Efeitos da rufiação e da manipulação do sistema genital sobre a fertilidade de éguas inseminadas

Estudaram-se os efeitos da rufiação e da manipulação do sistema genital (estocadas) sobre a fertilidade de éguas inseminadas com sêmen fresco (exp. 1) e resfriado/transportado a 14ºC/3,6 horas (exp. 2). No experimento 1 utilizaram-se 42 ciclos de 29 éguas, e no experimento 2, 148 ciclos de 100 éguas, distribuídos aleatoriamente em quatro grupos experimentais definidos por um esquema fatorial 2 x 2, com número desigual de repetições, de acordo com o tipo de rufiação utilizado (éguas rufiadas e não rufiadas) e o tipo de manipulação do sistema genital da fêmea no momento da inseminação (estocadas e não estocadas). Em ambos os experimentos não se observou efeito da estimulação sexual da égua sobre a fertilidade. No experimento 2 não se observou efeito da estimulação sexual sobre a dinâmica de crescimento folicular e ovulação.

Reduction in size of the ovulatory follicle reduces subsequent luteal size and pregnancy rate

We hypothesized that reducing the size of the ovulatory follicle using aspiration and GnRH would reduce the size of the resulting CL, reduce circulating progesterone concentrations, and alter conception rates. Lactating dairy cows (n=52) had synchronized ovulation and AI by treating with GnRH and PGF(2 alpha) as follows: Day -9, GnRH (100 mug); Day -2, PGF(2 alpha) (25 mg); Day 0, GnRH (100 mug); Day 1, AI. Treated cows (aspirated group; n=29) had all follicles > 4 mm in diameter aspirated on Days -5 or -6 in order to start a new follicular wave. Control cows (nonaspirated group; n=23) had no follicle aspiration. The size of follicles and CL were monitored by ultrasonography. The synchronized ovulation rate (ovulation rate to second GnRH injection; 42/52=80.8%) and double ovulation rate of synchronized cows (6/42=14.3%) did not differ (P > 0.05) between groups. Aspiration reduced the size of the ovulatory follicle (P < 0.0001; 11.5 +/- 0.2 vs 14.5 +/- 0.4 mm), and serum estradiol concentrations at second GnRH treatment (P < 0.0002; 2.5 +/- 0.4 vs 5.7 +/- 0.6 pg/mL). The volume of CL was less (P < 0.05) for aspirated than nonaspirated cows on Day 7 (2,862 +/- 228 vs 5,363 +/- 342 mm(3)) or Day 14 (4,652 +/- 283 vs 6,526 +/- 373 mm(3)). Similarly, serum progesterone concentrations were less on Day 7 (P < 0.05) and Day 14 (P < 0.10) for aspirated cows. Pregnancy rate per AI for synchronized cows was lower (P < 0.05) for aspirated (3/21=14.3%) than nonaspirated (10/21=47.6%) cows. In conclusion, ovulation of smaller follicles produced lowered fertility possibly because development of smaller CL decreased circulating progesterone concentrations. (C) 2001 by Elsevier B.V.

Influência do fator de crescimento semelhante à insulina I (IGF-I) adicionado ao meio fluido sintético de tuba uterina (SOF) sobre a maturação in vitro de oócitos caninos (Canis familiaris)

The follicular growth and oocyte maturation knowledge are very important to the development and improvement of new biotechnologies such as in vitro fertilization and somatic cell nuclear transfer. In order to the necessity of clarify the basic mechanisms related to canine oocyte maturation, this investigation focuses on the evaluation of the effect of insulin-like growth factor-i (IGF-I), added to synthetic oviductal fluid medium (SOF) on the in vitro maturation of domestic dog oocytes. Thirty-seven bitches undergoing ovariohysterectomy for castration or due to pathological conditions of the uterus were selected as oocytes' donors (n=875). The oocytes were allocated in the following groups: MO (stained in the collection's time), Control (72h in SOF) and Experimental (72h in SOF plus 100 ng IGF-I). After 72 hours of maturation the oocytes' nuclear status were assessed by Hoechst 33342 dye. The best results in terms of oocyte harvest were observed in those juvenile donors,, females in estrus, nuliparous and pure breeds. No significant differences were observed between treatments control (SOF) or experimental (IGF-I).

CYTOMORPHOLOGICAL AND IMMUNOCYTOCHEMICAL EVALUATION OF FINE NEEDLE ASPIRATES FROM HIGH-GRADE NON-HODGKINS-LYMPHOMAS

1. Fine needle aspirates from ten patients with high-grade malignant non-Hodgkin's lymphomas were analyzed by cytomorphology and immunocytochemistry.2. The following morphologic diagnoses were made: lymphoblastic lymphoma (3 cases), Burkitt's lymphoma (3 cases), mixed small and large cell lymphomas with predominance of large cells (2 cases), and centroblastic lymphoma (2 cases). Immunocytochemistry showed a B-cell phenotype in five cases and a T-cell phenotype in four. One case of lymphoblastic lymphoma was negative for the T and B cell markers used.3. The results of histological and immunohistochemical analyses performed on surgical biopsies from 8 patients confirmed the morphological diagnosis in all cases. Two cases of Burkitt's lymphoma were submitted only to cytological and immunological diagnosis.4. The high diagnostic accuracy of combined cytomorphology and immunocytochemical assessment of fine needle aspirate samples validates the use of the technique in the diagnostic work-up of high-grade non Hodgkin's lymphomas.

Risk factors for amenorrhea in juvenile systemic lupus erythematosus (JSLE): a Brazilian multicentre cohort study

We evaluated the prevalence and clinical associations of amenorrhea in 298 female juvenile systemic lupus erythematosus (JSLE) patients (ACR criteria) followed in 12 Brazilian Paediatric Rheumatology centres. Amenorrhea was observed in 35 patients (11.7%) with a mean duration of 7.2 +/- 3.6 months. The hormones were performed in 32/35 patients and none of them had FSH and LH levels above and estradiol below the normal range according to pubertal changes. JSLE patients with amenorrhea were younger (15.04 +/- 2.5 versus 17.8 +/- 3.1 years; P = 0.001), and had a shorter period of time between menarche and current age (3.4 +/- 2.9 versus 6.7 +/- 5.4 years; P = 0.001). Interestingly, the frequency, cumulative dose, number of pulses and duration of intravenous cyclophosphamide treatment were alike in patients with and without amenorrhea (P > 0.05). In contrast, patients with amenorrhea had significantly higher SLEDAI (P = 0.01) and SLICC/ACR-DI (P = 0.024) scores compared to those without this condition. Independent risk factors identified by multivariate analysis were higher SLEDAI (OR=1.059; CI=1.004-1.116; P=0.034) and SLICC/ACR-DI (OR=2.125; IC = 1.373-3.291; P = 0.001) scores. Our data suggest that in spite of imummosuppressive therapy, JSLE patients have an adequate ovarian follicular reserve and amenorrhea is particularly associated with disease activity and damage.

Epstein-Barr virus (EBV) nuclear antigen (EBNA)-4 mutation in EBV-associated malignancies in three different populations

Different ethnic groups with a high human leukocyte antigen (HLA)-A11 prevalence have been shown to experience a high rate of Epstein-Barr virus (EBV) infection, EBV-associated malignancies, and Epstein-Barr nuclear antigen (EBNA)-4 mutations. The epitopes 393-408 and 416-424 of EBNA-4 are major antigenic epitopes that elicit an HLA-A11 cytotoxic T lymphocyte (CTL) response to EBV infection. Mutations selectively involving one or more nucleotide residues in these epitopes affect the antigenicity of EBNA-4, because the mutant EBV strains are not recognized by the HLA-A11-restricted CTLs. To investigate these mutations in common EBV-associated malignancies occurring in different populations, we studied the mutation rate of epitopes 393-408 and 416-424 of EBNA-4 in 25 cases of EBV-associated Hodgkin's disease (HD), nine cases of AIDS-related non-Hodgkin's lymphoma, and 37 cases of EBV-associated gastric carcinoma (GC) from the United States, Brazil, and Japan. We found one or more mutations in these two epitopes in 50% (6/12) of United States HD, 15% (2/13) of Brazilian HD, 50% (6/12) United States GC and 28% (7/25) Japanese GC, and 22% (2/9) of United States AIDS-lymphoma. Similar mutations were found in 30% (3/10) of United States reactive, 0% (0/6) of Brazilian reactive, and 25% (2/8) Japanese reactive tissues. The most frequent amino acid substitutions were virtually identical to those seen in previously reported isolates from EBV-associated nasopharyngeal carcinomas and Burkitt's lymphomas occurring in high prevalence HLA-A11 regions. However, only 2/28 (7%) mutations occurred in HLA-A11-positive patients. Our studies suggest that: 1) EBNA-4 mutations are a common phenomenon in EBV-associated HD, GC, and AIDS-lymphoma; 2) the mutation rate does not vary in these geographic areas and ethnic groups; 3) EBNA-4 mutations in EBV-associated United States and Brazilian HD, United States and Japanese GC, and United States AIDS lymphomas are not related to patients' HLA-A11 status.

Transplante ovariano: aplicações na reprodução de animais domésticos, silvestres e humanos

Ovarian transplantation constitutes a technique in which one ovary or part of this is transferred from a donor to recipients that may be the same individual, other recipient of the same specie or no, near or far from the original anatomical site. This review covers important concepts for the elucidation of the main events of folliculogenesis and follicular maturation as well as the applications of ovarian transplantation and prospects of this technique in the breeding of domestic animals, wildlife and humans. Other studies are still necessary to better understanding of the events regarding to the ovarian development, interactions between donor and receptor tissues, their site of transplantation as well as individuals and involved species.

Población folicular y concentraciones plasmáticas de progesterona (P4) en ovejas sometidas a diferentes protocolos de sincronización

Los efectos de la prostaglandina (PGF2α) vs CIDR y eCG (gonadotrofina coriónica equina) en la dinámica de la población folicular y su relación con las concentraciones plasmáticas de P4 fueron investigadas en ovejas cíclicas. Fueron utilizadas 14 hembras ovinas de la raza Bergamascia; el Grupo 1 (Gl) se sometió a dos aplicaciones de PGF2α, y, el Grupo 2 (G2) tratado con CIDR durante 14 días siendo que, en el momento de su retirada, se administraron 500 UI de eCG. La dinámica folicular ovárica fue monitoreada por medio de ecógrafo. Se monitorearon todos los folículos ≥ mm y se gráfico su posición diariamente, observándose el desarrollo individual folicular. Desde el día anterior a la aplicación de la segunda dosis de PGF2α, (Gl) y desde la administración de eCG (G2) hasta el décimo día del ciclo estral se colectaron muestras de sangre para el análisis de P4. Hubo diferencia significativa (P<0,001) en las concentraciones plasmáticas de P4 entre los tratamientos. La sincronización del estro y ovulación utilizando CIDR + 500 UI de eCG, incrementó la cantidad de folículos reclutados, además de aumentar el diámetro máximo y la tasa de crecimiento de los folículos grandes en la primera onda folicular. En consideración a los resultados se puede concluir que la sincronización del estro y de la ovulación en hembras ovinas, utilizando el CIDR y 500 UI de eCG, incrementa la cantidad de folículos reclutados, además de aumentar el diámetro máximo y la tasa de crecimiento de los folículos grandes. La asociación CIDR+500 UI de eCG provoca aumentos significativos en las concentraciones plasmáticas de progesterona (P4) al inicio de la fase luteal en hembras ovinas.

Fibroblast Growth Factor-2 Regulation of Sprouty and NR4A Genes in Bovine Ovarian Granulosa Cells

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Reproductive cycles in Bos indicus cattle

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Productive and reproductive performance of heifers fed different levels of urea in the diet

The objective of the present study was to evaluate the nutrient intake, blood parameters, follicular diameter and performance of pre-puberty crossbred heifers fed isoproteic diets (14.1%CP) containing 0.0; 0.44; 0.88 and 1.32% urea on the total dry matter (DM) of the diet, with a 77:23 roughage:concentrate ratio. Twenty-four 18- month old heifers (Holstein x Zebu), 277.9 kg mean live weight (LW) were used, distributed in four treatments and six replications in a randomized complete design. The following were evaluated: dry matter intake (DM), crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF), ether extract (EE), hemicellulose (HEM), plasmatic ureic nitrogen (PUN), plasmatic glucose, plasmatic total cholesterol, follicular diameter and daily weight gain (DWG). No influence of the urea levels in the diet was observed on NDF and HEM intake. A maximum intake was obtained of DM (8.75 kg day(-1)), CP (0.88 kg day(-1)), ADF (2.5 kg day(-1)) and EE (0.17 kg day(-1)) respectively for the levels 0.7, 0.8, 0.7 and 0.7% urea in total DM. The 0.6%; 0.77% and 0.6% urea levels in diet were the critical points for obtaining maximum response for the PUN (10.96 mg dL(-1)) and plasmatic glucose (84.97 mg dL(-1)) concentrations and, for follicular diameter (11.08 mm) on the 40(th); 24(th) and 31(st) day, respectively. The plasmatic total cholesterol concentration and DWG were not influenced by the urea added to the diet, with averages of 119.39 mg dL(-1) and 1.66 kg day(-1), respectively. It was concluded that urea can be added up to 1.32% on the total DM of the diet for pre-puberty crossbred heifers.

Oocyte morphometry in female dogs (Canis familiaris, Linnaeus, 1758)

In the present study, the ovaries of 15 healthy bitches divided into three groups according to age were analysed by histology and morphometry: group 1 (1-3 years), group 2 (> 3-5 years) and group 3 (> 5-7 years). After ovariosalpingohysterectomy, the ovaries were fixed, routinely processed for embedding in paraffin and stained with haematoxylin-eosin. The following morphometric parameters were analysed: maximum and minimum diameter (mu m), perimeter (mu m), area (mu m(2)) and roundness of the cytoplasm and nucleus of oocytes from different types of follicles. Significant differences in the cytoplasmic and nuclear parameters of follicular oocytes between the experimental groups were determined by ANOVA and the Tukey test (5%). For the biotechnology of reproduction, the present results showed that in vitro maturation yielded the best performance for oocytes from primordial follicles in group 3 females and for oocytes embedded in secondary and tertiary follicles in group 2 females. In addition, the present findings will help in the diagnosis and prognosis of diseases affecting bitches, and will especially contribute to a better understanding of these cells by researchers in the field of histology and canine reproduction.

Fetal and maternal lesions of cyanide dosing to pregnant goats

The present work evaluated the pathological effects in both dams and their fetuses of cyanide ingestion during pregnancy using goat as animal model. From the Day 24 of pregnancy, three pregnant goats were dosed by gavage with 3.0 mg KCN/kg bw/day, and two others received only tap water. All dams were euthanized and necropsied at Day 120 of pregnancy. The fetuses were examined carefully for gross abnormalities. Determinations of cyanide and thiocyanate were performed in maternal and fetal blood and in amniotic fluid. Samples of several organs were collected for histopathologic evaluation. No clinical changes were seen in any animal throughout the pregnancy. No malformations or dead fetuses were observed: however, placenta from one treated goat presented increased cotyledon surface area occupied by vascular lumina. Histological lesions in KCN-treated dams consisted of vacuolation of hepatocytes and thyroid follicular cells, increased number of vacuoles on thyroid follicular colloid. and spongiosis of cerebral, internal capsule, and cerebellar peduncles tracts. Fetuses from these mothers showed vacuolation of hepatocytes and thyroid follicular cells, and spheroids in the cerebellar white matter. Levels of cyanide and thiocyanate were higher in maternal than fetal blood, which suggests that these substances were largely but not freely transferred from mothers to fetuses. (C) 2009 Elsevier B.V. All rights reserved.

Expression and Function of Fibroblast Growth Factor 18 in the Ovarian Follicle in Cattle

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Ultrastructural analysis of bovine oocytes from ovarian follicles with different diameters

In vitro embryo production is an important technique for facilitating the reproduction of animals with high genetic merit. The greatest challenge for the reproducibility of this technique is the quality of the oocyte that is submitted for in vitro maturation. The aim of this work was to evaluate the ultrastructural characteristics of oocytes from follicles of different diameters using transmission electron microscopy. The animals were divided into 2 groups and were given a single i.m. injection of 250 IU FSH (Pluset, Serono, Italy). To synchronize the follicular growth, all follicles > 2 mm were aspirated during the estrous cycle, which was considered day zero (D0). Group 1 (G1; n = 4) received FSH on day 1 (D1), and the 2- to 5-mm follicles were aspirated on day 2 (D2). The animals in group 2 (G2; n = 5) received FSH on day 2 (D2), and their 10- to 15-mm follicles were aspirated on day 5 (D5). After aspiration, the oocytes from both groups were fixed and prepared for ultrastructural analysis. The oocytes analyzed from both groups had similar ultrastructural characteristics. The presence and distribution of organelles in the cytoplasm of the oocytes did not differ between groups, suggesting that, in relation to the ultrastructural characteristics, oocytes from 2 to 5 mm and 10 to 15 mm follicles are similar.