Morphometric and ultrastructural characterization of Bos indicus preantral follicles

The aim of the present study was to characterize the ultrastructure of zebu cow preantral follicles (PAFs). Ovarian cortex samples were processed for light and transmission electron microscopy. Primordial follicles consisted of an oocyte surrounded by one layer of flattened or flattened-cuboidal granulosa cells. The oocyte contained a large and usually eccentric nucleus. Most organelles were located at the perinuclear ooplasm. Round shaped mitochondria, which contained electron-dense granules, smooth and rough endoplasma reticulum and a Golgi apparatus were also observed. Vesicles and coated pits were often observed in the cortical ooplasm. In primary follicles, the oocyte was surrounded by one layer of cuboidal granulosa cells. Short microvilli were observed on the oolema. Secondary follicles consisted of an oocyte surrounded by a variable number of layers of cuboidal granulosa cells. Small secondary follicles had an ultrastructure very similar to that observed in primary follicles. At this follicular stage, the zona pellucida was beginning to form around the oocyte. In large secondary follicles, the zona pellucida was totally developed around the oocyte. Several granulosa cell projections could be detected that were encroaching into the zona pellucida and protruding towards the oocyte, where gap junctions were observed between oocyte and granulosa cell membranes. Organelles within the oocyte were located at the periphery of the ooplasm, and clusters of cortical granules were observed. Round mitochondria were abundant in all developmental stages. In conclusion, this study described the ultrastructure of zebu cow PAFs, and some unique characteristics could be observed as compared with what has been reported for follicles of Bos taurus cattle

Effects of lowered temperatures and media on short-term preservation of zebu (Bos indicus) preantral ovarian follicles

The maintenance of follicle quality during the transportation of ovaries is essential for the successful cryopreservation and in vitro development of preantral follicles. The objective of this study was to evaluate the effect of cooling ovarian tissue on the conservation of zebu cow preantral follicles. Ovarian pieces were immersed in saline or coconut water (CW) solutions and maintained at 4 or 20 °C for 6, 12, or 18 h. Preantral follicles were evaluated by histology and transmission electron microscopy. Storage of ovarian pieces at 20 °C for 12 or 18 h significantly reduced the percentage of morphologically normal follicles compared to controls. In contrast, conservation at 4 °C for up to 18 h and at 20 °C for up to 6 h kept the percentage of normal follicles similar to controls. However, the type of solution that the ovaries were immersed in had little effect on the results. Decreased cellular metabolism probably accounted for better preservation of preantral follicles at 4 °C. In conclusion, zebu cow ovaries were successfully stored at 4 °C for up to 18 h with no morphological damage to preantral follicles. However, at 20 °C, ovaries could only be stored for 6 h

Effect of different cryoprotectants on the structural preservation of follicles in frozen zebu bovine (Bos indicus) ovarian tissue

Cryopreservation of ovarian tissue is a new and promising technique for germ-line storage. The objective of this study was to evaluate the effect of four cryoprotectants (at two concentrations each) on the preservation of zebu bovine preantral follicles after ovarian cryostorage. Strips of ovarian cortex were cryopreserved using glycerol (GLY; 10 or 20%), ethylene glycol (EG), propanediol (PROH) or dimethylsulphoxide (DMSO; 1.5 or 3 M). In addition, a toxicity test was performed for each cryoprotectant by exposing the ovarian tissue to them without freezing. Tissues were analyzed by histology and transmission electron microscopy. Ovarian tissue frozen in either concentration of DMSO or PROH or in 10% GLY retained a higher percentage of morphologically normal follicles (73–88%) than tissue frozen in 20% GLY or in either concentration of EG (16–52%). In the toxicity test, exposure of tissues to DMSO, PROH or GLY resulted in higher percentages of normal follicles (80–97%) than exposure to EG (49%). Electron microscopy revealed damage to the ultrastructure of follicles frozen in 10% GLY, while follicles cryopreserved in DMSO and PROH at either concentration exhibited normal ultrastructure. In conclusion, DMSO and PROH were the most effective cryoprotectants for zebu ovarian tissue, preserving the structural integrity of somatic and reproductive cells within the ovary

Testicular biometric characteristics in Santa Inês sheep submitted to protein supplementation

Para avaliar as características biométricas testiculares de carneiros da raça Santa Inês, 24 cordeiros machos, inteiros, com peso médio inicial de 24,5 kg ± 2,88, aos quatro meses de idade, foram distribuídos em quatro tratamentos: APv (animais vermifugados + concentrado com alta proteína), APn (animais não-vermifugados + concentrado com alta proteína), BPv (animais vermifugados + concentrado com baixa proteína), BPn (animais não vermifugados + concentrado com baixa proteína) durante 8 meses e meio. Antes do abate, realizaram-se as medições testiculares in situ: perímetro escrotal (PE), comprimento (COMP), largura testicular (LARG) e a forma dos testículos, bem como o volume testicular (cilindro e prolato esferóide). Após o abate, determinaram- se o volume testicular real (VOLR), o comprimento (COMPV) e a largura testicular (LARGV). Observou-se que os testículos dos animais eram simétricos e que o uso do paquímetro é uma forma altamente confiável para se realizar as medições testiculares in situ. O formato dos testículos afetou diretamente seu volume. O valor médio das equações do prolato esferóide e cilíndrica é a forma mais acurada para se estimar o volume testicular real em ovinos Santa Inês. O alto teor de proteína da dieta e o tratamento antiparasitário possibilitaram uma melhoria do peso corporal dos ovinos, sendo o principal fator de variação para as características biométricas testiculares. _____________________________________________________________________________________ ABSTRACTBiometric testicular traits were evaluated on 24 entire male Santa Inês lambs, with mean initial weight of 24.5 kg ± 2.88. These were distributed in four treatments (n=6): HPv (animals drenched + high protein concentrate), HPn: (animals not drenched + high protein concentrate), LPv: (animals drenched + low protein concentrate), LPn: (animals not drenched + low protein concentrate) for eight and a half months. Before slaughter testicular measurements were taken in situ. Scrotal perimeter (SC) was measured using tape as well as length (LENG) and width (WID) using calipers. Testicular shape and volume (using prolate and cylinder equation) were calculated. After slaughter the measurements were taken in vitro. Real testicle volume (VOLR) was measured using water displacement. Length (LENGV) and width (WIDV) were measured with calipers after skin removal. The testicles were symmetrical and calipers were an accurate means of in situ measurement of the testicle size. The form of the testicles affected the volume. The average of the prolate sphere and cylinder equations gave the best estimate for real testicle volume. High protein levels in the diet accompanied by antihelminth treatment led to higher body weight in the sheep, this being the main factor for variation in testicle biometric traits

Ultrastructural characterization of porcine oocytes and adjacent follicular cells during follicle development : lipid component evolution

The objective of this study was to characterize the morphometry and ultrastructure of porcine preantral and antral follicles, especially the lipid component evolution. Ovarian tissue was processed for light microscopy. Ovarian tissue and dissected antral follicles (< 2, 2–4, and 4–6 mm) were also processed for transmission electron microscopy using routine methods and using an osmium-imidazole method for lipid detection. Primordial follicles (34 ± 5 μm in diameter, mean ± SD) had one layer of flattened-cuboidal granulosa cells around the oocyte, primary follicles (40 ± 7 μm) had a single layer of cuboidal granulosa cells around the oocyte, and secondary follicles (102 ± 58 μm) had two or more layers of cuboidal granulosa cells around the oocyte. Preantral follicle oocytes had many round mitochondria and both rough and smooth endoplasmic reticulum. In oocytes of primordial and primary follicles, lipid droplets were abundant and were mostly located at the cell poles. In secondary and antral follicles, the zona pellucida completely surrounded the oocyte, whereas some microvilli and granulosa cells projected through it. Numerous electron-lucent vesicles and vacuoles were present in the oolemma of secondary and antral follicles. Based on osmium-imidazole staining, most of these structures were shown to be lipid droplets. As the follicle developed, the appearance of the lipid droplets changed from small and black to large and gray, dark or dark with light streaks, suggesting that their nature may change over time. In summary, although porcine follicles and oocytes had many similarities to those of other mammalian species, they were rich in lipids, with lipid droplets with varying morphological patterns as the follicle developed

Zebu (Bos indicus) ovarian preantral follicles : morphological characterization and development of an efficient isolation method

Preantral follicles are a major source of oocytes, and their utilization as an important tool to store large number of female gametes for future use in reproductive programs has been investigated. The increasing importance of studies in this subject, together with the important role of Zebu cattle in the economy of tropical and subtropical countries as well as their well-known differences from European cattle, led to this research. The present study aims to determine the best size interval for sectioning ovarian tissue to isolate preantral follicles from Zebu cows using a tissue chopper and to evaluate the follicular quality after isolation. Furthermore, it aims to provide information about the Zebu cow preantral follicle population and use this data (as a control) to evaluate the effectiveness of the tested isolation method. Testing eight different tissue sectioning size intervals, it was possible to conclude that the 125-pm-section interval is shown to be better than the intervals of 25, 50, 175 and 200 pm to isolate preantral follicles from Zebu cow ovaries. The 125- pm interval allowed the recovery of 26,050±1611 (meaniS.E.M.) preantral follicles per one-half ovary, while the number of preantral follicles in situ estimated by evaluation of histological sections was 35,28812342 per one-half ovary. Thus, the mean (iS.E.M.) recovery rate (=[number of preantral follicles isolated/number of preantral follicles in situ in the same ovary] x 100) was 74.3±4.3%. The morphometrical analysis showed that Bos indicus preantral follicles are similar to B. taurus preantral follicles based on previous reports. In conclusion, this study showed that a simple, mechanical method can be used effectively to isolate a large number of intact preantral follicles from Zebu cow ovaries, with a high recovery rate

Light microscopical and ultrastructural characterization of goat preantral follicles

Goat ovarian preantral follicles were morphologically and ultrastructurally described in this work. Primordial follicles are oocytes surrounded by one layer of squamous or squamouscuboidal granulosa cells; primary follicles have a single layer of cuboidal granulosa cells, and secondary follicles are oocytes surrounded by two or more layers of cuboidal granulosa cells. At all developmental stages a thick layer of glycoproteins, the basement membrane, surrounded the preantral follicles. The quiescent oocyte is spherical or oval and it has a large eccentrically located nucleus with a conspicuous nucleolus. The organelles were uniformly distributed in the cytoplasm. A large number of vesicles were spread throughout the cytoplasm in all the oocytes. The cytoplasm of oocytes also contains numerous rounded mitochondria besides the usual organelles. As the follicle develops, the mitochondria become elongated. The communication between the oocyte and the granulosa cells is apparently mediated through endocytosis as indicated by the abundant coated pits and vesicles noted in the cortical cytoplasm of the oocyte. The oocyte plasma membrane presented projections that penetrated between adjacent granulosa cells and a few short microvilli lying parallel to the oocyte surface. In secondary follicles, patches of zona pellucida material were observed. Overall, the results indicate that the morphological and ultrastructural organization of caprine preantral follicles resembles that of other mammals. However, some particularities were observed, and that may indicate species specific differences

Avaliação morfológica dos testículos de ovinos jovens Santa Inês submetidos a diferentes regimes de suplementação alimentar e vermifugação

ABSTRACT: The present study investigated the effect of protein supplementation and parasite load in relation to testicular morphology, in young Santa Inês male sheep. Twentyfour male lambs, non-castrated, were distributed in four treatments: HPd (dewormed animals + diet with high protein), HPn (animals non dewormed + diet with high protein), LPd (dewormed animals + diet with low protein), LPn (animals not dewormed + diet with low protein) during eight months. At the end of the experiment histological cuts of testicles were taken and cell count analyzed by computer and the program IMAGE PRO-PLUS was used for evaluation of measurements of the seminiferous tubules. The following results were found: LPn presented larger number of spermatogonias, but a reduced number of the other cells parameters (Sertoli cells, spermatocytes, spermatids and spermatozoa) and smaller diameter and circumference of the seminiferous tubules and shorter seminiferous epithelium when compared to other treatments. There was a significant correlation among tubules diameter of the epithelium height and tubules circumference. Correlations between lumen diameter and lumen circumference, as well as epithelium height and tubules circumference also presented high and positive correlations (r>0.70). When the external testis parameters were compared with internal parameters no significant correlation was observed (r0,70); comparando-se os parâmetros testiculares externos com os internos, observa-se que não houve correlação significativa (P<0,50). Conclui-se que a alta proteína influencia positivamente a espermatogênese, e a não-vermifugação associada a uma dieta pobre em proteínas pioram substancialmente a maioria dos parâmetros reprodutivos de carneiros jovens Santa Inês

Effect of Braun-Collins and Saline solutions at different temperatures and incubation times on the quality of goat preantral follicles preserved in situ

The present work has investigated the efficiency of Braun-Collins and Saline (0.9%) solutions in the conservation of goat preantral follicles in situ, at different temperatures and incubation times. For each animal the ovarian pair was divided into 19 fragments. One ovarian fragment was taken randomly and immediately fixed (control). The other 18 ovarian fragments were randomly distributed in tubes containing Braun-Collins or Saline (0.9%) solutions at 4, 20 or 39°C for 4, 12 or 24 h. A total of 3385, 372 and 191 primordial, primary and secondary follicles were examined, respectively. The quality of preantral follicles was evaluated by histology and transmission electron microscopy. The storage of ovarian fragments in Saline (0.9%) or Braun-Collins solutions at 4°C did not reduce significantly the percentage of morphologically normal follicles when compared with the control. The histological analysis revealed a morphological integrity of goat preantral follicles stored at 4°C for up to 24 h in both solutions, but these results were not confirmed by ultrastructural analysis. The transmission electron microscopy revealed that only preantral follicles stored at 4°C for a maximum of 12 h in both solutions were ultrastructurally normal. In conclusion, this study shows for the first time that goat preantral follicles can be stored in situ successfully at 4°C in Saline (0.9%) or Braun-Collins solution for up to 12 h

Mechanical isolation of caprine preantral follicles at different reproductive stages and ovarian situation

O presente trabalho descreve os efeitos do estágio reprodutivo (animais impúberes, adultos não-gestantes e gestantes), posição anatômica do ovário (direito e esquerdo) presença de corpo lúteo funcional, peso do ovário e número de folículos antrais superficiais (FAS) sobre o número de folículos pré-antrais (FPA) isolados mecanicamente. Os FPA isolados foram divididos em 3 classes a saber: primordiais, primários e secundários. O número médio (± EP) de FPA isolados por ovário foi de 1.324 ± 193; 866 + 170 e 779 ± 139, respectivamente para ovários oriundos de animais impúberes, não-gestantes e gestantes, sendo observada diferença significativa somente entre animais impúberes e gestantes. A posição anatômica do ovário (exceto para os animais não-gestantes) e a presença ou ausência de corpo lúteo não exerceram nenhum efeito sobre o número e distribuição dos FPA isolados. De um modo geral, o número de folículos primários e secundários foi negativamente correlacionado com o peso ovariano. Já o número de folículos primordiais e secundários foi, respectivamente, positiva e negativamente correlacionado com o número de FAS. Analisando as categorias de animais isoladamente, observou-se que, nos animais impúberes, o número de folículos primordiais foi positivamente correlacionado com o número de FAZ, enquanto que o número de folículos primários e secundários foi negativamente correlacionado com o número de FAS. No tocante à categoria de animais gestantes, o número de folículos primários foi negativamente correlacionado com o peso ovariano. Dependendo da categoria de ovários estudada, foram observadas associações significativas entre o número de folículos primordiais, primários e secundários. _________________________________________________________________________________ ABSTRACTThe aïm of this study was to describe the effects of reproductive stages (pre-pubertal, non-pregnant and pregnant adult animals), ovarian anatomic position (left or right), presence of functional corpus luteum, ovarian weight and number of superficial antral follicles on the number of preantral follicles mechanically isolated. The mean number (mean ± SEM) of isolated preantral follicles per ovary was 1,324 ± 193, 866 ±170 and 779 ± 139, respectively for prepubertal, non-pregnant and pregnant adult animals. Anatomic position (except for nonpregnant animals) and the presence or absence of corpus luteum dia nof affect the number and distribution of isolated preantral follicles. In general, the number of primary and secondary follicles was negatively correlated with the ovarian weight. The numbers of primordial and secondary follicles were, respectively, positively and negatively correlated with the number of superficial antral follicles. Analyzing animal categories separately, it was observed that in prepurbetal animals, the number of primordial follicles was positively correlated with the number of superficial antral follicles. However the numbers of primary and secondary follicles were negatively correlated with the number of superficial antral follicles. In the category of pregnant animals, the number of primary follicles was negatively correlated with the ovarian weight. According to ovary category analyzed, were observed significant correlation between the number of primordial, primary and secondary isolated follicles