Reproductive biology, morphology, and morphometry of ovaries and oviducts of Trachemys scripta elegans in Brazilian Cerrado / Biologia reprodutiva, morfologia e morfometriade ovários e ovidutos de Trachemys scripta elegans no Cerrado Brasileiro

Although Trachemys scripta elegans is an exotic species popular as a pet in Brazil, studies on reproductive biology and capacity are non-existent in the Brazilian Cerrado. This study analyzed ovarian and oviduct characteristics and the egg production capacity of T. scripta elegans grown in this biome. The findings will associate with the size of the specimens and the sexual maturity, aiming at comparisons with native and exotic populations, as well as interspecific and contributing to the understanding of its impact on the invaded ecosystems and the establishment of eradication programs. Thus, 39 females had evaluated the body biometry and the morphology and morphometry of the ovaries and oviducts. G2 (N=20): with Class I (>5-10mm) follicles, with Class I and Class II (>10-fold) follicles, 25mm) and G3 (N=9) with Class I, Class II and Class III (>25mm) follicles. Analysis of variance, Scott-Knott's test, and Pearson's correlation analysis showed that there was no significant difference between the groups in body biometry; in the mean gonadosomatic index and gonadal morphometry, only the width of the oviducts in the right antimer and the mass and width in the left antimer were higher in G3, the only one that presented eggs. There was positive and harmonic development between body mass, carapace, and plastron, and gonadal growth occurred concomitantly with body growth, indicating a higher reproductive potential and a positive relationship between the size of the litter and the female litter. The gonadosomatic index proved to be an excellent reproductive indicator, and the ovarian evaluation was a better indicator of sexual maturity than the maximum carapace length. Ovaries were irregular structures, without delimitation between the cortical and medullary regions and filled with vitelogenic follicles of different diameters, atresic follicles, and corpora lutea, which reflected the ovarian complexity of the species and the presence of follicular hierarchy. In the scarce stroma, two germinative beds were observed per ovary and the presence of gaps very close to the follicles and associated with the blood vessels. Analysis of gonadal tissue revealed three types of oocytes according to cytoplasmic characteristics: homogeneous, vesicular or vesicular in the cortex with apparent granules. Oviducts were functional and separated, joining only in the final portion to form the cloaca and subdivided into infundibulum, tuba, isthmus, uterus, and vagina. The structure of the uterine tube was composed of serosa, muscular and mucous, which was full of glands. The presence of eggs in the oviducts indicated that the specimens can reproduce in the Brazilian Cerrado. This study provides necessary and relevant information on the reproductive biology and capacity of T. scripta elegans in the Brazilian Cerrado and can contribute to the understanding of its impact on the invaded ecosystems and the establishment of eradication programs. The extraction of females with capacity can reduce the annual reproductive yield of the species and decrease its effect on local biodiversity.(AU)

Embora Trachemys scripta elegans seja uma espécie exótica popular como animal de estimação no Brasil, estudos sobre biologia e capacidade reprodutivas são inexistentes no Cerrado brasileiro. Este estudo analisou características ovarianas e do oviduto e a capacidade de produção de ovos em T. scripta elegans criadas neste bioma, correlacionando estes achados ao tamanho dos espécimes e a maturidade sexual, visando comparações com populações nativas e exóticas, bem como interespecíficas e contribuir para a compreensão de seu impacto nos ecossistemas invadidos e com o estabelecimento de programas de erradicação. Assim, 39 fêmeas tiveram avaliadas a biometria corporal e a morfologia e morfometria dos ovários e ovidutos. De acordo com o tamanho dos folículos ovarianos as fêmeas foram separadas em G1 (N= 10): com folículos Classe I (>5-10 mm), G2 (N= 20): com folículos Classe I e Classe II (>10-25 mm) e G3 (N= 9) com folículos Classe I, Classe II e Classe III (>25 mm). À análise de variância, teste de Scott-Knott e à análise de correlação de Pearson verificou-se que não houve diferença significativa entre os grupos na biometria corporal; no índice gonadossomático médio e na morfometria gonadal, apenas a largura dos ovidutos no antímero direito e a massa e a largura no antímero esquerdo foram maiores no G3, o único que apresentou ovos. Houve desenvolvimento positivo e harmônico entre massa corporal, carapaça e plastrão e o crescimento gonadal ocorreu concomitante ao crescimento corporal, indicando maior potencial reprodutivo e relação positiva entre o tamanho da ninhada de ovos e o da fêmea. O índice gonadossomático mostrou-se um bom indicador reprodutivo e a avaliação ovariana um melhor indicador da maturidade sexual que o comprimento máximo da carapaça. Ovários foram estruturas irregulares, sem delimitação entre a região cortical e medular e repletos de folículos vitelogênicos de diferentes diâmetros, folículos atrésicos e corpos lúteos, que refletiram a complexidade ovariana da espécie e a presença de hierarquia folicular. No estroma escasso foram observados dois leitos germinativos por ovário e a presença de lacunas muito próximas aos folículos e associadas aos vasos sanguíneos. A análise do tecido gonadal revelou três tipos de oócitos de acordo com as características do citoplasma: homogêneo, vesicular ou vesicular no córtex com grânulos aparentes. Ovidutos eram funcionais e separados, unindo-se apenas na porção final para formar a cloaca e subdividiam-se em infundíbulo, tuba uterina, istmo, útero e vagina. A estrutura da tuba uterina era constituída de serosa, muscular e mucosa, a qual era repleta de glândulas. A presença de ovos nos ovidutos indicou que os espécimes podem se reproduzir no cerrado brasileiro. Este estudo fornece informações básicas e relevantes da biologia e capacidade reprodutivas de T. scripta elegans no Cerrado brasileiro e pode contribuir com a compreensão de seu impacto nos ecossistemas invadidos e com o estabelecimento de programas de erradicação, uma vez que a extração de fêmeas com capacidade reprodutiva pode contribuir com a diminuição do rendimento reprodutivo anual da espécie e diminuir seu efeito sobre a biodiversidade local.(AU)

Effect of somatotropin on survival and diameter of bovine preantral follicles / Efeito da somatotropina na sobrevivência e no diâmetro de folículos pré-antrais de bovinos

The aim of this study was to evaluate the effect of Recombinant bovine somatotropin (rbST) on survival and diameter of bovine preantral ovarian follicles (PAOF) cultured in vitro. Ovaries were collected from adult cows and fragments of ovarian cortex were immediately fixed (non-cultured control) or cultured in vitro in α-MEM+ alone or containing 10, 50, 100 or 1,000ng/mL rbST. The fragments were processed for Classical Histology and Transmission Electron Microscopy. After one and seven days of culture, the percentage of normal follicles in the non-cultured control was superior (P< 0.05) to the follicles cultured in α-MEM+ alone or with different rbST concentrations. The oocyte and follicular mean diameter did not increase during the culture for one and seven days, both in media containing rbST and in the medium without this hormone. The only medium in which there was no reduction in follicular diameter with the time of culture was the medium without rbST. Ultrastructural damage in PAOF cultured in vitro was found. It is concluded that the use of rbST at different concentrations in in situ culture of bovine preantral follicles has no beneficial effects on survival and growth of bovine PAOF.(AU)

O objetivo deste trabalho foi avaliar o efeito da somatotropina recombinante bovina (rbST) sobre a sobrevivência e o diâmetro de folículos ovarianos pré-antrais (FOPA) bovinos cultivados in vitro. Ovários foram coletados de vacas adultas e fragmentos do córtex ovariano foram imediatamente fixados (controle não cultivado) ou cultivados in vitro em α-MEM + sozinho ou contendo 10, 50, 100 ou 1.000ng/mL de rbST. Os fragmentos foram processados para histologia clássica e microscopia eletrônica de transmissão. Após um e sete dias de cultivo, o percentual de folículos normais no controle não cultivado foi superior (P<0,05) aos cultivados em α-MEM + sozinho ou acrescido de diferentes concentrações de rbST. Os diâmetros médios oocitário e folicular não aumentaram durante o cultivo por um e sete dias, tanto nos meios contendo rbST, como no meio sem esse hormônio (α-MEM + ). O único meio em que não houve redução no diâmetro folicular com o tempo de cultivo foi o sem rbST. Verificaram-se ainda danos ultraestruturais em FOPA cultivados in vitro. Conclui-se que o uso de rbST em diferentes concentrações no cultivo in situ de folículos pré-antrais bovinos não tem efeitos benéficos na sobrevivência e no crescimento de FOPA bovinos.(AU)

First characterization of preantral follicles in the ovaries of adult alpaca / Primera caracterización de folículos preantrales de ovario de alpaca adulta

It is a fact that while even basic reproductive information on alpacas is unavailable, the normal ovarian reserve of this species in comparison to other species is also unidentified. In this study, the ovarian preantral follicles in healthy adult alpacas were characterized in order to establish a general model to in vitro studies. Ten ovaries were collected from five adult alpacas. The ovarian cortex samples were fixed with paraformaldehyde and histological analysis was done. Normal and degenerated follicles percentages were determined. The normal follicles were measured and classified in primordial, transitional, primary and secondary stages. Most of the preantral follicles present in the ovarian cortex of alpacas were primordial and transitional stages; primary (6.10%) and secondary (0.37%) follicles were rarely found. The primary and secondary follicles were larger in diameter when compared with the primordial and transitional follicles. The largest oocyte diameter was recorded in the secondary follicles (P < 0.05). This study serves to establish a biological model for future reproduction studies in Alpacas or as possible biological model for studies of folliculogenesis in humans(AU)

Es un hecho que, si bien no se dispone de información reproductiva básica sobre las alpacas, la reserva ovárica normal de esta especie en comparación con otras especies tampoco está identificada. En este estudio, se caracterizaron los folículos preantrales ováricos en alpacas adultas sanas. Se recogieron diez ovarios de cinco alpacas adultas. Las muestras de la corteza ovárica se fijaron con paraformaldehído y se realizó un análisis histológico. Se determinaron los porcentajes de folículos normales y degenerados. Los folículos normales se midieron y clasificaron en estadios: primordiales, de transición, primarios y secundarios. La mayoría de los folículos preantrales presentes en la corteza ovárica de las alpacas eran estadios primordiales y de transición; Raras veces se encontraron folículos primarios (6.10%) y secundarios (0.37%). Los folículos primarios y secundarios tenían un diámetro mayor en comparación con los folículos primordiales y de transición. El mayor diámetro de ovocitos se registró en los folículos secundarios (P <0.05). Este estudio sirve para establecer un modelo biológico para futuros estudios de reproducción en alpacas o como posible modelo biológico para estudios de foliculogénesis en humanos(AU)

Growth and Differentiation Factor-9 Supplementation Affects Viability and Morphology of Preantral Follicles in Equine Ovarian Fragments During Short-term in vitro Culture

Abstract This study aimed to evaluate different concentrations of growth and differentiation factor-9 (GDF-9) on the development and maintenance of equine preantral follicle morphology during short-term in vitro culture. Ovaries (n=5) from five mares were collected from a local slaughterhouse and transported to the laboratory, where nine fragments (5x5x1mm) were procured from each ovary. One fragment from each was immediately fixed and submitted for histological analysis (control group; D0). The other eight fragments were cultured in situ for two (D2) or six (D6) days in MEM+ or MEM+ supplemented with GDF-9 at different concentrations (i.e., 50, 100 and 200 ng/mL the GDF-9). After culturing with different concentrations of GDF-9 for 2 or 6 days, the fragments were processed for histological analysis. After two days of cultivation, we observed an increase in the percentage of developing follicles for 0 (MEM+), 50, 100 and 200 ng/mL GDF-9 compared to control (D0; P<0.05). When we evaluated all treatments that preserved follicular integrity, the GDF-9 concentration of 100 ng/mL presented results superior to those of the other cultures (P<0.05). While, at six days of culture, the concentration of 200 ng/mL of GDF-9 appeared to be more efficient in providing development compared to MEM+ (P<0.05). The percentage of morphologically intact follicles in the 6 days culture samples treated with 50 ng/mL of GDF-9 indicated that this concentration was effective in maintaining the integrity of the follicle (P<0.05). We conclude, therefore, that graduated GDF-9 addition to the medium ensure follicular development and is sufficient maintain the architecture.

Estimation, morphometry and ultrastructure of ovarian preantral follicle population in agouti (Dasyprocta leporina) / Morfometria, estimativa e ultraestrutura da população folicular ovariana preantral de cutia (Dasyprocta leporina)

The aim of this study was to characterize the preantral ovarian follicular population in agoutis (D. leporina) by estimating the number of follicles at each developmental category, and also describe the morphometry and the specific features of the follicle and the oocyte by using light and transmission electron microscopy. The length of each ovary was measured using a caliper rule, longitudinally sectioned into two halves and both were immediately fixed to perform the estimation of follicular population and ultrastructural analysis. The mean (±S.E.M.) population of follicular per pair of ovary was estimated at 4419.8±532.26 and 5397.52±574.91 for right and left ovaries, respectively, but no differences were observed between them. The diameters for follicles, oocyte and nuclei were: 18.62±3.40µm, 12.28±2.37µm and 6.10±0.93µm for primordial, 23.75±5.70µm, 14.22±3.00µm and 6.70±1.24µm for primary and 88.55±17.61µm, 52.85±17.56µm and 22.33±17.61µm for secondary follicles, respectively. The most of the follicles found belonged to the primordial category (86.63%), followed by primary (13.01%) and secondary (0.35%) one. Additionally, polyovular follicles were observed in all the animals and they represented 7.51% of the total follicles counted. The ultrastructural analysis showed that the oocyte presented a central and regular nuclei, displaying a homogenous mass. Among the organelles, the mitochondria were the most abundant and the oocyte Golgi apparatus was rarely observed. In conclusion, this work shows for the first time the characterization of the population of preantral follicles in the ovary of Dasyprocta leporina. Those information will be useful for further development and adaptation of biotechniques such as germplasm cryopreservation and in vitro gametes manipulation.(AU)

O objetivo deste trabalho foi caracterizar a população folicular ovariana pré-antral em cutias (D. leporina) estimando o número de folículos em cada categoria de desenvolvimento, e também descrever a morfometria e as características específicas do folículo e oócito usando microscopia de luz e eletrônica de transmissão. O comprimento de cada ovário foi medido utilizando um paquímetro, seccionados longitudinalmente em duas metades e ambos foram imediatamente fixados para realizar a estimativa da população folicular e análise ultraestrutural. A média (±S.E.M.) da população folicular por par de ovário foi estimada em 4419,8±532,26 e 5397,52±574,91 nos ovários direito e esquerdo, respectivamente, mas não foram observadas diferenças entre eles. Os diâmetros dos folículos, oócito e núcleos, respectivamente, foram: 18,62±3,40µm, 12,28±2.37µm e 6,10±0,93µm para primordial, 23,75±5,70µm, 14,22±3,00µm e 6,70±1,24µm para primário e 88,55±17,61µm, 52,85±17,56µm e 22,33±17,61µm de folículos secundários. A maioria dos folículos encontrados pertencia à categoria primordial (86,63%), seguido pelo primário (13,01%) e um secundário (0,35%). Adicionalmente, os folículos poliovulares foram observados em todos os animais e representavam 7,51% do total de folículos contados. A análise ultra-estrutural mostrou que o oócito apresentou núcleos centrais e regulares, exibindo uma massa homogênea. Dentre as organelas, as mitocôndrias foram as mais abundantes e o aparelho de Golgi do oócito foi raramente observado. Em conclusão, este trabalho mostra pela primeira vez a caracterização da população de folículos pré-antrais do ovário da Dasyprocta leporina. Essas informações serão úteis para o desenvolvimento e adaptação de biotécnicas, como a criopreservação de germoplasma e manipulação de gametas in vitro.(AU)

Evaluation of ovarian structures using computerized microtomography

ABSTRACT Visualization and clear understanding of the ovarian structures are important in determining the stage of oestrus, helping to diagnose several pathologies and supporting advances in reproductive technologies. In this research, computerized microtomography (microCT) was used to explore and characterize the ovarian structure of seven mammalian species. Ovaries of rats, female dog, queens, cows, mares, sows and a female donkey were used. After microCT scanning, the same samples were prepared for histologic evaluation, used here as a validation criterion. It was possible to distinguish regions of the cortex and medulla, visualize the morphology and distribution of blood vessels, clearly observe corpus luteum and antral follicles, and visualize oocytes inside some antral follicles. This is the first report using microCT to explore and compare ovarian structures in several domestic mammals. MicroCT revealed great potential for the evaluation of ovarian structures. This research open prospects for the use of computerized tomography (CT) as a non-invasive approach to studying ovarian structures in live animals, which may be especially attractive for scientific study of development of ovarian structures and/or ovarian pathologies in small animals' models.

Morphologic and morphometric analysis of the ovary of black bengal goat (Capra hircus) / Análisis morfológico y morfométrico del ovario de la cabra black bengal (Capra hircus)

Ovary plays the vital role in the reproductive biology and biotechnology of female animals. With the aim to study the ovarian morphometry of Black Bengal goat, both right and left ovaries were collected from the slaughter houses of different Thanas under Mymensingh district. For each of the specimens, gross parameters such as weight, length and width were recorded. Then they were processed and stained with H & E for histomorphometry. Our study revealed that the right ovary (0.53±0.02 g) was heavier than the left (0.52±0.02 g). The length of the right ovary (1.26±0.04 cm) was lower than the left (1.28±0.02 cm) but the width of the right (0.94±0.02 cm) was greater than the left (0.90±0.03 cm). The diameter of ovarian follicles in the cortex was measured as primordial 39.6±6.61 µm, primary single layer 54.0±4.06 µm, primary multi layer 147.6±11.04 µm, secondary with C-shaped antrum 449.5±75.71 µm and tertiary follicle of ovary 1.3±0.20 mm. In the graffian follicle, the thickness of granulosa cell layer was 79.2±11.04 µm, theca interna 75.76±6.82 µm, theca externa 130.07±12.53 µm and the oocyte diameter was 109.8±5.75 µm. These results will be helpful to manipulate ovarian functions in small ruminants.

El ovario desempeña un papel fundamental en la biología de la reproducción y la biotecnología de las hembras. Con el objetivo de estudiar la morfometría de ovario de la cabra Black Bengal se obtuvieron ovarios de ambos lados de diferentes mataderos del distrito de Mymensingh. Para cada uno de los especímenes, se registraron los parámetros de peso, longitud y ancho. Luego fueron procesados y teñidos con H & E para histomorfometría. El ovario derecho (0,53±0,02 g) fue más pesado que el izquierdo (0,52±0,02 g). La longitud del ovario derecho (1,26±0,04 cm) fue inferior al izquierdo (1,28±0,02 cm), pero el ancho del derecho (0,94±0,02 cm) fue mayor que el izquierdo (0,90±0,03 cm). El diámetro de los folículos ováricos en la corteza se midió como primordial 39,6±6,61 µm, de una capa primaria 54,0±4,06 µm, de múltiples capas primarias 147,6±11,04 µm, secundarias con antro en forma de C 449,5±75,71 µm y folículo terciario del ovario 1,3±0,20 mm. En el folículo terciario del ovario, el espesor de la capa de células de la granulosa fue 79,2±11,04 µm, teca interna 75,76±6,82 µm, teca externa 130,07±12,53 µm y el diámetro de los ovocitos fue 109,8±5,75 µm. Estos resultados serán útiles para manipular las funciones de ovario en los pequeños rumiantes.

Effects of natural environmental conditions on the ovarian histometric dynamics of five striped squirrels (Funambulus pennanti) / Efectos de las condiciones ambientales naturales sobre la dinámica histométrica de ovarios en la ardilla de las palmeras (Funambulus pennanti)

The present study was designed to determine the morphological and histological variations in the female palm squirrel's reproductive organs which inhabit croplands of central Punjab. 16 specimens were collected from 3 different locations of Faisalabad district during different months in order to collect their ovaries samples in order to determine the breeding phenology of this species. Body weight and external body measurements were recorded. Two adult females were autopsied each month and their ovaries were extracted. Tissue were prepared by the tissue paraffin technique and stained by H&E. Seasonal variations in the follicular size and shape were observed. Statistical analysis revealed that values of all parameters of ovaries were significantly (P<0.01) higher during peak folliculogenic phase (January to May) as compared to low folliculogenic phase (November­December). Three reproductive phases were observed according to follicular development i.e., quiescent period (November- December), recrudescence period (January) and peak reproductive phase (February­March). In conclusion of this study, different phases of sexual activities are determined which are controlled by seasonal variations. These variations are in accordance to different physical factors which influences gross anatomical and histological changes of ovaries and folliculogensis of female five-striped northern palm squirrel (Funambulus pennanti) for adjustments in breeding phenology.

El presente estudio fue diseñado para determinar las variaciones morfológicas e histológicas en los órganos reproductores de la ardilla de las palmeras que habita en las tierras de cultivo del centro de Punjab. Dieciséis muestras fueron recolectadas en 3 lugares diferentes del distrito de Faisalabad durante diferentes meses con el fin de obtener los ovarios y determinar la fenología reproductiva de esta especie. Se registraron el peso corporal y las medidas corporales externas. Se realizaron autopsias de dos hembras adultas cada mes y sus ovarios fueron extraídos. Se prepararon los tejidos por técnica de parafina y tinción H & E y se observaron las variaciones estacionales en el tamaño y forma folicular. El análisis estadístico reveló que los valores de todos los parámetros de los ovarios fueron significativamente más altos durante la fase foliculogénica (enero a mayo), en comparación con la fase baja (noviembre-diciembre) (P <0,01). De acuerdo al desarrollo folicular se observaron tres fases reproductivas, el período de reposo (noviembre-diciembre), el período de recrudecimiento (enero) y la fase peak reproductiva (febrero-marzo). En conclusión las diferentes fases de las actividades sexuales definitivamente están controladas por variaciones estacionales. Estas variaciones se ajustan a diferentes factores físicos que influyen en los cambios anatómicos e histológicos de los ovarios y la folículogénesis de la ardilla hembra de las palmeras (Funambulus pennanti) para realizar ajustes en la fenología de su reproducción.

Vitrification of bovine preantral follicles with dimethylsulfoxide and sucrose plus alfa-tocopherol / Vitrificação de folículos pré-antrais bovinos com dimetilxulfoxido e sacarose adicionado de alfa-tocopherol

The objective of this study was to evaluate the vitrification of bovine preantral follicles with dimethylsulfoxide (D) and sucrose (S) plus α-tocopherol 5mmol/L (T5) or 10mmol/L (T10) and, evaluate the thawed with minimal essential medium (m) with or without sucrose (s). Ovaries of cows were collected from slaughterhouse for the experiment I (n=66) and II (n=51). In the laboratory ovarian fragments were randomly assigned either to fresh control and 8 vitrification treatments (Controle and Dm; Dms, DSm; DSms; DST5m; DST5ms; DST10m; DST10ms). Ovarian fragments were placed in vitrification solution (5 min) and immersed in liquid nitrogen (-196°C), after a week, the fragments were thawed and analyzed. In the experiments I, preantral follicles were morphologically observed for histological evaluation, (normal; degenerated and developing of stage). In the experiment II, preantral follicles were mechanically isolated from ovarian tissue and examined with trypan blue, where dead and live corresponded to stained or non-stained. The treatments DSm, DSms and DST10m were effective in preserving the morphology in situ. However, the viability of isolated preantral follicles after vitrification remained high only in treatment DST10m. Thus, DST10m preserves survival rates and morphological integrity during vitrification of bovine preantral follicles.

Os objetivos deste estudo foram avaliar a vitrificação de folículos pré-antrais bovinos com dimetilsulfóxido (D) e sacarose (S) adicionando α-tocoferol 5mmol/L (T5) ou 10mmol/L (T10) e, avaliar o aquecimento com meio essencial mínimo (m) com ou sem sacarose (s). Ovários de fêmeas bovinas foram coletados de abatedouro, para o experimento I (n= 66) e II (n= 51). No laboratório fragmentos ovarianos foram distribuídos aleatoriamente para o controle fresco e 8 tratamentos de vitrificação (Controle e Dm; Dms, a DSm; DSms; DST5m; DST5ms; DST10m; DST10ms). Os fragmentos ovarianos foram colocados na solução de vitrificação (5 min) e imersos em nitrogênio líquido (-196°C). Após uma semana os fragmentos foram aquecidos e analisados. No experimento I, folículos pré-antrais foram observados morfologicamente para avaliação histológica (normal, degenerados e estádio de desenvolvimento). No experimento II, folículos pré-antrais foram mecanicamente isolados do tecido ovariano e examinados com o azul de trypan, observando mortos e vivos corados e não corados respetivamente. Os tratamentos a DSm, DSms e DST10m foram eficazes na preservação da morfologia in situ. No entanto, a viabilidade de folículos pré-antrais isolados após a vitrificação manteve-se elevada apenas no tratamento DST10m. Assim, DST10m preservou as taxas de sobrevivência e integridade morfológica durante a vitrificação de folículos pré-antrais bovinos.

Histological study of rat ovaries cryopreserved by vitrification or slow freezing and reimplanted in the early or late postmenopausal stage

PURPOSE: To compare two rat ovary cryopreservation techniques (vitrification vs. slow freezing) and two postmenopausal stages (early vs. late) with regard to graft take. METHODS: Thirty-three Wistar rats were submitted to bilateral oophorectomy. One ovary was submitted to histological analysis while the other was cryopreserved by slow freezing or vitrification. The cryopreserved ovary was thawed and reimplanted in the greater omentum one week (early menopause) or one month (late menopause) after oophorectomy. One month after ovary reimplantation, the graft take was evaluated macroscopically and histologically. RESULTS: Six of the animals were used ascontrols and seven died. The histological findings of 20 animals included atretic follicles (n=4), primordial follicles (n=2), and corpus luteum with primordial follicles (n=3). No ovarian tissue was found in 11 animals. Vitrification resulted in a higher graft take rate than slow freezing (50% vs. 38.5%), but the difference was not statistically significant. However, the graft take rate was 9.3 times higher in the early than in the late postmenopausal stage (61.5% vs. 14.3%) (p=0.043). CONCLUSION: Vitrification was superior to slow freezing as ovarian cryopreservation technique, and grafting was significantly more successful when the ovary was reimplanted in the late postmenopausal stage. .

Culture of bovine ovarian follicle wall sections maintained the highly estrogenic profile under basal and chemically defined conditions

Follicle cultures reproduce in vitro the functional features observed in vivo. In a search for an ideal model, we cultured bovine antral follicle wall sections (FWS) in a serum-free defined medium (DM) known to induce 17β-estradiol (E2) production, and in a nondefined medium (NDM) containing serum. Follicles were sectioned and cultured in NDM or DM for 24 or 48 h. Morphological features were determined by light microscopy. Gene expression of steroidogenic enzymes and follicle-stimulating hormone (FSH) receptor were determined by RT-PCR; progesterone (P4) and E2 concentrations in the media were measured by radioimmunoassay. DM, but not NDM, maintained an FWS morphology in vitro that was similar to fresh tissue. DM also induced an increase in the expression of all steroidogenic enzymes, except FSH receptor, but NDM did not. In both DM and NDM, there was a gradual increase in P4 throughout the culture period; however, P4 concentration was significantly higher in NDM. In both media, E2 concentration was increased at 24 h, followed by a decrease at 48 h. The E2:P4 ratio was higher in DM than in NDM. These results suggest that DM maintains morphological structure, upregulates the expression of steroidogenic enzyme genes, and maintains steroid production with a high E2:P4 ratio in FWS cultures.

Follicular fluid composition in relation to follicular size in pregnant and non pregnant dromedary camels (Camelus dromedaries)

aim of this study was to evaluate the effects of the reproduct ive status of female dromedary camels ( pregnant vs. non - pregnant ) on the chemical composition, hormonal profile and antioxidant capacity of follicular fluid collected from different sized ovarian follicles during the breeding season . One hundred ovaries we re collected at slaughter house from fifty female dromedary camel s . The ovaries were collected in pairs from each animal and allocated into two groups according to the reproductive status of the females ; 25 pairs were obtained from pregnant females and 25 p airs were obtained from non - pregnant animals . The follicles on each ovary were categorized according to their diameter into three categories; small ( 1 - 3 mm), medium (4 - 9 mm) and large ( 10 - 20 mm). Follicular fluid (FF) aspirated from each follicle category from each pair of ovaries w as analyzed . The results showed that the average number of follicles per ovary was greater ( P < 0.05) in the ovaries obtained from non - pregnant females compared to those collected from pregnant ones ( 6.4 ± 1.2 vs . 3.6 ± 0.9, resp ectively). Progesterone concentrations were significantly higher in the follicular fluid collected from all follicle categories in pregnant animals than those obta ined from non - pregnant animals . Glucose concentration s were higher (P < 0.05) in the follicul ar fluid collected from large follicles in the non - pregnant group (64.9 ± 6.1 mg/d l ) than those obtained from the same follicle category in the pregnant ovaries (45.4 ± 4.0 mg/d l ). C oncentrations of malondialdehyde (MDA) were higher (P < 0.05) in the FF co llected from small, medium and large follicles in pregnant ovaries than non - pregnant ones . In conclusion , these data indicate that FF composition differ s according to the reproductive status of the female . In pregnant camels, the p resence of the corpus lut eum on the ovar ies could play an important role not only in the process of follicle growth and development , but also in the concentrations of biochemical metabolites and hormonal profiles in the FF of dromedary camels.

Ovarian folliculogenesis in collared peccary: Pecari tajacu (Artiodactyla: Tayassuidae)

The sustainability and production of collared peccary (Pecari tajacu) has been studied in the last few years; however, further information on its reproduction is necessary for breeding systems success. Understanding folliculogenesis aspects will contribute to effective reproductive biotechniques, which are useful in the preservation and production of wildlife. The aim of this study was to evaluate the ovarian folliculogenesis in collared peccary. Ovaries from six adult females of collared peccary were obtained through ovariectomy and analyzed. These were fixed in aqueous Bouin’s solution and sectioned into 7μm slices, stained with hematoxilin-eosin and analyzed by light microscopy. The number of pre-antral and antral follicles per ovary was estimated using the Fractionator Method. The follicles, oocytes and oocyte nuclei were measured using an ocular micrometer. Results showed that the length, width, thickness, weight, and the gross anatomy of the right and left ovaries were not significantly different. However, the mean number of corpora lutea was different between the phases of the estrous cycle (p<0.05), with the highest mean in the luteal phase. Primordial follicles were found in the cortex; the oocytes were enveloped by a single layer of flattened follicular cells. In the primary follicles, proliferation of the follicular cells gave rise to cuboidal cells (granulosa cells). The secondary follicle was characterized by two or more concentric layers of cuboidal cells (granulosa), beginning of antrum formation, and the presence of pellucid zone and theca cells. Antral follicles were characterized by a central cavity (antrum), the presence of cumulus oophorus and theca layers (interna and externa). In the right ovary, the values of the primordial and primary follicles were similar, but significantly different from the secondary ones (p<0.05). In the left ovary, significant differences were observed between all follicles in the follicular phase (p<0.05); the mean number of primordial and primary follicles was similar in the luteal phase. The mean number of pre-antral follicles and antral follicles in the follicular phase was higher in the left ovary (p<0.05). The mean number of antral follicles in the luteal phase was similar in both ovaries. We also found significant differences in mean diameter of preantral follicles, oocyte, granulosa layer and oocyte nucleus during the estrous cycle. In the antral follicles a significant difference was observed only in follicular diameter (p<0.05). The predominance of active primordial and primary follicles was found in both phases; otherwise the secondary follicles and antral follicles showed a high degree of degeneration. The results obtained in the present work will strengthen the development of biotechnology programs to improve the productive potential and conservation of the collared peccary.

La sustentabilidad y la producción de pecarí de collar (Pecari tajacu) han sido estudiados en los últimos años, sin embargo, más información sobre su reproducción es necesaria para el éxito de los sistemas de crianza . La comprensión de los aspectos relacionados con la foliculogénesis contribuirá con la aplicación de biotécnicas de reproducción, las cuales son útiles en la preservación y la producción de la vida silvestre. El objetivo de este estudio fue obtener datos sobre la población folicular del ovario de pecarí de collar. En relación con la población folicular en el ovario derecho, los valores de los folículos primordiales y primarios fueron similares, pero se observó que había una diferencia significativa (p<0.05) con el secundario. En el ovario izquierdo, la fase folicular presentó diferencias significativas (p<0.05) entre todos los folículos, y en la fase lútea el número medio de folículos primordiales y primarios fueron similares. Ahora bien, con respecto a la población de folículos antrales, en la fase folicular, se observaron diferencias significativas entre los ovarios (p<0.05), y de forma similar en la fase lútea. También se encontraron diferencias significativas en el diámetro medio de los folículos preantrales, oocitos, la capa granulosa y el núcleo del oocito durante las fases del ciclo estral. Asimismo, en los folículos antrales se observó diferencia estadísticamente significativa (p<0.05) en el diámetro folicular. En ambas fases del ciclo estral, se encontró el predominio de folículos primordiales y primarios en desarrollo, por otro lado, los folículos secundarios y los folículos antrales mostraron un alto grado de degeneración. Los resultados aquí presentes son necesarios para el desarrollo de los programas de mejoramiento y conservación.

Assessment of gonadal follicle size in the invading bivalve Limnoperna fortunei (Mytilidae)

Limnoperna fortunei is an invasive gonochoristic and byssate freshwater bivalve originary from Southeast Asia. It shows great adaptive-reproductive ability, so knowledge of the gonadal cycle is an important factor for the prevention and control of this bioinvasion. This species is highly damaging to natural and human environments. We analyzed the distribution and maturity state of reproductive follicles in the mantle of both male and females. Male results are not shown but, in general, they followed the same pattern as that of females. Routine histological techniques included serial longitudinal sections and transversal sections in three body regions (anterior, middle and psoterior). Oocytes with conspicuous nucleoli were measured on both types of sections to estimate the maturity stage in the different regions. ANOVA indicates that there were no significant differences in maturity ratio between the studied regions, so that a small number of sections would render precise results to assess maturity

Assessment of gonadal follicle size in the invading bivalve Limnoperna fortunei (Mytilidae)

Limnoperna fortunei is an invasive gonochoristic and byssate freshwater bivalve originary from Southeast Asia. It shows great adaptive-reproductive ability, so knowledge of the gonadal cycle is an important factor for the prevention and control of this bioinvasion. This species is highly damaging to natural and human environments. We analyzed the distribution and maturity state of reproductive follicles in the mantle of both male and females. Male results are not shown but, in general, they followed the same pattern as that of females. Routine histological techniques included serial longitudinal sections and transversal sections in three body regions (anterior, middle and psoterior). Oocytes with conspicuous nucleoli were measured on both types of sections to estimate the maturity stage in the different regions. ANOVA indicates that there were no significant differences in maturity ratio between the studied regions, so that a small number of sections would render precise results to assess maturity

Ovarian folliculogenesis in the smallnose fanskate sympterygia bonapartii (Müller & Henle, 1841) (Chondrichthyes, Rajidae) / Foliculogénesis ovárica en la raya marmorada, sympterygia bonapartii (Müller & Henle, 1841) (Chondrichthyes, Rajidae)

Chondrichthyes have become an important economic resource in the last decades, with Argentina as one of the countries that exploits more sharks and skates, even at levels that exceed de limits of many species. However, there is a scarce knowledge of the reproductive biology of this group, particularly from species inhabiting the Southern hemisphere. This work shows the most relevant facts during folliculogenesis in Sympterygia bonapartii. Results show that germinal cells are present in immature and maturing females. The most important facts that vary along de follicular development are the number of types and layers of follicular cells, the establishment of thin projections from the follicular cells and the degree of development of the thecae. Follicular cells are, at least, of two different types and both of them emit projections that break through the zona pellucida. The outer theca shows signs of synthetic activity. Atretic follicles of different sizes are present in exemplars of all the reproductive stages. These results are discussed in a physiological and adaptive context.

Los Condrictios se han convertido en un recurso económico importante en las últimas décadas, siendo Argentina uno de los países que más explota tiburones y rayas, incluso a niveles que exceden los límites de varias especies. A pesar de esto, es poco lo que se conoce sobre la biología reproductiva de este grupo, particularmente en especies del Hemisferio Sur. En este trabajo se estudian los estadios más relevantes de la foliculogénesis en Sympterygia bonapartii. Los resultados muestran que las ovogonias están presentes tanto en ejemplares inmaduros como subadultos. Las características más importantes que varían a lo largo del desarrollo folicular son el número de capas y tipos celulares que constituyen el epitelio folicular, el desarrollo de proyecciones de las células de la granulosa y el grado de desarrollo de las tecas. Las células foliculares son, al menos, de dos tipos y ambos emiten proyecciones que atraviesan la zona pelúcida. La teca externa presenta características compatibles con la actividad sintética. Folículos atrésicos de distintos tamaños están presentes en ejemplares de todos los estadios de madurez sexual. Estos resultados se discuten en un marco fisiológico y adaptativo.

Follicular-stromal interaction in mare ovary during the reproductive cycle / Interacción estroma-folículo en el ovario de la yegua durante el ciclo reproductivo

Ovogenesis and foliculogenesis in the mare differ from other farm animals and therefore, gamete manipulation, in vitro fertilization and embryo transfer have been very difficult. The histologic traits of ovaries from 12 mares, in estrus and diestrus were studied. Number and size of follicules and corpora lutea were recorded. The ovarian stroma was evaluated using the pricosirius technique for collagen. A simple morphometric analysis was done using computerized scanner programs. During late estrus, one dominant follicle (46 +/- 4mm) is seen. The surrounding stroma contains collagen I and III. By the end of the estrus, one hemorrhagic follicle is seen, plus one or two small follicle (2 mm in size). In early diestrus there is a corpus luteum (43-60 mm) and some antral follicles (6 +/- 1). Collagen I forms strands inside the corpus luteum and predominates in the perifollicular ovarian stroma. Modifications of the extracellular matrix may change cell function by way of integrines, the matrix being in turn modified by hormones and other tissular factors. Estrogens can be related to collagen III predominance in ovarian stroma whereas progesterone is associated with increased collagen I. Therefore, follicular-stromal interactions are important in ovarian histophysiology.

La ovogénesis y foliculogénesis en la yegua, difiere de otros animales de granja, y por lo tanto, la manipulación de gametos, fertilización in vitro y transferencia de embriones ha sido muy dificultoso. Se estudiaron las características histológicas de ovarios de 12 yeguas durante el estro y el diestro. Se registró el número y tamaño de los folículos y cuerpos lúteos. Se evaluó el estroma ovárico usando la técnica picrosirius para colágeno. Se realizó un análisis morfométrico simple utilizando un programa escáner computarizado. Durante el estro tardío, se observó un folículo dominante (46 +/- 4 mm). El estroma circundante contiene colágeno I y III. Para el final de este período, se aprecia un folículo hemorrágico, más uno o dos pequeños folículos (2 mm de tamaño). En diestro temprano, hay un cuerpo lúteo (43 - 60 mm) y algunos folículos antrales (6 +/- 1). El colágeno I forma filamentos dentro del cuerpo lúteo y predomina en el estroma ovárico perifolicular. Modificaciones en la matriz extracelular pueden cambiar la función celular vía integrinas, la matriz está a su vez modificada por las hormonas y otros factores tisulares. Los estrógenos se pueden asociar a la predominancia de colágeno III en el estroma ovárico, mientras que la progesterona se asocia con un aumento de colágeno I. Por lo tanto, las interacciones estroma-folículo son importantes en la histofisiología del ovario.

Característica histológica, ultra-estrutural e produção de nitrito de folículos pré-antrais caprinos cultivados in vitro na ausência ou presença de soro / Histological and ultrastructural feature and nitrite production of caprine preantral follicles in vitro cultured in the presence or absence of serum

Avaliou-se o efeito da adição de diferentes tipos e concentrações de soro sobre o desenvolvimento e a sobrevivência de folículos ovarianos pré-antrais (FOPA) caprinos in vitro. Além disso, verificou-se a relação entre as concentrações de nitrito presentes no meio de cultivo e a viabilidade folicular. Cada par ovariano foi dividido em 29 fragmentos, sendo um destinado ao controle. Os fragmentos foram cultivados por um ou sete dias em meio essencial mínimo suplementado (MEM+) ou MEM+ com diferentes concentrações (10 ou 20 por cento) de soro fetal bovino (SFB), soro de cabra em estro (SCE) ou soro de cabra em diestro (SCD). Na análise morfológica após sete dias, apenas o tratamento com 10 por cento de SFB apresentou percentual de FOPA normais similar ao MEM+ (P>0,05). A análise ultra-estrutural dos folículos cultivados por sete dias com MEM+ ou MEM+ com 10 por cento de SFB mostrou danos oocitários, porém células da granulosa normais. A análise do meio de cultivo revelou correlação positiva entre a viabilidade folicular e a produção de nitrito. A suplementação com soro não melhorou a viabilidade de FOPA e a concentração de nitrito no meio de cultivo funcionou como um indicador da viabilidade das células da granulosa de FOPA caprinos cultivados in vitro.

The effect of the addition of different types and concentrations of sera on the viability and development of caprine preantal follicles (PAF) in vitro cultured was analyzed. In addition, it was evaluated the correlation between nitrite concentrations in culture medium and folicular viability. Each ovarian pair was divided in 29 fragments and one was used as control. The fragments were cultured for one or seven days in minimal essential medium (MEM+) or MEM+ with different concentrations of (10 or 20 percent) bovine fetal serum (BFS), estrous goat serum (EGS), or diestrous goat serum (DGS). After seven days, the morphological analysis showed that only the treatment with 10 percent BFS maintained the percentage of normal PAF similar to MEM+ (P>0.05). The ultrastructural analysis of follicles cultured for seven days in MEM+ or MEM+ with 10 percent BFS showed some oocyte damage, although the granulosa cells were normal. Analysis of culture medium revealed a positive correlation between follicular viability and nitrite production. Supplementation with serum did not improve the viability of PAF and nitrite levels in culture medium served as an indicator of viability of granulose cells from caprine PAF in vitro cultured.

Structural and ultrastructural aspects of folliculogenesis in Didelphis albiventris, the South-American opossum / Aspectos estructurales y ultraestructurales de la foliculogénesis de Didelphis albiventris, la zarigüeña sudamericana

The ovarian histology, the structural and the ultrastructural characteristics of the folliculogenesis in Didelphis albiventris were described in detail. Recent studies suggest that methatherian mammals have unusual reproductive cycle but there are few informations regarding the marsupials reproductive life. Despite of the opossum folliculogenesis pattern resembles methatherian and eutherian pattern in many aspects, the analysis shows some peculiar features of the oocyte structure and ultrastructure that make available new data on the reproductive biology of marsupials.

Fueron descritas con detalles la histología ovárica, las características estructurales y ultra-estructurales de la foliculogénesis del Didelphys albiventris. Estudios recientes sugieren que mamíferos metaterios tienen un ciclo reproductivo inusual, pero existen pocas informaciones en relación a la vida reproductiva de los marsupiales. A pesar de que el modelo de foliculogénesis de la zarigüeya se parece al modelo metaterio y euterio en muchos aspectos, el análisis muestra algunos rasgos peculiares de las estructura y ultra-estructura del oocito, que colocan a disposición nuevos datos en la biología reproductiva de los marsupiales.

Teste de toxicidade e criopreservação de folículos pré-antrais ovinos isolados utilizando Glicerol, Etilenoglicol, Dimetilsulfóxido e Propanodiol / Toxicity test and cryopreservation of sheep isolated preantral follicles using glycerol, ethylen glycol, dimethil sulfoxyde and propanediol

O objetivo deste estudo foi avaliar foliculos pré-antrais (FOPA) ovinos isolados após sua exposição e criopreservação utilizando glicerol (GLI), etilenoglicol (EG), propanodiol (PROH) ou dimetilsulfóxido (DMSO) a 1,5 e 3,0 M. Cada par ovariano de 5 ovelhas sem raça definida foi coletado em abatedouro local e submetido ao isolamento folicular. Da suspensão obtida, uma aliquota foi imediatamente destinada à análise da viabilidade folicular com o auxílio do corante vital azul de trypan. O restante da suspensão foi dividida em 16 aliquotas de 0,9 mL, suspensas (v/v) em MEM+ com EG, DMSO, GLI ou PROH a 1,5 ou 3,0 M, para teste de toxicidade e criopreservação. Após o término de cada tratamento, a viabilidade folicular foi analisada e os FOPA considerados viáveis se não corados ou não viáveis, quando corados. A análise dos dados mostrou que após o teste de toxicidade e criopreservação, em todos os crioprotetores e em ambas as concentrações, a percentagem de FOPA viáveis foi significativamente reduzida quando comparada ao controle. No teste de toxicidade, quando os crioprotetores foram comparados entre si nas mesmas concentrações, foram observadas percentagens signifIcativamente menores de FOPA viáveis no PROH 3,0 M (38,9%), apresentando-se, portanto, mais tóxico quando comparado aos demais crioprotetores. Após criopreservação, obteve-se percentagens significativamente maiores de foliculos pré-antrais viáveis quando o EG e o DMSO foram utilizados. Em conclusão, FOPA ovinos isolados podem ser criopreservados com sucesso utilizando-se D MSO e EG a 1,5 e 3,0 M.

The aim of this study was to evaluate isolated sheep preantral follicles (PF) after exposure and cryopreservation using glycerol (GLI), ethylene glycol (EG), propanediol (PROH) or dimethyl sulfoxide (DMSO) at 1.5 and 3.0 M. Each ovarian pair from 5 mixed breed adult sheeps was obtained at a local slaughterhouse and submited to follicular isolation. From the obtained suspension, one aliquot was immediately analysed with trypan blue. The remaining suspension was divided in 16 aliquots of 0.9 mL, suspended in (v /v) in MEM+with EG, DMSO, GLI or PROH at 1.5 or 3.0 M to the toxicity test and cryopreservation. After the end of each treatment, the follicular viability was analysed and the PF were classified as viable if not dyed or not viable if dyed with trypan blue. The analysis of the results showed that after the toxicity test and cryopreservation, using all cryoprotectants and at both concentrations, the percentage of viable PF was significandy reduced when compared to the control. At the toxicity test, when the cryoprotectants were compared at the same concentrations, the lowest percentage of viable preantral follicles was obtained when 3.0 M PRO H (38,9%) was used, being, more toxic when compared to the others cryoprotectants. After cryopreservation, significantly higher percentual of viable PF was observed when the EG and DMSO were used. In conclusion, sheep PF can be cryopreserved successfully using DMSO and EG at 1.5 and 3.0 M.