Reproductive toxicity following in utero and lactational exposure to a human-relevant phthalate mixture in rats.

This rodent (Wistar rats) study examined reproductive effects of in utero/lactational exposure to a mixture of 6 antiandrogenic phthalates (PMix): diisobutyl phthalate, di-n-butyl phthalate, diisopentyl phthalate, butylbenzyl phthalate, di-2-ethylhexyl phthalate, and diisononyl phthalate. The PMix was defined based on exposure data from pregnant women in Brazil. Experimental groups were established by extrapolating the estimated human dose to rats (0.1 mg/kg/day), followed by up to 3 additional doses corresponding to 5, 1000, and 5000 times the starting rat dose: 0 (control), 0.1, 0.5, 100, and 500 mg/kg/day. The fetal experiment assessed gestational exposure effects on fetal gonads, whereas the postnatal experiment evaluated reproductive parameters in males and females after in utero and lactational exposure. Prenatal exposure decreased fetal testicular testosterone production at 0.5 and 500 mg/kg/day. PMix 500 also reduced mRNA expression of steroidogenesis-related genes, upregulated transcript expression of the retinoic acid-degrading enzyme Cyp26b1, and increased multinucleated gonocytes incidence in fetal testes. Postnatal assessment revealed antiandrogenic effects at the highest dose, including reduced anogenital distance, nipple retention, and decreased weight of reproductive organs. Early puberty onset (preputial separation) was observed at the lowest dose in males. In contrast, females did not show significant changes in fetal and adult endpoints. Overall, the PMix recapitulated early and late male rat phthalate syndrome phenotypes at the highest dose, but also induced some subtle changes at lower doses, which warrant confirmation and mechanistic assessments. Our data support the use of epidemiologically defined mixtures for exposure risk assessments over traditional toxicological approaches.

Time-Budget and Welfare Indicators of Stabled Horses in Three Different Stall Architectures: A Cross-Sectional Study.

Keeping horses in single stalls can lead to the development of abnormal and stereotypic behaviors (ASB). Opportunities for social interactions and stall architecture can influence behavior. The current study aimed to identify how three different stall architectures influenced time-budget and physiological parameters in horses. Stall types included: (1) 3.2 × 3.7 m with tactile contact between horses (B1); (2) 2.6 × 3.5 m with visual contact between horses and outside view (B2); (3) 2.3 × 3.4 m with visual contact and outside view (B3). Ten horses from B1 and B3, and nine from B2 were randomly selected, filmed for 24 hours and the video was analyzed with continuous behavioral sampling. Nine horses from B1, 8 from B2 and 7 from B3 were randomly selected for blood sampling used to determine cortisol levels, cortisol circadian rhythm (CCR), white blood cells (WBC) and neutrophil/lymphocyte ratio. The effects of different stall architectures were analyzed by ANOVA and post hoc Tukey test. B1 had higher social interaction time (2.77% of the time-budget) (P = .020), with no other differences. Time spent eating represented a low proportion of horses' time-budget (14.31%) and all horses demonstrated ASB behaviors (21.10% of the time-budget). Twenty-five percent of the horses presented low WBC, 38% of the horses had high cortisol levels, and 29% of the horses had an altered CCR. Those alterations along with high prevalence of ASB indicate that horses were in a state of chronic stress. The stalls' architecture did not affect the presence of abnormal behaviors or indicators of stress.

Effects of Different Hay Feeders, Availability of Roughage on Abnormal Behaviors and Cortisol Circadian Rhythm in Horses Kept in Dry Lots.

Free choice forage could be the best option regarding horses' welfare but can lead to increased body weight (BW), and waste of hay. Automatic box feeders (BF) and slow feeders (SF) decrease food waste, but it is unknown how these affect the horses' time-budget (TB). This study compared the effects of feeding free choice hay (FC), to a SF and an automated BF on the horses' cortisol circadian rhythm (CCR) and behavior by 24-hours continuous behavioral sampling (CBS). The study was designed as a 3 × 3 Latin square design with 15 polo horses divided into 3 groups, for 15 days on each treatment. Every 15 days, BW was assessed, blood collected for CCR analysis, the behavior recorded during the last 24 hours of the last day of each treatment and the video analyzed with CBS. Time spent on all behaviors was evaluated and used for the determination of the animals' TB. The effects of the different feeders were analyzed with ANOVA. FC horses consumed and wasted more hay daily (16.6 ± 0.5kg) (P < .001), compared with BF (10.4 ± 0.5 kg), and SF (9.30 ± 0.45 kg). FC horses had higher weight gain (P < .001, 23.5 ± 4.6kg), compared to BF (1.2 ± 5.7 kg) and SF (0.37 ± 4.6) kg. FC and SF horses spent more than 50% of the TB foraging, generating a TB similar to grazing horses. BF horses spent less time eating (P < .001), increasing time spent standing, sniffing the ground, and practicing coprophagy (P < .050). BF horses showed the highest aggression (P < .043). CCR was not different among treatments.

The Analgesic Dipyrone Affects Pregnancy Outcomes and Endocrine-Sensitive Endpoints in Female and Male Offspring Rats.

Dipyrone is an analgesic and antipyretic drug commonly used in many countries. Although generally not recommended during pregnancy, it is known that many women use dipyrone during the gestational period. In this study, we investigated the endocrine and reproductive effects of dipyrone in female and male offspring rats exposed in utero from gestational days 10-21. Pregnant rats were treated with dipyrone at 25, 75, and 225 mg/kg/day via oral gavage. Developmental landmarks-anogenital index (AGI), number of nipples, vaginal opening, first estrus, and preputial separation-were evaluated in the offspring. Reproductive parameters, including estrous cycle regularity, daily sperm production, weight and histopathology of reproductive organs, steroid hormone levels, and gene expression of selected markers of reproductive function were assessed at adulthood. At the highest dose, dipyrone induced a significant increase in postimplantation losses/fetal death and delayed parturition in dams. Offspring exposed in utero to the highest dose also exhibited significant changes in some early life markers of endocrine disruption, in particular increased AGI in females, indicating a proandrogenic effect, and increased rate of retained nipples in males, indicating an antiandrogenic response. No changes were observed in markers of puberty onset or reproductive parameters at adulthood. These results suggest that exposure to therapeutically relevant doses of dipyrone may induce mild endocrine disruptive effects that can be detected in late pregnancy and early life. Such effects may be relevant considering dipyrone use by pregnant women and the possibility of coexposures with other endocrine disruptors.

The endocrine disrupting effects of sodium arsenite in the rat testis is not mediated through macrophage activation.

Arsenic (As) is an endocrine disrupting chemical that can disturb the male reproductive system. In a previous study, it was suggested that testicular macrophages could display a role in endocrine disruption induced by As exposure. This work aimed to evaluate the effects of chronic As exposure in the testis function of Wistar rats and examine the participation of macrophage activation and inflammatory response in these processes. We examined gene expression of steroidogenic machinery and immunological markers by RT-QPCR, plasma testosterone concentrations, sperm count and morphology, and histomorphometrical parameters after 60-days exposure to 1 or 5 mg.kg-1.day-1 of sodium arsenite, combined or not with 50 µg.kg-1 of LPS administered one day before euthanasia. We have demonstrated that As exposure reduced the weight of androgen-dependent organs and induced changes in spermatogenesis, in particular at the highest dose. LPS and As co-exposure promoted a decrease in testosterone synthesis, but did not increase the overexpression of markers of macrophage activation seen in LPS-only rats. Our results suggest that As does not alter the testicular macrophage function, but under immunological challenges LPS and As can display a complex interaction, which could lead to endocrine disruption.

Prenatal diclofenac exposure delays pubertal development and induces behavioral changes in rats.

Diclofenac is a non-steroidal anti-inflammatory drug widely used by the general population and, although generally contraindicated during pregnancy, it is also used by some pregnant women. This study investigated endocrine, reproductive and behavioral effects of diclofenac in male and female offspring rats exposed in utero from gestational days 10-20. Pregnant rats were treated with diclofenac at doses of 0.2, 1 and 5 mg/kg/day via oral gavage. Anogenital distance (AGD), number of nipples, and developmental landmarks of puberty onset - vaginal opening (VO), first estrus (FE) and preputial separation (PPS) - were evaluated in the offspring. At adulthood, behavioral and reproductive parameters were assessed. Male and female rats were tested in the elevated plus maze test to assess locomotor activity and anxiety-like behaviors, while male rats were also evaluated in the partner preference test. No significant effects were observed on AGD and number of nipples in both males and females. Diclofenac treatment induced an overall delay in developmental landmarks of puberty onset in male and female offspring, which reached statistical significance for PPS at the lowest diclofenac dose. Prenatal exposure to all tested doses abolished the preference of male rats for an estrous female, suggesting an impairment of brain masculinization. No changes were observed on male or female reproductive parameters at adulthood. Overall, our results indicate that prenatal exposure to therapeutically relevant doses of diclofenac may have an impact in the pubertal development of rats and negatively affect male partner preference behavior.

First assessment of hoary fox (Lycalopex vetulus) seasonal ovarian cyclicity by non-invasive hormonal monitoring technique.

Reproduction is key to species survival, and reproductive physiology represents a high priority investigative area for conservation biology, as it provides a basic understanding of critical life-history traits, information that is helpful for the establishment of management strategies. Here, we generated knowledge about the reproductive endocrinology of the hoary fox (Lycalopex vetulus), a small canid (2.5-4 kg) endemic to open areas of the Brazilian Cerrado and listed in the Brazilian National List of Endangered species. Specifically, we utilized non-invasive hormone monitoring methods to assess oestrogen and progestagen metabolites from eight female hoary foxes housed in five zoological institutions in the state of São Paulo-Brazil. We observed the elevations of oestrogen and progestagen metabolites between July and September in six of the eight females. No significant evidence of ovarian activity was observed during other months. Two females, who shared the same enclosure, did not show a pattern of reproductive cyclicity. Based on these characteristics, we concluded that captive hoary foxes are seasonal monoestric, with the beginning of the oestrus cycle occurring mainly in July followed by 2 months of the luteal phase when conception does not occur. We suggest the dosage of faecal metabolites of estradiol and progesterone could be used to differentiate the reproductive period from a non-reproductive period in Lycalopex vetulus females, providing relevant information about their reproductive biology that may contribute to species conservation and management strategies, such as increased ex situ reproductive success.

Evaluation of Mn exposure in the male reproductive system and its relationship with reproductive dysfunction in mice.

Manganese (Mn) is essential for animal development and homeostasis. However, anthropogenic activities increase the concentration of Mn in the environment and lead to increased risk of exposure to high doses of the metal. Thus, this study aimed to evaluate the effect of high doses of Mn on the male reproductive system of swiss mice. The 22-day old mice were randomly sorted into four groups and exposed to 0 (control), 15, 30 and 60 mg of MnCl2/kg/day, via daily gavages for 45 days. After the exposure, the mice were euthanized and sperm, hormonal and oxidative stress endpoints were evaluated in the testis, seminal vesicle and hypothalamus. Exposure to Mn promoted weight reduction of androgen-dependent organs, as well as alteration of the levels of fecal androgenic metabolites. Sperm parameters were drastically affected in all treated groups and the antioxidants tested (catalase and glutathione-disulfide reductase activities, and non-protein thiols content) decreased in the testis. However, only a few endpoints were altered in the seminal vesicle. For the hypothalamus, there was a reduction in acetylcholinesterase activity, suggesting a neurotoxic potential of Mn. In conclusion, Mn may affect the hypothalamic-gonadal axis by impairing the development of androgen-dependent organs, testicular redox status and Leydig cell maturation.

Effects of silymarin on angiogenesis and oxidative stress in streptozotocin-induced diabetes in mice.

The present study evaluated the effects of acute treatment with silymarin, an extract that is obtained from Silybum marianum, on angiogenesis, oxidative stress, and inflammation in normoglycemic and diabetic mice. Diabetes was induced by streptozotocin (80 mg/kg, intraperitoneal) in male Swiss mice, 6 weeks of age. A polyether-polyurethane sponge was surgically implanted in the back of the mice as a model of healing in both diabetic and normoglycemic animals that were treated with oral silymarin or water for 10 days. The pancreas, liver, kidneys, blood, and sponges were collected and analyzed. Diabetes led to impairments of antioxidant defenses, reflected by a reduction of pancreatic superoxide dismutase and hepatic and renal catalase and an increase in pancreatic lipoperoxidation. An inflammatory process was observed in diabetic mice, reflected by an increase in pancreatic tumor necrosis factor α (TNF-α) and the infiltration of inflammatory cells in islets. The number of vessels was lower in the implanted sponges in diabetic mice. Silymarin treatment attenuated this damage, restoring antioxidant enzymes and reducing pancreatic TNF-α and inflammatory infiltration. However, silymarin treatment did not restore angiogenesis or glycemia. In conclusion, treatment with silymarin red uced oxidative stress and inflammation that were induced in the model of streptozotocin-induced diabetes in several organs, without apparent toxicity. Silymarin may be a promising drug for controlling diabetic complications.

Supplementation with Pfaffia glomerata (Sprengel) Pedersen does not affect androgenic-anabolic parameters in male rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Paffia spp (Amaranthacea) has a widespread use of in Brazil as a possible hormonal supplement and a substitute of Panax ginseng, although information on its reproductive effects is missing. AIM OF THE STUDY: To evaluated possible anabolic-androgenic or anti-androgenic effects of Pfaffia glomerata (PG) extract using intact eight-months-old male rats and pre-pubertal castrated rats. MATERIALS AND METHODS: Three different dose levels of PG (8.5, 30 and 85 mg/kg/day) were administered to eight-months-old rats for 28 days or to castrated males for 7 days (Hershberger assay). In the experiment with intact animals, 24h fecal samples were collected for quantification of fecal metabolites of androgens throughout treatment. At the end of the treatment period, animals were euthanized for evaluation of serum testosterone, reproductive organ weights, number of spermatids per testis, diameter of seminiferous tubules and cross-sectional area of soleus muscle fibers. In the Hershberber assay, androgenic or anti-androgenic effects were evaluated by the weights of androgen-dependent tissues: ventral prostate, seminal vesicle, glans penis and levator ani muscle/bulbocavernosus muscle. RESULTS: No effects were observed in the concentrations of fecal metabolites of androgens monitored during the treatment of intact eight-months-old rats. Moreover, at the end of treatment, no changes were seen in any of the investigated parameters. In the Hershberger assay, the PG extract did not induce androgenic or anti-androgenic effects at the dose levels tested. Significant effects were only observed in animals treated with testosterone and testosterone plus flutamide, which were used as positive controls for androgenicity and anti-androgenicity, respectively. CONCLUSIONS: At the dose levels tested, PG extract does not induce anabolic-androgenic or anti-androgenic effects in rats.

Validation of a fecal glucocorticoid metabolite assay for collared peccaries (Pecari tajacu).

The possibility of assessing endogenous adrenal activity in the collared peccary (Pecari tajacu) was tested by using an adrenocorticotropic hormone (ACTH) challenge in a fecal glucocorticoid metabolite (FGM) assay. Feces were collected from 12 captive adult male peccaries beginning 48 hr prior to challenge; six of these animals received the challenge as an ACTH injection and the other six were injected with saline solution. Feces collection ended 120 hr after injections. As a control, feces were collected for eight consecutive days from another six adult male peccaries that remained in their original mixed-sex herds in semiconfined paddocks. All feces samples were freeze-dried, extracted by an ethanol vortex method, and assayed for glucocorticoids by means of an enzyme immunoassay. FGM concentrations were compared between the treatments by a repeated measures analysis of variance (ANOVA) followed by a post hoc Tukey test. The assay is reliable but, instead of the usual proportion of 1:50 in ethanol (fecal mass:solvent), 1:10 is recommended for best extraction of FGM. Baseline FGM concentrations were similar among the ACTH, saline, and control treatments (29.7 +/- 11.2 ng/g(-1) dry feces) during the 48 hr before the challenge. The ACTH group reached an FGM excretion peak at 24 hr post-treatment, followed by a decline, while in the control and saline groups FGM levels remained relatively constant. Therefore, the fecal glucocorticoid metabolite assay reflects endogenous adrenal activity in the collared peccary and is a powerful tool for noninvasive stress monitoring in peccaries.

Annual pattern of fecal corticoid excretion in captive Red-tailed parrots (Amazona brasiliensis).

Annual patterns of fecal corticoid excretion were analyzed in the threatened Red-tailed parrot (Amazona brasiliensis) in captivity. Corticoid concentration over the 15 months of the study (mean +/- standard error, 12.6 +/- 0.32 ng g(-1), n = 585) was lowest around May (the southern Fall), and greatest around September (late winter), just prior to their normal breeding period. Corticoid excretion follows a seasonal pattern best explained by reproductive cycles rather than climate, although climate may be involved in the timing of corticoid excretion. Fecal corticoids also show promise as a tool to measure stress levels. We demonstrate that fecal corticoid measurement is a simple, yet efficient method for monitoring adrenocortical activity in captive, and perhaps wild, parrots. Monitoring adrenocortical activity can inform researchers about imposed stress in captivity, whether pair-bonds are forming in captive birds, and of the timing of breeding both in captivity and in nature.