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.

First attempt to monitor luteinizing hormone and reproductive steroids in urine samples of the Amazonian manatee (Trichechus inunguis).

The aims of this study were to validate an enzyme immunoassay (EIA) for the measurement of luteinizing hormone (LH) in urine samples of Amazonian manatees (Trichechus inunguis; Mammalia: Sirenia) and to monitor urinary LH and reproductive steroids during the ovarian cycle in this species. Urine samples were collected from two captive males following a hormonal challenge with a gonadotropin-releasing hormone (GnRH) analogue. The urinary LH results from hormonal challenge were compared with urinary androgens for the purpose of EIA validation. Furthermore, urine samples were collected daily, over a 12-wk period, from two captive adult females, for 2 consecutive yr. The urinary LH pattern from females was compared with the patterns of urinary progestagens and estrogen conjugates throughout the ovarian cycle. An LH peak was observed in both male Amazonian manatees after the hormonal challenge, occurring prior to or together with peak androgen levels. In the females, the ovarian cycle ranged from 40 to 48 days (mean of 43.7 days). Two distinct peaks of estrogen conjugates were observed across all cycles analyzed, and the urinary LH peaks observed were accompanied by peaks of urinary estrogen conjugates. The EIA was validated as a method for the quantification of urinary LH from Amazonian manatees, as it was able to detect variations in the levels of LH in urine samples. These results suggest that T. inunguis exhibits a peculiar hormonal pattern during the ovarian cycle. Therefore, further studies are desirable and necessary to clarify the relationship between this hormonal pattern and morphological changes, as well as mating behavior, in Amazonian manatee.

Function of Cryopreserved Cat Ovarian Tissue after Autotransplantation.

The aim of this study was to assess a slow-freezing protocol of cat ovarian tissue cryopreservation using autotransplantation. Four adult queens were ovariohysterectomized and the ovaries were fragmented and cryopreserved. After one week, the grafts were thawed and autografted to the subcutaneous tissue of the dorsal neck of each queen, then randomly removed after 7, 14, 28, 49, and 63 days after transplantation. Percentages of morphologically normal primordial and growing follicles (MNFs) were 88% and 97%, respectively, in fresh tissue samples (fresh controls), and 74% and 100%, respectively, immediately after thawing (cryo D0). No MNFs were found after 49 days of transplantation. In both fresh control and cryo D0 fragments, granulosa cells were frequently in proliferation. Two morphologically normal antral follicles were detected in one queen on Day 28 post-transplantation. Connective tissue fibers increased, suggesting replacement of active ovarian cortex by fibrous tissue. Tissue vascularization was observed at 7 days after grafting, and wide blood vessels were clearly visible on Days 49 and 63. In conclusion, although follicular survival was low after cryopreservation and grafting of cat ovarian tissue, follicles were able to develop up to the antral stage, which is an encouraging outcome.

Restoration of fresh cat ovarian tissue function by autografting to subcutaneous tissue: A pilot study.

Ovarian tissue transplantation could be a valuable technique for the preservation of endangered animals. The domestic cat affords an adequate experimental model for studies aimed at wild felids due to its phylogenetic similarity. Thus, this pilot study evaluated the efficacy of cat ovarian tissue autotransplantation to a peripheral site. Three adult queens were submitted to ovariohysterectomy. The ovaries were fragmented into eight pieces; two were fixed as a control and six were transplanted to subcutaneous tissue of the dorsal neck. Grafts were monitored weekly by ultrasound and fecal samples collected daily in order to monitor estradiol levels. Grafts were recovered on Days: 7, 14, 28, 49 and 63 post-transplantation for histological analyses. One graft was maintained in one animal for 8 months. A total of 2466 ovarian follicles were analyzed: 1406 primordial and 1060 growing follicles. All animals presented antral follicles in one or more of the grafts. The percentage of morphologically normal primordial follicles was always higher than 80%, except for Day 7 transplants. Although the proportion of growing follicles increased after transplantation, there was a general decrease in the percentage of morphologically normal growing follicles from Day 7 onwards. All animals demonstrated at least three estradiol peaks during the 63-day period, and one animal exhibited estrous behaviour on three occasions. Hormonal peaks directly correlated with the visualization of antral follicles (by ultrasound and/or histology) and the observation of estrous behaviour. Long-term results on one female showed the concentration of 37.8 pg/mL of serum estradiol on Day 233 post-grafting and the female exhibited estrous behaviour on several occasions. This graft showed one antral follicle, one luteinized follicle and two preantral follicles. In conclusion, cat ovary autotransplantation to the subcutaneous tissue restored ovarian function, with hormone production and antral follicle development, over both short and long term periods. This could be a valuable technique in the evaluation of ovarian cryopreservation methods in felids. Once the technique is shown successful, it may be applied in allografts or xenografts between different feline species.

Morphology and Ultrastructure of the Amazon River Dolphin (Inia geoffrensis) Spermatozoa.

The spermatozoa from seven adult Amazon river dolphins (Inia geoffrensis, CETACEA: INIIDAE) were analyzed by light and electron microscopy. The spermatozoa showed an elongated ellipsoid shaped head and a long tail with a well distinguishable midpiece. The head spermatozoa have a smooth surface like other odontocetes examined, with the exception of the Delphinidae family. The mean dimensions of the spermatozoa were within the range already reported for other cetaceans. The spermatozoa midpiece, as in other cetaceans, showed a random pattern of mitochondria, different from that described for other mammals. Further studies of sperm morphology of a wider spectrum of cetacean families could help to better understand the reproductive biology of these animals and the intergeneric and intrageneric relationships among them, as well as, among other mammals. Anat Rec, 300:1519-1523, 2017. © 2017 Wiley Periodicals, Inc.

Endocrine monitoring of the ovarian cycle in captive female Amazonian manatees (Trichechus inunguis).

The Amazonian manatee (Trichechus inunguis; Mammalia: Sirenia), a threatened aquatic mammal endemic to the Amazon basin, is the only sirenian that lives exclusively in fresh water. Information about the reproductive endocrinology of the Amazonian manatee is scarce; therefore, the aim of this study was to monitor salivary progesterone and estradiol patterns during the ovarian cycle in T. inunguis. Salivary samples were collected daily during a 12-week period of two consecutive years from two captive adult females. The salivary estradiol and progesterone were measured by enzyme immunoassay. The results were analyzed in an iterative process of excluding values that were higher than the mean plus 2 standard deviations until the basal values were determined. The interval between two peaks of salivary estradiol followed by a rise of progesterone was considered as one complete cycle for the calculation of the cycle length. We observed only three complete cycles in all samples analyzed. The cycle length ranged from 42 to 48 days (mean of 44.67 days). We also observed two distinct salivary estradiol peaks during all cycles analyzed, with the first peak occurring before the rise in salivary progesterone and the second occurred followed by a return to basal progesterone levels. This is the first in-depth study of the ovarian cycle in Amazonian manatees. Our results demonstrate that salivary samples can be a useful tool in the endocrine monitoring of this species and suggest that T. inunguis shows a peculiar hormonal pattern during the ovarian cycle, a finding that may have physiological and ecological significance in the reproductive strategy of these animals.