Priscila Viau

Noninvasive Monitoring of Androgens in Male Amazonian Manatee (Trichechus inunguis): Biologic Validation

Abstract The Amazonian manatee (Trichechus inunguis) is endemic in the Amazonian basin and is the only exclusively fresh water sirenian. Historically hunted on a large scale, this species is now considered endangered, and studies on the reproductive physiology are critical for the improvement of reproductive management of captive and wild populations of manatees. The aim of this study was to verify the viability of androgen measurement in saliva, lacrimal, urine, and fecal samples of the Amazonian manatee by conducting a hormone challenge. Two adult male manatees (A-1 and A-2) were submitted to an experimentation protocol of 12 day (D1 to D10). On D0, the animals received an intramuscular injection of gonadotropin-releasing hormone (GnRH)-analogue. Salivary, lacrimal, urinary, and fecal samples were collected daily (between 0800 hours and 0900 hours) and frozen at −20°C until assayed. Fecal samples were lyophilized, extracted with 80% methanol, and diluted in buffer before the radioimmunoassay (RIA). Urine samples underwent acid hydrolysis and were diluted in depleted bovine serum. Salivary and lacrimal samples were assayed without the extraction step. Hormonal assays were conducted with a commercial testosterone RIA kit. An androgen peak (>median + 2 interquartile range [IQR]) was observed in all matrices of both animals, although it was less prominent in the lacrimal samples of A-2. However, the fecal androgen peak (A-1 peak  =  293.78 ng/g dry feces, median [IQR]  =  143.58 [32.38] ng/g dry feces; A-2 peak  =  686.72 ng/g dry feces, median [IQR]  =  243.82 [193.16] ng/g dry feces) occurred later than urinary (A-1 peak  =  648.16 ng/mg creatinine [Cr], median [IQR]  =  23.88 [30.44] ng/mg Cr; A-2 peak  =  370.44 ng/mg Cr, median [IQR]  =  113.87 [117.73] ng/mg Cr) and salivary (A-1 peak  =  678.89 pg/ml, median [IQR]  =  103.69 [119.86] pg/ml; A-2 peak  =  733.71 pg/ml, median [IQR]  =  262.92 [211.44] pg/ml) androgen peaks. These intervals appear to be correlated with the long digesta passage time in this species. The salivary and urinary peaks were closely associated. These results demonstrate that androgen concentrations in saliva, urine, or feces samples reflect reliably physiologic events and are a powerful tool for noninvasive reproductive monitoring of Amazonian manatees.

Effect of a new device for sustained progesterone release on the progesterone concentration, ovarian follicular diameter, time of ovulation and pregnancy rate of ewes

This study evaluated the effectiveness of a new progesterone intravaginal device (DPR) in ewes through four experiments: Experiment 1 compared the circulating progesterone concentration of ovariectomized ewes that received either a new or a re-used DPR. Experiment 2 compared the progesterone concentration between DPR-estrous-synchronized ewes and naturally estrous-cycling ewes. Experiment 3 evaluated the effect of new and re-used DPRs on ovarian follicular dynamics and time of ovulation of estrous cycling ewes. Experiment 4 compared the pregnancy rate after the use of a DPR and Controlled Internal Drug Releasing Device (CIDR). The mean concentration of progesterone released by the DPR device during its first use (New Group: 5.1 ± 0.5 ng/ml) was greater than that during the second use (Re-used Group: 2.4 ± 0.3 ng/ml). There was no difference between the animals that received DPR devices for first and second use in terms of ovulatory follicle diameter, follicular wave emergence day for ovulatory follicle and period of ovulatory wave of ovarian follicular development. However, there was a significant difference between groups regarding the time between DPR device removal and first ovulation (New Group: 71.7 ± 2.5h and Re-Used Group: 63.9 ± 2.7h). Pregnancy rates were similar between ewes with DPR and CIDR devices. It was concluded that DPR is effective in increasing and maintaining progesterone concentrations, controlling follicular dynamics, promoting synchronized times of ovulation from healthy follicles, promoting development of a competent corpus luteum and when used results in pregnancy rates similar to that with use of the CIDR.

Validação laboratorial e fisiológica de conjunto comercial para a quantificação de corticóides fecais em chimpanzé (Pan troglodytes) e orangotango (Pongo pygmaeus), cativos e submetidos a enriquecimentos ambientais

Neste trabalho foi realizado estudo comparativo dos niveis de corticoides fecais (CF) de chimpanze (Pan troglodytes) e orangotango (Pongo pygmaeus). Foram analisadas amostras coletadas em duas fases distintas, relacionadas com a introducao de tecnicas de enriquecimento ambiental, a saber: Base (antes da introducao) e Habituacao (imediatamente apos). Realizamos as validacoes do conjunto comercial para radioimunoensaio ImmunuChemTM Double Antibody Corticosterone da MP Biomedicals, para mensuracao de CF. A validacao laboratorial dos conjuntos diagnosticos para uso em extrato fecal de primatas foi realizada pelo metodo de paralelismo, no qual, para cada especie, concentracoes conhecidas de corticosterona foram adicionadas a um pool de extratos fecais, sendo estas amostras analisadas em seguida. As inclinacoes das curvas obtidas nestes ensaios e da curva padrao do ensaio foram entao comparadas. Os resultados obtidos para chimpanze e orangotango, foram respectivamente, Y= 17,23+1,31*X;R^2=0,98 e Y=11,14+1,29*X; R^2=0,99. Para a validacao fisiologica, foi utilizada a introducao de tecnicas de enriquecimento ambiental como causador de aumento dos niveis de CF, consequentes a inducao de resposta do tipo estresse. Os resultados foram expressos em medias e erros-padrao da media. As concentracoes medias destes corticoides foram: chimpanzes: Base (5,90 +/-2,41x10³ ng/g de fezes), Habituacao (14,92 +/- 4,66x10³ ng/g de fezes) e para o orangotango: Base (91,1 +/- 30,0x10³ ng/g de fezes), Habituacao (185,1 +/- 57x10³ng/g de fezes). Houve diferenca significativa (P<0,05) para os valores destes CF para ambas as especies entre as duas fases estudadas.

Seasonal variation in urinary and salivary reproductive hormone levels in Amazonian manatees (Trichechus inunguis)

The Amazonian manatee (Trichechus inunguis) is a threatened aquatic mammal endemic to the Amazon basin. The aim of this study was to evaluate the urinary and salivary reproductive hormone levels of captive Amazonian manatees collected during two seasons of the year. Salivary samples from four males and urinary and salivary samples from three females were collected during two seasons (March-June and September-November) over two consecutive years. Salivary testosterone in males was measured by radioimmunoassay and reproductive hormones in females (salivary progesterone and oestradiol and urinary progestogens, oestrogens and luteinising hormone) were measured by enzyme immunoassay. The data were analysed in a 2×2 factorial design, where the factors were year and season. There was no effect of year or season for salivary testosterone. All female hormones showed a seasonal effect (higher hormone levels during March-June than September-November) or an interaction between year and season (P<0.05). These results strongly indicate the existence of reproductive seasonality in Amazonian manatees; however, apparently only females exhibit reproductive quiescence during the non-breeding season. Further long-term studies are necessary to elucidate which environmental parameters are related to reproductive seasonality in T. inunguis and how this species responds physiologically to those stimuli.

Vitamin C supplementation has no effect on American bullfrog's immune response.

American bullfrog (Lithobates catesbeianus) is the only species raised in Brazilian commercial frog farms, and the intensive culture of these animals has gained great popularity in Brazil over the past several years. Stress is one of the major obstacles in frog culture. To minimise this problem, the aim of the present study was to investigate the role of added vitamin C in the diet of American bullfrogs as an antistress factor through the assessment of plasma corticosterone, leucocyte levels and macrophage activation in animals raised in confinement. The experimental design was entirely randomised, with six treatments (supplementation of 0, 250, 500, 750, 1000 and 2000 mg of vitamin C/kg of feed) and four replications. The plasma corticosterone level, leucocyte level and macrophage activation were evaluated. It was concluded that vitamin C had no influence on the evaluated parameters due to the possible adaptation of the animals to life in captivity (domestication). The results of this research indicate that farmers should not increase the levels of vitamin C in commercial feeds because this would only enhance production costs.