Caracterização de biotérios, legislação e padrões de biossegurança

The use of laboratory animals in conjunction with research on the human organism provides a basis for on understanding of several important physiological and pathological processes. Besides, the results of experimental studies enable technical and safety improvements to be made in surgical techniques used in the medical clinic. As living biological material is involved, we should guarantee its physical well-being, taking into account microbiological contamination and the genetics, nutrition and correct manipulation of the animals, in order to avoid incorrect conclusions from the experiments or unnecessarily large numbers of animals being used. In parallel with the concerns and legislation on the use of laboratory animals, there is also a growing preoccupation with the welfare and safety of those who handle the laboratory animals, since they run the risk of acquiring occupational diseases through contact with zoonotic pathogens or developing allergies. Prevention requires the application of modern technological advances in the design of the animal house and in the work routines. Unfortunately, few establishments in Brazil possess staff with adequate training and a basic infrastructure of research that includes the laboratory animal breeding centers, equivalent to those existing ones in the United States and Europe.

An overview of functional and stereological evaluation of spermatogenesis and germ cell transplantation in fish

Although there are almost thirty-thousand species of fish living in a great variety of habitats and utilizing vast reproductive strategies, our knowledge of morphofunctional and quantitative aspects of testis structure and spermatogenesis is still incipient for this group of vertebrates. In this review, we discuss aspects that are important to better understanding of testis structure and function, and of the development of germ cells (GC) during spermatogenesis. To achieve this, we have recently completed a number of studies presenting morphometric and functional data related to the numbers of GC and Sertoli cells (SC) per each type of spermatogenic cyst, the number of spermatogonial generations, the SC efficiency, and the magnitude of GC loss that normally occurs during spermatogenesis. We also investigated SC proliferation and the relationship of this important event to early spermatogenic cysts. The available data strongly suggest that SC proliferation in sexually mature tilapia is the primary factor responsible for the increase in testis size and for determination of the magnitude of sperm production. The influence of temperature on the duration of spermatogenesis in tilapia was also evaluated and we have used this knowledge to deplete endogenous spermatogenesis in this teleost, in order to develop an experimental system for GC transplantation. This exciting technique results in new possibilities for investigation of spermatogenesis and spermatogonial stem cell biology, creating also an entirely new and promising scenario in biotechnology - transgenic animal production and the preservation of the genetic stocks of valuable animals or endangered species. © Springer Science+Business Media B.V. 2008.

LH surge in Nelore cows (Bos indicus), after induced estrus or after ovarian superestimulation

Considering that there is limited information about the preovulatory LH surge in Zebu cattle (Bos indicus), the purpose of the present work was to assess the LH surge in Nelore cows during the estrous cycle and after ovarian superestimulation of ovarian follicular development with FSH. This information is particularly important to improve superovulatory protocols associated with fixed-time artificial insemination. Nelore cows (n = 12) had their estrus synchronized with an intravaginal device containing progesterone (CIDR-B ®) associated with estradiol benzoate administration (EB, 2.5 mg, i.m., Day 0). Eight days later all animals were treated with PGF2α (Day 8) in the morning (8:00 h) and at night, when CIDR devices were removed (20:00 h). Starting 38 h after the first PGF2α injection, blood sampling and ovarian ultrasonography took place every 4 h, during 37 consecutive hours. Frequent handling may have resulted in a stress-induced suppression of LH secretion resulting in only 3 of 12 cows having ovulations at 46.7 ± 4.9 and 72.3 ± 3.8 h, respectively, after removal of CIDR-B. Thirty days later, the same animals received the described hormonal treatment associated with FSH (Folltropin ®, total dose = 200 mg) administered twice a day, during 4 consecutive days, starting on Day 5. Thirty-six hours after the first injection of PGF2α, to minimize stress, only seven blood samples were collected at 4 h interval each, and ultrasonography was performed every 12 h until ovulation. In 11 of 12 cows (92%) the LH surge and ovulation were observed 34.6 ± 1.6 and 59.5 ± 1.9 h, respectively, after removal of progesterone source. The maximum values for LH in those animals were 19.0 ± 2.6 ng/ml (mean ± S.E.M.). It is concluded that, in Nelore cows submitted to a ovarian superstimulation protocol, the LH surge occurs approximately 35 h after removal of intravaginal device containing progesterone, and approximately 12 h before the LH surge observed after an induced estrus without ovarian superstimulation. © 2008 Elsevier B.V. All rights reserved.

Semen variables of sheep (Ovis aries) experimentally infected with Toxoplasma gondii

The influence of Toxoplasma gondii on semen variables and sperm morphology of sheep was evaluated in eight reproductive males distributed into three experimental groups: GI, three sheep inoculated with 2.0 × 105 of P strain oocytes; GII, three sheep infected with 1.0 × 106 of RH strain tachyzoites and; GIII two control sheep. Clinical (rectal temperature, cardiac and respiratory frequencies), parasite and serology exams (IIF) were realized. Sperm variables (volume, motility, vigor and concentration) and semen morphology for each sheep were also evaluated. Thus, semen and blood collections were assessed on post-inoculation days (PIDs)-1,3,5,7,11,14 and weekly thereafter up to PID 70. Clinical alterations were observed (hypothermia and anorexia) in infected sheep from groups GI and GII. Parasitic outbreaks were detected in five sheep. All the infected sheep produced antibodies against T. gondii from PID 5 onwards, reaching a peak of 4096 and 8192 for group GI and GII sheep, respectively. Differences (P < 0.05) were observed regarding the ejaculate volume between the inoculated groups (oocytes and tachyzoites) and control. Even though experimental toxoplasmic infection resulted in clinical symptomology in the inoculated sheep, the minimal alterations in sperm pathologies could not be directly attributed to T. gondii. © 2008 Elsevier B.V. All rights reserved.

Evaluation of creatine kinase (CK) and aspartate aminotransferase (AST) activities after laparoscopic or conventional ovariectomy in queens

Creatine kinase (CK) and aspartate aminotransferase (AST) are mainly muscle-specific enzymes, which can be associated with muscle tissue damage. The aim of this study was to assess the activities of CK and AST during the postoperative period, after conventional (G1) and videolaparoscopic ovariectomy (G2), in queens. A further group (G3) was subjected to anaesthesia only. Results demonstrate that there were significant differences between groups. The highest levels of CK were recorded in Gl, however at a confidence level of p < 0.05 there was no significant difference between groups during the first 6 hours after surgery. A significant (p < 0.05) increase of CK values was identified between 0h and 3h in both groups (Gl and G2). Regarding AST activity there was no significant variation between groups, but again there was a significant difference between values at 0h and 3h after surgery. In conclusion, ovariectomy performed by videolap-aroscopy seems to cause less muscle damage when compared to the conventional method. © 2009 by Verlag Hans Huber, Hogrefe AG, Bem.

Influence of alcohol consumption on alveolar bone level associated with ligature-induced periodontitis in rats

Alcohol consumption is a risk indicator for periodontal disease. The purpose of this study was to morphometrically evaluate the influence of alcohol consumption on alveolar bone level associated with ligature-induced periodontitis in rats. Thirty-six female rats (Wistar, 120 days-old) were randomly divided into three groups that received a daily administration of a water diet (control, n = 12), a 10% alcohol diet (10% ethanol, n = 12) or a 20% alcohol diet (20% ethanol, n = 12). Four weeks after the onset of the experiment, cotton ligatures were placed around the cervix of the upper right second molar in six rats. The other 6 rats in each group remained unligated. The rats were sacrificed four weeks after ligature placement. The maxillary bones were removed and alveolar bone loss was analyzed by measuring the distance between the cementoenamel junction and the alveolar bone crest at 2 buccal and 2 palatal sites on the upper right second molar. Analyses between the ligated and unligated groups showed that the presence of ligature induced alveolar bone loss (p < 0.05). Unligated groups showed no significant differences between each other (p > 0.05). In the ligated groups, rats receiving 20% ethanol showed significantly greater bone loss compared to control rats or rats receiving 10% ethanol. These results demonstrate that alcohol consumption may increase alveolar bone loss in female rats in a dosedependent manner.

Occurrence and expression of p53 suppressor gene and c-Myc oncogene in dog eyelid tumors

Purpose: To detect the occurrence and expression of the suppressor gene p53 and of the oncogene c-Myc in eyelid tumors of dogs using the PCR, RT-PCR, PCR-ELISA and RT-PCR-ELISA techniques. These genes have not been described in dog eyelid tumors before. Methods: Nine samples of eyelid or third eyelid epithelial tumors were obtained from the archives of the Department of Veterinary Pathology. Tumor diagnosis was confirmed by evaluation of hematoxylin-eosin stained sections, and immunohistochemistry for cytokeratin AE1/AE3 and vimentin V9. A canine mammary tumor was used for positive control. Agarose gel electrophoresis, PCR-ELISA and RT-PCR-ELISA were used to detect p53 and c-Myc genes. Results: The occurrence of p53 was detected in most of the eyelid tumors and third eyelid tumors studied (88.8%, n = 8) and was expressed in 75% of the positive samples, as indicated by ELISA. The c-Myc gene was found in 77.7% (n = 7) of the samples and was expressed in eight samples. Conclusions: Eyelid and third eyelid tumors of dogs express both the p53 and the c-Myc genes as shown by PCR and RT-PCR. However, PCR ELISA and RT-PCR ELISA were more efficient in assessing occurrence and expression of these genes because they identified amplified products that were not detected by agarose gel electrophoresis. © 2010 American College of Veterinary Ophthalmologists.

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.

Neither plasma progesterone concentrations nor exogenous eCG affects rates of ovulation or pregnancy in fixed-time artificial insemination (FTAI) protocols for puberal Nellore heifers

The objective was to evaluate the effects of plasma progesterone (P4) concentrations and exogenous eCG on ovulation and pregnancy rates of pubertal Nellore heifers in fixed-time artificial insemination (FTAI) protocols. In Experiment 1 (Exp. 1), on Day 0 (7 d after ovulation), heifers (n = 15) were given 2 mg of estradiol benzoate (EB) im and randomly allocated to receive: an intravaginal progesterone-releasing device containing 0.558 g of P4 (group 0.5G, n = 4); an intravaginal device containing 1 g of P4 (group 1G, n = 4); 0.558 g of P4 and PGF2α (PGF; 150 μg d-cloprostenol, group 0.5G/PGF, n = 4); or 1 g of P4 and PGF (group 1G/PGF, n = 3). On Day 8, PGF was given to all heifers and intravaginal devices removed; 24 h later (Day 9), all heifers were given 1 mg EB im. In Exp. 2, pubertal Nellore heifers (n = 292) were treated as in Exp. 1, with FTAI on Day 10 (30 to 36 h after EB). In Exp. 3, pubertal heifers (n = 459) received the treatments described for groups 0.5G/PGF and 1G/PGF and were also given 300 IU of eCG im (groups 0.5G/PGF/eCG and 1G/PGF/eCG) at device removal (Day 8). In Exp. 1, plasma P4 concentrations were significantly higher in heifers that received 1.0 vs 0.588 g P4, and were significantly lower in heifers that received PGF on Day 0. In Exp. 2 and 3, there were no significant differences among groups in rates of ovulation (65-77%) or pregnancy (Exp. 2: 26-33%; Exp. 3: 39-43%). In Exp. 3, diameter of the dominant ovarian follicle on Day 9 was larger in heifers given 0.558 g vs 1.0 g P4 (10.3 ± 0.2 vs 9.3 ± 0.2 mm; P < 0.01). In conclusion, lesser amounts of P4 in the intravaginal device or PGF on Day 0 decreased plasma P4 from Days 1 to 8 and increased diameter of the dominant follicle on Day 9. However, neither of these nor 300 IU of eCG on Day 8 significantly increased rates of ovulation or pregnancy. © 2011.

Description and genetic analysis of three sets of monozygotic twins resulting from transfers of single embryos to recipient mares

Case Description-3 sets of monozygotic twins resulting from transfers of single embryos to recipient mares were examined. Clinical Findings-In all 3 recipient mares with twin pregnancies, only 1 embryonic vesicle was detected before day 25 of gestation. In 1 recipient mare, 2 apparent adjacent vesicles, each containing an embryo with a heartbeat, were visualized on ultrasonographic examination on day 37 of gestation. The other 2 recipient mares underwent ultrasonographic examination on day 30 of gestation, at which time only 1 vesicle and embryo was identified. In these latter 2 recipient mares, however, a thorough ultrasonographic examination for a second conceptus on day 30 had not been performed, as only 1 embryo had been transferred and visualized on early ultrasonographic examination. Treatment and Outcome-All twin pregnancies resulted in death of both fetuses. Genetic analysis confirmed that each set of monozygotic twins originated from the transferred embryo. Clinical Relevance-Monozygotic twin pregnancy may occur after embryo transfer; thus recipient mares should be examined thoroughly for multiple conceptuses, especially between 25 and 30 days of gestation. At this time, the allantoides of monozygotic twins should be visible ultrasonographically and effective management may still be possible.

Fatores de risco da transmissão de zoonoses por costumes da população de Ilha Solteira, Brasil

Objective Determining conditions regarding possible zoonosis transmission risk based on Ilha Solteira-São Paulo citizens' habits aimed at establishing concrete recommendations for the corresponding local authorities to reduce some risk factors. Methods100 focalized interviews were held on Ilha Solteira's urban perimeter during April 2008. The people interviewed were adults who lived or worked in houses in the study area. Results This research found a significant number of cat and/or dog owners who allowed their pets to stay in internal areas of their houses. They did not define a specific place for animals to defecate and/or urinate or did not arrange appropriate final disposal of such waste. Conclusion Local authorities must make greater efforts at educating Ilha Solteira pets' owners and providing them with information and encouraging greater citizen commitment and awareness to improve habits related to caring for pets/animal sand reducing zoonosis transmission risk factors.

First defection of the equine herpesvirus 1 neuropathogenic variant in Brazil

This report describes the first detection of an equine herpesvirus 1 (EHV-1) neuropathogenic variant (G 2254/D 752) in Brazil from a case of fatal equine herpesvirus myeloencephalopathy (EHM) in a mare. The results of nucleotide sequencing of the EHV-1 ORF30 gene showed that two other Brazilian EHV-1 isolates from EHM cases are representatives of the non-neuropathogenic variant (A 2254/N 752), suggesting that other unidentified factors are probably also involved in the neuropathogenicity of EHV-1 in horses. These findings will contribute to the epidemiological knowledge of EHV-1 infection in Brazil.

Effect of superstimulatory treatments on the expression of genes related to ovulatory capacity, oocyte competence and embryo development in cattle.

Multiple ovulation (superovulation) and embryo transfer has been used extensively in cattle. In the past decade, superstimulatory treatment protocols that synchronise follicle growth and ovulation, allowing for improved donor management and fixed-time AI (FTAI), have been developed for zebu (Bos indicus) and European (Bos taurus) breeds of cattle. There is evidence that additional stimulus with LH (through the administration of exogenous LH or equine chorionic gonadotrophin (eCG)) on the last day of the superstimulatory treatment protocol, called the 'P-36 protocol' for FTAI, can increase embryo yield compared with conventional protocols that are based on the detection of oestrus. However, inconsistent results with the use of hormones that stimulate LH receptors (LHR) have prompted further studies on the roles of LH and its receptors in ovulatory capacity (acquisition of LHR in granulosa cells), oocyte competence and embryo quality in superstimulated cattle. Recent experiments have shown that superstimulation with FSH increases mRNA expression of LHR and angiotensin AT(2) receptors in granulosa cells of follicles >8 mm in diameter. In addition, FSH decreases mRNA expression of growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) in oocytes, but increases the expression of both in cumulus cells, without diminishing the capacity of cumulus-oocyte complexes to generate blastocysts. Although these results indicate that superstimulation with FSH is not detrimental to oocyte competence, supplementary studies are warranted to investigate the effects of superstimulation on embryo quality and viability. In addition, experiments comparing the cellular and/or molecular effects of adding eCG to the P-36 treatment protocol are being conducted to elucidate the effects of superstimulatory protocols on the yield of viable embryos.