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Vitrification is essential for successful tissue cryopreservation and biobanking in wild cats. This study aimed to compare different methods of vitrification (Ovarian Tissue Cryosystem-OTC, Straws-STW, and Solid Surface vitrification-SSV) for testicular fragment vitrification in tom cats. Testicular fragments were recovered from five adult tom cats and subjected to equilibrium vitrification using different cryovials and methods under the same conditions of vitrification solutions and cryoprotectants. The efficiencies of the methods were evaluated using histological analysis of spermatogonia and Sertoli cell nuclei, seminiferous tubular basement membrane detachment, and the gonadal epithelium shrinkage score scale. Cell viability was assessed using Hoechst PI and Terminal deoxynucleotidyl transferase nick end labeling (TUNEL) assay. The results showed that OTC is an effective vitrification method for maintaining the distinction between spermatogonia and Sertoli cells. OTC was similar to the control for basal membrane detachment parameters (p = 0.05). Epithelial shrinkage was low in the SSV group, which showed the highest percentage of viable cells among the vitrified groups (p = 0.0023). The OTC and SSV vitrification methods were statistically similar in terms of the percentage of TUNEL-positive cells (p = 0.05). Therefore, OTC and SSV provide favorable conditions for maintaining viable cat testicular tissue cells after vitrification.

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ABSTRACT: The present study aimed to analyze the casuistry of xenarthrans treated at the veterinary hospital of the Federal University of Pará to quantify the most important cases for clinical care and identify the main species, sex, age group, origin, and destination of the animals admitted. The data were collected from January 2013 to August 2020. The variables analyzed were the number of animals by species, sex, age group, weight (kilograms), form of admission (rescue or guardianship), person responsible for the admission of the animal, origin, circumstances of admission, length of stay (days), and evolution or destination of the animal (release, captivity, death, euthanasia, or escape). Fisher's exact test was used to determine whether disease incidence was related to sex and whether animal mortality was influenced by sex and disease. The chi-square test was used to determine whether mortality differed by age group. Ninety-two (92) xenarthrans were included: 66 Bradypus variegatus, 10 Choloepus didactylus, 15 Tamandua tetradactyla, and one Dasypus novemcinctus. The animals were mainly from the municipality of Castanhal (70%) and were acquired through rescue (88%, B. variegatus; 80%, C. didactylus; 100%, T. tetradactyla), a significant proportion of which was per natural persons (88%, B. variegatus; 80%, C. didactylus; 100%, T. tetradactyla). The most important circumstances that led to the admission of the animals were as follows: animals for examination (42%, n=39), young animals at risk (9%, n=8), hit by a vehicle (8%, n=7), respiratory disease (7%, n=6), fall from a tree (5%, n=5), and electrotrauma (5%, n=5). Release was the most common destination of animals after hospitalization (59% B. variegatus, 50% C. didactylus, and 66% T. tetradactyla). The sex of the animals did not affect the incidence of disease or mortality in any species. In contrast, mortality of B. variegatus was significantly higher in animals admitted with disease (p=0.000). Mortality in cubs of this species was higher than that in juveniles and adults (p=0.003).

RESUMO: O objetivo deste trabalho foi analisar a casuística dos Xenarthras atendidos no Hospital Veterinário da Universidade Federal do Pará (UFPA), a fim de quantificar as principais ocorrências para atendimento clínico, identificar as principais espécies, o sexo, a faixa etária, a procedência e destinação dos animais recebidos. Os dados foram obtidos entre janeiro de 2013 a agosto de 2020. As variáveis analisadas foram número de animais por espécie, sexo, faixa etária, peso (quilogramas), forma de recebimento (resgate, tutela), responsável pelo encaminhamento do animal ao hospital, procedência, circunstância de admissão, tempo de internação (dias) e evolução ou destinação do animal recebido (soltura, cativeiro, morte, eutanásia, fuga). O teste de Fisher foi utilizado para avaliar se a ocorrência de doenças estava relacionada ao sexo, bem como se a mortalidade animal foi influenciada pelo sexo e por enfermidades. O teste Qui-quadrado foi utilizado para avaliar se a mortalidade diferiu em relação à faixa etária. Um total de 92 animais Xenarthras foi admitido, composto por 66 Bradypus variegatus, 10 Choloepus didactylus, 15 Tamandua tetradactyla e um Dasypus novemcinctus. Os animais foram procedentes principalmente do município de Castanhal (70%), sendo o resgate a principal forma de aquisição dos animais (88%, B. Variegatus; 80%, C. Didactylus; 100%, T. tetradactyla), realizado em maior parte por pessoa física (88%, B. Variegatus; 80%, C. Didactylus; 100%, T. tetradactyla). O sexo dos animais não influenciou a ocorrência de doenças ou mortalidade em nenhuma espécie. Por outro lado, a mortalidade de B. variegatus foi significativamente maior naqueles animais admitidos com alguma enfermidade (p=0,000). A mortalidade em filhotes desta espécie foi maior quando comparada com as categorias jovem e adulta (p=0,003).

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This study aimed to describe the viability of domestic feline spermatozoa after epididymal tail vitrification. For this, 10 pairs of testis-epididymis complexes were used. The epididymal tails were vitrified using the solid-surface vitrification (SSV) method, in which two vitrification media containing ethylene glycol (EG) 40% or glycerol (GLY) 40% were tested. Vitrification with the presence of EG resulted in better results for all sperm motility parameters (motility, vigour and CASA) compared with GLY (P < 0.05). There were no statistical differences for sperm viability and acrosome integrity, plasma membrane integrity, or overall health of morphologically normal sperm before or after vitrification among experimental groups. In conclusion, epididymal tail vitrification appears to be a suitable method for long-term storage of cat sperm, especially if the procedure is performed with EG as the cryoprotectant.

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Saimiri collinsi is used as an animal model in biotechnology research for conservation of species from the genus Saimiri. However, the development of biotechnologies depends on a proper knowledge of the sperm morphology to understand the basic aspects of sperm physiology, as potential male fertility depends on different cellular sperm structures. With this purpose, this study characterized the micromorphological and ultrastructural characteristics of squirrel monkeys (Saimiri collinsi) sperm using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). SEM electromyography revealed that a normal Saimiri collinsi sperm measures 71.7 ± 0.7 µm with lateral tail insertion, a paddle-shaped flattened head and an acrosome occupying most of the head. TEM also showed that the middle piece is characterized by a central 9 + 2 microtubule axoneme surrounded by nine dense fibres, and that the mitochondria were juxtaposed, forming the mitochondrial sheath. Here we provide the first micromorphological and ultrastructure description of S. collinsi sperm.

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The establishment of protocols for the control of the ovarian function of collared peccaries is recommended for the development of assisted reproductive techniques. The goals were to (1) compare a gonadotropin combination with prostaglandin analogue to synchronize timing of onset of estrus among animals, and (2) elucidate the effects of the most desirable protocol for performing an artificial insemination study and macroscopic evaluation of the ovaries. Three of five females treated with a double administration of 120 µg prostaglandin (cloprostenol) at a 9-day interval expressed symptoms of estrus 9 days after the second injection. One female presented estrus after 6 days, whereas other did not respond to the treatment. All females (5/5) treated with a single dose containing 400 IU eCG and 200 IU hCG manifested estrus 6 days after the hormone injection. In a second experiment, ten females that were estrous synchronized using eCG/hCG, were artificially inseminated with fresh semen and monitored for pregnancy every 30 days. Although there was no detection of fetuses by ultrasonic examination, seven females (7/10) had greater than basal progesterone values for 60 days after the treatments were imposed. Ovaries from two females treated with eCG/hCG were collected 6 days post-injection. There was confirmation of an ovarian stimulation as a result of the presence of 88 and 25 antral follicles, as well as three and eight hemorrhagic structures in ovaries of each female, respectively. It, therefore, is proposed that eCG/hCG can be used as an effective treatment for estrous synchronization in collared peccaries.

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Bone fractures in birds are usually diagnosed with the aid of traditional radiography. However, this technique remains limited because of the difficulties associating this examination with real-time procedures. The aim of this study was to describe the use of B-mode ultrasound to assess the long bones of two avian orders. For the study, we used carcases of birds from the orders Falconiformes (n = 9) and Strigiformes (n = 12), with weights ranging from 108 to 1020 g. An ultrasound device with a 5-12 MHz linear probe was employed to produce images of the long bones (humerus, radius, ulna, femur and tibiotarsus). Ultrasound (US) measurements and physical measurements using a caliper were applied to compare the diameter of the bones. Images were also recorded from the US examination performed in two live patients attending the hospital with suspected bone fractures. No statistical difference was found between the two methods of measurement in carcases weighing up to 267 g (P > 0.01). The US examination provided relevant clinical information about the bone cortex and assisted in real-time surgical procedures. RESEARCH HIGHLIGHTS Long bones of Falconiformes and Strigiformes birds were assessed with B-mode ultrasound. Ultrasound analysis is a relevant tool in clinical orthopaedics for avian species. Ultrasound of the bone might be applied for monitoring of healing processes.

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We aimed to evaluate the effect of three extracellular cryoprotectants on the morphology of vitrified feline preantral follicles. Feline ovarian fragments (0.5 × 2 × 2 mm) collected from five domestic adult cats subjected to ovariohysterectomy for routine castration were vitrified with ethylene glycol (EG) 40% combined or not with sucrose (0.1 or 0.5 M), trehalose (0.1 or 0.5 M), or raffinose (0.1 M). After vitrification using the solid-surface method and warming of the tissues, cryoprotectants were washed out of the ovarian tissues, which were fixed for histological analysis. The percentages of normal follicles were similar to the control (fresh) (62.9 ± 4.1%) only for tissues exposed and cryopreserved with EG + trehalose at concentrations of 0.1 (35.8 ± 8.3%) and 0.5 M (33.4 ± 5.4%). All the other sugars decreased the percentages of morphologically normal follicles as compared to the control group and the trehalose groups. Based on the results of the present study, we recommend the use of trehalose as the extracellular cryoprotectant for the vitrification of feline ovarian tissue.

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The aim of the present study was to evaluate the development of fresh and vitrified agouti ovarian tissue after xenografting to C57Bl/6 severe combined immunodeficiency (SCID) female mice. Ovaries were obtained from five female agoutis and divided into 16 fragments. Five fragments were transplanted immediately to ovariectomised SCID mice and the others were vitrified, stored for 2 weeks and transplanted only after rewarming. Tissue fragments were transplanted under the kidney capsule in recipients. The return of ovarian activity in recipients was monitored by the observation of external signs of oestrus and vaginal cytology over a period of 40 days after transplantation, after which the grafts were removed and evaluated for morphology, cell proliferation and the occurrence of DNA fragmentation. Ovarian activity returned in four of five mice that received fresh ovarian tissue from agoutis and in one of six mice that had received vitrified tissue a mean (±s.e.m.) 20.6±8.6 days after xenotransplantation. After graft removal, a predominance of primordial and primary follicles was observed in all grafts. Vitrification reduced cell proliferation and increased the occurrence of DNA fragmentation in grafted agouti ovarian tissue. In conclusion, the present study demonstrates that xenografted agouti ovarian tissue, fresh or vitrified, is able to promote the return of ovarian activity in ovariectomised SCID C57B1/6 mice. However, improvements to vitrification protocols for agouti ovarian tissue are necessary.

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We describe morphological and morphometrical characteristics of preantral ovarian follicles from three recently recognized Saimiri species: S. macrodon, S. cassiquiarensis and S. vanzolinii; the last one a threatened species. Ovaries from four adult monkeys were evaluated: one pair from a pregnant S. macrodon, two ovarian pairs from S. cassiquiarensis females (one of them pregnant), and one left ovary from a senile S. vanzolinii, applying classical histology. Follicular preantral population was quantified and morphology and morphometry of primordial, primary and secondary follicles were evaluated. Follicular preantral population varied among species, being 347,153 in the ovaries of the S. macrodon, 270,342 and 278,376 in the ovaries of both adult non-pregnant and pregnant S. cassiquiarensis females, and 28,149 in the ovary from a senile S. vanzolinii. Most follicles were at primordial or transition stages, except for the senile S. vanzolinii female, which presented the lowest percentages of primordial and transition follicles when compared with primary and secondary ones. Most preantral follicles (>70%) were morphologically normal in the ovaries from all studied S. macrodon and S. cassiquiarensis females, but the ovary of the senile S. vanzolinii female presented a significant decrease in the percentage of normal follicles (primordial: 61%, transition: 52%, primary: 54%, and secondary: 48%). In general, follicular diameter increased significantly from primordial to transition, and subsequently from primary to secondary follicles.

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Ovarian agenesis is an unusual anomaly with traumatic or congenital origin. In the present case report, we describe our findings in a senile S. vanzolinii female. As this neotropical primate species is listed as vulnerable, with limited geographic distribution in the Brazilian Amazonia, ovarian agenesis may be an important finding to be reported.

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