses of a mixture of chemicals, are only recently emerging. Biosolids derived from human waste represent the environmental chemical mixtures humans are exposed to in real life. Prior studies in sheep have shown aberrant reproductive and metabolic phenotypes in offspring after maternal biosolids exposure. OBJECTIVE: To determine if exposure to biosolids perturbs the maternal metabolic milieu of pregnant ewes, in a fetal sex-specific manner. METHODS: Ewes were grazed on inorganic fertilizer (Control) or biosolids-treated pastures (BTP) from before mating and throughout gestation. Plasma from pregnant ewes (Control n = 15, BTP n = 15) obtained mid-gestation were analyzed by untargeted metabolomics. Metabolites were identified using Agilent MassHunter. Multivariate analyses were done using MetaboAnalyst 5.0 and confirmed using SIMCA. RESULTS: Univariate and multivariate analysis of 2301 annotated metabolites identified 193 differentially abundant metabolites (DM) between control and BTP sheep. The DM primarily belonged to the super-class of lipids and organic acids. 15-HeTrE, oleamide, methionine, CAR(3:0(OH)) and pyroglutamic acid were the top DM and have been implicated in the regulation of fetal growth and development. Fetal sex further exacerbated differences in metabolite profiles in the BTP group. The organic acids class of metabolites was abundant in animals with male fetuses. Prenol lipid, sphingolipid, glycerolipid, alkaloid, polyketide and benzenoid classes showed fetal sex-specific responses to biosolids. DISCUSSION: Our study illustrates that exposure to biosolids significantly alters the maternal metabolome in a fetal sex-specific manner. The altered metabolite profile indicates perturbations to fatty acid, arginine, branched chain amino acid and one­carbon metabolism. These factors are consistent with, and likely contribute to, the adverse phenotypic outcomes reported in the offspring.

Foetal and post-natal exposure of sheep to sewage sludge chemicals disrupts sperm production in adulthood in a subset of animals.

Exposure to ubiquitous, environmental chemicals (ECs) has been hypothesized as a cause for declining male reproductive health. Understanding the long-term effects of EC exposure on reproductive health in humans requires animal models and exposure to 'real life', environmentally relevant, mixtures during development, a life stage of particular sensitivity to ECs. The aim of this study was to evaluate the effects of in utero and post-natal exposure to environmentally relevant levels of ECs, via sewage sludge application to pasture, on the adult male sheep testis. Hormones, liver concentrations of candidate ECs and Sertoli and germ cell numbers in testes of adult rams that were exposed to ECs in sewage sludge in utero, and until weaning via maternal exposure, and post-weaning via grazing pastures fertilized with sewage sludge, were quantified. Evaluated as a single group, exposure to sludge ECs was without significant effect on most parameters. However, a more detailed study revealed that 5 of 12 sludge-exposed rams exhibited major spermatogenic abnormalities. These consisted of major reductions in germ cell numbers per testis or per Sertoli cell and more Sertoli cell-only tubules, when compared with controls, which did not show any such changes. The sludge-related spermatogenic changes in the five affected animals were significantly different from controls (p < 0.001); Sertoli cell number was unaffected. Hormone profiles and liver candidate EC concentrations were not measurably affected by exposure. We conclude that developmental exposure of male sheep to real-world mixtures of ECs can result in major reduction in germ cell numbers, indicative of impaired sperm production, in a proportion of exposed males. The individual-specific effects are presumed to reflect EC effects on a heterogeneous population in which some individuals may be more susceptible to adverse EC effects. Such effects of EC exposure in humans could have adverse consequences for sperm counts and fertility in some exposed males.

Masculinization of the distal tubular and external genitalia in female sheep with prenatal androgen exposure.

Prenatal exposure to endogenous or exogenous androgens alters the development of the female reproductive tract. Although lesions in ovaries and external genitalia of androgenized female sheep have been reported, lesions of the tubular genitalia have not. Testosterone propionate (TP) or dihydrotestosterone (DHT) was administered by intramuscular injection twice weekly to 32 ewes from 30 to 90 days of pregnancy. The ewes lambed normally. The reproductive tracts from 24 treated and 13 control postpubertal female offspring were examined at 10 months of age. The ovaries, oviducts, and uteri were grossly and histologically normal in both TP- and DHT-exposed sheep. However, in the DHT-treated sheep, the uterus connected to a misshapen, saccular vagina that opened into the urethra; in the TP-treated sheep, it ended in a blind sac. In both TP- and DHT-treated sheep, the urethra was approximately 5 times longer than that of control sheep, and it resembled a male urethra with bilateral male accessory genital glands. The urethra terminated in a fully developed penis in both TP- and DHT-treated sheep, and a scrotal sac was present (without testes). These results show that prenatal exposure of female sheep to exogenous androgens results in masculinization of the tubular and external genitalia.

Effects of exposure to environmental chemicals during pregnancy on the development of the male and female reproductive axes.

There is a large body of literature describing effects of environmental chemicals (ECs), many of them anthropogenic with endocrine-disrupting properties, on development in rodent laboratory species, some of which lead to impaired reproduction and adverse health. This literature joins extensive human epidemiological data and opportunistic wildlife findings on health effects of ECs. In contrast, the effect of endocrine disruption on foetal development and reproductive performance in domestic species is less extensively documented. This applies both to domestic farm and to companion species even though the former is critical to food production and the latter share our homes and many aspects of the modern developed human lifestyle. In domestic species, the nature of chemicals exposure in utero and their consequences for animal health and production are poorly understood. A complication in our understanding is that the pace of development, ontogeny and efficiency of foetal and maternal hepatic and placental activity differs between domestic species. In many ways, this reflects the difficulties in understanding human exposure and consequences of that exposure for the foetus and subsequent adult from epidemiological and largely rodent-based data. It is important that domestic species are included in research into endocrine disruption because of their (i) wide variety of exposure to such chemicals, (ii) greater similarity of many developmental processes to the human, (iii) economic importance and (iv) close similarities to developed world human lifestyle in companion species.

Maternal and fetal tissue accumulation of selected endocrine disrupting compounds (EDCs) following exposure to sewage sludge-treated pastures before or after conception.

Liver concentrations of selected pollutant classes were determined in groups of sheep fetuses and their dams, at 55 (Experiment 1) and 110 (Experiment 2) days of gestation (term = 145 d) following exposure, throughout their breeding lives and after mating, to pasture treated with either inorganic fertiliser (control, CC) or with sewage sludge (treated, TT). In a unique study designed to separate the respective contributions of environmental sources and mobilised tissue to the available EDC burden, in additional groups of animals, pollutant burdens at 110 days gestation were assessed following exposure to the respective treatments, either throughout their breeding lives until mating, but not thereafter (TC), or only between mating and slaughter (CT) (Experiment 3). With very few exceptions, maternal and fetal liver concentrations of diethylhexyl phthalate (DEHP) and selected polychlorinated biphenyls (PCBs), and polybrominated diphenyl ethers (PBDE) and polycyclic aromatic hydrocarbons (PAHs) were not significantly affected by sludge exposure in any group. In some cases, maternal and fetal tissue EDC concentrations were different but the differences were not consistent, and maternal and fetal concentrations of none of the classes of chemical were significantly correlated. It was not possible to identify a single chemical, or class of chemical, that may be responsible for previously observed physiological effects of exposure to sludge-treated pastures. It is concluded that exposure of sheep to pastures fertilised with sewage sludge was not associated with increased liver concentrations of EDCs, irrespective of the stage of development at which they were measured and of maternal tissue mobilisation and EDC release during gestation. Thus, retrospective measurements of EDC tissue burdens could not be used to accurately assess earlier fetal EDC insults.

Neutering affects mRNA expression levels for the LH- and GnRH-receptors in the canine urinary bladder.

Neutering a bitch increases the incidence of acquired urinary incontinence (AUI) 20-fold. Mechanistically this effect is thought to be related to altered steroid/reproductive hormone concentrations and a recent study showed that gonadotrophin releasing hormone (GnRH) analogue treatment improved continence in bitches with AUI. The aim of this study was to examine mRNA expression levels for luteinizing hormone (LH)- and GnRH-receptors in the canine bladder and the correlation between these and in vitro contractility of the bladder using age matched entire and neutered, male and female canines and canines with AUI. Biopsies from the dome of the bladder were dissected post mortem with informed owner consent. mRNA expression for LH- and GnRH-receptor was quantified by rtPCR (relative to beta-actin). Contractility was assessed (cumulative concentration response curve for carbachol) in strips of bladder muscle using standard protocols. Analysis of variance (Tukey post-test) demonstrated thet neutering was associated with significantly increased levels of expression of LH- and GnRH-receptor mRNA in both sexes (P<0.01). mRNA expression for both receptors was significantly higher in female versus male canines. Neither effect was affected by animals' age and/or weight. A significant inverse correlation (Spearman's test) was found between bladder contractility and mRNA expression for both receptors. This effect was most pronounced in canines with AUI which demonstrated the highest mRNA expression levels yet had the lowest contractility of all animals studied. This suggests that increased LH- and GnRH-receptor mRNA expression is associated with changes in bladder function that increase an animal's predisposition to develop AUI.

Variations in serum concentration of phenobarbitone in dogs receiving regular twice daily doses in relation to the times of administration.

The laboratory records of 1427 client-owned dogs on chronic phenobarbitone treatment were analysed. They were divided into two groups: the 918 dogs from which blood samples were collected at the trough, that is, within two hours before the next dose of phenobarbitone, and the 509 dogs from which samples were taken during the non-trough period. There were no significant differences between the mean serum concentrations of phenobarbitone in the trough and non-trough samples from dogs receiving doses ranging from 2 mg/kg per day to more than 10 mg/kg per day. However, the higher doses of phenobarbitone were associated with progressively lower phenobarbitone concentrations in the trough group relative to the non-trough group, and this difference was significant at doses of more than 10 mg/kg per day.

Peripubertal GnRH and testosterone co-treatment leads to increased familiarity preferences in male sheep.

Chronic gonadotropin-releasing hormone agonist (GnRHa) treatment is effective for the medical suppression of the hypothalamic-pituitary-gonadal axis in situations like central precocious puberty and gender dysphoria. However, its administration during the peripubertal period could influence normal brain development and function because GnRH receptors are expressed in brain regions that regulate emotions, cognition, motivation and memory. This study used an ovine model to determine whether chronic peripubertal GnRHa-treatment affected the developmental shift from preference of familiarity to novelty. Experimental groups included Controls and GnRHa-treated rams. To differentiate between effects of altered GnRH signaling and those associated with the loss of sex steroids, a group was also included that received testosterone replacement as well as GnRHa (GnRHa + T). Preference for a novel versus familiar object was assessed during 5-min social isolation at 8, 28 and 46 weeks of age. Approach behavior was measured as interactions with and time spent near the objects, whereas avoidance behavior was measured by time spent in the entrance zone and attempts to escape the arena via the entry point. Emotional reactivity was measured by the number of vocalizations, escape attempts and urinations. As Control and GnRHa-treated rams aged, their approach behaviors showed a shift from preference for familiarity (8 weeks) to novelty (46 weeks). In contrast, relative to the Controls the GnRHa + T rams exhibited more approach behaviors towards both objects, at 28 and 46 weeks of age and preferred familiarity at 46 weeks of age. Vocalisation rate was increased in GnRHa treated rams in late puberty (28 weeks) compared to both Control and GnRHa + T rams but this effect was not seen in young adulthood (46 weeks). These results suggest that the specific suppression of testosterone during a developmental window in late puberty may reduce emotional reactivity and hamper learning a flexible adjustment to environmental change. The results also suggest that disruption of either endogenous testosterone signalling or a synergistic action between GnRH and testosterone signalling, may delay maturation of cognitive processes (e.g. information processing) that affects the motivation of rams to approach and avoid objects.

Circadian rhythms of melatonin and behaviour in juvenile sheep in field conditions: Effects of photoperiod, environment and weaning.

Entrainment of circadian rhythms (CR) to the light dark cycle has been well described under controlled, experimental conditions. However, studies in rodents have reported that rhythms in the laboratory are not always reproduced under field conditions. The aim of this study was to characterise the CR of sheep maintained under conditions of standard UK farm animal husbandry and to investigate the effects of environmental challenges presented by season, weaning and changes in housing on CR. Male sheep (n = 9) were kept at pasture, or group housed in barns, under natural photoperiod for one year. CR in locomotor activity were monitored using accelerometry, and 24 h patterns in plasma cortisol and melatonin were measured every 4 h by ELISA. CR was measured before and after weaning, in summer and winter, and at pasture and by barn housing. Cosinor analysis revealed high amplitude, diurnal rhythms in locomotor activity that were disrupted by weaning and by barn housing. Rhythms in winter showed an interrupted night time activity pattern, but only when the sheep were kept at pasture. Cortisol and melatonin secretion followed typical circadian patterns in winter and summer. The CR of the sheep under the field conditions of this study were strikingly robust under basal conditions, but easily disrupted by environmental challenges. Interrupted patterns of activity during the long nights of wintertime, not previously reported for sheep kept in experimental conditions were recorded. Based on these findings, we propose that animals require exposure to more complex environments than the laboratory in order to exhibit their true circadian phenotype.

Distribution of galanin receptor-2 immunoreactive neurones in the ovine hypothalamus: no evidence for involvement in the control of gonadotrophin-releasing hormone secretion.

Galanin is a small neuropeptide that mediates its effects via three receptor isoforms: galanin receptor-1, galanin receptor-2 and galanin receptor-3 (Gal-R1, Gal-R2 and Gal-R3). Galanin is thought to be an important intermediate in signalling in the hypothalamic-pituitary-gonadal axis and has been widely detected in the ovine hypothalamus. The expression of galanin and Gal-R1 has been reported to fluctuate during the reproductive cycle. Although the distribution of Gal-R1 has been determined in the ovine hypothalamus, the distribution of Gal-R2 was hitherto unknown. Using immunohistological and immunofluorescence techniques, we have mapped the distribution of Gal-R2 in the ovine hypothalamus, collected during the follicular phase of the oestrous cycle and examined colocalisation of Gal-R2 with oestrogen receptor alpha (ERalpha) and gonadotrophin-releasing hormone (GnRH). Gal-R2 was expressed in several regions of the hypothalamus (supraoptic nucleus, paraventricular nucleus, ventromedial nucleus, arcuate nucleus) but not as widely expressed as Gal-R1. Areas of Gal-R2 expression overlapped with those reported for Gal-R1. We observed that, in certain defined regions of the hypothalamus, up to 50% of neurones that express Gal-R2 also express ERalpha. No neurones coexpressed Gal-R2 and GnRH. Thus, we conclude that, in follicular phase animals, this receptor plays little or no role in direct intermediary signal transmission in GnRH-mediated control of the reproductive cycle.

Postnatal exposure to octylphenol decreases semen quality in the adult ram.

The aim of this experiment was to determine if maternal exposure to octylphenol pre- and/or postnatally influenced FSH concentrations and semen quantity and quality in postpubertal rams. Rams were born to ewes that received twice-weekly s.c. injections of octylphenol equivalent to 1000microg/kg/day for one of the following periods: (1) day 70 of gestation (D70) to weaning (at 20 weeks postnatally; n=4); (2) D70 to birth (n=6); (3) birth to weaning (n=7), controls received corn oil from D70 to weaning (n=5). Rams were blood-sampled weekly and semen characteristics were evaluated at 1 year of age. Maternal exposure to octylphenol, pre- and/or postnatally did not affect FSH concentrations, semen volume, concentration, percentage live, motility or IVM/IVF characteristics. However, exposure to octylphenol from birth to weaning increased the number of morphologically abnormal sperm cells in the ejaculates of these rams.

'The effect of different genres of music on the stress levels of kennelled dogs'.

Classical music has been shown to reduce stress in kennelled dogs; however, rapid habituation of dogs to this form of auditory enrichment has also been demonstrated. The current study investigated the physiological and behavioural response of kennelled dogs (n=38) to medium-term (5days) auditory enrichment with five different genres of music including Soft Rock, Motown, Pop, Reggae and Classical, to determine whether increasing the variety of auditory stimulation reduces the level of habituation to auditory enrichment. Dogs were found to spend significantly more time lying and significantly less time standing when music was played, regardless of genre. There was no observable effect of music on barking, however, dogs were significantly (z=2.2, P<0.05) more likely to bark following cessation of auditory enrichment. Heart Rate Variability (HRV) was significantly higher, indicative of decreased stress, when dogs were played Soft Rock and Reggae, with a lesser effect observed when Motown, Pop and Classical genres were played. Relative to the silent period prior to auditory enrichment, urinary cortisol:creatanine (UCCR) values were significantly higher during Soft Rock (t=2.781, P<0.01) and the second silent control period following auditory enrichment (t=2.46, P<0.05). Despite the mixed response to different genres, the physiological and behavioural changes observed remained constant over the 5d of enrichment suggesting that the effect of habituation may be reduced by increasing the variety of auditory enrichment provided.

Do glucocorticoids predict fitness? Linking environmental conditions, corticosterone and reproductive success in the blue tit, Cyanistes caeruleus.

Glucocorticoids, including corticosterone (CORT), have been suggested to provide a physiological link between ecological conditions and fitness. Specifically, CORT, which is elevated in response to harsh conditions, is predicted to be correlated with reduced fitness. Yet, empirical studies show that CORT can be non-significantly, positively and negatively linked with fitness. Divergent environmental conditions between years or study systems may influence whether CORT is linked to fitness. To test this, we monitored free-living blue tits (Cyanistes caeruleus) during breeding over 3 years. We quantified foraging conditions during brood rearing, and examined whether they were correlated with parental baseline CORT and reproductive success. We then tested whether CORT predicted fitness. Elevated parental CORT was associated with lower temperatures, greater rainfall and lower territory-scale oak density. Whereas asynchrony with the caterpillar food peak was correlated with reduced nestling mass and fledging success, but not parental CORT. Only low temperatures were associated with both reduced nestling mass and elevated parental CORT. Despite this, parents with elevated CORT had lighter offspring in all years. Contrarily, in 2009 parental CORT was positively correlated with the number fledged. The absence of a direct link between the foraging conditions that reduce nestling quality and elevate parental CORT suggests that parental CORT may provide a holistic measure of conditions where parents are working harder to meet the demands of developing young. As the positive correlation between parental CORT and fledging success differed between years, this suggests that contrasting conditions between years can influence correlations between parental CORT and fitness. Ultimately, as CORT concentrations are intrinsically variable and linked to the prevalent conditions, studies that incorporate environmental harshness will improve our understanding of evolutionary endocrinology.

A reduction in long-term spatial memory persists after discontinuation of peripubertal GnRH agonist treatment in sheep.

Chronic gonadotropin-releasing hormone agonist (GnRHa) administration is used where suppression of hypothalamic-pituitary-gonadal axis activity is beneficial, such as steroid-dependent cancers, early onset gender dysphoria, central precocious puberty and as a reversible contraceptive in veterinary medicine. GnRH receptors, however, are expressed outside the reproductive axis, e.g. brain areas such as the hippocampus which is crucial for learning and memory processes. Previous work, using an ovine model, has demonstrated that long-term spatial memory is reduced in adult rams (45 weeks of age), following peripubertal blockade of GnRH signaling (GnRHa: goserelin acetate), and this was independent of the associated loss of gonadal steroid signaling. The current study investigated whether this effect is reversed after discontinuation of GnRHa-treatment. The results demonstrate that peripubertal GnRHa-treatment suppressed reproductive function in rams, which was restored after cessation of GnRHa-treatment at 44 weeks of age, as indicated by similar testes size (relative to body weight) in both GnRHa-Recovery and Control rams at 81 weeks of age. Rams in which GnRHa-treatment was discontinued (GnRHa-Recovery) had comparable spatial maze traverse times to Controls, during spatial orientation and learning assessments at 85 and 99 weeks of age. Former GnRHa-treatment altered how quickly the rams progressed beyond a specific point in the spatial maze at 83 and 99 weeks of age, and the direction of this effect depended on gonadal steroid exposure, i.e. GnRHa-Recovery rams progressed quicker during breeding season and slower during non-breeding season, compared to Controls. The long-term spatial memory performance of GnRHa-Recovery rams remained reduced (P<0.05, 1.5-fold slower) after discontinuation of GnRHa, compared to Controls. This result suggests that the time at which puberty normally occurs may represent a critical period of hippocampal plasticity. Perturbing normal hippocampal formation in this peripubertal period may also have long lasting effects on other brain areas and aspects of cognitive function.

Spatial memory is impaired by peripubertal GnRH agonist treatment and testosterone replacement in sheep.

Chronic gonadotropin-releasing hormone agonist (GnRHa) is used therapeutically to block activity within the reproductive axis through down-regulation of GnRH receptors within the pituitary gland. GnRH receptors are also expressed in non-reproductive tissues, including areas of the brain such as the hippocampus and amygdala. The impact of long-term GnRHa-treatment on hippocampus-dependent cognitive functions, such as spatial orientation, learning and memory, is not well studied, particularly when treatment encompasses a critical window of development such as puberty. The current study used an ovine model to assess spatial maze performance and memory of rams that were untreated (Controls), had both GnRH and testosterone signaling blocked (GnRHa-treated), or specifically had GnRH signaling blocked (GnRHa-treated with testosterone replacement) during the peripubertal period (8, 27 and 41 weeks of age). The results demonstrate that emotional reactivity during spatial tasks was compromised by the blockade of gonadal steroid signaling, as seen by the restorative effects of testosterone replacement, while traverse times remained unchanged during assessment of spatial orientation and learning. The blockade of GnRH signaling alone was associated with impaired retention of long-term spatial memory and this effect was not restored with the replacement of testosterone signaling. These results indicate that GnRH signaling is involved in the retention and recollection of spatial information, potentially via alterations to spatial reference memory, and that therapeutic medical treatments using chronic GnRHa may have effects on this aspect of cognitive function.

Timing of Maternal Exposure and Foetal Sex Determine the Effects of Low-level Chemical Mixture Exposure on the Foetal Neuroendocrine System in Sheep.

We have shown that continuous maternal exposure to the complex mixture of environmental chemicals (ECs) found in human biosolids (sewage sludge), disrupts mRNA expression of genes crucial for development and long-term regulation of hypothalamic-pituitary gonadal (HPG) function in sheep. The present study investigated whether exposure to ECs only during preconceptional period or only during pregnancy perturbed key regulatory genes within the hypothalamus and pituitary gland and whether these effects were different from chronic (life-long) exposure to biosolid ECs. The findings demonstrate that the timing and duration of maternal EC exposure influences the subsequent effects on the foetal neuroendocrine system in a sex-specific manner. Maternal exposure prior to conception, or during pregnancy only, altered the expression of key foetal neuroendocrine regulatory systems such as gonadotrophin-releasing hormone and kisspeptin to a greater extent than when maternal exposure was 'life-long'. Furthermore, hypothalamic gene expression was affected to a greater extent in males than in females and, following EC exposure, male foetuses expressed more 'female-like' mRNA levels for some key neuroendocrine genes. This is the first study to show that 'real-life' maternal exposure to low levels of a complex cocktail of chemicals prior to conception can subsequently affect the developing foetal neuroendocrine system. These findings demonstrate that the developing neuroendocrine system is sensitive to EC mixtures in a sex-dimorphic manner likely to predispose to reproductive dysfunction in later life.

Progesterone can block the preovulatory gonadotropin-releasing hormone/luteinising hormone surge in the ewe by a direct inhibitory action on oestradiol-responsive cells within the hypothalamus.

Elevated oestradiol concentrations during the follicular phase stimulate a surge in gonadotropin-releasing hormone (GnRH) and luteinising hormone (LH) concentrations, which leads to ovulation. Progesterone can block the oestradiol-induced GnRH/LH surge, but the mechanism that is involved is unclear. We examined the effect of progesterone on oestradiol-induced activation of cells within the ovine hypothalamus/preoptic area (POA) to determine: (i) in which regions progesterone acts to block the GnRH/LH surge and (ii) whether progesterone directly or indirectly prevents activation of oestradiol-responsive cells. Cellular activation was assessed by measuring the number of cells that expressed Fos (an immediate early gene). Exposure to increased oestradiol concentrations in the absence of progesterone (which normally stimulates a LH surge) did not cause any region-specific changes in hypothalamic Fos expression during the activation stage of the LH surge-induction process (Experiment 1). The same treatment significantly increased cellular activation within the POA, lateral septum (LS), and arcuate nucleus at the time of surge onset (Experiment 2). Concurrent exposure to increased oestradiol and progesterone concentrations during the activation stage of the surge-induction process (which normally blocks the LH surge) was associated with significantly reduced cellular activation within the ventromedial hypothalamus and anterior hypothalamic area, relative to the positive controls (oestradiol increment alone) and arcuate nucleus relative to the negative controls (no increment in oestradiol) during the activation stage (Experiment 1). At the time of surge onset (Experiment 2), exposure to progesterone during the activation period prevented the oestradiol-induced increase in cellular activation that occurred in the POA, LS and arcuate nucleus of the positive controls. These results demonstrated that oestradiol and progesterone induced differential region- and time-specific effects on cellular activation within the regions of the ovine brain that generate the preovulatory GnRH/LH surge. Moreover, the lack of cellular activation within the POA, LS and arcuate nucleus at the time of surge onset in animals exposed to progesterone during the activation stage is consistent with the hypothesis that progesterone can block the preovulatory surge by direct inhibition of oestradiol-induced cellular activation in these areas.

'Four Seasons' in an animal rescue centre; classical music reduces environmental stress in kennelled dogs.

On admission to rescue and rehoming centres dogs are faced with a variety of short- and long-term stressors including novelty, spatial/social restriction and increased noise levels. Animate and inanimate environmental enrichment techniques have been employed within the kennel environment in an attempt to minimise stress experienced by dogs. Previous studies have shown the potential physiological and psychological benefits of auditory stimulation, particularly classical music, within the kennel environment. This study determined the physiological/psychological changes that occur when kennelled dogs are exposed to long-term (7 days) auditory stimulation in the form of classical music through assessment of effects on heart rate variability (HRV), salivary cortisol and behaviour. The study utilised a cross over design in which two groups were exposed to two consecutive 7 day treatments; silence (control) and classical music (test). Group A was studied under silent conditions followed by 7 days of test conditions during which a fixed classical music playlist was played from 10:00-16:30 h. Group B received treatment in the reverse order. Results showed that auditory stimulation induced changes in HRV and behavioural data indicative of reduced stress levels in dogs in both groups (salivary cortisol data did not show any consistent patterns of change throughout the study). Specifically, there was a significant increase in HRV parameters such as µRR, STDRR, RMSSD, pNN50, RRTI, SD1 and SD2 and a significant decrease in µHR and LF/HF from the first day of silence (S1) to the first day of music (M1). Similarly, examination of behavioural data showed that dogs in both groups spent significantly more time sitting/lying and silent and less time standing and barking during auditory stimulation. General Regression Analysis (GRA) of the change in HRV parameters from S1 to M1 revealed that male dogs responded better to auditory stimulation relative to female. Interestingly, HRV and behavioural data collected on the seventh day of music (M2) was similar to that collected on S1 suggesting that the calming effects of music are lost within the 7 days of exposure. A small '9-Day' study was conducted in attempt to determine the time-scale in which dogs become habituated to classical music and examination of the results suggests that this occurs within as soon as the second day of exposure. The results of this study show the potential of auditory stimulation as a highly effective environmental enrichment technique for kennelled dogs. However, the results also indicate the requirement for further investigations into the way in which auditory stimulation should be incorporated within the daily kennel management regime in order to harness the full physiological and psychological benefits of music.

Does a short loop feedback mechanism for the control of luteinizing hormone secretion exist in the ewe?

It is not known whether a short loop feedback mechanism for the regulation of LH exists in sheep. This study on ovariectomized ewes investigated whether a bolus injection (10, 1, and 0.1 microg LH or 1 microg BSA; n 4) or a 3-h continuous infusion of exogenous LH (100 or 1 ng/min; n = 7) into the third ventricle through a permanent indwelling cannula could influence the activity of the GnRH pulse generator, as determined by measurement of endogenous LH secretion. To assess the potential for involvement in a LH short loop feedback system and to estimate the level of LH in the hypothalamic milieu, the concentrations of LH in the peripheral circulation, portal circulation, and third ventricle were measured during an estradiol-induced preovulatory LH surge (n = 4). Neither the bolus nor continuous administration of LH into the third ventricle had any effect on the mean interpulse interval, nadir, pulse amplitude, or circulating level of systemic LH. Furthermore, despite portal LH concentrations being more than 20-fold higher than jugular LH concentrations, LH levels in third ventricular cerebrospinal fluid remained barely detectable and did not reflect dynamic secretory events in the peripheral or hypothalamo-hypophyseal portal blood. These data demonstrate that in ewes, little pituitary LH reaches the third ventricle, and the small amount that does is unable to affect peripheral gonadotropin release. Our study suggests, therefore, that a short loop feedback system for LH does not exist in the ewe.