Genetic analysis and cAMP measurement: comparison between lean and obese anovulating mice

PURPOSE: To evaluate genes differentially expressed in ovaries from lean (wild type) and obese (ob/ob) female mice and cyclic AMP production in both groups.METHODS: The expression on messenger RNA levels of 84 genes concerning obesity was analyzed through the PCR array, and cyclic AMP was quantified by the enzyme immunoassay method.RESULTS: The most downregulated genes in the Obesity Group included adenylate cyclase-activating polypeptide type 1, somatostatin, apolipoprotein A4, pancreatic colipase, and interleukin-1 beta. The mean decrease in expression levels of these genes was around 96, 40, 9, 4.2 and 3.6-fold, respectively. On the other hand, the most upregulated genes in the Obesity Group were receptor (calcitonin) activity-modifying protein 3, peroxisome proliferator activated receptor alpha, calcitonin receptor, and corticotropin-releasing hormone receptor 1. The increase means in the expression levels of such genes were 2.3, 2.7, 4.8 and 6.3-fold, respectively. The ovarian cyclic AMP production was significantly higher in ob/ob female mice (2,229±52 fMol) compared to the Control Group (1,814±45 fMol).CONCLUSIONS: Obese and anovulatory female mice have reduced reproductive hormone levels and altered ovogenesis. Several genes have their expression levels altered when leptin is absent, especially adenylate cyclase-activating polypeptide type 1.

Comparative pathogenicity of bovine herpesvirus 1 (BHV-1) subtypes 1 (BHV-1.1) and 2a (BHV-1.2a)

The study aimed to examine the capacity of two bovine herpesvirus type 1 (BHV-1) isolates of different subtypes (EVI 123/96, BHV-1.1; SV265/98, BHV-1.2a) to induce respiratory disease in calves. These two isolates are representative of the BHV-1 subtypes prevalent in Brazil. Viral subtypes were confirmed by monoclonal antibody analysis and by restriction enzyme digestion of viral genomes. The viruses were inoculated intranasally into seven 3 months old calves (four with BHV-1.1, three with BHV-1.2a). Three other calves of identical age and condition were kept as uninfected controls. In both groups of infected calves, the clinical signs observed were consistent with typical infectious bovine rhinothracheitis (IBR), including pyrexia, apathy, anorexia, nasal and ocular mucopurulent discharges, erosions on the nasal mucosa, conjunctivitis, lachrymation, redness of nasal mucosa, dyspnoea, coughing, tracheal stridor and enlargement of retropharingeal, submandibular and cervical lymphnodes. No significant differences were observed between the clinical scores attributed to both groups. Virus shedding in nasal and ocular secretions were also similar, apart from a significant difference in nasal virus shedding on day 1 to 3 post-inoculation, which was higher for BHV-1.1 than for BHV-1.2a. Following corticosteroid induced reactivation of the latent infection, recrudescence of clinical signs was also observed, with no significant differences on both groups. It was concluded that both subtypes BHV-1.1 and BHV-1.2a were able to induce clinically undistinguishable respiratory disease in calves, either subsequent to a primary infection or following reactivation.

Novel Functions of ErbB4 and NRG-1 in Development and Cancer

Cells communicate, or signal, with each other constantly to ensure proper functioning of tissues and organs. Cell signaling is often performed by interplay of receptors and ligands that bind these receptors. ErbB receptors (epidermal growth factor receptors, EGFR, HER) bind extracellular growth factors and transduce these signals inside of cells. ErbB dysfunction promotes carcinogenesis, and also results in numerous defects during normal development. This study focused on the functions of one member of the ErbB receptor family, ErbB4, and growth factor, neuregulin-1 (NRG-1), that can bind and activate ErbB4. This study aimed to find novel functions of ErbB4 and NRG-1. Hypoxia, or deficiency of oxygen, is common in cancer and ischemic conditions. One of the key findings of the work was the identification and characterization of a cross-talk between ErbB4 and Hypoxia-inducible factor 1α (HIF-1α), the central mediator of hypoxia signaling. ErbB4 activation by NRG-1 was found to increase HIF-1α activity. Interestingly, this regulation occurred in reciprocal manner as HIF-1α was also able to increase protein levels of NRG-1 and ErbB4. Moreover, expression of NRG-1 and ErbB4 was associated with HIF activity in vivo in human clinical samples and in mice. Reduction of functional ErbB4 in developing zebrafish embryos resulted in defects in development of the skeletal muscles. To study ErbB4 functions in pathological situation in humans, clinical samples of serous ovarian carcinoma were analyzed using tissue microarrays and real-time RT-PCR. A specific isoform of ErbB4, CYT-1, was associated with poor survival in serous ovarian cancer and increased anchorage independent growth of ovarian cancer cells in vitro. These observations demonstrate that ErbB4 and NRG-1 are essential regulators of cellular response to hypoxia, of development, and of ovarian carcinogenesis.

Ultrasonographic and radiographic determination of egg development of jurarás (Kinosternon scorpioides) in captivity

Kinosternon scorpioides (Linnaeus, 1766), with its common name of jurará, is a quite variable species of turtles, and many different names have been applied to populations throughout its range. Currently, however, four subspecies are considered valid as K. scorpioides arises from southern Panama over most of northern South America and is found in Ecuador, northern Peru, southern Bolivia, northern Argentina, eastern Guyana and Brazil. Thus, an ultrasonographic and radiographic study was performed in order to describe the morphology and development of eggs of 20 female jurará mud turtles K. scorpioides, from September 2005 to August 2006. In the first month, the ovarian cycle was characterized by absence of vitellogenic follicles, atresic follicles or oviduct eggs. From October 2005 to March 2006 on, ultrasonographic scanning allowed to establish the growing vitellogenic follicles. Vitellogenic follicles were observed with spherical to ovoid shapes, with a surrounding echogenic yolk, a nonechogenic albumin layer, and a high echogenic shell. The oviduct eggs were identified by radiography just 180 days after beginning the experiment, when the shell became enough mineralized to impress the radiographic film. This experiment allowed to obtain by means of the 7.5 MHz linear probe images with adequate resolution and penetration for visualization of follicles. Successive ultrasonographic examinations of 20 K. scorpioides females allowed to access initial stages of vitellogenic follicles and oviduct eggs, and radiographic examination revealed to be an easy technique to assess oviduct eggs and to allow evaluation of egg development in jurarás, from 6 months on.

Genital immunization of heifers with a glycoprotein Edeleted, recombinant bovine herpesvirus 1 strain confers protection upon challenge with a virulent isolate

Venereal infection of seronegative heifers and cows with bovine herpesvirus type 1.2 (BoHV-1.2) frequently results in vulvovaginitis and transient infertility. Parenteral immunization with inactivated or modified live BoHV-1 vaccines often fails in conferring protection upon genital challenge. We herein report an evaluation of the immune response and protection conferred by genital vaccination of heifers with a glycoprotein E-deleted recombinant virus (SV265gE-). A group of six seronegative heifers was vaccinated with SV265gE- (0,2mL containing 10(6.9)TCID50) in the vulva submucosa (group IV); four heifers were vaccinated intramuscularly (group IM, 1mL containing 10(7.6)TCID50) and four heifers remained as non-vaccinated controls. Heifers vaccinated IV developed mild, transient local edema and hyperemia and shed low amounts of virus for a few days after vaccination, yet a sentinel heifer maintained in close contact did not seroconvert. Attempts to reactivate the vaccine virus in two IV vaccinated heifers by intravenous administration of dexamethasone (0.5mg/kg) at day 70 pv failed since no virus shedding, recrudescence of genital signs or seroconversion were observed. At day 70 pv, all vaccinated and control heifers were challenged by genital inoculation of a highly virulent BoHV-1.2 isolate (SV56/90, 10(7.1)TCID50/animal). After challenge, virus shedding was detected in genital secretions of control animals for 8.2 days (8-9); in the IM group for 6.2 days (4-8 days) and during 5.2 days (5-6 days) in the IV group. Control non-vaccinated heifers developed moderate (2/4) or severe (2/4) vulvovaginitis lasting 9 to 13 days (x: 10.7 days). The disease was characterized by vulvar edema, vulvo-vestibular congestion, vesicles progressing to coalescence and erosions, fibrino-necrotic plaques and fibrinopurulent exudate. IM vaccinated heifers developed mild (1/3) or moderate (3/4) genital lesions, lasting 10 to 12 days (x: 10.7 days); and IV vaccinated heifers developed mild and transient vulvovaginitis (3/4) or mild to moderate genital lesions (1/4). In the IV group, the clinical signs lasted 4 to 8 days (x: 5.5 days). Clinical examination of the animals after challenge revealed that vaccination by both routes conferred some degree of protection, yet IV vaccination was clearly more effective in reducing the severity and duration of clinical disease. Furthermore, IV vaccination reduced the period of virus shedding in comparison with both groups. Taken together, these results demonstrate that SV265gE- is sufficiently attenuated upon IV vaccination in a low-titer dosis, is not readily reactivated after corticosteroid treatment and lastly, and more importantly, confers local protection upon challenge with a high titer of a virulent heterologous BoHV-1 isolate. Therefore, the use of this recombinant for genital immunization may be considered for prevention of BoHV-1-associated genital disease in the field.

Effect of bone morphogenetic protein-7 (BMP-7) on in vitro survival of caprine preantral follicles

This study was conducted in order to verify the effect of different concentrations of BMP-7 in the in vitro survival and development of caprine preantral follicles. Fragments of caprine ovarian cortical tissue were cultured for 1 or 7 days in Minimum Essential Medium (MEM+) supplemented with different concentrations of BMP-7 (1, 10, 50 or 100ng/ml). Non-cultured fragments or those cultured for 1 or 7 days were processed for classical histology and transmission electron microscopy (TEM). Parameters such as follicular survival, activation and growth were evaluated. The results showed that, after 1 or 7 days of culture, the percentage of morphologically normal follicles was significantly reduced in all treatments when compared with fresh control, except at 1ng/ml of BMP-7 for 1 day. In addition, the concentration of 10ng/ml of BMP-7 significantly increases follicular diameter from day 1 to 7 of culture. There was no influence of the other concentrations of BMP-7 regarding to the follicular and oocyte diameter. Ultrastructure studies confirmed follicular integrity after 7 days of culture in 1ng/ml BMP-7. In conclusion, small concentrations of BMP-7 can improve the survival and growth of caprine preantral follicles during in vitro culture.

Bone Morphogenetic Protein-6 (BMP-6) induces atresia in goat primordial follicles cultured in vitro

This study investigated the effects of bone morphogenetic protein 6 (BMP-6) on in vitro primordial follicle development in goats. Samples of goat ovarian cortex were cultured in vitro for 1 or 7 days in Minimum Essential Medium (control medium) supplemented with different concentrations of BMP-6. Follicle survival, activation and growth were evaluated through histology and transmission electron microscopy (TEM). After 7 days of culture, histological analysis demonstrated that BMP-6 enhanced the percentages of atretic primordial follicles when compared to fresh control (day 0). Nevertheless, BMP-6 increased follicular and oocyte diameter during both culture periods. As the culture period progressed from day 1 to day 7, a significant increase in follicle diameter was observed with 1 or 50ng/ml BMP-6. However, on the contrary to that observed with the control medium TEM revealed that follicles cultured for up to 7 days with 1 or 50ng/ml BMP-6 had evident signs of atresia. In conclusion, this study demonstrated that BMP-6 negatively affects the survival and ultrastructure of goat primordial follicles.

Records of performance and sanitary status from a dairy cattle herd in southern Brazil

Over the last decades, the emphasis on the health of dairy cows has changed from an individual to a herd level. In this scenario, the role played by the recording system and its interpretation by veterinarians has gained primordial importance. The records of productive and reproductive performance and of sanitary status from a southern Brazilian dairy cattle herd have been presented and discussed. The period of study was 2000-2009. Mean values per lactation period were 349D 8436M 290F 275P 201SCS (D: days in lactation, M: kg of milk yield, F: kg of fat, P: kg of protein and SCS: somatic cell score in 1000 cells/ml of milk). Major indexes of reproductive efficiency included age at first calving (31 months), services per conception (2.1), intercalving interval (428 days), calving to conception interval (146 days), mean annual rates of parturitions (76.2%), fetal losses (9.8-19.0%), and stillbirths (3.6%), apart of voluntary waiting period (94 days). Main information on sanitary status of the herd was associated with the mean prevalence of common disorders of dairy cattle such as anaplasmosis (29.8%), mastitis (27.8%), digital diseases (26.3%), ovarian cysts (21.3%), placental retention (19.7%), postpartum uterine infections (10.6%), and calf diarrhea (23.7%) and pneumonia (16.8%), among others. In addition, culling reasons (low reproductive performance [56.3%] and udder/mastitis problems [33.6%]), causes of cattle deaths (anaplasmosis [16.4%] and leukosis [11.4]), and the impact of cattle diseases such as tuberculosis, leukosis, and neosporosis on the herd have also been presented and succinctly discussed. Numbers between brackets represent rates accumulated in the 10-year period.

Progesterone and Follicle Stimulating Hormone interact and promote goat preantral follicles survival and development in vitro

We investigated the effects of progesterone and follicle stimulating hormone (FSH) on survival and growth of caprine preantral follicles. Pieces of ovarian tissue were cultured for 1 or 7 days in minimum essential medium (MEM) alone or containing progesterone (1, 2.5, 5, 10 or 20ng/mL), FSH (50ng/mL) or the interaction between progesterone and FSH. Fresh (non-cultured control) and cultured ovarian tissues were processed for histological and ultrastructural studies. After 7 days the addition of FSH to all progesterone concentrations maintained the percentage of normal follicles similar to fresh control. At day 7 of culture, a higher percentage of developing follicles was observed only in 2.5ng/ml of progesterone associated with FSH or 10ng/ml of progesterone alone when compared with control. From day 1 to day 7 of culture, a significant increase in the percentage of developing follicles was observed in MEM and 2.5ng/ml of progesterone + FSH. In addition, after 7 days, in all treatments, there was a significant increase in follicular diameter when compared with control, except for MEM alone and in 5ng/ml of progesterone + FSH or 10ng/ml of progesterone alone. Ultrastructural studies confirmed follicular integrity after 7 days of culture in 2.5ng/ml of progesterone with FSH. In conclusion, this study demonstrated that the interaction between progesterone and FSH maintains ultrastructural integrity, stimulates primordial follicles activation and further growth of cultured caprine preantral follicles.

Morphological aspects of the ovaries of turtle Kinosternon scorpioides raised in captivity

The swear turtle "jurará" (Kinosternon scorpioides) is a mud turtle of the Amazon region exposed to disordering capture in the rural areas of Maranhão, Brazil. Despite its popularity in these areas, little meaningful information regarding the reproductive morphology is currently available, fact that impedes the adoption of policies for preservation of the species. To obtain more information, we studied the ovarian morphology adult jurará females kept in captivity by morphological and morphometric analysis in the dry and rainy season. The results revealed that all females were sexually mature and were in a vitellogenic period. The ovaries are two irregular structures composed by follicles in different stages of development (primary, secondary and tertiary) scattered in a stroma of loose connective highly vascularized tissue. The ovary weight was 6.25±4.23g and 2.27±1.42g, for the right and left one respectively. The gonadosomatic indexes were 2.06% for the dry season and 1.79% for the rainy season. The average of the follicles was 29.83 units per ovary. Microscopically, the mature ovaries revealed a basal layer composed by four cellular layers: the inner and outer theca, stratum granulosum with perivitelline membrane and zona radiata with vitelline membrane. No significant differences were observed in the ovaries either in the dry or wet period.

Morphological and morphometric evaluation of prepubertal gilt ovaries, uterine tubes and uterus at different oestrus cycle stages

Studies are performed in developing techniques/procedures that provide greater reproductive performance in farm animals, including pigs. In this sense, the study of gilts reproductive organs at different oestrus cycle stages for assessing the presence of abnormalities and/or other parameters that may affect the future animal fertility is important. In order to evaluate the morphological, morphometric and histomorphometric features of ovaries, uterus and uterine tubes (UTs) characteristics of prepubertal gilts at different oestrus cycle stages, reproductive tracts from 48 animals immediately after slaughter were obtained. After, the structures were dissected and removed, and the ovaries were used for classification of oestrus cycle stage of each gilt in follicular phase (FP) and luteal phase (FL). Then, morphometric evaluations of ovaries, UTs, uterine horns and uterine body were performed. Besides that, medial segments of UTs and uterus were fixed in Bouin solution, processed and included in paraffin, when histological sections of 5.0 micrometers (µm) were obtained and stained with Hematoxylin and Eosin. Histomorphometric analyzes using image capture system and specific software were performed. Afterwards, data were submitted to Student's t test for assessment the statistical differences (P<0.05) between the two different oestrus cycle stages (FP × LP) and between the placement of reproductive structures (right × left antimer). Among the gilts evaluated, 35 were in the FP and 13 in LP. There was no difference (P>0.05) between morphometric parameters of ovaries, UTs and uterus of gilts in FP and LP. Likewise, in respect to the placement of reproductive structures, both in the oestrus cycle stages, as in the general average, there was no difference (P>0.05). Regarding the histomorphometric variables, gilts classified in FP presented a higher (P<0.05) height of glandular and UT epithelium compared to animals in LP. On the other hand, the diameter of endometrial glands was higher (P<0.05) in gilts at LP compared to FP. Furthermore, gilts in LP presented a higher (P<0.05) proportion of endometrium occupied by glands, whereas animals in FP had a higher (P<0.05) proportion of connective tissue and blood vessels. In conclusion, in prepubertal gilts, the histomorphometric parameters as endometrial glands diameter, the height of glandular epithelium and of UT epithelium and the proportion of endometrium occupied by connective tissue, besides the glands and blood vessels varies through the oestrus cycle, possibly under the influence of ovarian steroids.

Influence of Insulin-like Growth Factor I (IGF-I) on the survival and the in vitro development of caprine preantral follicles

The aim of this study was to investigate the effects of the insulin-like growth factor -I (IGF-I) on survival, activation (transition from primordial to primary follicles) and growth of caprine preantral follicles cultured in vitro. Fragments of ovarian cortex were cultured for one and seven days in the absence or presence of IGF-I (0, 50 and 100ng/ml). The non-cultured and cultured tissues were processed and analyzed by histology and transmission electron microscopy. The culture for one day in a medium with 100ng/ml of IGF-I showed 86.7% of morphologically normal follicles. These results were similar (P>0.05) to the percentage of normal follicles found in the control (96.7%). It was also found that this medium increased the percentage of follicular activation (developing follicles) with one day of culture. The oocyte and follicular diameters remained similar to the control by culturing for one day in a medium containing 100ng/ml of IGF-I. The ultrastructural analysis did not confirm the integrity of the follicular fragments in a medium containing IGF-I (100ng/ml) after one and seven days of culture. In conclusion, this study demonstrated that the addition of 100 ng/ml of IGF-I in the culture medium enables the development of preantral follicles of goats with one day of culture. However, it is not sufficient to maintain the follicular integrity and the follicular survival rate after seven days of culture.

Estrus cycle monitoring of captive collared peccaries (Pecari tajacu) in semiarid conditions

Collared peccaries (Peccary tajacu) are among the most hunted species in Latin America due the appreciation of their pelt and meat. In order to optimize breeding management of captive born collared peccaries in semiarid conditions, the objective was to describe and correlate the changes in the ovarian ultrasonographic pattern, hormonal profile, vulvar appearance, and vaginal cytology during the estrus cycle in this species. During 45 days, females (n=4) were subjected each three days to blood collection destined to hormonal dosage by enzyme immunoassay (EIA). In the same occasions, evaluation of external genitalia, ovarian ultrasonography and vaginal cytology were conducted. Results are presented as means and standard deviations. According to hormonal dosage, six estrous cycles were identified as lasting 21.0 ± 5.7 days, being on average 6 days for the estrogenic phase and 15 days for the progesterone phase. Estrogen presented mean peak values of 55.6 ± 20.5 pg/mL. During the luteal phase, the high values for progesterone were 35.3 ± 4.4 ng/mL. The presence of vaginal mucus, a reddish vaginal mucosa and the separation of the vulvar lips were verified in all animals during the estrogenic peak. Through ultrasonography, ovarian follicles measuring 0.2±0.1 cm were visualized during the estrogen peak. Corpora lutea presented hyperechoic regions measuring 0.4±0.2 cm identified during luteal phase. No significant differences (P>0.05) between proportions of vaginal epithelial cells were identified when comparing estrogenic and progesterone phases. In conclusion, female collared peccaries, captive born in semiarid conditions, have an estral cycle that lasts 21.0±5.7 days, with estrous signs characterized by vulvar lips edema and hyperemic vaginal mucosa, coinciding with developed follicles and high estrogen levels.

Mode B ultrasonography and abdominal Doppler in crab-eating-foxes ( Cerdocyon thous )

Abstract: Annually hundreds of crab-eating foxes (Cerdocyon thous) are referred to rehabilitation centers and zoos in Brazil. The ultrasonographic study of wildlife species is an important tool for a non-invasive and accurate anatomical description and provides important information for wildlife veterinary care. The aim of the present study was to determine the characteristics of the main abdominal organs as well as the vascular indexes of the abdominal aorta and renal arteries of crab-eating foxes (Cerdocyon thous) using mode B ultrasonography and Doppler ultrasonography, respectively. Ultrasonographic features of the main abdominal organs were described and slight differences were noticed between ultrasound imaging of abdominal organs of crab-eating foxes and other species. The bladder presented wall thickness of 12±0.01mm, with three defined layers. Both, the right and left kidneys presented corticomedullary ratio of 1:1 and similarly to the adrenals and the liver, they were homogeneous and hypoechoic compared to the spleen. The spleen was homogeneous and hyperechoic compared to the kidneys. The stomach presented 3 to 5 peristaltic movements per minute, wall thickness of 39±0.05mm and lumen and mucosa with hyperechoic and hypoechoic features, respectively. Small and large intestines presented 2 to 3 peristaltic movements per minute, wall thickness of 34±0.03mm and three defined layers with hyperechogenic (submucosa and serosa) and hypoechogenic (muscular) features. Ovaries of the female crab-eating fox were hypoechoic compared to the spleen and with heterogeneous parenchyma due to the presence of 2x2mm ovarian follicles. Prostates of the six males were regular and with a well defined boundary, with a homogeneous and hyperechoic parenchyma compared to the spleen. Vascular indexes of the abdominal aorta (PSV: 25.60±0.32cm/s; EDV: 6.96±1.68cm/s; PI: 1.15±0.07 e RI: 0.73±0.07) and right (PSV: 23.08±3.34cm/s; EDV: 9.33±2.36cm/s; PI: 1.01±0.65 e RI: 0.65±0.16) and left renal arteries (PSV: 23.74±3.94cm/s; EDV: 9.07±3.02cm/s; PI: 1.04±0.31 e RI: 0.64±0.10) were determined. Thus, conventional and Doppler ultrasonographic imaging provides basic information that can be used as reference for the species as well for other wild canids and it is a precise and non-invasive method that can be safely used to evaluate and diagnose abdominal injuries in these patients.

Cytoskeletal proteins in the follicular wall of normal andcystic ovaries of sows

The expression of cytoskeletal proteins was evaluated immunohistochemically in 36 normal ovaries sampled from 18 sows and 44 cystic ovaries sampled from of 22 sows, was evaluated. All sows had history of reproductive problems, such as infertility or subfertility. The immunohistochemically stained area (IHCSA) was quantified through image analysis to evaluate the expression of these proteins in the follicular wall of secondary, tertiary, and cystic follicles. Cytokeratins (CK) immunoreactivity was strong in the granulosa cell layer (GC) and mild in the theca interna (TI) and externa (TE) of the normal follicles. There was severe reduction of the reaction to CK in the GC in the cystic follicles, mainly in the luteinized cysts. The immunoreactivity for vimentin was higher in the GC from normal and cystic follicles in contrast with the other follicular structures. In the luteinized cysts, the IHCSA for vimentin was significantly higher in TI and in both observed cysts, the labeling was more accentuated in TE. Immunohistochemical detection of desmin and α-SMA was restricted to the TE, without differences between the normal and cystic follicles. The results of the current study show that the development of ovarian cysts in sows is associated to changes in the expression of the cytoskeletal proteins CK and vimentin.