Rosana Nogueira de Morais

Seasonal analysis of semen characteristics, serum testosterone and fecal androgens in the ocelot (Leopardus pardalis), margay (L. wiedii) and tigrina (L. tigrinus).

Captive adult male ocelots (Leopardus pardalis, n = 3), margays (L. wiedii, n = 3) and tigrinas (L. tigrinus, n = 4) in two locations in southern Brazil were studied for 14 consecutive months to evaluate the effect of season on testicular function. Reproductive evaluations, including testicular measurements, electroejaculation and blood collection were conducted monthly. Fecal samples were collected weekly for androgen metabolite analysis to assess testicular steroidogenic activity. Ocelots had the highest number of motile spermatozoa in the ejaculate (114.7+/-15.8 x 10(6); P < 0.05), the highest percentage of morphologically normal spermatozoa (82.4+/-1.2%; P < 0.05) and the highest concentration of fecal androgens (1.71 vs. 0.14 microg/g; P < 0.05). Margays and tigrinas had lower numbers of motile spermatozoa (23.4+/-2.8 x 10(6), 74.2+/-8.9 x 10(6), respectively), lower percentages of morphologically normal spermatozoa (57.4+/-2.8, 59.2+/-3.5%, respectively), and lower fecal androgen concentrations (0.15+/-0.01, 0.23+/-0.01 microg/g, respectively). Serum testosterone concentrations were similar among the three species. Fecal androgen concentrations were not affected by season, with the exception of the ocelot where concentrations were higher (P < 0.05) in the summer. Ejaculates were collected throughout the year; however, peaks in average sperm production were observed during the summer for all species. In summary, this study has identified several species differences in male testicular traits among ocelots, margays and tigrinas. Results of longitudinal reproductive assessments suggest males of each species are capable of breeding throughout the year.

Direct Versus Indirect Measurement of Digit Ratio (2D:4D) A Critical Review of the Literature and New Data

Digit ratio (2D:4D: the relative lengths of the second and fourth digits) is widely used as a correlate of prenatal sex steroids. There are two common methods of measuring 2D:4D, the direct method and the indirect method. The modern interest in 2D:4D began 16 years ago when finger lengths were measured directly, but many studies now report 2D:4D calculated from indirectly measured fingers from photocopies or scans. However, there are concerns about the accuracy of the latter in comparison to the former. The purpose of this article was twofold: to review these concerns and to add new data to the debate. Our review shows that in 2005, directional effects in indirect 2D:4D were reported such that direct 2D:4D > indirect 2D:4D. This finding was challenged by a 2006 report that direct 2D:4D was lower (not higher) than indirect 2D:4D for male right-hand 2D:4D. Two further studies from the same group have claimed that indirect 2D:4D may be lower, higher, or comparable to direct 2D:4D. More recent comparisons of direct 2D:4D versus indirect 2D:4D and a meta-analysis of Chinese studies have replicated the finding of direct 2D:4D > indirect 2D:4D. We considered an additional sample and found significant direct 2D:4D > indirect 2D:4D for three of four ratios. The overall literature is discussed within the context of standards of research (sample size) and publishing (clarity of report). It is concluded that direct 2D:4D does tend to be greater than indirect 2D:4D. Implications for comparative studies and other aspects of research in 2D:4D are discussed.

Coadministration of active phthalates results in disruption of foetal testicular function in rats

The reproductive effects of the coadministration of di-2-(ethylhexyl) phthalate (DEHP) and di-butyl phthalate (DBP) were studied in both foetal and adult male rat offspring exposed in utero. Pregnant Wistar rats were treated by oral gavage from gestation day 13 to 21 with vehicle control, 150 mg DEHP/kg body weight (bw)/day, 100 mg DBP/kg bw/ or a combination of the two compounds (DEHP 150 + DBP 100 mg/kg bw/day). An additional group of dams received 500 mg DBP/kg bw/day. A significant decrease in foetal testicular testosterone levels was observed in animals exposed to 500 mg DBP/kg/day or the phthalate mixture. Similarly, histological analysis of the foetal testis revealed that the coadministration of DEHP and DBP was able to increase the diameter of seminiferous cords and induce gonocyte multinucleation at doses that individually had no significant effects on these variables. However, in the phthalate mixture group, no significant changes were observed in anogenital distance and nipple retention, variables that are used to indicate possible anti-androgenic effects. Also, the adult endpoints investigated, that included reproductive organ weights and the number of spermatids per testis, were unaffected by any treatment regimen. Overall, coadministration of DEHP and DBP in utero significantly reduced testicular testosterone levels and resulted in misshapen seminiferous cords and gonocyte multinucleation in rat foetal testis. Our results also confirm that these foetal endpoints seem to be the most sensitive markers of prenatal phthalate exposure.

Toxicity of artemisinin [Artemisia annua L.] in two different periods of pregnancy in Wistar rats

Artemisinin compounds are important for treating multidrug-resistant malaria; however, the possible resorption and abnormalities observed in animal reproduction studies may contraindicate artemisinin use during the first trimester. To evaluate whether artemisinin interferes with developmental outcomes at different periods of pregnancy, Wistar rats were treated by gavage with increasing doses of 7, 35 and 70 mg/kg/day from gestational day [GD] 7 to 13 or 14 to 20. Viable embryos and post-implantation losses, and progestagens and testosterone levels, were monitored in the former treatment group and pregnancy and outcomes data, post-implantation losses and male and female developmental endpoints of the offspring were evaluated in the latter treatment group. Results indicate toxicity for both periods of treatment, with lower sensitivity at later stages of pregnancy. The results showed that dosing with 35 or 75 mg/kg of artemisinin caused high percentages of post-implantation losses that correlated with a trend to lower maternal progestagens and a significant maternal testosterone decrease. These findings demonstrate that oral administration of artemisinin can adversely effect post-implantation development and pregnancy in the rat.

Testicular development evaluation in rats exposed to 60 Hz and 1 mT electromagnetic field

Society has been increasingly exposed to low‐frequency electromagnetic fields (EMF), mainly from electricity distribution networks and electro‐electronic devices. Aiming to clarify the extension of possible interactions between EMF and testicular development, this study evaluated the effects of exposure to 60 Hz and 1 mT EMF in the maturation of testicular components. Wistar rats were exposed to EMF three times per day for 30 min, between the 13th day of gestation and the 21st postnatal day. Results showed a decrease in the following parameters: tubular diameter and seminiferous tubules area; seminiferous epithelium height; total volume of seminiferous tubule; tubular lumen; seminiferous epithelium; and Leydig cells. On the other hand, an increase was observed in connective tissue cells and blood vessels volume. Plasma testosterone, Sertoli cells population, tubular length and gonadosomatic index did not change when exposed to EMF. Histomorphometric analysis showed that exposure to EMF can promote a delay in testicular development. Copyright

Evaluation of testicular degeneration induced by low-frequency electromagnetic fields

The population exposure to electromagnetic fields (EMF) has been growing in recent decades. The generation, distribution and use of electric energy can generate low‐frequency electromagnetic fields. The present study investigates the effects of EMF (60 Hz and 1 mT) on spermatogenesis of rats during different periods of maturation. Wistar rats were exposed to EMF from day 13 of gestation to postnatal day 21 or 90 in three daily applications of 30 min. Plasma testosterone concentration was not changed by EMF exposure; however, histopathological and histomorphometrical analyses of the testes showed testicular degeneration in a subset of animals exposed to EMF. The magnitude of the degenerative process varied between those individuals affected, indicating different individual sensitivity to EMF. The main alterations observed through transmission electron microscopy were highly electron‐dense mitochondria with loss of their organization and cristae. Exposure to 60 Hz and 1 mT EMF can disturb spermatogenesis and may produce subfertility or infertility. Copyright

Adrenal activity in maned wolves is higher on farmlands and park boundaries than within protected areas

In this study we measured excreted fecal corticoid metabolites (FCM) in maned wolves (Chrysocyon brachyurus) living within a protected reserve, on farmlands or in a boundary zone between the two habitats, and determined the impacts of season and reproductive status on adrenal activity. Feces were collected within a national park (n=191 samples), a park boundary zone (n=39) and on nearby farmlands (n=27), processed and analyzed by enzyme immunoassay. FCM amounts from samples collected on farmlands were higher (P<0.05) than in those collected inside the reserve and from the boundary zone. In relation to seasonality, FCM were elevated (P<0.05) in spring (September-November) when wolf pairs were raising young. We then divided the samples collected during breeding season (March-August) into cycling females and male/non-cycling females based on fecal progesterone: fecal testosterone ratio. FCM concentrations of the former collected inside the park were higher than (P<0.05) than the latter group. However, there were no differences in FCM levels between the two groups for samples collected in the boundary zone and on farmlands. Furthermore, FCM concentrations of male/non-cycling females samples collected on farmlands were 2- to 5-fold higher (P<0.05) than in counterparts collected inside the park. The consistently high FCM concentrations in samples collected on farmlands indicate that, in addition to seasonality, gender and reproductive status, anthropogenic pressures also contribute to elevating adrenal steroid for individuals living in altered habitat.

Perinatal exposure to fluoxetine via placenta and lactation inhibits the testicular development in male rat offspring

Due to the widespread use of fluoxetine to treat depression, including pregnant and nursing women, the present study aimed to investigate the effects of in utero and lactational exposure to fluoxetine in rat offspring at post natal day 22. Wistar rat dams were orally treated with fluoxetine (5, 10, and 20 mg/kg) from day 13 gestation to day 21 lactation. Exposure to 10 and 20 mg/kg fluoxetine reduced the body and testis weights. The volume of the seminiferous tubules and epithelium were also reduced following 20 mg/kg fluoxetine exposure. The length of the seminiferous tubules and the population of Sertoli cells changed in offspring exposed to fluoxetine. The amount of seminiferous tubules lacking tubular lumen was higher in rats exposed to 20 mg/kg fluoxetine. Plasma testosterone showed no significant change. In conclusion, fluoxetine exposure via the placenta and lactation may inhibit and delay testicular development, adversely affecting several testicular parameters important for the establishment of sperm production in adulthood.

Testicular testosterone: Estradiol ratio in domestic cats and its relationship to spermatogenesis and epididymal sperm morphology

The phenomenon of teratozoospermia in felids is not fully understood. In this study, we investigated the testicular androgen:estrogen balance in domestic cats and correlated these data with epididymal sperm morphology and the degree of spermatogenic activity. During spring and summer, testes and blood samples were obtained from 37 mixed-breed domestic cats (12 to 48 mo). The epididymal sperm were harvested and evaluated for sperm counts, motility, and morphology. Distal cytoplasmic droplets were not considered a defect, and samples were considered normozoospermic if they contained more than 60% normal sperm (N = 25) or teratozoospermic if they contained less than 45% normal sperm (N = 12). The testicular and serum concentrations of testosterone (T) and 17β-estradiol (E2) were determined with an enzyme immunoassay. The gonadosomatic index and epididymal sperm numbers and motility did not differ between groups. The percentage of normal sperm was higher in normozoospermic (74.3 ± 2.0, mean ± SEM) than in teratozoospermic samples (43.1 ± 1.4). The most prevalent sperm defects in the teratozoospermic group were abnormal acrosomes (9.7 ± 2.0) and bent midpieces (12.2 ± 2.0) or tails (24.0 ± 2.7) with cytoplasmic droplets. Histomorphometric data were similar between groups, although there was a lower Leydig cell nuclear volume in teratozoospermic samples. Normozoospermic samples contained a higher percentage of haploid cells and had a higher index of total spermatogenic transformation than teratozoospermic samples. Serum concentrations of T (0.5 ± 0.1 vs. 0.8 ± 0.4 ng/mL) and E2 (9.5 ± 1.2 vs. 11.4 ± 2.3 pg/mL) and testicular T concentrations (471.6 ± 65.3 vs. 313.4 ± 57.6 ng/g) were similar between groups. However, compared with normozoospermic samples, teratozoospermic samples had higher testicular E2 concentrations (8.5 ± 3.6 vs. 5.4 ± 0.5 ng/g) and a lower T:E2 ratio (31.8 ± 4.1 vs. 87.2 ± 11.6). There were significant correlations between testicular E2 values and percentages of normal sperm (r = -0.55) as well as those with primary sperm defects (r = 0.58) or abnormal acrosomes (r = 0.64). The T:E2 ratio was also correlated with meiotic index (r = 0.45) and percentage of normal sperm (r = 0.58). In conclusion, a high testicular E2 concentration and a reduced T:E2 ratio were significantly associated with higher ratios of abnormal sperm types, suggesting that the balance between androgens and estrogens is an important endocrine component in the genesis of teratozoospermia in felids.

Fluoxetine induces changes in the testicle and testosterone in adult male rats exposed via placenta and lactation.

Abstract Fluoxetine is a selective serotonin reuptake inhibitor used to treat depression in pregnant and nursing women. However, recent studies have shown adverse effects in the male reproductive system after fluoxetine treatment. Aiming to analyze the extent of damage caused by fluoxetine in the testicle and safe doses for treatment during the perinatal period, the present study analyzed the effects of in utero exposure and exposure during lactation to fluoxetine in spermatogenesis of male rat offspring in adulthood. Wistar rat dams were orally treated with fluoxetine (5, 10, and 20 mg/kg) from 13 days of gestation to lactation day 21 and their offspring were analyzed at 90 days old. Results showed a reduction in the weight of testes (16%), epididymis (28%), and seminal glands (18%) in animals exposed to fluoxetine 20 mg/kg compared to the control. Seminal gland weight was also reduced 25% and 30% in animals exposed to 5 mg/kg and 10 mg/kg fluoxetine, respectively. Body weight of animals exposed to 20 mg/kg fluoxetine was reduced from post-natal day 9 to 36 compared to controls but from the post-natal day 9 to 36 there was no statistical difference. The volume of seminiferous epithelium reduced 17% and the total volume of Leydig cells reduced 30% in the group exposed to fluoxetine at 20 mg/kg. Furthermore, Leydig cells volume reduced 29% in the 5 mg/kg group. The length of the seminiferous tubules reduced 17% and daily sperm production per testicle also reduced 18% in animals exposed to the highest dose of fluoxetine compared to controls. The individual area of Leydig cells increased 7% and plasma testosterone increased 49% in animals exposed to fluoxetine at 20 mg/kg. In conclusion, exposure to 20 mg/kg fluoxetine via the placenta and during lactation may change testosterone and testicular parameters important for sperm production and male fertility in adulthood.