Desenvolvimento morfolôgico dos ovários em embriões e fetos bovinos da raça Nelore

The morphologic events related to the prenatal development of the ovaries in 81 Nelore breed embryos and fetuses gathered in a local slaughterhouse, with age range from 26 to 240 days following fecundation were studied. The age of fetuses was estimated from measures taken in the cranium-caudal direction. The sex was identified from macroscopic observations and using Polymerase Chain Reaction (PCR) technique. For histology the gonads were fixed in Bourn's fluid for 24 hours and 5 μm thick section's were stained with hematoxylin-eosin. Formation of gonadal ridge and presence of germinal cells were found within it at 29 and 34 days, respectively. Oogonia and primordial follicles, unlike the growing follicles, exhibited significant differences in diameter in the various periods studied. Positive correlation (P<0.05) was found between the diameter of oogonia and their nucleus as well as between primordial and growing follicles with their oocytes and respective nuclei. The gonad was fully formed at 40 days. Primordial follicles, in the growing stage, and antral follicles first appeared, approximately at 95, 140, and 180 days, respectively. Despite the onset and duration of oogenesis being similar to that of taurine breeds, folliculogenesis initiates at an early stages in the Nelore breed.

Utilização do primeiro ciclo ovulatório da estação reprodutiva para produção de embriões em éguas sob condições tropicais

The efficacy of the first ovulation of the breeding season was determined through the response of first pre-ovulatory follicle of the breeding season to hCG, embryo recovery rate, viability of recovered embryos, serum concentrations of progesterone, and response of first CL to PGF 2α. Thirteenth mares that were in vernal transition were accompanied until the first pre-ovulatory follicle was detected. At this moment, the ovulation was induced with 2,500 IU hCG (IV) and the mares were inseminated every other day until ovulation. Seven days after the ovulation, embryo recovery was performed and the progesterone concentration was determined. After detection of the first pre-ovulatory follicle of breeding season with ≥ 25mm, it took 14.92 ± 10.80 days for the follicle to reach the preovulatory size and 18.00 ± 11.08 days to ovulation. After administration of hCG, 11/13 mares ovulated in 48 hours. These follicles growth 2.19 ± 0.86 mm/day on average. Nine of 13 mares (69.2%) produced embryos and all were considered viable after morfological evaluation and fluorescence exams. The CL appeared competent producing 7.39 ± 2.11 ng/ml P 4 on average, and responding to PGF 2α. According to these results the first ovulatory cycle of the year can be utilized to produce viable embryos.

Follicular dynamics and plasma FSH and progesterone concentrations during follicular deviation in the first post-ovulatory wave in Nelore (Bos indicus) heifers

The objective of the present study was to characterize ovarian follicular dynamics and hormone concentrations during follicular deviation in the first wave after ovulation in Nelore (Bos indicus) heifers. Ultrasonographic exams were performed and blood samples were collected every 12 h from the day of estrus until 120-144 h after ovulation in seven females. Deviation was defined as the point at which the growth rate of the dominant follicle became greater than the growth rate of the largest subordinate follicle. Deviation occurred approximately 65 h after ovulation. Growth rate of the dominant follicle increased (P < 0.05) after deviation, while growth rate of the subordinate follicle decreased (P < 0.05). Diameter of the dominant follicle did not differ from the subordinate follicle at deviation (approximately 5.4 mm). The dominant follicle (7.6 mm) was larger (P < 0.05) than the subordinate follicle (5.3 mm) 96 h after ovulation or 24 h after deviation. Plasma FSH concentrations did not change significantly during the post-ovulatory period. The first significant increase in mean plasma progesterone concentration occurred on the day of follicular deviation. In conclusion, the interval from ovulation to follicular deviation (2.7 days) was similar to that previously reported in B. taurus females, but follicles were smaller. Diameters of the dominant follicle and subordinate follicle did not differ before deviation and deviation was characterized by an increase in dominant follicle and decrease in subordinate follicle growth rate. Variations in FSH concentrations within 12-h intervals were not involved in follicular deviation in Nelore heifers. © 2006 Elsevier B.V. All rights reserved.

The reproductive biology and cycle of Rioraja agassizi (Müller and Henle, 1841) (Chondrichthyes: Rajidae) in southeastern Brazil, SW Atlantic Ocean

Specimens of Rioraja agassizi were collected at Guarujá pier, São Paulo State, Brazil, from March 2005 to March 2006. A total of 275 males were captured. Based on the analysis of the clasper length, gonad weight, clasper gland length, alar thorns and lobule diameter, the size-at-maturity was estimated to be 32 cm. There were 1049 females in the record. Size-at-maturity was calculated in 40 cm. Sexual resting females were observed. Gonadosomatic and hepatosomatic indexes varied significantly in both sexes throughout the year. However, ovulation, egg-laying and presence of sperm in the seminal vesicle were observed all year round. For this reason, an annual cycle with at least one peak in the sexual activity is proposed for this species.

Efeitos de diferentes sistemas de cultivo in vitro sobre o crescimento de folículos pré-antrais isolados de ovários de fetos bovinos

The objective of this study is to use different in vitro culture systems of preantral follicles from Nelore breed bovine fetuses in the last gestation quarter. The evaluation of treatments considered the time of growth of isolated follicles. Preantral follicles were mechanically isolated and submitted to the individual culture, for 9 days, in media no supplemented or supplemented with fetal calf serum (FCS), bovine serum albumin (BSA) or synthetic defined supplement substitute of serum KnockoutSR (KNO). We have also evaluated the effects of collagen gel or fetal calf fibroblast monolayer as substratum for in vitro cultures. The increase on the follicular diameter was followed in the first day (0 h), at the 72 h, 144 h and 216 h. Considering cultures of isolated follicles, the results have shown that the association between media supplemented with FCS and collagen gel was significantly more efficient on the increase of the follicular diameter than other treatments. It is not still established a system of appropriate cultivation that sustains the differentiation and multiplication of the granular cells and that maintains the contact of the same ones with the oocyte to provide molecules and factors that supply the metabolic demand. We also understand that our results also represent another promising step on the search for the ultimate system of in vitro culture of preantral follicles from bovines.

Sexual development and reproductive cycle of the Eyespot skate Atlantoraja cyclophora (Regan, 1903) (Chondrichthyes: Rajidae: Arhynchobatinae), in southeastern Brazil

Specimens o/Atlantoraja cyclophora were collected monthly from commercial fishing landings at Guarujá, São Paulo State, Brazil, from March 2005 to April 2006 at depths between 10 and 146 m. Males ranged from 13.3 to 58.5 cm TL (n = 396). Both the smallest mature male and the largest immature male were 47.0 cm long. Males' size-at-50% maturity was calculated to be 46.3 cm. Females ranged from 11.5 to 68.0 cm (n = 401). The smallest mature and the largest immature female were 51.6 and 53.0 cm long respectively. For the females, size-at-50% maturity was calculated to be 53.2 cm. In the males the hepatosomatic andgonadosomatic indices varied between 0.48 (August) and 3.54 (November) and between 0.15 (November) and 1.45 (June) respectively, with no significant variation for the fourteen-month period. In the females the hepatosomatic and gonadosomatic indices varied from 1.55 and 6.30 3.54 (both for April 2006) and from 0.08 (December) to 4.41 (October) respectively, with no significant difference among months. Egg-bearing females were found in all months with proportions varying from 0.03 (March) to 0.67 (April). Both males and females undergo an annual cycle, with slight seasonal variations in reproductive activity and a peak in the proportion of egg bearing females between April and July.

Análise histopatológica de órgãos linfóides de bovinos e camundongos intoxicados experimentalmente por Baccharis coridifolia: Caracterização imunoistoquímica de linfócitos B e T

Baccharis coridifolia is one of the most important poisonous plants to cattle in the South of Brazil. The plant provokes necrotic lesions in the lymphoid tissues and in the gastrointestinal tract of cattle. Experimental administration to mice produces most of the lesions seen in the lymphoid tissues of cattle. This study was conducted to search possible differences in the susceptibility of T and B lymphocyte subpopulations. Lymph nodes, spleen, thymus and gut-associated lymphoid tissue (GALT) of cattle and mice experimentally poisoned were evaluated. The results were evaluated based on cell populations affected or remaining in the organs. Immunostaining for B lymphocytes (anti-BLA-36) identified the germinal center of follicles of the lymph node, spleen and GALT in both species. Immunostaining for T lymphocyte (anti-CD3) identified the paracortical area of the germinal centers of the lymph nodes and GALT, the periarteriolar area of the spleen, and the whole thymus both in cattle and mice. Experimentally poisoned cattle and mice shows necrosis of the germinal center of secondary follicles of the lymph nodes, spleen and GALT, where necrotic cells were immunostained for B and less often for T lymphocyte. Necrotic cells in the paracortical region of the lymph node were less often and were immunostained. Necrotic lesions of the thymus were seen only in mice, with positively stained for T lymphocyte. The distribution of the lesions in the lymphoid tissues and the immunostaining in necrotic cells suggested that the active principles of the plant are cytotoxic to B and T cells.

Análise morfológica dos folículos primordiais de ovários de ovino criopreservados em EG, DMSO e sua associaçã o

Objective: Compare the cryoprotectants Dimethyl Sulphoxide (DMSO), Ethylene Glycol (EG) and their association for cryopreservation of sheep ovarian cortex. Methodology: Fragments collected from ovaries were divided into 3 parts. 1. One part from sample was destined for analysis of fresh material. 2. The second part was incubated with solution of freezing having 1,5M EG or 1,5M DMSO or 1,5MEG + 1,5M DMSO and washed for dilution of the cryoprotectants. 3. The third part was submitted to cryopreservation using the same cryoprotectans (EG 1,5M; DMSO 1,5M and EG + DMSO 1,5M) and cryopreserved. In all groups, one part of sample was submitted to pre-antral follicles isolation and the remainder was destined to ultra-structural analysis. Results: After isolation of fresh primordial follicles (control), the percentage of viable follicles was 78,9%. The percentage of viable follicles only exposed to cryoprotectants 1,5M EG, 1,5M DMSO and 1,5M EG + 1,5M DMSO was 77,1%, 68,4% and 60,7% respectively. After cryopreservation were 75%, 60% and 55,6% respectively. Ultra-structural analysis of the primordial follicles derived from fresh ovarian fragments or from fragments just exposed to the cryoprotectants showed similar morphology. However, in frozen samples, alterations of mitochondria were observed in all groups. Despite this, the integrity of the remained organelles was preserved in follicles cryopreserved with EG, while that in others groups (DMSO and association) an excess of vacuolizaton in cytoplasm of oocytes and swelling of nuclear membrane was observed indicating degeneration. Conclusion: The Ehilene Glycol seems to be the cryoprotector more adequated for cryopreservation of sheep ovarian tissue.

Avaliaçãeo ultrassonográfica da dinâmica folicular ovariana de jumenta (equus asinus) durante o ciclo estral

The ovarian activity was accessed by ultrasound throughout 10 estrous cycles from Jennies (Marchador Brasileira). Daily ultrasound examinations were performed and the dada was retrospectively studied based on daily identification of each follicle detected. Blood samples were collected every 24 hours from ovulation (D0) until the next identified ovulation. The follicles measuring 11mm were detected and their mean diameter was registered daily using an ovarian map and permitting a retrospective evaluation of the dada, which represented the follicular growth dynamics. One and two major follicular waves were detected in six (60%) and four (40%) cycles respectively from ten estrous cycles in the present study. The primary wave emergency and follicle deviation from the cycles observed during the present study with one major follicular wave occurred at day 10.2 ± 0.75 and at day14.1 ± 0.81 and for cycles with two major waves, those events occurred at 9.0 ± 2.0 and 14.0 ± 1.15 days after ovulation. The maximum diameter of the dominant follicle at the cycles with one and two follicular waves were 37.2 ± 3.35 mm and 37.3 ± 1.1, respectively. The mean intervals from two ovulations were 23.0 ± 1.79 and 22.3 ± 1.26 days when observed in cycles with one and two major follicular waves. The maximum diameter of the dominant follicle was slightly smaller when double ovulations were observed if compared with the single ovulations (P > 0.05). It can be concluded that wave emergency period is observed around 9.6 days of the estrous cycle and the occurrence of deviation can be observed on day 14 of the estrous cycle.

Correlações entre morfometria ovariana e hormônios em vacas zebus não prenhes

The study of ovarian morphometry is directly related to its practical applications to interpret the examination of the reproductive tract of the cow. The objective of this work was to study the ovarian morphometry collected in slaughterhouse and correlations between corpus luteum area, progesterone and cortisol in no pregnant zebu cows. One hundred fourteen pairs of ovaries were collected from slaughterhouse, measured in thickness, length, width and volume, diameter and volume of the follicle, diameter and area of the corpus luteum. Significative difference (p<0.05) were observed for widths of (1.95 cm and 1.83 cm) and volume (p<0.05) of 7.26 ml and 6.23 ml of left and right ovaries respectively. For size and volume of follicles, and diameter and area of the corpus luteum, there was no significative difference between the sides. There was correlation of 0.46 (p<0.01) between left ovary volume × luteum corpus area, in the presence of follicles with diameters equal or greater than 9 mm, the corpus luteum of the massive and protuberant type were present in 23 (43.39%) of 53 ovaries, predominating in relation to type of massive and incluse 16 (30.18%). There were no significative correlation between corpus luteum area × progesterone, corpus luteum area × cortisol and progesterone × cortisol. In conclusion, the presence of the included corpus luteum in zebu cows could result in error during the rectal palpationexam to estimate the ovarian activity.

Fatores preditivos do sucesso da inseminação intrauterina: A morfologia espermática de acordo com o novo manual de laboratório da Organização Mundial de Saúde

Objective: The aim of our study was to assess the likelihood of IUI success as a function of the previously described predictive factors, including sperm morphology according to the new reference values defined by WHO. Material and Methods: This retrospective study enrolled 300 couples which underwent IUI. Regression analyses were used to correlate maternal age, number of preovulatory follicles on the day of hCG administration, number of inseminated motile sperm, and normal sperm morphology with clinical pregnancy. Results are expressed as odds ratio (OR) with 95% of confidence intervals (CI). Results: Women older than 35 years showed a lower pregnancy rate (6.5% vs 18.2%, p=0.017). Logistic regression models confirmed the lower chance of pregnancy occurrence for older women (OR: 0.39; CI: 0.16-0.96; p=0.040). The presence of two or more preovulatory follicles on the day of hCG administration resulted in higher pregnancy rate when compared to cases in which only one preovulatory follicle was present (18.6% vs 8.2%, p=0.011). The regression model showed a more than two fold increase on probability of pregnancy when two or more preovulatory follicles were detected (OR: 2.58; CI: 1.22-5.46, p=0.013). The number of inseminated motile sperm positively influenced pregnancy occurrence (OR: 1.47; CI: 0.88-3.14, p=0.027). Similar pregnancy rates were observed when semen samples were classified as having normal or abnormal morphology (10.6% vs 10.2%, p=0.936). Conclusion: Our results demonstrate that sperm morphological normalcy, according to the new reference value, has no predictive value on IUI outcomes. © Todos os direitos reservados a SBRA - Sociedade Brasileira de Reprodução Assistida.

Cellular localization of androgen synthesis in equine granulosa-theca cell tumors: Immunohistochemical expression of 17α-hydroxylase/17,20-lyase cytochrome P450

Elevated blood testosterone concentrations, often accompanied by male-typical behaviors, is a common signalment of mares with granulosa-theca cell tumors (GCTCs), but no definitive information exists regarding the cellular differentiation of tumors associated with androgen secretion. This study was conducted to localize and thereby define the cellular expression of 17α-hydroxylase/17,20-lyase cytochrome P450 (P450c17), the enzyme most directly responsible for androgen synthesis, in 30 GTCTs and control tissues (gonads and adrenal glands) using immuno-histochemistry (IHC). Immuno-reactivity for P450c17 was evident in approximately half of 30 specimens examined, was most consistent in the interstitial cells surrounding existing or developing cysts, and was less intense in cells within cysts in the smaller proportion of specimens where this was observed. In control tissues, the expression of P450c17 was localized primarily in theca interna of normal ovarian follicles, in theca-lutein cells of some corpora lutea, but not in granulosa-lutein cells. Testicular interstitial cells and islands of adreno-cortical cells located in the adrenal medulla of the adrenal cortex further established the specificity of the antisera used. These data provided the first substantive evidence that polyhedral cells identified previously in GTCTs by histopathology have the potential to synthesize and secrete androgens, similar to theca interna and theca lutein cells in normal equine ovaries. © 2010 Elsevier Inc.

Physiological bases for understanding estrous cycle differences between bos taurus and bos indicus

Background: Although there is some information in the literature discussing differences of the estrous cycle of Bos taurus and Bos indicus cattle, most of the data derive from studies performed in temperate climate countries, under environmental and nutritional conditions very different than those found in tropical countries. Moreover, the physiological basis for understanding the differences between Bos taurus and Bos indicus estrous cycles are still unknown. This review explores the physiological and metabolic bases for understanding the key differences between the Bos taurus and Bos indicus estrous cycle. Moreover, it presents recent results of studies that have directly compared reproductive variables between Zebu and European cattle. Review: The knowledge of reproductive physiology, especially the differences between Bos taurus and Bos indicus, is important for the development and application of different techniques of reproductive management in cattle. In this regard, overall, Bos indicus have a greater number of small ovarian follicles and ovulatory follicles are smaller as compared to Bos taurus. Consequently, Zebu cattle also have smaller corpus luteum (CL). Nevertheless, circulating concentrations of steroid and metabolic hormones are not necessarily higher in European cattle. In fact, some studies have shown that despite ovulating smaller follicles and having smaller CL, Bos indicus cows or heifers have higher circulating concentrations of estradiol, progesterone, insulin and IGF-I compared to Bos taurus females. In addition, there are also substantial differences between Bos indicus and Bos taurus cattle in relation to follicle size at the time of selection of the dominant follicle. Conclusion: Data from very recent studies performed in Brazil have corroborated results from previous reports that have observed substantial differences in the estrous cycle variables of Bos indicus versus Bos taurus cattle. Those differences are probably related to distinct metabolism and metabolic hormone concentrations between Zebu and European cattle. This increased knowledge will allow for the establishment of more adequate reproductive management protocols in both breeds of cattle.

Locus no cromossomo 19 influencia positivamente o número de oócitos recuperados em ciclos estimulados

Objective: To evaluate if identified loci associated with normal age of menopause variation and early menopause can account for the poor response to controlled ovarian stimulation. Methods: A total of 71 patients, with age ≤ 35 years old, undergoing intracytoplasmic sperm injection were tested for three of the four newly identified genetic variants associated with normal variation in menopausal age and early menopause. Patients were divided into two groups: poor responder group (PR group, n=21) and normoresponder group (NR group, n=50). The influence of risk allele frequency on the response to controlled ovarian stimulation (COS) was evaluated. Logistic regression models were used. Results: There was no significant difference in the incidence of the genetic variants between NR and PR group. The risk allele for chromosome 19 variant (rs4806660) demonstrated a protective effect. The presence of a risk allele, either in homozygosis or in heterozygosis, was associated with an increased response to COS, resulting in an elevated number of follicles (Coef: 2.54, P= 0.041) and retrieved oocytes (Coef: 1.41, P= 0.041). Conclusions: Genetic variants rs244715, rs9379896 and rs4806660 are not risk factors for poor ovarian response. Instead, rs4806660 is associated with higher number of follicles and retrieved oocytes. It could be hypothesized that rs4806660 is associated with an increased response to gonadotrophin stimulus. © Todos os direitos reservados a SBRA - Sociedade Brasileira de Reprodução Assistida.

Mare's folliculogenesis: Assessment of ovarian and perifollicular vascular perfusion by doppler ultrasound

Background: Ultrasound Doppler is a new technology that has recently been used in large animal reproduction. As the conventional ultrasound (B-mode) it is a noninvasive technique, but with the advantage of allows the assessment of the hemodynamic of reproductive tract in real time. The observation of important features of the vascularization and changes that occur during physiological processes that were not previously seen on B-mode encourage a reassessment of the concepts already established on the events of the reproductive physiology of animals and their applications. Review: In attempt to re-understand the equine reproductive physiology and finding practical uses to this new technique, authors showed that, during the follicular deviation, features are observed by Doppler before being observed under B-mode ultrasound like changes in the speed of the blood flow two days before deviation of the follicle size and one day before the increase in blood flow area of the follicular wall. According to another study ovulation is characterized by a decreased blood flow of the follicular wall in the last four hours preceding it, as well as the serration of the granulosa layer and formation of a non vascularized apex, but in our ongoing study, the ability to decrease the vascularity was not found. Very vascularized follicles are associated with higher rates of oocyte maturation and pregnant that does less vascularized follicles in the preovulatory phase. Those follicles that have septated evacuation (or prolonged) showed more vascularization and serration of the granulosa one hour before ovulation than follicles that ovulate normally, and this vascularization includes the apex of the follicle, the follicular wall portion that is not vascularized in normal ovulation. Another study reported that hemorrhagic follicles have better vascularization of the follicular wall on the days preceding ovulation than follicles destined to ovulate. Some authors also showed that anovulatory follicles grow in size at the same rate as ovulatory follicles, but the percentage of vascularization of its wall is much smaller at 35 mm. Another study characterized that the vascular wall of the follicle that results in the first ovulation of the year is much smaller on the day before ovulation than the number of vessels present in a follicle that will ovulate in the middle of the breeding season. In these cases, the use of Doppler ultrasound can help to prevent economic losses as insemination of mares in cycles that are not able of resulting in pregnancy. This review aims to gather the information found in the literature about the characteristics of follicular hemodynamic of mares taking into account moments of deviation in follicle size, ovulation, ovulation failure and follicular viability. Conclusion: The Doppler technology has the potential to provide important information about the follicular environment and thus be used in practice in search of the perfect equine reproductive management, achieving better utilization of genetic material and increasing the financial return. The use of this new tool opens a large area for several interesting studies that will contribute to the knowledge of the physiological events of the mare for that this technique can soon be effectively applied.