Search

SummaryPrimordial germ cells transplantation is a unique approach for conservation and reconstitution of endangered fish species. This study aimed to establish techniques to culture dechorionated embryos in different incubation systems and also to determine anaesthetic concentration for fish recipients in the larval stage for subsequent primordial germ cell transplantation. Intact and dechorionated embryos were divided into three incubation systems: (1) a control group with manual replacement of the solution; (2) a closed environment with high oxygen with manual replacement of the solution; and (3) constant solution recirculation. This combination resulted in six treatments. For the evaluation of anaesthetics for larvae, the concentrations evaluated were 19.5 mM, 24.4 mM, 29.3 mM, and 34.2 mM of 2-phenoxyethanol. Anaesthesia concentration and recovery at different stages were evaluated. For transplantation, primordial germ cells of Astyanax altiparanae were transplanted into anaesthetised larvae (1 dph) of Prochilodus lineatus. Better results were obtained in the recirculation system for dechorionated embryos of P. lineatus for hatching (54.18%) and normal morphology (50.06%). The 2-phenoxyethanol anaesthetic with a dose of 29.3 mM resulted in shorter induction times, in addition to the recovery time between 5 and 10 min. By using this anaesthetic concentration at transplantation, GFP-positive cells were seen in two recipients, but the cells did not proliferate. This study established an effective incubation system for the development of the dechorionated embryo and determined an effective anaesthetic concentration for P. lineatus larvae. In addition, micromanipulation and transplantation of primordial germ cells in neotropical species were conducted for the first time.

Primordial germ cells (PGCs) are responsible for the generation of all germ cells, and therefore, they are essential targets to be used as a tool for the production of germline chimera. The labeling and route of PGCs were evaluated during the initial embryonic development of Pseudopimelodus mangurus, using whole-mount in situ hybridization (WISH) and mRNA microinjection in zygotes. The WISH method was synthesized a specific RNA antisense probe using part of the coding region from P. mangurus nanos3 cDNA. The RNA microinjection was performed using the GFP gene reporter regulated by translation regulatory P. mangurus bucky ball and nanos3 3’UTR sequences, which are germline-specific markers used to describe in vivo migration of PGCs. The nanos3 and bucky ball gene expressions were evaluated in tissues for male and female adults and initial development phases and larvae from first to seventh days post-hatching. The results from the WISH technique appointed to the origin of PGCs in P. mangurus from the aggregations of nanos3 mRNA in the cleavage grooves and the signals obtained of nanos3 probes corresponded topographically to the migratory patterns of the PGC reported for other fish species. The microinjection of GFP-nanos3 3'UTR mRNA in the zygotes of P. mangurus was not successful for PGC labeling, but it was possible to identify three embryos injected, presenting 3 to 5 evidence of possible PGC labeled in the hatching phase. The nanos3 and bucky ball gene expression was reported in the female gonads and from fertilized eggs until the blastula phase. These results provide information about the PGC migration of P. mangurus and the possible use of PGCs for the future generation of germline chimera to be applied in the conservation efforts of Neotropical Siluriformes species, contributing to the establishment of genetic banks, manipulation of organisms, and assisting in biotechnologies such as the transplantation of germ cells in fish.

This study aimed to evaluate the ploidy and survival of larvae resulting from crosses between tetraploid females and diploid males of yellowtail tetra

Made available in DSpace on 2022-04-29T08:30:50Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-01 Gynogenesis is a chromosomal manipulation technique where an offspring is generated exclusively from maternal inheritance. In the yellowtail tetra (Astyanax altiparanae) almost all gynogenetic fish were females, suggesting a genetic sex determination system of XX/XY. However, due to environmental (e.g. temperature) or unknown genetic factors, the rise of males are described. Therefore, the aim of this study was to analyze the reproductive parameters of spontaneously gynogenetic males. The parameters analyzed were spermatozoon viability, motility and ploidy. Additionally, the gonads were processed for histology and the genetic inheritance confirmed by genotyping. Sperm parameters of gynogenetic males did not show differences when compared with semen of normal males. These data supports that gynogenetic males presents fertilization capacity and can be used for future studies of chromosome manipulation or possible production of monosex populations in A. altiparanae. Laboratory of Fish Biotechnology National Center for Research and Conservation of Continental Fish Chico Mendes Institute of Biodiversity Conservation Aquaculture Center Sao Paulo State University Universidade Federal Rural de Pernambuco (UFRPE) Unidade Acadêmica de Serra Talhada Aquaculture Center Sao Paulo State University

Made available in DSpace on 2021-06-25T11:57:48Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-02-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) The aim of this study was to evaluate different post-shock temperatures for tetraploid induction in the yellowtail tetra Astyanax altiparanae. Newly fertilized eggs were divided into four groups, three were submitted to heat shock (40 degrees C for 2 min) at 24 min post-fertilization (mpf) and another group remained without shock (control). Groups submitted to temperature shock were further separated at the following temperatures: 22 degrees C, 26 degrees C and 28 degrees C. Survival among embryonic development was counted and at hatching the ploidy was analyzed by flow cytometry. The results showed that the post-shock temperature affects the parameters analyzed and, therefore, must be considered for optimization of the production of tetraploid in A. altiparanae. Those data are innovative and could be used in future studies of basic biology in this species. Fed Univ Grande Dourados UFGD, BR-79825070 Dourados, MS, Brazil Sao Paulo State Univ, Aquacuiture Ctr, Via Acesso Prof Paulo Donato Castaane S-N, BR-14884900 Jaboticabal, SP, Brazil Chico Mendes Inst Biodivers Conservat, Natl Ctr Res & Conservat Continental Fish, Lab Fish Biotechnol, Rodovia Pref Euberto Nemesio Pereira de Godoy, BR-13630970 Pirassununga, SP, Brazil Sao Paulo State Univ, Aquacuiture Ctr, Via Acesso Prof Paulo Donato Castaane S-N, BR-14884900 Jaboticabal, SP, Brazil FAPESP: 2010/17429-1 FAPESP: 2011/11664-1 FAPESP: 2016/12383-0 CNPq: 471140/2012-0

Made available in DSpace on 2020-12-12T01:59:40Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-06-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) The streaked prochilod Prochilodus lineatus is an important fish species from the Neotropical region. In this study, a protocol of triploidization was established by temperature shock. Fertilized eggs were shocked at 2 min post-fertilization at 0, 38, 40 and 42°C. At 0°C, the embryos were maintained for 30 min, while the rest for 2 min. Heat shocked embryos and control (during all experiment) were incubated at 27°C. The ploidy status was confirmed by flow cytometry, erythrocyte diameter, conventional cytogenetics (Giemsa staining), chromosome banding and fluorescence in situ hybridization using 5S and 18S rDNA probes. Heat-shock at 40°C produced 96.7% of triploids and such a procedure did not reduce the fertilization, hatching rate and the percentage of normal embryos. The use of chromosome banding and FISH gave rise to effective procedure to identify triploids. The data obtained here is innovative for the species and bring new information for basic and applied studies. Laboratory of Fish Biotechnology National Center for Research and Conservation of Continental Fish Chico Mendes Institute of Biodiversity Conservation Department of Veterinary Medicine – FZEA University of São Paulo Aquaculture Center São Paulo State University Federal Rural University of Rio de Janeiro Faculdade de Ciências Bauru UNESP – Univ. Estadual Paulista Aquaculture Center São Paulo State University Faculdade de Ciências Bauru UNESP – Univ. Estadual Paulista FAPESP: 2010/17429-1 FAPESP: 2011/11664-1

The transplantation of primordial germ cells (PGCs) is a valuable tool for gene-banking and reconstitution by means of a germline chimera. For this technology, studies regarding developmental stages and traceability of PGCs are necessary. The objective of this study was to develop a micromanipulation procedure for the future establishment of cryobanks of PGCs in migratory characins. Incubation temperatures were evaluated at 22 ° C, 26 ° C, and 30 ° C in order to synchronize developmental stages. The highest hatching rates and the lowest abnormality rate arose at 26° C, which was considered to be the best incubation temperature. Enzymatic removal of the chorion was determined to be best using 0.05% pronase, in which the embryos presented better survival rates. In order to visualize PGCs in vivo, artificial GFP-nos1 3’UTR mRNA was injected and the migration route was observed in vivo as PGCs were visualized firstly at the segmentation stage (6 to 13 somites). The number of GFP positive cells ranged from 8 to 20 per embryo (mean of 13.8; n = 5). After hatching, GFP-positive cells increased to 14 to 27 embryos (mean of 19.8; n = 5). Visualization of the GFP-positive cells was possible at 10 days post hatching, and at this stage, the cells were positioned in the yolk extension region. This is the first report on PGC visualization in vivo in Neotropical fish; the obtained data provide information on the identification and migration of PGCs. The information presented in this work brings new insights in gene banking in Neotropical species and subsequent reconstitution through a germinal germline chimera.

Triploidization plays an important role in aquaculture and surrogate technologies. In this study, we induced triploidy in the matrinxã fish (Brycon amazonicus) using a heat-shock technique. Embryos at 2 min post fertilization (mpf) were heat shocked at 38°C, 40°C, or 42°C for 2 min. Untreated, intact embryos were used as a control. Survival rates during early development were monitored and ploidy status was confirmed using flow cytometry and nuclear diameter analysis of erythrocytes. The hatching rate reduced with heat-shock treatment, and heat-shock treatments at 42°C resulted in no hatching events. Optimal results were obtained at 40°C with 95% of larvae exhibiting triploidy. Therefore, we report that heat-shock treatments of embryos (2 mpf) at 40°C for 2 min is an effective way to induce triploid individuals in B. amazonicus.

Made available in DSpace on 2022-04-29T08:29:02Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-01-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) In freshwater fish with external fertilization, sperm sampling can be contaminated with urine, which triggers motility and gives rise to decreased fertilization success. The maintenance of freshwater fish in hyperosmotic conditions may reduce urine production and improve sperm quality. Thus, the aim of this work was to verify if acute exposure to various NaCl concentrations improves sperm quality in the yellowtail tetra Astyanax altiparanae. Spermiation was induced using a single dose of carp pituitary gland (5 mg kg-1) and the males were maintained at various NaCl concentrations: NaCl 0.00% (control), NaCl 0.45% (hypoosmotic), NaCl 0.9% (isosmotic) and NaCl 1.0% (hyperosmotic) for 6 h at 26 °C. Sperm was collected and verified for activation by urine and motility traits. At 0.00%, 0.45%, and 0.90%, the sperm was motile just after sampling, indicating activation by urine. Surprisingly, at hyperosmotic conditions, no activation was observed. Other sperm and motility parameters did not show any statistical differences, including sperm viability (P = 0.7083), concentration (P = 0.9030), total motility (P = 0.6149), VCL (curvilinear velocity; P = 0.1216), VAP (average path velocity; P = 0.1231) and VSL (straight-line velocity; P = 0.1340). Our results indicate that acute maintenance at hyperosmotic conditions eliminates sperm activation by urine and maintains sperm quality. Such a new procedure is interesting for both basic and applied sciences, including reproductive practice in fish. Universidade Estadual de São Paulo Departamento de Zoologia Instituto Chico Mendes de Conservação da Biodiversidade Centro Nacional de Pesquisa e Conservação da Biodiversidade Aquática Continental Laboratório de Biotecnologia de Peixes Universidade de São Paulo Faculdade de Medicina Veterinária e Zootecnia Departamento de Reprodução Animal Universidade Estadual de São Paulo Centro de Aquicultura Universidade Estadual de São Paulo Departamento de Zoologia Universidade Estadual de São Paulo Centro de Aquicultura FAPESP: 2010/17429-1 FAPESP: 2011/11664-1

Made available in DSpace on 2020-12-10T20:07:47Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-08-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) AES Tiete (Research & Development Program ANEEL) Flow cytometry is a valuable tool in biomedical and animal sciences. However, equipment used for such analysis presents limitations at field conditions, suggesting then preservation procedures for future analysis at laboratory conditions. In this study, freezing at low (-20 degrees C), ultra-low (-80 degrees C) and cryogenic temperatures (-196 degrees C, i.e. liquid nitrogen) were used as preservation procedures of fish tissue. Samples were maintained in 0.9% NaCl or lysing solution, and stored at the temperatures above for 0 (fresh control), 60, 120 and 180 days of storage. After storage, the samples were thawed and proceeded to flow cytometric analysis. Storage at low temperatures (-20 degrees C), both in lysing and 0.9% NaCl, exhibited poor results when analyzed after 60, 120 and 180 days, showing noisy peaks, deviation in the DNA content and absence of peaks. Ultralow (-80 degrees C) and cryogenic (-196 degrees C) temperatures, both in lysing solution and 0.9% NaCl, showed good results and high quality of histograms. Both storage procedures gave similar histograms and DNA content in comparison with control group (fresh) even after 60, 120 and 180 days of storage, exhibiting the main peak at 2C content from diploid cells and a secondary peak at 4C derived from dividing cells. In conclusion, samples may be stored for 180 days at -80 degrees C and -196 degrees C in both, 0.9% NaCl or lysing solution. As cryogenic temperatures in liquid nitrogen permits indefinite storage, this procedure should be used for long-term preservation. Univ Sao Paulo, Dept Anim Reprod FMVZ, Ave Prof Dr Orlando Marques de Paiva 87, BR-05508270 Sao Paulo, SP, Brazil Chico Mendes Inst Biodivers Conservat, Lab Fish Biotechnol, Natl Ctr Res & Conservat Continental Fish, Rodovia Pref Euberto Nemesio Pereira de Godoy, BR-13630970 Pirassununga, SP, Brazil Sao Paulo State Univ Julio de Mesquita Filho, Inst Biosci, Rua Prof Dr Antonio Celso Wagner Zanin 250, BR-18618689 Botucatu, SP, Brazil Univ Sao Paulo, Dept Vet Med FZEA, Ave Duque Caxias Norte 225, BR-13630080 Pirassununga, SP, Brazil Sao Paulo State Univ, Aquaculture Ctr, Via Acesso Prof Paulo Donato Castellane S-N, BR-14884900 Jaboticabal, SP, Brazil AES Tiete, Br-153,0 Km 139 Ctr, BR-16370000 Promissao, SP, Brazil Sao Paulo State Univ Julio de Mesquita Filho, Inst Biosci, Rua Prof Dr Antonio Celso Wagner Zanin 250, BR-18618689 Botucatu, SP, Brazil Sao Paulo State Univ, Aquaculture Ctr, Via Acesso Prof Paulo Donato Castellane S-N, BR-14884900 Jaboticabal, SP, Brazil FAPESP: 2010/17429-1 FAPESP: 2011/11664-1 AES Tiete (Research & Development Program ANEEL): 0064-1042/2014

Made available in DSpace on 2020-12-10T20:04:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-01-01 AES Tiete/ANEEL (Research & Development Project) This study aimed to describe a procedure for sampling, reproduction, and first feeding of the Neotropical catfish Pseudopimelodus mangurus, an endangered fish species. Wild adult P. mangurus specimens were collected in the Mogi Guassu River and subsequently induced to spawn in laboratory conditions. After hand-stripping, the females, the average weight of the oocytes was 143 +/- 1.6 g, with a fecundity of 718 +/- 49.8 oocytes g(-1). The diameter of the oocytes non-hydrated was 1,226.3 +/- 47.7 mu m to 1,761.2 +/- 26.4 mu m after hydration. The fertilization rates were 98.00 +/- 0.63%, and the hatching rate was 68.94 +/- 11.83%. The first feeding was made three days post-hatching with six different treatments, in which the best results arose with sequential feeding with Artemia nauplii, Astyanax altiparanae and Prochilodus lineatus larvae. This condition resulted in a final length of 2,012.7 +/- 44.8 mu m by the 10th day of the experiment, higher survival (65 +/- 0.9%) and lower cannibalism rate (14 +/- 0.3%). The data obtained in this study is important for the establishment of reproductive biotechniques, mass production of P. mangurus, and future establishment of ex-situ GenBank. Sao Paulo State Univ, Inst Biosci, Botucatu, SP, Brazil Natl Ctr Res & Conservat Continental Fish, Lab Fish Biotechnol, Chico Mendes Inst Biodivers Conservat, Pirassununga, SP, Brazil Sao Paulo State Univ, Aquaculture Ctr, Jaboticabal, SP, Brazil AES Tiete, Promissao, SP, Brazil Dept Vet Med FZEA, Pirassununga, SP, Brazil Sao Paulo State Univ, Inst Biosci, Botucatu, SP, Brazil Sao Paulo State Univ, Aquaculture Ctr, Jaboticabal, SP, Brazil AES Tiete/ANEEL (Research & Development Project): 0064-1052/2014

Made available in DSpace on 2020-12-10T19:50:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-04-15 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Tetraploid fish are an important source of diploid gametes for polyploid production, and they can be induced by inhibition of the first mitotic cell division. Dispite its potential, very few protocols regarding tetraploidization and subsequent application to mass production of triploid fish for aquaculture exist. In the yellowtail tetra Astyanax altiparanae, triploids present sterility and increased carcass yield, suggesting the generation of tetraploid for large-scale production of such triploids. In this study, the efficacy of heat shock treatments (40 degrees for 2 min) at 16, 18, 20, 22, 24, and 26 min post-fertilization (mpf) at an incubation temperature of 22 degrees C were compared on tetraploid induction of A. altiparanae. Ploidy status was confirmed by flow cytometry, karyotyping, and nuclear diameter of erythrocytes. Afterwards, sperm parameters and fertility capacity of tetraploid males were evaluated. The results showed that heat shock decreased the hatching rate, especially at 22 (19.1 +/- 9.6%), 24 (16.0 +/- 9.1%), and 26 mpf (10.3 +/- 6.2%) in comparison with the control group (62.2 +/- 7.7%). The flow cytometric analysis showed that the control group and the treatments with heat shock at 16 and 18 mpf presented only diploid individuals. However, from 20 mpf onwards, tetraploid individuals were observed, and the highest percentage arose at 26 mpf (94.55%). When mature, tetraploid males are capable of producing diploid spermatozoa with fertilization capacity, generating a 100% triploid offspring. The results indicate that the heat shock treatment at 26 mpf (40 degrees C for 2 min) is optimum for tetraploid induction in the A. altiparanae. Additionally, tetraploid mature males generated diploid spermatozoa and can be used to mass production of triploid fish. The current data are the first report of tetraploids in family Characidae, presenting an application in basic and applied sciences. Sao Paulo State Univ, Aquaculture Ctr, Via Acesso Prof Paulo Donato Castellane S-N, BR-14884900 Jaboticabal, SP, Brazil Chico Mendes Inst Biodivers Conservat, Natl Ctr Res & Conservat Continental Fish, Lab Fish Biotechnol, Rodovia Pref Euberto Nemesio Pereira de Godoy, BR-13630970 Pirassununga, SP, Brazil Univ Fed Rural Rio de Janeiro, BR-465 Km 7, BR-23897000 Seropedica, RJ, Brazil Hokkaido Univ, Fac Fisheries Sci, 3-1-1 Minato Cho, Hakodate, Hokkaido 0418611, Japan Sao Paulo State Univ, Aquaculture Ctr, Via Acesso Prof Paulo Donato Castellane S-N, BR-14884900 Jaboticabal, SP, Brazil FAPESP: 2010/17429-1 FAPESP: 2011/11664-1 FAPESP: 2016/12383-0 CNPq: 471140/2012-0

Made available in DSpace on 2020-12-10T19:40:33Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-01-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Gynogenesis is a chromosome manipulation technique applicable on the production of all-female populations, generation of inbred or clonal lines and identification of genetic sex determination mechanisms in fish. Therefore, the aim of this study was induce meiotic gynogenesis in Astyanax altiparanae and determine the genetic basis of sex determination. Firstly, we evaluated the best UV-irradiation dosage in order to inactivate sperm genome, preserving the capacity of egg activation without the genetic material contribution to the offspring. After the fertilization using the inactivated sperm, diplodization was achieved by heat-shock (40 degrees C; 2 min) by suppression of the 2nd polar body extrusion. Diploid control and gynogenetic individuals were raised until 240 days. The gynogenetic offspring were confirmed by genotyping using microsatellite markers and the sex ratio of gynogenetic progenies suggests that most data fit to a XX/XY model of sex determination. However, unexpected sex ratios were also observed and possible reasons are discussed. This was the first protocol of induced gynogenesis in a Neotropical fish and such a procedure may be used to produce clonal linage of fish and for future mass production of monosex populations in A. altiparanae and other related species. Sao Paulo State Univ, Aquaculture Ctr, Via Acesso Prof Paulo Donato Castellane S-N, BR-14884900 Jaboticabal, SP, Brazil Chico Mendes Inst Biodivers Conservat, Natl Ctr Res & Conservat Continental Fish, Lab Fish Biotechnol, Rodovia Pref Euberto Nemesio Pereira de Godoy, BR-13630970 Pirassununga, SP, Brazil Univ Fed Rural Rio de Janeiro, BR 465 Km 7, BR-23897000 Seropedica, RJ, Brazil Univ Sao Paulo, Dept Vet Med FZEA, Lab Theriogenol Dr OJ Ginther, Ave Duque de Caxias Norte 225, BR-13630080 Pirassununga, SP, Brazil Sao Paulo State Univ, Aquaculture Ctr, Via Acesso Prof Paulo Donato Castellane S-N, BR-14884900 Jaboticabal, SP, Brazil FAPESP: 2010/17429-1 FAPESP: 2011/11664-1 FAPESP: 2016/12383-0 CNPq: 471140/2012-0

Made available in DSpace on 2018-11-26T17:54:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-04-01 AES Tiete The aim of this study was to describe the effect of temperature on the fertilization, early developmental stages, and survival rate of two Neotropical catfishes Pimelodus maculatus and Pseudopimelodus mangurus. After fertilization, the eggs were incubated at 22 degrees C, 26 degrees C, and 30 degrees C, which resulted in fertilization rates of 96.95 +/- 1.79%, 98.74 +/- 0.76%, and 98.44 +/- 0.19% for P. maculatus and 96.10 +/- 1.58%, 98.00 +/- 0.63%, and 94.60 +/- 2.09% for P. mangurus, respectively. For P. maculatus, hatching occurred after 22 h 30 min post-fertilization at 22 degrees C, 16 h 30 min at 26 degrees C, and 11 h 20 min at 30 degrees C, and the hatching rates were 43.87 +/- 7,46%, 57.57 +/- 17.49%, and 53.63 +/- 16.27%, respectively. For P. mangurus, hatching occurred after 28 h 30 min post-fertilization at 22 degrees C and 17 h 30 min at 26 degrees C with respective hatching rates of 45.4 +/- 21.02% and 68.1 +/- 12.67%. For this species, all embryos incubated at 30 degrees C died before hatching. Additionally, for P. maculatus, the larvae from the lower (22 degrees C) and higher temperatures (30 degrees C) presented increased abnormality rates, as observed in the head, tail and yolk regions. The lowest abnormality rate was detected at 26 degrees C, which was considered the optimal incubation temperature for both species. The developed protocol enables the manipulation of embryonic development, which is important for the application of reproductive biotechniques, including chimerism and chromosome-set manipulation. The data obtained here are also important for the surrogate propagation of this species as P. mangurus was recently categorized as an endangered fish species. Chico Mendes Inst Biodivers Conservat, Natl Ctr Res Conservat Continental Fish, Lab Fish Biotechnol, Rodovia Pref Euberto Nemesio Pereira de Godoy, Pirassununga, Brazil Sao Paulo State Univ, Inst Biosci, Rua Prof Doutor Antonio Celso Wagner Zanin S-No, BR-18618689 Botucatu, SP, Brazil Sao Paulo State Univ, Aquaculture Ctr, Via Acesso Prof Paulo Donato Castellane S-N, BR-14884900 Jaboticabal, SP, Brazil AES Tiete, Br-153,Rod,0 Km 139 Ctr, BR-16370000 AES Tiete, SP, Brazil FZEA, Dept Vet Med, Ave Duque Caxias Norte 225, BR-13639080 Pirassununga, SP, Brazil UNIFESSPA Fed Univ South Southeast Para, Inst Hlth Biol Studies IESB, Folha 31,Quadra 7,Lote Especial S-N, BR-68507590 Maraba, PA, Brazil Sao Paulo State Univ, Inst Biosci, Rua Prof Doutor Antonio Celso Wagner Zanin S-No, BR-18618689 Botucatu, SP, Brazil Sao Paulo State Univ, Aquaculture Ctr, Via Acesso Prof Paulo Donato Castellane S-N, BR-14884900 Jaboticabal, SP, Brazil

Two performance studies were conducted to investigate the effects of 3 different sources of Cu on production parameters of piglets. A total of 256 piglets weaned at 24 ± 2 d were randomly allocated into 4 treatments with 10 or 8 replicates per treatment of 4 or 3 piglets per pen in Exp. 1 and 2, respectively. The experimental period was divided into 3 feeding phases: Phase 1 (24 to 35 d), Phase 2 (36 to 49 d), and Phase 3 (50 to 70 d). Treatments included a Control group (fed 10 mg/kg of Cu from CuSO4), a group fed 160 mg/kg of either CuSO4 (CuSO4-160) or tri-basic copper chloride (TBCC), and a group fed Cu methionine hydroxy analogue chelated (Cu-MHAC) at 150, 80, and 50 mg/kg in Phases 1, 2, and 3, respectively. The methionine value of Cu-MHAC was accounted during diet formulation to achieve the same levels of methionine across treatments. Phases 1 and 2 diets contained 2,200 and 1,500 ppm of ZnO, respectively; and antibiotics were used as growth promoters. Performance parameters were analyzed as completely randomized block design, in which each experiment was considered as a block. In trial 2, blood serum and mucosal samples, from the fundic region of the stomach, were collected from 1 piglet per replicate at day 70 and tested for serum growth hormone levels (GH) and ghrelin mRNA expression, respectively. The contrast between Cu-MHAC vs. CuSO4-160 + TBCC showed that piglets fed Cu-MHAC exhibited better feed conversion ratio (FCR) in all feeding phases compared with feeding inorganic Cu (P < 0.05). Overall, feeding Cu-MHAC improved body weight (BW), BW gain, feed intake (FI), and FCR vs. Control diet fed piglets; yet, it improved BW and FCR vs. TBCC fed piglets, and improved BW, BW gain, and FI vs. CuSO4-160 fed piglets (P < 0.05). Feeding TBCC promoted similar performance than feeding CuSO4-160, regardless of age (P > 0.05). Both ghrelin expression and growth hormone serum levels were significantly increased by feeding Cu-MHAC vs. Control diet fed animals (P < 0.01). Feeding CuSO4-160 upregulated ghrelin expression vs. Control (P < 0.01) while GH serum levels and ghrelin expression did no change by feeding TBCC compared with Control diet fed animals (P > 0.05). It was concluded that feeding Cu-MHAC at the levels tested herein can improve growth performance of piglets beyond feeding 160 ppm of either CuSO4 or TBCC, which may be partially explained by the increased expression of ghrelin and GH serum levels.

We evaluated the competence of bovine oocytes via morphological selection associated with follicular diameter and by brilliant cresyl blue (BCB) staining, in order to improve oocyte selection and the efficiency of in vitro embryo production. Follicles with small (2 < 4 mm), large (4–8 mm) or mixed (2–8 mm) diameters were aspirated from slaughterhouse ovaries. Next, the morphologically categorized oocytes were stained and evaluated with 26 ?M of BCB (90 min), followed by an assessment of in vitro maturation (MII) and blastocyst production. Oocytes from the large (61.1 ± 3.9%) and mixed (52.9 ± 4.6%) follicular groups showed comparable (p > 0,05) BCB+ rates, but both differed (p < 0.05) as compared to the small-diameter group (41.0 ± 4.5%). Between the BCB+ and BCB – oocytes, there was no difference (p > 0,05) in MII rate in the mixed (respectively 86.7 ± 3.4 and 75.8 ± 2.2), large (respectively 89.4 ± 2.6 and 79.7 ± 2.3) and small (respectively 76.7 ± 2.9 and 67.2 ± 2.5) diameter groups. Comparing BCB (+ and -) oocytes, we observed (p > 0,05) no change in the percentage of embryos in the large group (respectively 67.3 ± 3.3 and 53.5 ± 5.8) and in the small group (respectively 43.8 ± 5.3 and 33.9 ± 2.2), but there was a difference (p < 0.05) in the mixed group (respectively 57.8 ± 3.9 and 39.6 ± 7.5). Furthermore, when we compared between follicular groups, the BCB+/large-diameter group (61.1 ± 3.9%) had a significant higher percentage of embryos (p < 0.05) than the small-diameter BCB+ (43.8 ± 5.3) groups. To conclude, selection procedures that use follicle size and/or BCB staining may be used as an additional tool to improve the in vitro production of embryos. Avaliou-se a competência de oócitos bovinos através da seleção morfológica associada ao diâmetro folicular e pela coloração azul cresil brilhante (BCB), a fim de melhorar a seleção de oócitos e a eficiência da produção in vitro de embriões. Folículos com diâmetro pequeno (2 < 4 mm), grande (4-8 mm) ou misto (2-8 mm) foram aspirados de ovários de matadouro. Em seguida, os oócitos classificados morfologicamente foram corados e avaliados com 26 ?M de BCB (90 min), seguido de uma avaliação da maturação in vitro (MII) e produção de blastocisto. Os oócitos dos grupos folículo grande (61,1 ± 3,9%) e misto (52,9 ± 4,6%) apresentaram taxas de BCB+ comparáveis (p > 0,05), mas ambos diferiram (p < 0,05) em relação ao grupo folículo pequeno (41,0 ± 4,5%). Entre os oócitos BCB+ e BCB–, não houve diferença (p > 0,05) na taxa de MII nos grupos mistos (respectivamente 86,7 ± 3,4 e 75,8 ± 2,2), grande (respectivamente 89,4 ± 2,6 e 79,7 ± 2,3) e pequeno (respectivamente 76,7 ± 2,9 e 67,2 ± 2,5). Comparando os oócitos BCB (+ e -), não observamos alteração (p > 0,05) na porcentagem de embriões no grupo grande (respectivamente 67,3 ± 3,3 e 53,5 ± 5,8) e no grupo pequeno (respectivamente 43,8 ± 5,3 e 33,9 ± 2,2), mas há houve diferença (p < 0,05) no grupo misto (respectivamente 57,8 ± 3,9 e 39,6 ± 7,5). Além disso, quando comparamos os grupos foliculares, o grupo BCB+/grande (61,1 ± 3,9%) apresentou uma porcentagem significativamente maior de embriões (p < 0,05) do que o grupo BCB+/pequeno (43,8 ± 5,3). Para concluir, os procedimentos de seleção que usam tamanho do folículo e/ou coloração com BCB podem ser usados como uma ferramenta adicional para melhorar a produção in vitro de embriões.

Wild fish domestication can be considered a strategic approach to endangered species conservation, supporting studies and reducing economic and environmental costs. Three of the most important strategies in the domestication processes of fish are the adaptation of wild fish to captivity, the reproduction of the adapted fish and the production and maintenance of the young individuals. That being said, the present study is divided in three experiments: the 1st aimed to adapt wild Pseudopimelodus mangurus to captivity environment using different feeding approaches and a prophylactic strategie; the 2nd aimed to reproduce the adapted individuals from the 1st experiment; and the 3rd aimed to train the P. mangurus juveniles to accept commercial diets. The 1st and 2nd experiments were successful at the maintenance and artificial reproduction of P. mangurus kept in tanks between the reproductive seasons. The results suggest that the reproductive performance of animals kept in captivity (initial relative fertility-IRF = 609.25 ± 36.6 eggs/g) was similar (p > 0,05) to the performance found in wild individuals (IRF = 679.21 ± 45.66 eggs/g). Feed training of P. mangurus juveniles (3rd experiment) was also conducted, evaluating three feeding treatments with different concentrations of bovine heart and ration. At the end of the experiment, the treatment containing half bovine heart and half commercial feeding resulted in the highest values of weight gain (0.10 ± 0.16 g), specific growth rate (0.37 ± 0.11 mm), length (47.78 ± 2.35 mm) and growth (2.15 ± 2.27 mm), suggesting reasonable acceptability to artificial diets in the cultivation of this species. As conclusion, the present study contributes with the development of techniques for the domestication of fresh water fish species with commercial value or andangered of extinction, showing the domestication and reproduction of wild P. mangurus in captivity. However, more studies have to be conducted in order to improve the acceptance of artificial feeding by juveniles and to increase their survival rate. Resumo A domesticação de peixes selvagens pode ser considerada uma abordagem estratégica para a conservação de espécies ameaçadas, apoiando estudos e reduzindo custos econômicos e ambientais. Três das estratégias mais importantes para o processo de domesticação de peixes são a adaptação dos peixes ao cativeiro, a reprodução dos peixes adaptados e a produção e manutenção dos indivíduos jovens. O presente estudo está dividido em três partes: a 1ª objetivou adaptar Pseudopimelodus mangurus selvagens ao ambiente de cativeiro usando diferentes abordagens alimentares e uma estratégia profilática; o 2º objetivou reproduzir os indivíduos adaptados do 1º experimento; e o 3º teve como objetivo treinar os juvenis de P. mangurus para aceitar dietas comerciais. O 1o e 2o experimento obteveram sucesso na manutenção e reprodução artificial de P. mangurus mantidos em tanques entre as estações reprodutivas. Os resultados sugerem que o desempenho reprodutivo dos animais mantidos em cativeiro (fertilidade inicial relativa-FIR = 609,25 ± 36,6 ovos / g) foi similar (p> 0,05) ao dos indivíduos selvagens (FIR = 679,21 ± 45,66 ovos / g). O treinamento alimentar de P. mangurus juvenis (3º experimento) também foi realizado avaliando-se 3 tratamentos alimentares com diferentes concentrações de coração bovino e ração. Ao final do experimento, o tratamento contendo metade coração bovino e metade ração gerou os maiores valores de ganho de peso (0,10 ± 0,16 g), taxa de crescimento específico (0,37 ± 0,11 mm), comprimento (47,78 ± 2,35 mm) e crescimento (2,15 ± 2,27 mm), sugerindo razoável aceitabilidade para dietas artificiais no cultivo desta espécie. Como conclusão, o presente estudo contribui com o desenvolvimento de técnicas para a domesticação espécies de peixes de água doce, de interesse comercial ou ameaçados de extinção, mostrando a domesticação e reproduçao de em cativeiro P. Mangurus selvagens. No entanto, mais estudos devem ser conduzidos no intuito de aumentar a aceitação de dietas comerciais pelos juvenis e melhorar sua taxa sobrevivência.

Made available in DSpace on 2021-06-25T11:55:29Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-03-30 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) This study aims to describe the early development of Piaractus mesopotamicus in detail at 22 degrees C, 26 degrees C, and 30 degrees C and describe primordial germ cell migration in vivo by using artificial GFP-nos1 3'UTR mRNA for future application in surrogate technology. The highest hatching rate was observed at 26 degrees C with 52.9 +/- 18.9%, which was considered the best incubation temperature. At 22 degrees C, the embryos survived only until the blastula stage. The first visualization of PGCs occurred in the segmentation period when the embryos presented between 6 and 16 somites. The number of GFP-positive cells varied from 7 to 18 per embryo (mean 11.2; n = 15). After hatching, the GFP-positive cells ranged from 7 to 22 per embryo (mean 14.6; n = 15). The PGCs were traced until the 9th day after hatching. The obtained data allow for the incubation temperature manipulation and early development and traceability of PGCs, which are important for reproductive biotechniques, establishment of cryobanks, and subsequent reconstitution through a germline chimera. Sao Paulo State Univ, Inst Biosci, Botucatu, SP, Brazil Chico Mendes Inst Biodivers Conservat, Natl Ctr Res & Conservat Continental Fish, Lab Fish Biotechnol, Pirassununga, SP, Brazil Univ Fed Rural Rio De Janeiro, Inst Zootecn, Rio De Janeiro, Brazil Univ Los Llanos, Inst Acuicultura Los Llanos, Villavicencio, Colombia, Brazil Hokkaido Univ, Fac Fisheries Sci, Hakodate, Hokkaido, Japan Sao Paulo State Univ, Inst Biosci, Botucatu, SP, Brazil FAPESP: 2010/17429-1 FAPESP: 2011/116641

Salmonella enterica serovar Heidelberg is a human pathogen also found in broilers. A strain (UFPR1) has been associated with field reports of resistance to short-chain organic acids (SCOA) in broilers in the South of Brazil, but was susceptible to a Bacillus subtilis-based probiotic added in feed in a related study. This work aimed to (i) report clinical symptoms caused by SH UFPR1 in broilers, (ii) study its susceptibility to some antibiotics in vitro, and (iii) SCOA in vivo; and (iv) relate these phenotypic observations with its genome characteristics. Two in vivo trials used 1-day-old chicks housed for 21 days in 8 sterilized isolated negative pressure rooms with 4 battery cages of 12 birds each. Birds were challenged or not with 107 CFU/bird of SH UFPR1 orally and exposed or not to SCOA in a 2 × 2 factorial design. Zootechnical parameters were unaffected (P > 0.05), no clinical signs were observed, and few cecal and hepatic histologic and immune-related alterations were seen, in birds challenged with SH. Formic and propionic acids added together in drinking water, fumaric and benzoic acid in feed (Trial 1), and coated calcium butyrate in feed (Trial 2) did not reduce the SH isolation frequencies seen in cecum and liver in broilers after SH challenge (P > 0.05). SH UFPR1 was susceptible to amikacin, amoxicillin + clavulanate, ceftiofur, cephalexin, doxycycline and oxytetracycline; and mildly susceptible to ampicillin + sulbactam, cephalothin, ciprofloxacin, enrofloxacin, and gentamycin in an in vitro minimum inhibitory concentration model using Mueller–Hinton agar. The whole genome of SH UFPR1 was sequenced and consisted of a circular chromosome, spanning 4,760,321 bp with 52.18% of GC-content encoding 84 tRNA, 22 rRNA, and 4,427 protein-coding genes. The comparison between SH UFPR1 genome and a multidrug-resistant SL476 strain revealed 11 missing genomic fragments and 5 insertions related to bgt, bgr, and rpoS genes. The deleted genes codify proteins associated with cell cycle regulation, virulence, drug resistance, cellular adhesion, and salt efflux which collectively reveal key aspects of the evolution and adaptation of SH strains such as organic acids resistance and antibiotic sensitivity and provide information relevant to the control of SH in poultry.

Ovum pick up (OPU) associated with in vitro production (IVP) of embryos has been shown as an important tool in cattle breeding to increase the number of descendants from animals of high genetic value. In herds maintained distant from the laboratory, collecting cumulus-oocyte complexes (COCs) and transporting them to the laboratory may take several hours and decrease COCs viability, representing a challenge for commercial settings. In this study, a prematuration culture to induce temporary meiosis block was evaluated in a commercial scale IVP setting as a strategy to transport bovine OPU-derived COCs from Nelore and Brangus donors. Effects on embryo yield and pregnancy rates were assessed. Viable COCs from each donor were destined to one of the experimental groups (control, blocks 1 and 2). Control group COCs were placed in cryotubes with 1 mL TCM199-HEPES. In block groups (1 and 2), COCs were placed in cryotubes with 300 μL TCM 199 + 12 μM butyrolactone I (block medium). All groups were gassed and kept in a thermos bottle for 4 hours at 36 °C. Next, COCs in the control group were transferred to IVM medium and block 1 group to block medium, and cultured for 22 hours and 15 hours, respectively, at 38.5 °C and 5% CO2 in air. Block 2 COCs were kept in the cryotubes and in the thermos bottle for another 15 hours at 36 °C to simulate long-term transport conditions. After meiosis block in prematuration culture, blocks 1 and 2 COCs were matured in vitro for 22 hours as for the control group. After IVM, COCs in all groups were submitted to IVF and IVC, and blastocyst rates were evaluated on day 7. Embryos were transferred and pregnancy rates evaluated at 60 days of gestation. The mean total number of COCs retrieved by OPU did not differ between Nelore and Brangus donors (16.8 and 17.2, respectively, P0.05), but Nelore donors produced more viable COCs than Brangus (10.1 and 7.6, respectively, P0.05) and more embryos/cow (3.8 and 2.7, respectively, P0.05). Blastocyst rates were similar for control (40.2% and 36.7%), block 1 (37.3% and 34.5%), and block 2 groups (34.7% and 33.6%) for Nelore and Brangus cattle, respectively (P0.05). Pregnancy rates did not differ regardless of breed or treatment (36.7%, P0.05). In conclusion, temporary meiosis block during prematuration culture did not affect embryo development or pregnancy rates; therefore, this strategy may be used to transport bovine COCs in a commercial IVP setting.

Cows treated with single doses of follicle stimulating hormone (FSH) and ovum pick up (OPU) after 24 hours were evaluated for oocyte recovery, in vitro production of embryos (IVPE), and transferred embryos. To begin evaluations, the ovarian follicles larger than three millimeters in diameters were removed from all cows used in the study. Two days after OPU, 200 milligrams of FSH was given in a single dose in 6 cows (treated). Twenty-four hours after application of FSH, the cows underwent a new OPU session for oocyte retrieval. These procedures were repeated three consecutive times without interval. In control (FSH-free) cows the OPU were performed at intervals of one week or oocyte retrieval. The viable oocytes were submitted to IVPE, and the blastocysts were transferred to the recipients. The mean number of oocytes did not differ (p > 0.05) between control cows (12.1±2.8) and those treated (10.9±1.6). There were also no differences (p > 0.05) in the number (6.6±1.7 and 7.1±0.9, respectively) or in the percentage (54.4±3.3 and 64.5±3.1%, respectively) of viable oocytes between control cows and those treated. The percentage of IVPE and pregnancy did not differ (p > 0.05) between control cows (39.8±2.6% and 44.7±4.8%) and those treated (37.8±2.5% and 39.5±4.1%), respectively. The treatment with single doses of FSH did not significantly alter the evaluated results for oocyte recovery, IVPE and transferred embryos, and, therefore, did not promote significant improvements for the IVPE as a whole.

The level of hydrogen peroxide (H 2 O 2 ) in plants signalizes the induction of several genes, including that of ascorbate peroxidase (APX-EC 1.11.1.11). APX isoenzymes play a central role in the elimination of intracellular H 2 O 2 and contribute to plant responses to diverse stresses. During the infection process in Theobroma cacao by Moniliophthora perniciosa oxidative stress is generated and the APX action recruited from the plant. The present work aimed to characterize the T. cacao APX involved in the molecular interaction of T. cacao – M. perniciosa . The peroxidase activity was analyzed in protein extracts from cocoa plants infected by M. perniciosa and showed the induction of peroxidases like APX in resistant cocoa plants. The cytosolic protein of T. cacao (GenBank: ABR68691.2 ) was phylogenetically analyzed in relation to other peroxidases from the cocoa genome and eight genes encoding APX proteins with conserved domains were also analyzed. The cDNA from cytosolic APX was cloned in pET28a and the recombinant protein expressed and purified (r Tc -cAPX). The secondary structure of the protein was analyzed by Circular Dichroism (CD) displaying high proportion of α-helices when folded. The enzymatic assay shows stable activity using ascorbate and guaiacol as an electron donor for H 2 O 2 reduction. The pH 7.5 is the optimum for enzyme activity. Chromatographic analysis suggests that r Tc -cAPX is a homodimer in solution. Results indicate that the r Tc -cAPX is correctly folded, stable and biochemically active. The purified r Tc -cAPX presented biotechnological potential and is adequate for future structural and functional studies.

The aim of this study was to assess the effects of different antioxidants on the levels of reactive oxygen species (ROS) and glutathione (GSH) in oocytes during in vitro maturation (IVM), as well as on the production of embryos. Oocyte of slaughterhouse-derived cattle ovaries were placed in IVM with different antioxidants: quercetin (2 μM), cysteamine (100 μM), carnitine (0.5 mg/ml), vitamin C (50 μg/ml) or resveratrol (2 μM). Oocytes matured without any antioxidant supplementation were used as control. The oocytes were assessed for maturation rates and for ROS and GSH levels by fluorescence staining in 2',7'-dichlorodihydrofluorescein diacetate and Cell Tracker Blue, respectively. Embryo production was assessed in terms of cleavage, blastocysts and hatching rates and embryo cell numbers. The results expressed in arbitrary fluorescence units showed ROS reduction (p .05) in the groups with quercetin (27.5 ± 3.4), vitamin C (27.1 ± 3.0) or resveratrol (28.1 ± 4.7), in comparison with those with cysteamine (34.9 ± 4.5), carnitine (34.6 ± 3.8) or to the control group (36.5 ± 5.2). GSH levels increased (p .05) in cysteamine (63.5 ± 5.5) or carnitine (60.8 ± 4.4) groups in comparison with quercetin (52.7 ± 5.1), vitamin C (53.0 ± 3.8), resveratrol (53.1 ± 4.4) or to the control (49.6 ± 4.5). Nuclear maturation cleavage and hatched blastocysts rates did not differ (p .05) between groups. However, blastocyst rates after in vitro fertilization in quercetin (53.5 ± 3.9%), vitamin C (52.1 ± 3.1%) resveratrol (54.2 ± 4.0%), cysteamine (52.4 ± 2.7%) or carnitine (54.2 ± 3.1%) groups were higher (p .05) than in the control (47.2 ± 2.7%). Total cell numbers in embryos from the vitamin C, resveratrol, cysteamine or carnitine groups were higher than in quercetin and control groups, which were similar to each other. The results suggest that using antioxidants during IVM may reduce oxidative stress either by decreasing ROS levels directly or by increasing GSH levels in oocytes, depending on the type of antioxidant used. Overall, oxidative stress control during IVM with the antioxidants examined here improved blastocyst development with similar efficacy.

Made available in DSpace on 2018-11-26T15:30:06Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-08-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Universidade de Sao Paulo (USP/FZEA) Gene expression profiling of in vivo- and in vitro-matured bovine oocytes can identify transcripts related to the developmental potential of oocytes. Nonetheless, the effects of in vitro culturing oocytes are yet to be fully understood. We tested the effects of in vitro maturation on the transcript profile of oocytes collected from Bos taurus indicus cows. We quantified the expression of 1488 genes in in vivo-and in vitro-matured oocytes. Of these, 51 genes were up-regulated, whereas 56 were down-regulated (>= 2-fold) in in vivo-matured oocytes in comparison with in vitro-matured oocytes. Quantitative real-time polymerase chain reaction (PCR) of nine genes confirmed the microarray results of differential expression between in vivo- and in vitro-matured oocytes (EZR, EPN1, PSEN2, FST, IGFBP3, RBBP4, STAT3, FDPS and IRS1). We interrogated the results for enrichment of Gene Ontology categories and overlap with protein-protein interactions. The results revealed that the genes altered by in vitro maturation are mostly related to the regulation of oocyte metabolism. Additionally, analysis of protein-protein interactions uncovered two regulatory networks affected by the in vitro culture system. We propose that the differentially expressed genes are candidates for biomarkers of oocyte competence. In vitro oocyte maturation can affect the abundance of specific transcripts and are likely to deplete the developmental competence. Univ Norte Parana, Arapongas, Brazil Agr Laffranchi, POB 45, BR-86125000 Tamarana, Parana, Brazil Univ Sao Paulo, Pirassununga, Brazil Auburn Univ, Auburn, AL USA Univ Estadual Mato Grosso Sul, Aquidauana, Brazil Univ Estadual Paulista, Botucatu, SP, Brazil Univ Norte Parana, Escola Med Vet, Arapongas, PR, Brazil Agr Laffranchi, Lab Reprod Anim, Tamarana, PR, Brazil Univ Sao Paulo, Dept Ciencias Basicas, Pirassununga, SP, Brazil Auburn Univ, Dept Anim Sci, Auburn, AL USA Univ Estadual Mato Grosso Sul, Escola Zootecnia, Aquidauana, MS, Brazil Univ Estadual Paulista, Dept Tecnol, Botucatu, SP, Brazil Univ Estadual Paulista, Botucatu, SP, Brazil Univ Estadual Paulista, Dept Tecnol, Botucatu, SP, Brazil FAPESP: FAPESP 05/58702-4 FAPESP: 05/59694-5

The use of recombinant proteins has increased in diverse commercial sectors. Various systems for protein production have been used for the optimization of production and functional protein expression. The mammary gland is considered to be a very interesting system for the production of recombinant proteins due to its high level of expression and its ability to perform post-translational modifications. Cows produce large quantities of milk over a long period of lactation, and therefore this species is an important candidate for recombinant protein expression in milk. However, transgenic cows are more difficult to generate due to the inefficiency of transgenic methodologies, the long periods for transgene detection, recombinant protein expression and the fact that only a single calf is obtained at the end of each pregnancy. An increase in efficiency for transgenic methodologies for cattle is a big challenge to overcome. Promising methodologies have been proposed that can help to overcome this obstacle, enabling the use of transgenic cattle as bioreactors for protein production in milk for industry.

Acyl-CoA binding protein (ACBP) is a housekeeping protein and is an essential protein in human cell lines and in Trypanosoma brucei. The ACBP of Moniliophthora perniciosa is composed of 104 amino acids and is possibly a non-classic isoform exclusively from Basidiomycetes. The M. perniciosa acbp gene was cloned, and the protein was expressed and purified. Acyl-CoA ester binding was analyzed by isoelectric focusing, native gel electrophoresis and isothermal titration calorimetry. Our results suggest an increasing affinity of ACBP for longer acyl-CoA esters, such as myristoyl-CoA to arachidoyl-CoA, and best fit modeling indicates two binding sites. ACBP undergoes a shift from a monomeric to a dimeric state, as shown by dynamic light scattering, fluorescence anisotropy and native gel electrophoresis in the absence and presence of the ligand. The protein's structure was determined at 1.6 A resolution and revealed a new topology for ACBP, containing five alpha-helices instead of four. alpha-helices 1, 2, 3 and 4 adopted a bundled arrangement that is unique from the previously determined four-helix folds of ACBP, while alpha-helices 1, 2, 4 and 5 formed a classical four-helix bundle. A MES molecule was found in the CoA binding site, suggesting that the CoA site could be a target for small compound screening.

O presente trabalho teve como objetivos a clonagem, expressão e purificação da proteína HGPRT de Leishimania tarentolae, para a caracterização e cristalização dessa enzima, a fim do seu estudo estrutural e funcional. O gene da HGPRT foi amplificado a partir de uma biblioteca genômica de Leishmania tarentolae da cepa UC Lambda ZAP Express BamHI-Sal3A I. Esse gene foi clonado no vetor de expressão pET29a(+) e usado na transformação da bactéria Escherichia coli BL21 (DE3). Esse sistema de expressão apresentou uma superexpressão da proteína recombinante, que foi purificada em cromatografia de forma iônica, com o uso da coluna de troca anônica POROS 20HQ. A proteína purificada foi utilizada para sua caracterização, como a determinação das constantes cinéticas (Km, Vmax e Kcat) para os diferentes substratos. A proteína foi encontrada como dímero em solução e o pI de aproximadamente 8,2. Além disso, essa proteína foi utilizada nos ensaios de cristalização pelo método de difusão de vapor em gotas suspensas. Os cristais obtidos foram utilizados nos testes da difração de raios-X sendo coletado um conjunto de dados no Laboratório Nacional de Luz Síncroton. Os dados de difração de raios-X foram processados com o uso do pacote de programa HKL. A resolução da estrutura cristalográfica foi obtida pelo método de substituição molecular através do programa AmoRe. Para o refinamento da estrutura foram utilizados os programas de refinamento automático CNS e REFMAC, e a manipulação na estação gráfica foi realizada através do programa O. A estrutura da HGPRT foi resolvida a 2,1 &#197 de resolução com os fatores de concordância Rwork e Rfree finais de 17,34 e 21,53%, respectivamente. The aims of this work were the cloning, expression and purification of the protein HGPRT from Leishimania tarentolae, for the characterization and crystallization of the enzyme for it structural and functional study. The full-length hgprt gene was isolated from a leishmania tarentolae UC strain Lambda ZAP Express BamHI-Sal3A I genomic library. This gene was cloned in the expression vector pET29a+, and used in the transformation of the bacteria Escherichia coli BL21(DE3). This expression system presented a super-expression of the recombinant protein, which was purified by ionic change chromatography, utilizing the anionic change column POROS 20HQ. The purified protein was utilized for its characterization, including its kinetics constants (Km, Vmax e Kcat) for different substrates. The protein was found to be a dimmer in solution with pI close to 8.2. Besides, this protein was used in the crystallization assays by the steam diffusion in suspended drop technique. The obtained crystals were utilized in the x-ray diffraction studies. The data collection was performed in the Laboratório Nacional de Luz Síncroton. The X-ray diffraction data were processed utilizing the package program HKL. The crystallographic structure resolution was obtained by the molecular replacement method from the AmoRe program. For the structure refinement, the automatic refinement programs CNS and REFMAC were used, and the graphic manipulation was made through the O program. The structure of the HGPRT was resolved with 2,1 &#197 of resolution and final agreement factors Rwork and Rfree of 17,34% and 21,53%, respectively.

Prior to generating transgenic animals for bioreactors, it is important to evaluate the vector constructed to avoid poor protein expression. Mammary epithelial cells cultured in vitro have been proposed as a model to reproduce the biology of the mammary gland. In the present work, three lentiviral vectors were constructed for the human growth hormone (GH), interleukin 2 (IL2), and granulocyte colony-stimulating factor 3 (CSF3) genes driven by the bovine β-casein promoter. The lentiviruses were used to transduce mammary epithelial cells (MAC-T), and the transformed cells were cultured on polystyrene in culture medium with and without prolactin. The gene expression of transgenes was evaluated by PCR using cDNA, and recombinant protein expression was evaluated by Western-blotting using concentrated medium and cellular extracts. The gene expression, of the three introduced genes, was detected in both induced and non induced MAC-T cells. The human GH protein was detected in the concentrated medium, whereas CSF3 was detected in the cellular extract. Apparently, the cellular extract is more appropriate than the concentrated medium to detect recombinant protein, principally because concentrated medium has a high concentration of bovine serum albumin. The results suggest that MAC-T cells may be a good system to evaluate vector construction targeting recombinant protein expression in milk.

The phytocystatins regulate various physiological processes in plants, including responses to biotic and abiotic stresses, mainly because they act as inhibitors of cysteine proteases. In this study, we have analyzed four cystatins from Theobroma cacao L. previously identified in ESTs libraries of the interaction with the fungus Moniliophthora perniciosa and named TcCYS1, TcCYS2, TcCYS3 and TcCYS4. The recombinant cystatins were purified and subjected to the heat treatment, at different temperatures, and their thermostabilities were monitored using their ability to inhibit papain protease. TcCYS1 was sensitive to temperatures above 50°C, while TcCYS2, TcCYS3, and TcCYS4 were thermostable. TcCYS4 presented a decrease of inhibitory activity when it was treated at temperatures between 60 and 70°C, with the greater decrease occurring at 65°C. Analyses by native gel electrophoresis and size-exclusion chromatography showed that TcCYS4 forms oligomers at temperatures between 60 and 70°C, condition where reduction of inhibitory activity was observed. TcCYS4 oligomers remain stable for up to 20 days after heat treatment and are undone after treatment at 80°C. TcCYS4 presented approximately 90% of inhibitory activity at pH values between 5 and 9. This protein treated at temperatures above 45°C and pH 5 presented reduced inhibitory activity against papain, suggesting that the pH 5 enhances the formation of TcCYS4 oligomers. A variation in the titratable acidity was observed in tissues of T. cacao during the symptoms of witches’ broom disease. Our findings suggest that the oligomerization of TcCYS4, favored by variations in pH, is an endergonic process. We speculate that this process can be involved in the development of the symptoms of witches’ broom disease in cocoa.

Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) (EC 2.4.2.8) is an important enzyme involved in the recycling of purine nucleotides in all cells. Parasitic protozoa of the order Kinetoplastida are unable to synthesize purines de novo and use the salvage pathway for the synthesis of nucleotides; therefore, this pathway is an attractive target for antiparasitic drug design. The hgprt gene was cloned from a Leishmania tarentolae genomic library and the sequence determined. The L. tarentolae hgprt gene contains a 633-nucleotide open reading frame that encodes a 23.4-kDa protein. A pairwise alignment of the different HGPRT's sequences revealed a 26%-53% sequence identity with the Leishmania sequences and 87% identity to the HGPRT of Leishmania donovani. A recombinant protein was expressed in Escherichia coli, purified to homogeneity and found to retain enzymatic activity. The steady-state kinetic parameters were determined for the recombinant enzyme and the enzyme is active as a homodimer in solution. Single crystals were obtained for the L. tarentolae HGPRT representing the first Leishmania HGPRT crystallized and initial crystallographic data were collected. The crystals obtained belong to the orthorhombic space group (P2(1)2(1)2(1)) with unit cell parameters a=58.104 A, b=85.443 A and c=87.598 A and diffract to a resolution of 2.3 A. The availability of the HGPRT enzyme from Leishmania and its crystallization suitable for X-ray diffraction data collection should provide the basis for a functional and structural analysis of this enzyme, which has been proposed as a potential target for rational drug design, in a Leishmania model system.

Pharmaceutical industry started with the production of proteins for therapeutic purposes using the extraction from producing tissues, such as insulin derived from a pig’s pancreas and growth hormone from the pituitary gland. The proteins from these tissues often show low quality and increase in the risk of transmitting diseases such as HIV or those caused by prions. Recombinant DNA technology has allowed the heterologous proteins production using the bacterial system [1] that results in increased protein availability, therefore, improving quality and reducing the risk of disease transmission. Bacteria have been widely used as bioreactors because they are easily cultured. However, these microorganisms have limited ability to perform post-translational modifications of proteins necessary for the formation of biologically active molecules [2].

A quercetina é um flavonoide, amplamente encontrada em frutas, vegetais, grãos, flores, com elevada concentração no vinho tinto, e tem sido caracterizada funcionalmente pela atividade antioxidante. Para avaliação da maturação nuclear e do desenvolvimento embrionário bovino, os oócitos foram maturados por 22h na presença de quercetina (0,4, 2, 10 e 50µM), cisteamina (100µM) e na ausência dos antioxidantes. Os oócitos maturados foram corados com Hoechst para avaliação da maturação in vitro. Para avaliação do desenvolvimento embrionário, os oócitos foram fertilizados e cultivados in vitro, as taxas de desenvolvimento embrionário foram determinadas no sétimo dia de cultivo e o percentual de eclosão e o número de células dos embriões no oitavo dia. Os níveis de glutationa (GSH) dos oócitos foram mensurados por emissão de fluorescência com CMF2HC. A porcentagem de maturação nuclear (±89%) não diferiu entre os grupos. O desenvolvimento embrionário variou entre os tratamentos, o percentual de blastocisto foi superior (P0,05) entre os grupos (±63,0%). O número médio de células dos embriões também foi similar entre os grupos (±233). Os níveis intracelulares de GSH foram superiores nos oócitos maturados com cisteamina, mas similares entre os oócitos tratados com quercetina e o controle. A suplementação da maturação in vitro com antioxidantes melhora as taxas de blastocistos. A quercetina foi superior à cisteamina, que, por sua vez, foi superior ao controle. Mas os níveis de GSH foram superiores somente nos oócitos tratados com cisteamina. Quercetin is a flavonoid widely found in fruit, vegetables, grains and flowers, with a high concentration in red wine, and has been functionally characterized by its antioxidant activity. For assessment of nuclear maturation and bovine embryo, oocytes were matured for 22h in the presence of quercetin (0.4, 2, 10 and 50µM), cysteamine (100µM) and in the absence of antioxidants. The matured oocytes were stained with Hoechst to evaluate the in vitro maturation. To assess embryonic development, oocytes were fertilized and cultured in vitro and rates of embryo development were obtained in the seventh day of culture and the percentage of hatching and the number of cells on eighth day embryos. The levels of glutathione (GSH) of the oocytes were measured by fluorescence emission with CMF2HC. The percentage of nuclear maturation (±89%) did not differ between groups. Embryonic development varied between treatments, the percentage of blastocyst was higher (P0.05) between groups (±63.0%). The average number of embryo cells was also similar in both groups (±233). The intracellular GSH levels were higher in oocytes matured with cysteamine, but similar between the oocytes treated with quercetin and control. The supplementation of matured in vitro with antioxidants improves blastocyst rates. Quercetin was greater than cysteamine, which in turn was superior to the control. However, GSH levels were higher in oocytes treated only with cysteamine.

Recombinant coagulation factor IX must be produced in mammalian cells because FIX synthesis involves translational modifications. Human cell culture-based expression of human coagulation factor IX (hFIX) is expensive, and large-scale production capacity is limited. Transgenic animals may greatly increase the yield of therapeutic proteins and reduce costs. In this study, we used a lentiviral system to obtain transgenic cells and somatic cell nuclear transfer (SCNT) to produce transgenic animals. Lentiviral vectors carrying hFIX driven by 3 bovine β-casein promoters were constructed. Bovine epithelial mammary cells were transduced by lentivirus, selected with blasticidin, plated on extracellular matrix, and induced by lactogenic hormones; promoter activity was evaluated by quantitative PCR. Transcriptional activity of the 5.335-kb promoter was 6-fold higher than the 3.392- and 4.279-kb promoters, which did not significantly differ. Transgenic bovine fibroblasts were transduced with lentivirus carrying the 5.335-kb promoter and used as donor cells for SCNT. Cloned transgenic embryo production yielded development rates of 28.4%, similar to previous reports on cloned non-transgenic embryos. The embryos were transferred to recipient cows (N = 21) and 2 births of cloned transgenic cattle were obtained. These results suggest combination of the lentiviral system and cloning may be a good strategy for production of transgenic cattle.

Moniliophthora perniciosa is the causal agent of witches’ broom disease of cacao. Cyclophilins have been implicated in a wide variety of cellular processes, including the response to environmental stresses, cell cycle control, regulation of calcium signaling and transcriptional control. The involvement of cyclophilins in pathogenicity was described both in pathogenic fungi toward animals and plants. In Yeast, calcineurin and cyclophilin are involved in fungal morphogenesis and virulence. There is evidence that cyclophilin interacts with calcineurin in the absence of cyclosporin A (CsA), and it has been proposed that the activity of CsA acts in the regulatory interaction between cyclophilin and calcineurin; also, fungistatic or fungicide actions have been observed in this activity. The M. perniciosa cyclophilin gene from cDNA of the fruiting body in the vector pET28a has been cloned and the protein was expressed, purified and crystallized. The cyclophilin structures in the apo and bound with CsA forms were solved at 1.85 and 1.47 Co, respectively. Comparison of structures from different organisms indicates conserved structures of cyclophilins. Small differences were found in apo and bound forms; hence, it follows that cyclophilin structures do not have accentuated modifications in the presence of the ligand. However, various hydrogen bonds between side chains of amino acids and water molecules are broken in the ligand site when CsA is bound. The germination spore assays using CsA showed a low inhibitory activity in germination, but high inhibition of the germ tube growth. These results show that cyclophilin plays an important role in the growth process, but not in the germination, hence suggesting that cyclophilin is as potential target for the fungistatic action against M. perniciosa.

Epithelial cells from mammary gland tissue that are cultured in vitro are able to maintain specific functions of this gland, such as cellular differentiation and milk protein synthesis. These characteristics make these cells a useful model to study mammary gland physiology, development and differentiation; they can also be used for production of exogenous proteins of pharmaceutical interest. Bovine mammary epithelial cells were cultured in vitro after isolation from mammary gland tissue of animals at different stages of development. The cells were plated on Petri dishes and isolated from fibroblasts using saline/EDTA treatment, followed by trypsinization. Cells isolated on plastic were capable of differentiating into alveolus-like structures; however, only cells derived from non-pregnant and non-lactating animals expressed β-casein. Real-time qPCR and epifluorescence microscopy analyses revealed that alveolus-like structures were competent at expressing Emerald green fluorescent protein (EmGFP) driven by the β-casein promoter, independent of β-casein expression.

The present work reports the activities of urea cycle enzymes during the ontogenic development of the teleost pacu (Piaractus mesopotamicus). Urea cycle enzymes from the kidney and liver of adult fish were compared with those from the fish's embryonic phases. Samples were evaluated over all phases of embryonic development, the larval period and alevin. Ammonia and urea concentrations were determined during embryogenesis and in the plasma of adult fish. Except for carbamoyl phosphate synthetase-III (CPS-III), all enzymes of the urea cycle were expressed in the larvae and alevins as well as in the liver and kidney of adult fish. In spite of the low level of activity of the ornithine urea cycle (OUC) enzymes compared to those in mammals, and the low levels of tissue urea concentration compared to ammonia, the ureogenesis was evaluated in pacu. Ammonia seems to be the main nitrogenous waste during embryonic development. In this phase glutamine synthetase (GS) may play a role in ammonia detoxification, and the OUC enzymes can be individually involved in functions other than urea production. The presence of ornithine carbamoyl transferase (OCT) in all developmental phases of pacu and in the adult liver and kidney suggests that this enzyme is performing different metabolic pathways. OCT in the kidney, wherein the activity is less than in the liver, should work in the biosynthesis of polyamines and control the arginine plasma concentration given that renal arginase and argininosuccinate synthetase-argininosuccinate lyase are more active than from the liver. We suppose that OCT during the embryogenesis is a control step regulating the cellular concentration of ornithine for polyamines synthesis.

Background Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) (EC 2.4.2.8) is a central enzyme in the purine recycling pathway. Parasitic protozoa of the order Kinetoplastida cannot synthesize purines de novo and use the salvage pathway to synthesize purine bases, making this an attractive target for antiparasitic drug design. Results The glycosomal HGPRT from Leishmania tarentolae in a catalytically active form purified and co-crystallized with a guanosine monophosphate (GMP) in the active site. The dimeric structure of HGPRT has been solved by molecular replacement and refined against data extending to 2.1 Å resolution. The structure reveals the contacts of the active site residues with GMP. Conclusion Comparative analysis of the active sites of Leishmania and human HGPRT revealed subtle differences in the position of the ligand and its interaction with the active site residues, which could be responsible for the different reactivities of the enzymes to allopurinol reported in the literature. The solution and analysis of the structure of Leishmania HGPRT may contribute to further investigations leading to a full understanding of this important enzyme family in protozoan parasites.

Membros do gênero Ranavirus, pertencente à família Iridoviridae, têm sido responsáveis por epizootias em animais ectotérmicos em várias partes do mundo, pois representam patógenos emergentes capazes de infectar três classes de vertebrados: peixes ósseos, anfíbios e répteis. No Brasil, os relatos de surtos por ranavirose sassociados ao Frogvirus3 (FV3) ocorrem desde meados dos anos 2000, sendo cada vez mais frequentes principalmente em criações comerciais de rã-touro. Portanto, as infecções por ranavírus representam um risco para outros setores da aquicultura, como a piscicultura. Neste contexto, visando a produção de imunorreagentes para uso em testes de diagnóstico, os objetivos foram: produzir a proteína recombinante do gene MCP (Major capsid protein) do isolado brasileiro deRanavirus,FV3-símile; produzir anticorpos policlonais de coelho anti-rMCP e, por fim, padronizar um teste de ELISA-IB (Indirect-Blocking Enzyme Linked Immunosorbent Assay). Para isso, foi construído o plasmídeo pET28a/MCP, transformado em Rosetta (™) (DE3) para a expressão da proteína rMCP. Após a purificação, a produção de anticorpos policlonais anti-rMCPfoi conduzida em dois coelhos jovens através de três inoculações da proteína rMCP, por via intramuscular e subcutânea, em intervalos de 14 dias, sendo os soros analisados por Immunoblot. Para a padronização do teste ELISA-IB, foram amostrados soros de rãs-touro e realizada a infecção experimental de tilápias do Nilo, para a obtenção de soros controles. Destaque para os resultados com o sucesso da expressão da proteína rMCP de 50kDa,no entanto, em sua forma insolúvel sob purificação desnaturante seguida de diálise. A rMCP desencadeou a produção de anticorpos policlonais nos coelhos, já na segunda imunização. No ELISA-IB, a proteína rMCP foi utilizada como antígeno viral, seguida dos soros testes e anticorpos de bloqueio (policlonal anti-rMCP). Contudo, problemas com a obtenção de soros controles adequados dificultaram a padronização do teste, sendo possível, no entanto, identificar porcentagem de inibição em soro de rã. O uso dos imunorreagentes produzidos e o ELISA-IB demostraram potencial promissor para o diagnóstico laboratorial de infecção por ranavírus em peixes, bem como anfíbios, no intuito de colaborar para a melhoria das condições sanitárias da aquicultura brasileira. Além disso, os imunorreagentes obtidos podem propiciar novos estudos para a produção de imunógenos vacinais contra o Ranavirus. Members of the genus Ranavirus, belonging to the family Iridoviridae, have been responsible for epizootics in ectothermic animals in various parts of the world, as they represent emerging pathogens capable of infecting three classes of vertebrates: bony fish, amphibians and reptiles. In Brazil, reports of Frog virus 3 (FV3)-associated ranavirus outbreaks have occurred since the mid-2000s, and are increasingly common mainly in commercial bullfrog farming. Therefore, ranavirus infections pose a risk to other aquaculture sectors, such as fish farming. In this context, aiming at the production of immunoreagents for use in diagnostic tests, the objectives were: to produce the recombinant MCP (Major capsid protein) gene from the Brazilian Ranavirus isolate, FV3-like; produce rabbit anti-rMCP polyclonal antibodies and, finally, standardize an ELISA-IB (Indirect-Blocking Enzyme Linked Immunosorbent Assay) test. To this end, the Rosetta (™)transformed plasmid pET28a / MCP (DE3) was constructed for the expression of the rMCP protein. Following purification, the production of polyclonal anti-rMCP antibodies was conducted in two young rabbits by three intramuscular and subcutaneous immunizations with the rMCP protein at 14-day intervals and the sera analyzed by Immunoblot. For standardization of the ELISA-IB test, bullfrog sera were sampled and experimental infection of Nile tilapia was carried out to obtain control sera. It is worth mentioning for the results, the successful expression of the 50kDa rMCP protein, however, in its insoluble form, under denaturing purification followed by dialysis. The rMCP triggered the production of polyclonal antibodies in rabbits as early as the second immunization. In ELISA-IB, rMCP protein was used as viral antigen, followed by sera tests and blocking antibodies (anti-rMCP polyclonal). However, problems with obtaining adequate control sera made the standardization of the test difficult, but it was possible to identify inhibition percentage in frog serum. The use of the produced immunoreagents and the ELISA-IB show promising potential for laboratory diagnosis of ranavirus infection in fish, as well as amphibians, in order to contribute to the improvement of sanitary conditions in Brazilian aquaculture. In addition, the obtained immunoreagents may provide further studies for the production of vaccine immunogens against Ranavirus.

Mixosporídeos são cnidários cosmopolitas e endoparasitas obrigatórios, com cerca de 2.600 espécies descritas. Possuem um complexo ciclo biológico, caracterizado pela transmissão via esporos multicelulares. Podem ser encontrados em vários tecidos e órgãos de vertebrados, principalmente peixes, mas também em anfíbios, répteis, aves e mamíferos. Esses cnidários também são encontrados em invertebrados aquáticos (oligoquetas), estes atuam como hospedeiro definitivo e os vertebrados atuam como hospedeiros intermediário. Uma variada gama de espécies de mixosporídeos são importantes patógenos de peixes, e apresentam grande impacto econômico na aquicultura e na pesca extrativista, no mundo inteiro. No Brasil são descritas aproximadamente 140 espécies de mixosporídeos e algumas dessas em peixes nativos e de sistemas de criação têm se mostrado patogênicas ou com grande potencial para causar danos em seus hospedeiros, ocasionando impactos e prejuízos econômicos tanto na pesca quanto na aquicultura. Os métodos tradicionais de detecção desses parasitas em peixes e oligoquetas necessitam da captura dos hospedeiros, para a obtenção de cistos ou esporos e normalmente estes métodos demandam muito trabalho envolvendo uma série de etapas, e a necessidade de vários equipamentos. Ainda quando não há a presença visível de infecções ou altas taxas de mortalidades de peixes, a detecção dos parasitas pode ser dificultada. Este estudo teve como objetivo otimizar métodos para detectar a presença de DNA de mixosporídeos através da análise do SSrDNA em amostras ambientais através das técnicas de PCR Convencional (cPCR) e PCR em Tempo Real (qPCR), oferecendo métodos simples de detecção precoce, e que podem avaliar e quantificar a presença de mixosporídeos na água de pisciculturas. Foram coletadas 48 amostras de água de duas pisciculturas do interior do estado de São Paulo. As amostras contendo cada um 1 litro de água foram armazenadas em gelo e conduzidas para o laboratório. Posteriormente, as amostras foram processadas seguindo as etapas: filtração à vácuo em membrana de nitrato de celulose; extração de DNA de esporos reditos na membrana; Reação de cPCR e qPCR com primers descritos na literatura, e primers construídos para esse estudo. A metodologia utilizada para a filtração e extração de DNA mostraram-se eficientes para a obtenção de DNA de esporos de mixosporídeos presentes na água e as concentrações de DNA variaram entre 1,6 ng a 53,2 ng. A cPCR foi capaz de detectar até 0,1 x 10-3 ng de DNA e identificar 24 amostras positivas (50%). A qPCR detectou concentrações de até 0,1 x 10-4 ng de DNA de mixosporideos e 40 amostras coletadas (83%) foram positivas. Os resultados desse trabalho, mostraram que a qPCR é mais sensível que a cPCR sendo capaz de detectar a presença do SSrDNA de mixosporídeos em um número maior de amostras. Myxosporeans are cnidarians, mandatory cosmopotian, endoparasites, with about 2.600 species listed. They have a complex biological cycle, characterized by transmission via multicellular spores. Myxosporeans infect a variety of tissues and vertebrate organs, mainly fish, but also amphibians, reptiles, birds and mammals. Annelids serve as the invertebrate hosts in the complex life cycle of myxosporeans. The latter are invertebrate hosts. A wide range of myxosporeans species are important fish pathogens, and present a great economic impact in aquaculture and commercial fishing worldwide. In Brazil, 140 myxosporeans species were registered. Some listed in native fish and fish farming have been shown to be pathogenic or in great potential to cause damage to their hosts, causing impacts and economic losses in both fishing and aquaculture. The current methods of detecting these parasites require the capture of hosts to obtain samples, which is toilful and time consuming. Even when there is no visible infection or high fish mortality rates, the detection of parasites can be challenging. This present study aimed to optimize methods to detect the presence of myxosporeans DNA in environmental samples using conventional PCR (cPCR) and Real-Time PCR (qPCR) techniques, offering simple methods of early detection, which can evaluate and quantify the presence of myxosporeans in fish farms water. 48 water samples were collected from two fish farms in the countryside of São Paulo state. They were stored in ice and transported to the laboratory. After, the samples were processed according to the following steps: filtration, vacuum on cellulose nitrate membrane; DNA extraction from the membrane; Reaction of Conventional PCR and Real-Time PCR with primers described in the literature, and primers built for this study. The methodology used for DNA filtration and extraction proved to be efficient for obtaining DNA from myxosporeans present in water and the DNA concentrations varied from 1.6 ng to 53,2 ng. The cPCR was able to detect up to 0.1 x 10-3 ng of DNA and identify 24 positive samples (50%). The qPCR detected concentrations up to 0,1 x 10-4 ng of myxosporeans DNA and 40 samples collected (83%) were positive. The results of this work showing to be more sensitive qPCR than cPCR able to detect the presence of myxosporid SSrDNA in a larger number of sample.

Células neuronais cocultivadas com células-tronco derivadas do tecido adiposo (ADSC) podem induzir estas últimas à transdiferenciação neuronal. No entanto, os processos de comunicação celular envolvidos nessa indução, e a funcionalidade da ADSC transdiferenciada in vivo, são desconhecidos. Recentemente, um novo tipo de comunicação celular mediada por vesículas extracelulares são indicados na modulação de diferentes eventos celulares como a diferenciação. Portanto, a hipótese, nesta proposta, foi identificar se o processo de diferenciação celular é mediado por vesículas extracelulares e se as células diferenciadas são capazes de atuar na regeneração de tecidos nas lesões do sistema nervoso periférico. Para tal, essa pesquisa foi dividida em duas fases: a primeira consiste no processo in vitro, com o objetivo de observar o processo de transição da ADSC para a linhagem neuronal e analisar a função da comunicação celular no processo de diferenciação; a segunda consistiu na avaliação in vivo da possível funcionalidade das ADSC diferenciadas. O camundongo foi o modelo animal utilizado (C57BL/6 e FVB). As ADSC e as células neuronais foram isoladas, cultivadas em cultivo primário e cocultivadas durante três, sete e 14 dias. Para a comprovação das mudanças fenotípicas das ADSC, realizou-se a imunolocalização com beta tubulina III e SNAP25 e PCR em tempo real (RT-qPCR) dos genes Map2 e Snap25. Seguido de analises de genes relacionados com a neurogênese. Adicionalmente, as vesículas extracelulares foram isoladas e utilizados para análises in vitro da diferenciação e análises gênicas e funcionais. Como resultado, verificou-se que as ADSC em cocultivo com neurônios podem se diferenciar em neuronais-like. Além disso, comprovou-se a comunicação por vesículas extracelulares entre neurônios e ADSC, e as vesículas extracelulares foram correlacionadas neste processo, pelo transporte da proteína SNAP25. Após estes resultados prosseguiu-se para a segunda fase deste trabalho, a etapa in vivo, que incidiu na utilização das ADSC cocultivadas por sete dias e avaliação funcional local e sistêmica no processo de regeneração do nervo ciático após neurotmese. Como resultado desta etapa as células-tronco cocultivadas modularam a lesão, e proporcionaram uma melhoria na funcionalidade após lesão. Conclui-se, através desta pesquisa, que as ADSC diferenciadas em neuronais-like, sob indução dos neurônios e suas vesículas extracelulares podem ser uma fonte celular alternativa no auxílio na regeneração de nervos periféricos. Neuronal cells co-cultured with stem cells derived from adipose tissue (ADSC) can induce the latter to neuronal transdifferentiation. However, the cellular communication processes involved in this induction, and the functionality of the transdifferentiated ADSC in vivo, are unknown. Recently, a new type of cellular communication measured by extracellular vesicles was indicated in the modulation of different cellular events like differentiation. Therefore, the hypothesis in this proposal was to identify if the process of cellular differentiation is mediated by extracellular vesicles and if the differentiated cells are able to act in the regeneration of tissues in the lesions of the peripheral nervous system. For this, the research was divided in two phases: the first one consisted of the in vitro process, with the objective of observing the transition process of the ADSC to the neuronal lineage and analyzing the cellular communication function in the differentiation process; the second consisted in the in vivo evaluation of the possible functionality of the differentiated ADSCs. Murine was the animal model used (C57BL/6 and FVB). ADSCs and neuronal cells were isolated, cultured in primary culture and co-cultured for three, seven and 14 days. To confirm the phenotypic changes of ADSC, immunolocalization with beta tubulin III and SNAP25 and real-time PCR (RT-qPCR) of the Map2 and Snap25 genes was performed, followed by analysis of genes related to neurogenesis. In addition, extracellular vesicles were isolated and used for in vitro differentiation and gene and functional analysis. As a result, it has been found that ADSCs in co-culture with neurons can differentiate into neuronal-like. The communication by extracellular vesicles between neurons and ADSCs was verified, and the extracellular vesicles were correlated in the differentiation process by the transport of the protein SNAP25. After these results, the second phase of this work was continued, the in vivo step, which focused on the use of the co-cultivated ADSCs for seven days and functional local and systemic evaluation in the process of sciatic nerve regeneration after neurotmese. As a result of this step, the co-cultured stem cells modulated the lesion and provided an improvement in functionality after injury. It is concluded that ADSCs can transdifferentiate neuronal lines in co-culture with neurons, the extracellular vesicles play a certain role in this process and the transdifferentiated ADSC may be an alternative to aid in the regeneration of peripheral nerves.

O protozoário ciliado Ichthyophthirius multifiliis causador da doença dos pontos brancos em muitas espécies de peixes de água doce destaca-se como um importante patógeno por ser responsável por grandes perdas para a indústria de pescados mundial. Duas espécies de peixes de grande importância para a economia no Brasil foram selecionadas para este estudo, o pintado (Pseudoplatystoma corruscans (Spix & Agassiz, 1829) e a cachara (Pseudoplatystoma reticulatum (Eigenmann & Eigenmann, 1889). Com o objetivo de avaliar a resposta imune destes peixes frente ao parasita, inicialmente, o protozoário I. multifiliis foi isolado e repicado em P. corruscans para a obtenção da forma infectante (teronte) utilizada nas imunizações. A identificação molecular dos peixes foi realizada através da amplificação e sequenciamento do gene RAG2 mostrando que as espécies puras são homozigotas enquanto o híbrido é heterozigoto para este gene. Através de PCR foi realizada ainda a caracterização parcial dos genes arginase 1, arginase 2, IL-1β, IL-8 e TNF-α dos peixes pertencentes a família Pseudoplatystoma que resultaram em amplificações de sequências com aproximadamente 530, 815, 400, 280 e 300 pb, respectivamente, que foram usadas para a construção de primers específicos utilizados nas análises de expressão gênica por PCR em tempo real (qPCR). A expressão gênica em P. corruscans e P. reticulatum imunizados com terontes vivos de I. multifiliis foram verificadas 6, 12, 18 e 24 horas após imunização, sendo retirada amostras do baço, fígado e rim anterior dos peixes. No geral a expressão dos genes destes órgãos em P. corruscans apresentam um padrão semelhante de regulação. Um aumento significativo de expressão em relação ao período de imunização foi verificado para: a arginase 2 as 6 e 18 horas no baço, as 6 h no fígado e as 6, 18 e 24 horas no rim cranial; o gene da IL1-β as 24 h no fígado e 18 h e 24 h no rim cranial; e o gene da IL-8 somente as 18 h no baço. Já os genes arginase 1 e TNFα não tiveram regulação significativa nos períodos. Para P. reticulatum observou-se que somente o baço teve aumento na regulação com diferenças significativas nos períodos para arginase 2, IL-8 e TNFα, as 6 e 18 horas, já os genes arginase 1 e IL1-β não apresentaram alterações significativa. The ciliate protozoan Ichthyophthirius multifiliis has been reported in various freshwater fishes and stands out as an important pathogen for being responsible to severe losses to both food and aquarium fish production. Among the fish species explored economically in Brazil it can be highlight the pintado (Pseudoplatystoma corruscans (Spix & Agassiz, 1829) and cachara (Pseudoplatystoma reticulatum (Eigenmann & Eigenmann, 1889). At this study the parasite I. multifiliis was original isolated from a petshop infected fish and maintained by serial transmission on naive pintado (P. corruscans) to obtain the infecting form (teronte) used at immunizations. The molecular identification of fish was realized through the amplification and sequencing of RAG2 gene, were the pintado (P. corruscans) and cachara (P. reticulatum) were homozygous, while the hybrid between those fish was considered heterozygous for this gene. At the partial gene characterization from fishes belonging to the Pseudoplatystoma family, the PCR amplification result in sequences of 530, 815, 400, 280 and 300 pb of the genes arginase 1, arginase 2, IL1-β, IL-8 e TNFα respectively, which were used to construct of specific primers for the quantitative real-time PCR (qPCR). The gene expression at P. corruscans and P. reticulatum immunized with live theronts of I. multifiliis was evaluated 6, 12, 18 and 24 hours after the immunization, where it was collected samples of the spleen, liver and heady kideny. The gene expression of the organs of pintado (P. corruscans) showed similar pattern of expression. An up-regulation with statistical significance was observed for: arginase 2 at 6 and 18 hours at spleen, 6 h at kidney and 6, 18 and 24 h at head kidney; the gene of IL1-β at 24 h at spleen, 18 and 24 hours at head kidney; the gene of IL-8 at 18 h at spleen. The genes of arginase 1 and TNFα didn\'t showed statistical significant regulation at this experiment. At the gene expression of cachara (P. reticulatum) only the spleen had a statistical alteration after theronts immunization where the arginase 2, IL-8 and TNFα was up-regulated at 6 h and 18 h, while the arginase 1 and IL1-β didn\'t showed statistical alteration.

A clonagem de mamíferos por transferência nuclear de células somáticas (TNCS) ainda é afetada pela baixa eficiência. As modificações epigenéticas estabelecidas durante o processo de diferenciação celular estão entre os principais fatores. Uma vez que estas modificações atuam como barreiras epigenéticas restringindo a reprogramação do núcleo somático. Considerando isso, a maioria dos fatores que promovem a descondensação da cromatina, incluindo os inibidores das Histonas Deacetilases (HDACis), tem sido demonstrado aumentarem a eficiência da reprogramação nuclear, tornando o seu uso comum para melhorar as taxas da TNCS. Neste trabalho, nós testamos dois inibidores das histonas deacetilases: o Ácido Valpróico (VPA) e o Ácido β-Hidróxibutírico (BOHB), sendo um farmacológico e o outro um metabólito endógeno na TNCS. Nosso objetivo era testar se o tratamento de células doadoras de núcleo ou zigotos com estes HDACis melhoravam o desenvolvimento de embriões bovinos clonados. Em relação ao VPA, nós observamos que o tratamento de fibroblastos com o VPA aumentou a acetilação das histonas e a expressão de genes importantes para o desenvolvimento como IGF2R e PPARGC1A. Entretanto, quando as células tratadas foram usadas como doadoras de núcleo, não foi observado diferença nos níveis de acetilação das H3K9 entre os grupos. Além disso, as alterações foram rapidamente removidas após a transferência nuclear. Em relação as taxas de desenvolvimento, o uso de células tratadas como doadoras de núcleo não resultou em diferença durante o desenvolvimento pré- e pós-implantacional. Em relação ao BOHB, nós executamos um série de experimentos para testar se esta molécula age como um inibidor das HDACs em bovinos. Nós observamos que fibroblastos tratados com BOHB apresentam aumento nos níveis globais de H3K9ac. O tratamento altera a expressão de genes importantes como os transportadores de glicose e a enzima chave no metabolismo lipídico SCD. Também, nós demonstramos que este metabólito é capaz de alterar uma marca epigenética em zigotos clonados, que persiste até o estágio de blastocisto. Entretanto, o tratamento dos zigotos com este metabólito endógeno não aumentou a taxa de desenvolvimento pré-implantacional, embora tenha alterado a expressão de um fator de transcrição que protege embriões contra estresse oxidativo, o FOXO3a. O tratamento de zigotos partenogenéticos com BOHB não afetou o desenvolvimento embrionário, nem a produção de ATP, sugerindo que o BOHB não apresenta efeitos tóxicos como anteriormente pensado. Concluindo, os resultados deste trabalho mostram que a inibição das HDACs através de uma molécula farmacológica ou um metabólito endógeno, não aumenta a eficiência da TNCS em bovinos. Entretanto, aqui nós mostramos que o corpo cetônico BOHB pode ser um elo de ligação entre o ambiente extracelular e a regulação do metabolismo. E serve como um modelo in vitro para testar como distúrbios metabólicos como a cetose afeta o metabolismo e epigenoma celular e embrionário em bovinos Cloning of mammals by somatic cell nuclear transfer (SCNT) is still plagued by the low efficiency. The epigenetic marks established during the cell differentiation process are among the main cause. These modifications act as a barrier restricting the nuclear reprogramming process of somatic nuclei. Based on this, molecules that promotes chromatin decondensing, including Histone Deacetylases inhibitors (HDACis), has been demonstrated increase the efficiency of nuclear reprogramming, making their use common on SCNT procedure. Herein, we tested two histone deacetylase inhibitors: Valproic Acid and β-hydroxybutyric Acid, the former a pharmacological drug and the latter, an endogenous metabolite on SCNT. Our objective was to test whether the donor cells treatment or zygotes with these HDACis improve the bovine cloned embryos development. Regarding the VPA, we observed that fibroblasts treatment with VPA increases the histone acetylation and expression of developmentally important genes such as IGF2R and PPARGC1A. However, when treated cells were used as nuclear donors, we did not observe difference on H3K9ac levels between the groups. Moreover, the alterations were quickly removed after SCNT. Regarding the developmental rates, the use of treated cells as nuclear donors did not affect the pre- and post-implantation development. In the second experiment, we used the BOHB in a series of experiments to test whether this molecule acts as a histone deacetylase inhibitor in bovines. We observed that treatment of fibroblasts with BOHB increased the global levels of H3K9ac. Also, treatment alters the expression of important genes such as glucose transporters and a key enzyme regulating lipid synthesis. Additionally, we demonstrated that this metabolite affect at least one epigenetic mark in cloned zygotes, that lasts until the blastocyst stage. However, zygote treatment with this endogenous metabolite did not increase the pre-implantation developmental rates, albeit increased the expression of a transcription factor that protects cells against oxidative stress, the FOXO3a. Treatment of parthenogenetic embryos with BOHB did not affect the embryo development, neither ATP production, suggesting that BOHB is not toxic as previously believed. Concluding, the results presented here shows that the HDAC inhibition through a pharmacological compound or an endogenous metabolite did not increase the efficiency of bovine SCNT. However, here we showed that the ketone body BOHB might be a nexus between the extracellular environment and the cellular metabolism. Also, it can used as a in vitro model to interrogate questions about does metabolic disturbances such as ketosis in cattle affects the epigenome and cellular metabolism in bovines