Histological characterization of reproductive tract and fetal annexes of the West Indian Manatee (Trichechus manatus) from Brazil / Caracterização histológica do trato reprodutor e anexos fetais do peixe-boi marinho (Trichechus manatus) do Brasil

The West Indian manatee (Trichechus manatus) is one of the most threatened aquatic mammals in Brazil, and is currently classified as "endangered" (MMA). The objective of this study was to characterize histologically the reproductive tract and fetal annexes of stranded manatees in northeastern Brazil. Tissue samples were collected from the reproductive tract of 23 manatees, which were fixed in 10% buffered formalin, processed using standard histological protocols and stained with hematoxylin eosin. We qualitatively described the histological and histomorphometric characteristics of each structure. Six ovaries were analyzed. In four ovaries, we found a large number of primordial and primary follicles. Two ovaries were different from the others: one had inflammatory infiltration and the other had a thickening in the cortex and absence of follicles. We also analyzed seven uteri (of which four were in the proliferative phase, two in the secretory phase, and one in the recovery phase), four placentas, one vagina, six testes (four were in the immature phase, one in the pubertal phase, and one in the mature phase), two epididymides, two penises, and one umbilical cord. The histological and morphometric findings in our work will support future analyses of the reproductive tract of T. manatus from Brazil.(AU)

O peixe-boi marinho (Trichechus manatus) é um dos mamíferos aquáticos mais ameaçados do Brasil e, atualmente é classificado como "em perigo" (MMA). O objetivo deste estudo foi caracterizar histologicamente o trato reprodutor e os anexos fetais de peixes-bois marinhos encalhados no Nordeste do Brasil. Foram coletadas amostras de tecidos do trato reprodutor de 23 peixes-bois marinhos (T. manatus), que foram fixadas em formol tamponado a 10%, processados usando protocolos histológicos padrão e corados com hematoxilina eosina. Foi realizada a descrição qualitativa das características histológicas e histomorfométricas de cada estrutura. Foram analisados seis ovários. Em quatro ovários, foi encontrado um grande número de folículos primordiais e primários. Dois ovários eram diferentes dos outros: um tinha infiltração inflamatória e o outro tinha um espessamento no córtex e ausência de folículos. Também foram analisadas sete amostras uterinas (das quais quatro estavam na fase proliferativa, duas na fase secretória e uma na fase de recuperação), quatro placentas, uma vagina, seis testículos (quatro na fase imatura, um na fase puberal e um na fase madura), dois epidídimos, dois pênis e um cordão umbilical. Os achados histológicos e morfométricos em nosso trabalho apoiarão futuras análises do trato reprodutivo de T. manatus do Brasil.(AU)

Regional differences of testicular artery blood flow in post pubertal and pre-pubertal dogs.

BACKGROUND: Measurement of testicular artery blood flow is used in several species to evaluate reproductive function and testicular and scrotal pathology. In dogs there are inconsistent reports about normal flow in post-pubertal dogs and no information concerning pre-pubertal dogs. The aim of this study was to describe regional differences in testicular artery blood flow in clinically normal post-pubertal and pre-pubertal dogs with no history of reproductive tract disease. RESULTS: The post-pubertal dogs produced normal ejaculates throughout the study. In all dogs the three different regions of the artery were imaged and monophasic flow with an obvious systolic peak and flow throughout diastole was observed on every occasion. The highest peak systolic velocity (PSV) and end diastolic velocity (EDV) were measured within the distal supra-testicular artery and marginal artery whilst the lowest PSV and EDV were measured within the intra-testicular arteries. Flow measurements were not different between left and right testes and were consistent between dogs on different examination days. Calculated resistance index (RI) and pulsatility index (PI) were lowest in the intra-testicular arteries. The pre-pubertal dogs had significantly smaller testes than the post-pubertal dogs (p < 0.05) and were unable to ejaculate during the study. The three different artery regions were imaged at every examination time point, and flow profiles had a similar appearance to those of the post-pubertal dogs. PSV, EDV, RI and PI showed a similar trend to the post-pubertal dogs in that values were lowest in the intra-testicular arteries. Notably, values of PSV, EDV, RI and PI were significantly lower (p < 0.05) in pre-pubertal dogs compared with post-pubertal dogs. CONCLUSIONS: This study demonstrated important regional and pubertal differences in testicular artery blood flow of dogs, and form the basis for establishing baseline reference values that may be employed for the purposes of clinical diagnosis.

Triplex Doppler evaluation of the testes in dogs of different sizes / Avaliação Triplex Doppler dos testículos de cães de tamanhos diferentes

This study aimed to assess whether there are differences in Doppler velocimetry parameters between different sizes. Twenty dogs were equally divided into small and large groups used in this study. The dogs were evaluated using Triplex ultrasound. Testicular artery was located by Colour Doppler in the spermatic cord, marginal to the testes and intratesticular segments and then, spectral Doppler were used to calculate: peak systolic velocity (PSV), end diastolic velocity (EDV), resistance index (RI) and pulsatility index (PI). The mean testicular volume in the left side was significantly higher than the right side, in both groups. Doppler examination showed higher velocities (EDV) at spermatic cord in large dogs; marginal to the testes was observed higher velocities in small dogs; intratesticular region no differences were observed (P < 0.05) and within the groups differences between segments of the artery were also observed for each parameter. The results showed that there are differences in Doppler velocimetry parameters between different sizes.(AU)

Este trabalho teve como objetivo verificar se existem diferenças nos parâmetros dopplervelocimétricos entre cães de diferentes portes. Para tanto, foram utilizados 20 cães, sendo 10 cães de pequeno porte e 10 cães de grande porte. Foram avaliados pelo Triplex Doppler para localização da artéria testicular nos segmentos de cordão espermático, marginal ao testículo e intratesticular. Após isso, o Doppler espectral foi acionado para cálculo dos parâmetros de velocidade de pico sistólico (VPS), velocidade diastólica final (VDF) e índices de resistência (IR) e pulsatilidade (IP). O volume testicular médio do testículo esquerdo foi significativamente maior que o direito em ambos os grupos. Ao Doppler, foram observadas diferenças dos valores entre os portes, sendo as velocidades no cordão espermático superiores nos animais de grande porte (P < 0,05) e dentro dos grupos também foram observadas diferenças entre as regiões da artéria testicular. Diante do exposto, conclui-se que existem diferenças nos parâmetros dopplervelocimétricos quando comparados animais de diferentes portes, além disso, os parâmetros dopplervelocimétricos são diferentes dependendo da região em que são mensurados.(AU)

Ovarian and uterine periovulatory Doppler ultrasonography in bitches / Ultrassonografia Doppler ovariana e uterina em cadelas

This paper aims to describe the uterine and ovarian ultrasonographic characteristics and Doppler velocimetric features of their arteries in bitches during the periovulatory period. Fifteen estrous cycles in 10 animals were evaluated. The ultrasonographic characteristics, resistance indices (RI) and pulsatility indices (PI) of the uterus and ovaries in each animal were recorded 5 days before and after ovulation (D0). The data were statistically analyzed, and the results were expressed as the mean ± standard error of mean (P<0.05). In results the ultrasonographic features of the uterus were the same on all of the cycles and evaluated days. The uterus had an average diameter of 0.85±0.02cm. An increase in the volume of the ovaries and the diameter of the ovarian follicles were measured. Ovaries had a volume of 0.64±0.06cm³, and the follicles cavities had a diameter of 0.46 ± 0.01 cm on the day of ovulation. After ovulation, it was observed that some follicles not collapse in some cycles. Two days prior to ovulation, the uterine blood perfusion decreased. This decrease remained unchanged until ovulation. Following ovulation, we measured a gradual increase in the uterine perfusion and in the ovarian artery. This artery directed blood flow to the ovaries and increased the intra-ovarian perfusion on the day after ovulation. In conclusion, specific features are observed in the uterus and ovarian ultrasound image and Doppler values of their arteries presented on the periovulatory days and when associated allow to estimate more accurately the date of ovulation.

Este trabalho teve como objetivo descrever as características ultrassonográficas uterinas e ovarianas, e dopplervelocimétricas das suas artérias nos dias periovulatórios em cadelas. Quinze ciclos estrais em 10 animais foram avaliados. As características ultrassonográficas, índices de resistência (IR) e índices de pulsatilidade (IP) do útero e dos ovários em cada animal foram registrados 5 dias antes e depois da ovulação (D0). Os dados foram analisados estatisticamente e os resultados foram expressos em média ± erro padrão da média (P<0,05). Como resultados as características ultrassonográficas uterinas foram semelhantes em todos os ciclos e dias avaliados, tendo o corpo uterino um diâmetro médio de 0,85±0,02cm. Foi observado um aumento no volume dos ovários e nos diâmetros dos folículos, tendo os ovários um volume de 0,64±0,06cm³, e a cavidade dos folículos um diâmetro de 0,46±0,01cm no dia da ovulação. Após a ovulação, foi observado colapso dos folículos somente em alguns ciclos. A perfusão sanguínea uterina diminuiu dois dias antes da ovulação e permaneceu inalterada até a ovulação. Após a ovulação, houve um aumento gradual na perfusão das artérias uterinas e ovarianas, direcionando o fluxo de sangue para os ovários e aumentando a perfusão da artéria intra-ovariana um dia após a ovulação. Conclui-se que características específicas são observadas na imagem ultrassonográfica uterina e ovariana, e dopplervelocimétricas de suas artérias nos dias periovulatórios, e quando associadas permitem estimar com mais precisão o dia da ovulação.

Refrigeração do epidídimo canino a 4ºC e recuperação dos espermatozoides epididimários utilizando ACP-106c / Cooling of the canine epididymis at 4ºC and recovery of epididymal sperm using ACP-106c

Objetivou-se avaliar a qualidade dos espermatozoides recuperados da cauda do epidídimo após a refrigeração do complexo testículo-epidídimo (CTE) de cães usando o diluidor ACP-106c. Foram utilizados 60 cães machos adultos, com peso de 10-20 kg. Após a eutanásia, removeu-se o CTE que foi imerso em solução fisiológica 0,9% e transportado em caixa térmica ao laboratório a 30ºC. Para a refrigeração e recuperação dos espermatozoides epididimários, os 60 pares do CTE foram divididos em 4 grupos, de acordo com o tempo de refrigeração do CTE e posterior recuperação espermática: G0h, G6h, G12h e G18h, em que cada par do CTE permaneceu por zero, seis, doze ou dezoito horas a 4ºC, respectivamente. A recuperação dos espermatozoides da cauda do epidídimo foi realizada pela técnica de flutuação utilizando-se o diluidor ACP-106c ou Tris. Para cada epidídimo foi adicionado 1,0 mL de um dos dois diluidores, pré-aquecidos a 37ºC por 5 minutos. Em seguida foram centrifugados a 800g/5 minutos para remoção dos resíduos celulares. Avaliou-se a morfologia, funcionalidade e motilidade espermática total e progressiva, além de parâmetros obtidos pelo CASA. Os dados foram submetidos à ANOVA seguido do teste de Turkey (P < 0,05). Em todos os parâmetros avaliados, não houve diferença entre os diluidores testados (P>0,05). Os valores de motilidade total nos grupos G0h, G6h, G12h, e G18h para o ACP-106c foram 84,4±7,7; 81,6±11,6; 88,3±6,5 e 69,5±16,9, respectivamente, e para o Tris 85,2±8,7; 77,4±14,3; 79,0±17,8 e 65,4±17,9, respectivamente. Um decréscimo na qualidade espermática foi observado após 18 horas de refrigeração em ambos os diluidores. Dessa forma pode-se concluir que o ACP-106c pode ser utilizado para recuperar os espermatozoides epididimários refrigerados e podem ser viáveis por até 12h de refrigeração.

The study aimed to assess the quality of sperm recovered from the epididymal cauda after cooling the complex testis-epididymis (CTE) of dogs using ACP-106c extender. Sixty adult male dogs, weighing 10-20 kg were used. After euthanasia, CTE was removed and it was immersed in 0.9% saline and transported to the laboratory in cooler at 30ºC. For cooling and recovery of epididymal spermatozoa, the 60 pairs of CTE were divided into four groups, according to the refrigeration time of the CTE and subsequent sperm recovery: G0h, G6h, G12h and G18h, wherein each pair of CTE remained zero, six, 12 or 18 hours at 4ºC respectively. The recovery of sperm epididymal tail was conducted by flotation technique using ACP-106c or Tris extender. For each epididymis, it was added 1.0 mL of either extenders, preheated to 37ºC for 5 minutes. They were then centrifuged at 800g/5 minutes to remove the cell debris. Morphology, functionality and total and progressive motility, and parameters obtained by CASA were evaluated. Data were analyzed by ANOVA followed by Turkey test (P <0.05). In all parameters assessed, there was no difference between the extenders used (P>0.05). The values of total motility groups G0h, G6H, G12H, and G18h for ACP-106c were 84.4±7.7, 81.6±11.6, 88.3±6.5 and 69.5±16.9 respectively, and for Tris 85.2±8.7, 77.4±14.3, 79.0±17.8 and 65.4±17.9 respectively. A decrease in sperm quality was observed after 18 hours of cooling in both extenders. Thus it can be concluded that the ACP-106c may be used to recover the epididymal spermatozoa chilled and may be viable for up to 12h cooling.

Intersexuality in a crab-eating fox (cerdocyon thous) / Intersexualidade em uma raposa (cerdocyon thous)

Intersexuality is a reproductive pathology that has been described in wild animals in recent years. However, its occurrence and consequences remain obscure and therefore all aspects of this reproductive disorder deserve attention. The aim of this study is to report a case of intersexuality with probable absence of gonadal tissue in the crab-eating fox (Cerdocyon thous) native to Brazil. The animal has male external genitalia, but its prepuce and penis were both hypoplastic. Because of a clinical suspicion of bilateral cryptorchidism, a laparotomy was performed and the absence of prostate and gonads were revealed. The procedure also revealed vas deferentia, extending laterally from the each side of the bladder basis to the right and left abdominal wall muscles. The animal died one month later, and post mortem examination confirmed the absence of prostatic and gonadal tissues. Muscular structures similar to uterine horns and cervix were founded macroscopically and confirmed by optic microscopy. In addition, post mortem findings corroborate with penis hypoplasia, since penile bone presence was observed. The vasa deferentia had a normal tissue structure, although hypoplastic. In conclusion, the case of a crab-eating fox (Cerdocyon thous) reported here represents a proved intersexual animal with probable absence of gonadal tissue.

A intersexualidade é uma das patologias reprodutivas, hereditária e/ou congênita, que têm sido mais reportada em animais selvagens nos últimos anos. Entretanto, suas ocorrências e conseqüências permanecem obscuras, sendo que todos os aspectos relacionados a este distúrbio reprodutivo merecem atenção. O objetivo deste estudo foi relatar um caso de intersexualidade com provável ausência de tecido gonadal em uma raposa (Cerdocyon thous) do Brasil. O animal apresentava sexo fenotípico masculino, mas seu prepúcio e pênis eram pequenos. Devido a uma suspeita clínica de criptorquidismo bilateral, foi realizada uma laparotomia, sendo, no procedimento cirúrgico, revelada a ausência de próstata e gônadas. Os ductos deferentes foram localizados lateralmente à bexiga, entendendo-se desde a sua base, inserindo-se na musculatura da parede abdominal direita e esquerda. Um mês após, o animal morreu e os achados necroscópicos confirmaram a ausência de tecidos prostático e gonadal. Entretanto, uma estrutura muscular histologicamente semelhante a cornos e cérvix uterinos foram encontradas. Além disso, pelo mesmo método, ratificou-se a ausência de próstata e o pênis exibiu aspecto normal contendo osso peniano. Os ductos deferentes apresentaram estrutura tecidual normal, porém hipoplásicos. Concluiu-se que o caso reportado no presente trabalho representa um exemplo de intersexualidade com provável ausência de tecido gonadal em um animal da espécie Cerdocyon thous.

Cryopreservation of preantral ovarian follicles in situ from domestic cats (Felis catus) using different cryoprotective agents.

The objective of this investigation was to verify the structural characteristics of preantral follicles (PAF) of cat ovarian tissue after cryopreservation in 1.5 M glycerol or ethylene glycol, using a slow-freezing procedure. Ovaries (n = 10) from domestic cats were divided into fragments. One fragment was immediately preserved for classical histology (fresh control), and additional fragments were immersed in minimum essential medium plus 10% bovine fetal serum (MEM+BFS), or MEM+BFS supplemented with 1.5 M glycerol or ethylene glycol. The samples were frozen and plunged into liquid nitrogen. After 1 wk, the samples were thawed. A total of 600 PAF were evaluated. In the fresh control, there were 71.3% normal PAF. After thawing, the rates of normal PAF were 26.0, 39.3 and 58.0% for samples without cryoprotectant or with glycerol or ethylene glycol, respectively. We concluded that ethylene glycol was useful for the cryopreservation of feline PAF in situ.

Prognostic value of canine frozen-thawed semen parameters on in vitro sperm-oocyte interactions.

Combining the data from conventional semen analysis with oocyte penetration assays should improve the assessment of the fertilizing ability of a semen sample. Thus, the objective of the present study was to evaluate the prognostic value of various semen parameters on the in vitro interactions between frozen-thawed canine sperm and homologous oocytes. Ten ejaculates from five stud dogs (two ejaculates/dog) were collected by digital manipulation. Semen samples were evaluated, extended in Tris-egg yolk-glycerol, frozen and stored in liquid nitrogen, and thawed several weeks later. Samples were evaluated for motility and sperm populations by computer-aided semen analysis (CASA), plasma membrane integrity (carboxy-fluorescein diacetate and propidium iodide), and sperm morphology (Bengal Rose). Thawed spermatozoa were also incubated with homologous oocytes for 18 h in an atmosphere of 5% CO(2) and 95% air at 38 degrees C and sperm-oocyte interactions were evaluated. Simple linear regression models were calculated, with sperm parameters as independent variables and sperm-oocyte interactions as the dependent variable. There were significant associations between: percentage of oocytes bound to spermatozoa and beat cross frequency (BCF; R(2)=63%); percentage of oocytes that interacted with spermatozoa and BCF (R(2)=73%); and number of penetrated spermatozoa and velocity average pathway (VAP; R(2)=64%) and velocity straight line (VSL; R(2)=64%). Although plasma membrane integrity and sperm morphology had little prognostic value for in vitro interactions between canine frozen-thawed sperm and homologous oocytes, some motility patterns (evaluated by CASA) were predictive of in vitro sperm-oocyte interactions.

The potential for gamete recovery from non-domestic canids and felids.

Species are becoming extinct at a rate 100 times the natural background rates. Considering all mammalian orders, 24% of all Carnivora species are threatened. The goal of carnivore conservation is to reverse the decline in populations and to secure remaining populations in ways that will assure enduring public support. In this context, biotechnology is a tool with tremendous potential for assisting the conservation of endangered canid and felid species. As the first step for biotechnology development is the gamete obtainment, this review will discuss the potential of gamete recovery from non-domestic canids and felids, based on learning how to apply these procedures in the domestic carnivores. Thus, electroejaculation and obtaining both epidydimal spermatozoon and spermatogonial germ cells are indicated as techniques for male gametes recovery. In the female gametes retrieval, different methods for oocyte recovery from both antral and preantral follicles, and the possibility for ovarian tissue transplantation are discussed. Furthermore, the study discusses the responsibilities involved in the use of assisted reproduction in endangered species conservation.