Sperm characterization of the Amazonian freshwater cururu stingray Potamotrygon wallacei (Potamotryogonidae): basic knowledge for reproduction and conservation plans.

This study aimed to describe the morphology and sperm quality of free-living adult males of cururu stingray Potamotrygon wallacei, endemic from the Rio Negro basin, Brazilian Amazon. The sperm was collected in loco from the seminal vesicle region and fixed in buffered saline formaldehyde solution for further evaluation of morphometry, sperm plasma membrane integrity and sperm concentration. The spermatozoa presented a total length of 138.25 ± 1.82 µm with a helical shape and a long head. A high percentage of cells with intact membrane (98 ± 2%) and normal spermatozoa (92 ± 1%) were observed. The cell concentration was 0.34 ± 0.05 × 1010 spermatozoa/ml of semen. These observations are unprecedented for potamotrygonid species and will serve as a basis for future management and conservation strategies.

The effect of cryoprotectant agents on DNA methylation patterns and progeny development in the spermatozoa of Colossoma macropomum.

DNA methylation patterns are inherited from parents and are imperative for proper embryonic development; however, alterations in these patterns can compromise fertilization and development into a fully functioning adult animal because DNA methylation is part of a complex program of gene transcription. In this study, we investigated the impact of cryoprotectant agents (CPAs) on DNA methylation patterns in spermatozoa and the consequences on embryonic development and the survival rate of progeny. Global methylation was assessed by enzymatic reactions in Colossoma macropomum spermatozoa that were cryopreserved using dimethylsulfoxide, dimethylformamide, methanol, ethyl glycol and glycerol as CPAs. Fertilization was carried out to evaluate survival rates and abnormalities in embryonic development upon treatment with each of the CPAs. Fresh semen served as the control. Our results indicated that, compared to the control group, spermatozoa cryopreservation decreased the fertilization rate and delayed embryonic development from the midblastula stage. Furthermore, spermatozoa cryopreserved in all CPAs had lower methylation levels and exhibited more delays and abnormalities during embryonic development than did fresh semen. Methanol resulted in fertilization, hatching rates and embryonic development that were closer to the control but had lower methylation levels. In conclusion, ours results show significant alterations on spermatozoa DNA methylation patterns caused by CPAs that are used in the semen cryopreservation process. DNA methylation pattern alterations affected the viability of progeny (r=0.48); however, these effects can be minimized by choosing the CPA that will compose the freezing solution.

Viability of zebrafish (Danio rerio) ovarian follicles after vitrification in a metal container.

Cryopreservation of ovarian tissue has been studied for female germline preservation of farm animals and endangered mammalian species. However, there are relatively few reports on cryopreservation of fish ovarian tissue and especially using vitrification approach. Previous studies of our group has shown that the use of a metal container for the cryopreservation of bovine ovarian fragments results in good primordial and primary follicle morphological integrity after vitrification. The aim of this study was to assess the viability and in vitro development of zebrafish follicles after vitrification of fragmented or whole ovaries using the same metal container. In Experiment 1, we tested the follicular viability of five developmental stages following vitrification in four vitrification solutions using fluorescein diacetate and propidium iodide fluorescent probes. These results showed that the highest viability rates were obtained with immature follicles (Stage I) and VS1 (1.5 M methanol + 4.5 M propylene glycol). In Experiment 2, we used VS1 to vitrify different types of ovarian tissue (fragments or whole ovaries) in two different carriers (plastic cryotube or metal container). In this experiment, Stage I follicle survival was assessed following vitrification by vital staining after 24 h in vitro culture. Follicular morphology was analyzed by light microscopy after vitrification. Data showed that the immature follicles morphology was well preserved after cryopreservation. Follicular survival rate was higher (P < 0.05) in vitrified fragments, when compared to whole ovaries. There were no significant differences in follicular survival and growth when the two vitrification devices were compared.

Effect of cryoprotectants on the survival of cascudo preto (Rhinelepis aspera) embryos stored at -8 °C.

Cryopreservation of germplasm provides a promising method to preserve fish genetic material, which is of great importance in preservation of species diversity, aquaculture, and management of fish models used in biomedical research. In the present study, cryopreservation of Rhinelepis aspera embryos, a Brazilian endangered species, was studied for the first time using a short-term cooling protocol. Embryos at blastoporous closing stage were selected, placed in 6-ml glass vials and stored at -8 °C for 6 h in 10 different cryoprotectant solutions: S1 (17.1% sucrose + 9% methanol); S2 (17.1% sucrose + 9% DMSO); S3 (8.5% sucrose + 8.5% glucose + 9% methanol); S4 (8.5% sucrose + 8.5% glucose + 9% DMSO); S5 (17.1% sucrose + 9% ethylene glycol); S6 (8.5% sucrose + 8.5% glucose + 9% ethylene glycol); S7 (17.1% sucrose + 4.5% methanol + 4.5% DMSO); S8 (17.1% sucrose + 4.5% methanol + 4.5% ethylene glycol); S9 (17.1% sucrose + 4.5% DMSO + 4.5% ethylene glycol); and S10 (100% water). Embryo viability was assessed by hatching rate, counting live larvae and number of failed eggs under a stereomicroscope. The results showed that only the cryoprotectant solutions that contained methanol associated to sucrose (S1, S7 and S8) provided partial protection of Rhinelepis aspera embryos from cold damage (over 50% hatching rate in S1), while the use of DMSO and ethylene glycol, isolated or in combination, resulted in no hatching rate. Further studies are needed in order to extend the storage time and to improve the hatching rate for the species.

Injuries in pacu embryos (Piaractus mesopotamicus) after freezing and thawing.

Although the sperm cryopreservation of freshwater and marine teleosts has been feasible for years, the cryopreservation of some fish embryos still remains elusive. Thus, the objective of this experiment was to analyze the embryo morphology after freezing and thawing 40 embryos of Piaractus mesopotamicus immersed into methanol and ethylene glycol, both at 7, 10 and 13% plus 0.1 M sucrose for 10 min. Soon after thawing, three embryos were treated with historesin, stained with hematoxylin-eosin and analyzed under an optical microscope. From every treatment, one palette containing embryos was thawed and incubated, but none of the eggs hatched. Samples containing two embryos were immersed into 10% methanol or 10% ethylene glycol both in association with sucrose, and embryos immersed into only water or sucrose solution were frozen, processed and analyzed using scanning electron microscopy (SEM). In both cases, the control group was immersed into only water. Although the embryos had the chorion, vitello, yolk syncytial layer and blastoderm, all of them were found altered under the optical microscope and by SEM. The chorion was irregular and injured; there was no individuality in the yolk granules; the yolk syncytial layer had an irregular shape, thickness and size; the blastoderm showed injuries in the nucleus shape and sometimes was absent; the blastoderm was located in atypical areas and absent in some embryos. In conclusion, no treatment was effective in preserving the embryos, and none of the embryos avoided injury from intracellular ice formation. These morphological injuries during the freezing process made the P. mesopotamicus embryos unfeasible for hatching.

Use of Powdered Milk in Semen Cryopreservation Protocols for Fish: A Systematic Review.

This systematic review provides an overview of the history and current status of cryopreservation of fish sperm and a detailed evaluation of cryoprotocols using powdered milk. A literature search was performed in PubMed, Scopus, Web of Science, and SciELO databases. Twenty-nine articles were selected after excluding duplicate articles or articles that did not meet the eligibility criteria. Rhamdia quelen and Danio rerio were the most studied species. Slow freezing method, dry-shipper, freezing rate of -35.6°C/min, thawing in water bath (35.93°C ± 10°C), and 0.25 and 0.5 mL plastic straws were the main approaches evaluated. Methanol was the most used permeable cryoprotectant in combination with powdered milk, yielding the best results at 10% concentration. Motility rate was the main analysis performed after cryopreservation in virtually all studies, being subjectively evaluated by most authors. Powdered milk at 15% promoted the best results in the analyzed studies. For motility rate, the gains with the addition of powdered milk were observed in the orders Perciformes (Oreochromis mossambicus), Siluriformes (Pangasius pangasius, Pseudoplatystoma corruscans, and Pseudoplatystoma mataense), and Cypriniformes (Tor soro and Barbonymus gonionotus). For fertilization, gains were observed in the order Siluriformes (P. mataense) and Cypriniformes (T. soro). Sperm viability gains were observed in the orders Siluriformes (P. pangasius), Characiformes (Piaractus brachypomus), and Cypriniformes (B. gonionotus). The scientific evidence we present in this study may contribute and serve as a starting point for new and more refined studies to be developed in the field.

Catalase and Uric Acid Prevent Morphological Damage to the Sperm Flagella of Colossoma macropomum During 96 Hours at Low Storage Temperatures.

Oxidative stress is one of the main causes of loss of sperm function during chilled storage. The aim of the current study was to evaluate the effects of a fructose-based extender, which was supplemented with catalase or uric acid, on the motility, viability, morphological integrity, and lipid peroxidation (LPO) of Colossoma macropomum spermatozoa. Sperm was diluted in extenders containing catalase (0; 0.1; 0.8; and 1.5 kU/L) or uric acid (0; 0.25; 0.5; and 1.0 mmol/L) and then stored at 4.3 ± 0.6°C for 96 hours. The chilling storage time had more significant and pronounced effects on practically all the measured sperm quality parameters than the different concentrations of both antioxidants added to the extenders. This was true for sperm motility, motility duration, sperm viability, and the percentage of normal spermatozoa. In fact, for all these parameters, values were higher in the extenders supplemented with catalase or uric acid, than those not supplemented with these antioxidants, especially after 96 hours. The LPO process showed an antioxidant-dependent response. In catalase-supplemented extenders thiobarbituric acid reactive substance (TBARS) levels increased gradually and significantly with time, but remained stable during the 96 hours of chilled storage in all samples in which uric acid was added. Despite this, TBARS levels were lower in the extenders supplemented with both catalase and uric acid than in those not having these antioxidants. Inverse correlations were found between sperm motility and the damage in sperm flagella. Our findings suggest that the supplementation of an extender with catalase or uric acid is beneficial and protects fish sperm membranes from damage caused by oxidative stress during low-temperature storage. The extenders containing 0.1 kU/L of catalase and 0.25 mmol/L of uric acid provided effective antioxidant protection for the spermatozoa of this important Amazonian fish.

A study on the vitrification of stage III zebrafish (Danio rerio) ovarian follicles.

Attempts to cryopreserve fish embryos have been conducted over the past three decades, nevertheless successful cryopreservation protocol for long-term storage still remains elusive. Fish oocytes offer some advantages when compared to embryos, which may help in improving the chances of cryopreservation. In the present study, a series of cryo-solutions were designed and tested for their vitrifying ability using different devices (0.25ml plastic straw, vitrification block and fibreplug™). Toxicity of vitrification solutions was evaluated by assessing follicle membrane integrity with trypan blue staining. In addition, the effect of vitrification protocol on stage III zebrafish ovarian follicles was investigated by measuring the cytoplasmic ATP content and the mitochondrial distribution and activity using JC-1 probe and confocal microscopy. After vitrification, follicles showed membrane integrity of 59.9±18.4% when fibreplug and V16 (1.5M methanol+4.5M propylene glycol) solution were employed. When vitrified in V2 (1.5M methanol+5.5M Me2SO) the membrane integrity decreased to 42.0±21.0%. It was observed that follicles located in the middle of the fragments were more protected from injuries and some of them showed good morphological appearance 2h post-warming. Mitochondria integrity of granulosa cells layer was clearly damaged by the vitrification protocol and ATP level in the follicles declined significantly after warming. Vitrification of zebrafish follicles in ovarian tissue fragments and its effect at sub-cellular level is reported here for the first time. Information gained from this study will help in guiding development of optimal protocol for cryopreservation of fish oocytes.

Alterations in Gene Expression and the Fatty Acid Profile Impact but Do Not Compromise the In Vitro Maturation of Zebrafish (Danio rerio) Stage III Ovarian Follicles after Cryopreservation.

The vitrification of ovarian follicles is a strategic tool that may contribute to advances in aquaculture and the conservation of many important species. Despite the difficulties inherent to the cryopreservation of oocytes, some successful protocols have been developed for different species, but little is known about the capacity of oocytes to develop after thawing. Therefore, the profiles of the reproductive pathway genes and fatty acid membrane composition during the initial stages of development were analyzed in fresh ovarian follicles and follicles after the vitrification process. There were differences in the expression of the hypothalamic-pituitary-gonad axis genes during the follicular development in the control group as well as in the vitrified group. Similarly, alterations in the composition of fatty acids were observed after vitrification. Despite this, many alterations were observed in the vitrified group; more than half of the stage III ovarian follicles were able to grow and mature in vitro. Therefore, the vitrification of ovarian follicles may impact them at molecular and membrane levels, but it does not compromise their capability for in vitro maturation, which indicates that the technique can be a strategic tool for aquaculture.

Short-term preservation of Colossoma macropomum oocytes by different methods and chilling temperatures.

A preservation protocol has not been established for Colossoma macropomum oocytes, and its development may improve the production and breeding programs of this South American fish species. Thus, this study aimed to determine the effect of different methods and protocols for the preservation of C. macropomum oocytes. Seven experiments were conducted throughout the breeding season of this species. The oocytes were collected and stored in sterile conditions. Preserved oocytes were subjected to storage in the following treatments: room temperature (RT, 27 °C), centrifugation followed by ovarian fluid removal (Cen), vacuum (Vac), chilled temperature (ChT), centrifugation and vacuum (CV), vacuum and chilled temperature (VChT), and centrifugation, vacuum, and chilled temperature (CVChT) in dry sterilized plastic containers and plastic bags. Chilled storage was tested at 4 and 13 °C. Fertilization and hatching rates were assessed at 0, 30, 60, 90, and 120 min after stripping (MAS) for preservation protocols. The larval malformation rate was analyzed at 0 and 30 MAS for RT and ChT. Quantitation and identification (by mean of MALDI-TOF MS) of bacteria were performed at 0, 60, 90, and 120 MAS, and scanning electron microscopy (SEM) analyses were carried out at 0, 60, and 90 MAS. The fertilization and hatching rates decreased over preservation time and breeding season. RT samples fertilized at 0, 30, and 60 MAS yielded similar fertilization rates at both the beginning and end of the season. By the end of the season, oocytes from treatment ChT at 13 °C 30 MAS yielded higher fertilization and hatching rates, and a lower percentage of larvae malformation than RT 30 MAS. The treatment ChT at 4 °C triggered low a fertilization rate. The treatments ChT (13 °C) and Cen provided good fertilization rate when used alone and with other approaches, i.e., treatments VChT, CV, and CVChT. The treatment Vac presented inconsistent results, so no conclusion could be made. Bacterial colony counts were low (10-1.6 × 105 CFU-mL-1), and a total of 18 bacteria species were identified in all batches analyzed; however, the treatments did not influence the number of bacteria. C. macropomum female breeders presented distinct bacteria species in their oocytes and the presence of bacteria did not impair oocyte quality until 120 MAS. Moreover, SEM analyses showed that the micropyle was not occluded during oocyte storage, and ovarian fluid was observed on the surface of chilled oocytes. Therefore, Colossoma macropomum oocytes could be preserved under chilled storage at 13 °C for 30 min throughout its breeding season.

Silicon and mechanical damage increase polyphenols and vitexin in Passiflora incarnata L.

Passiflora incarnata L. is a species of global pharmacological importance, has not been fully studied in the context of cultivation and management. It is known that silicon acts on abiotic stress and promotes phenols synthesis. The practice of mechanical damage is widely used in P. incarnata crops, and its interaction with silicon can have a significant influence on plant metabolism. Therefore, our objective was to investigate the effects of silicon and mechanical damage on photosynthesis, polyphenols and vitexin of P. incarnata. The experiment was conducted in a factorial design with SiO2 concentrations (0, 1, 2, 3 mM) and presence or absence of mechanical damage. It was found that mechanical damage improved photosynthetic performance at lower concentrations or absence of silicon. Moreover, this condition promoted an increasing in vitexin concentration when SiO2 was not provided. The application of 3 mM Si is recommended to increase polyphenols and vitexin, without harming dry mass of aerial part. The interaction between silicon and mechanical damage could be a tool to increase agronomic yield and commercial value of the P. incarnata crop.

Sperm characterization of the endangered Amazonian fish Hypancistrus zebra: Basic knowledge for reproduction and conservation strategies.

Hypancistrus zebra is an ornamental fish endemic to the Xingu River (Brazilian Amazon) and is critically endangered by the construction of a hydroelectric plant in its habitat and illegal fishing. In an attempt to create a germplasm bank for conservation purposes, in the present study there was characterization of H. zebra sperm for the first time and assessment of sperm quality throughout the year after successive stripping. Semen was collected four times during a year, and there was similar (P > 0.05) high quality for all values of sperm variables evaluated. Hypancistrus zebra sperm had an average motility rate of 88.60 ± 2.49% and membrane integrity rate of 87.93 ± 1.88%. There was a peculiar characteristic for the species, with an intermediate sperm vigor (3.00 ± 0.13) and a long duration of motility (14.72 ± 1.31 min) which is uncommon for freshwater fish. Semen had an overall mean of 79.13 ± 9.78% normal spermatozoa and 20.96 ± 9.76% of sperm cells with some morphological abnormalities. The most frequent morphological abnormalities were a degenerated head, an isolated head and a coiled flagellum. The collection of good quality semen throughout the year allows for the possible use of artificial reproduction techniques and cryopreservation for development of a germplasm bank that could contribute to successful conservation of this endangered Amazonian fish.

Notes on natural history and behavior of Monodelphis glirina (Didelphimorphia, Didelphidae) and sympatry with congeners

Monodelphis glirina é endêmica da Amazônia e aspectos dos seus hábitos de vida e história natural são pouco conhecidos. Dados sobre a espécie foram coletados no norte de Mato Grosso, Brasil, incluindo observações sobre seu comportamento e simpatria com congêneres. Durante 10 expedições e com um esforço de 3.680 baldes-dia, foram capturados 29 M. glirina, três M. sacie um M. emiliae. Adultos representaram 82,8% das capturas de M. glirina e a razão sexual foi três machos para cada fêmea. A captura de jovens e fêmeas lactantes foi sazonal, uma vez que ambos foram capturados apenas na estação chuvosa. Observamos três eventos de M. glirina predando roedores em pitfall traps, todos com o mesmo comportamento de alimentação. Também registramos um individuo escalando uma árvore durante a noite, incluindo um video. Nosso trabalho contribui para uma melhor compreensão da ecologia desse gênero altamente diversificado e ainda pouco conhecido. (AU)

Removal of Capybara (Hydrochoerus hydrochaeris) population as measure to control Amblyomma tick population and reduce Brazilian Spotted Fever transmission risk in a Gated Community in Bragança Paulista (SP, Brazil) ­ Case Study / Remoção de capivaras (Hydrochoerus hydrochaeris) como ação para controle da população de carrapatos Amblyomma e redução do risco de transmissão da Febre Maculosa Brasileira em um Condomínio Residencial em Bragança Paulista (SP, Brasil) ­ Estudo de Caso

This study reports the factors which led a gated community located in Bragança Paulista (SP, Brazil), a non-endemic area for Brazilian Spotted Fever (BSF), to be classified as a Risk Area for transmission of this disease, showing that an increasing resident population of capybaras (Hydrochoerus hydrochaeris) in the area was likely responsible for a proliferation of Amblyomma sculptum ticks and acted as an amplifying host for Rickettsia rickettsii, the main etiologic agent of BSF. We report management actions proposed to control the local tick burden and reduce BSF risk, including measures to control parasitic and free-living tick populations and exclusion of the resident capybara population. Analyses of tick population data and R. rickettsii serology tests indicate that these measures were effective, greatly reducing the environmental burden of Amblyomma sculptum ticks and reducing the BSF transmission risk at the area.(AU)

Este estudo relata os fatores que levaram um Residencial localizado em Bragança Paulista (SP, Brasil), área não-endêmica para Febre Maculosa Brasileira (FMB), a ser classificado como Área de Risco para a doença, mostrando que uma crescente população residente de capivaras (Hydrochoerus hydrochaeris) na área era a provável responsável por uma proliferação de carrapatos Amblyomma sculptum e estava atuando como hospedeiro amplificador da bactéria Rickettsia rickettsii, principal agente etiológico da FMB. Relatamos as ações de manejo ambiental propostas para controlar a quantidade de carrapatos no local e reduzir o risco de transmissão da doença, incluindo medidas para o controle de populações de carrapatos parasíticas e no ambiente e a eliminação da população residente de capivaras. Análises de dados populacionais de carrapatos e testes serológicos para R. rickettsii indicaram que as medidas tomadas foram efetivas, causando grande redução da população de carrapatos no ambiente e reduzindo o risco de transmissão de FMB na área.(AU)

Morphological and morphometric analysis of skeletal muscle between male and female young adult Colossoma macropomum (Characiformes: Serrasalmidae)

This study aimed to evaluate muscle organization in tambaqui in order to describe the muscle growth process. We analyzed the morphometric pattern of fibers from white muscle of young-adults (300 days) by smaller diameter. The organization of white muscle exhibited a typical morphological pattern found in other fish species. Heavier animals showed higher frequency of larger diameter fibers (>50 m ) and smaller animals had higher frequency of smaller diameter fibers ( 20 m ) (P =0.005). However, both animals showed the same frequency of intermediate diameter fibers (20-50 m ). Body weight showed a positive correlation with muscle diameter fiber (r=0.45), being 20-50 m the diameters that contributed the most to animal weight (P 0.0001). A weak correlation between fiber diameter and animal sex was observed (r=0.2). Females showed higher frequency of large fiber diameters (>50 m ) than males. However, there was no difference between body weight and sex (P =0.8). Our results suggest that muscle growth is by hypertrophy and hyperplasia due to a mosaic appearance from different diameters fibers, which is characteristic of large size fish species.

O objetivo deste trabalho foi avaliar a organização muscular em tambaqui, a fim de descrever o processo de crescimento muscular. Foi analisado o padrão morfométrico das fibras do músculo branco de animais com 300 dias de idade usando o método de diâmetro menor. O músculo branco apresentou uma organização morfológica padrão encontrado em peixes. Animais de maior peso apresentaram maior frequência de fibras de maior diâmetro (> 50 m ) e os animais de menor peso apresentaram maior frequência de fibras de menor diâmetro ( 20 m ) (P = 0,005). Entretanto, ambos os animais, com maior e menor peso, apresentaram frequências semelhantes de fibras de diâmetro intermediário (20-50 m ). O parâmetro peso corporal mostrou correlação positiva com o diâmetro da fibra muscular (r = 0,45), sendo as fibras de diâmetro intermediários (20-50 m ) que mais contribuíram para o peso do animal (P 0,0001). Fêmeas apresentaram maior frequência de fibras de maior diâmetro (>50 m ) que machos. Observou-se uma fraca correlação entre o diâmetro da fibra e o sexo dos animais (r = 0,2). Apesar de fraca, a correlação estimada é corroborada pela fibras de grandes diâmetros (> 50 m ) serem mais frequente nas fêmeas que nos machos. No entanto, não houve diferença entre o peso corporal dos animais aos 300 dias de idade e sexo (P = 0,8). Os resultados encontrados sugerem que o crescimento muscular ocorre por hipertrofia e hiperplasia, caracterizado pela aparência em mosaico de fibras de diferentes diâmetros, característico de peixes de grande tamanho.

Uso de índices de reflectância das folhas para avaliar o nível de nitrogênio em grama-bermuda / Leaves reflectance index of the bermuda grass to evaluate the nutritional status in nitrogen

A reflectância da folha em determinados comprimentos de onda pode ser uma alternativa para estimar a concentração de nitrogênio (N) na planta, devido à relação entre o teor de clorofila e de N no tecido foliar. Este trabalho teve por objetivo avaliar índices da cor verde em grama-bermuda para predizer o nível de N na planta. O experimento foi conduzido em área comercial de produção de grama-bermuda, localizada na cidade de Capela do Alto/SP. O delineamento experimental utilizado foi de blocos ao acaso, com cinco tratamentos (0, 150, 300, 450 e 600kg de N ha-1) e quatro repetições. Foi avaliada a reflectância das folhas pelo uso de medidor de cor de grama, clorofilômetro, e por meio de análise da imagem digital. O matiz (H) e índice de cor verde escuro (ICVE), calculados com base nos índices de reflectância da imagem digital, o ICVE, obtido com medidor de cor de grama, e a intensidade de coloração verde (ICV), obtida com o clorofilômetro, apresentaram forte correlação positiva com a concentração de N e a taxa de cobertura do solo (TCS) da grama-bermuda, podendo ser utilizados como índices auxiliares na recomendação de adubação nitrogenada para a cultura. Os valores obtidos que podem servir como primeiros índices para avaliação do estado nutricional em N na grama-bermuda Celebration são: H de 88 a 109o e ICVE de 0,54 a 0,66 (imagem digital); H de 67 a 76o e ICVE de 0,41 a 0,44 (medidor de cor da grama TCM 500); e ICV de 374 a 471 (clorofilômetro CM 1000).

Leaves reflectance in a certain wavelength can be an alternative to estimate the N concentration in the plant because of the relationship between the chlorophyll content and N in the leaf tissue. This research aimed to evaluate the index of green color of Bermuda grass to predict the nutritional status in N. The experiment was installed and conducted in an area of grass commercial production, located in "Capela do Alto / SP". The experimental design was randomized blocks, with five treatments: 0, 150, 300, 450 and 600kg ha-1 and four replications. It was evaluated the leaves reflectance by using the grass color meter, chlorophyll meter and by the digital image analysis. The hue (H) and dark green color index (DGCI), calculated based on rates of reflectance of the digital image, the DGCI, obtained by grass color meter and intensity of green color obtained with chlorophyll meter presented strong positive correlation with N concentration and the soil cover rate of Bermuda grass which can be used as auxiliary index to assist in the recommendation of nitrogen fertilization. The values that can serve as the primary index for assessing the nutritional status of N in Celebration Bermuda grass are: H 88 to 109o and DGCI 0.54 to 0,66 (digital image); H 67 to 76o and DGCI 0.41 to 0.44 (TCM 500 color grass meter) and GCI 374 to 471 (CM 1000 chlorophyll metter).

Uso de lodo de esgoto na produção de tapetes de grama esmeralda / Use of sewage sludge in zoysiagrass production

Com objetivo de avaliar o efeito do lodo de esgoto na formação e qualidades de tapetes de grama, foi instalado, em Itapetininga, São Paulo (SP), o experimento utilizando grama Zoysia japonica Steud., conhecida como esmeralda. O delineamento experimental utilizado foi o de blocos ao acaso em parcelas subdivididas, com quatro repetições, sendo as parcelas principais constituídas de cinco doses de lodo (0, 10, 20, 30 e 40Mg ha-1, base seca), mais um tratamento com NPK, de acordo com as necessidades da cultura e as subparcelas com dois sistemas de manejo (com e sem o uso de escarificador utilizado para romper uma camada superficial compactada). Utilizou-se o lodo da Estação de Tratamento de Esgoto de Jundiaí, SP. As doses de lodo aplicadas correspondem às doses de 100, 200, 300 e 400kg ha-1 de nitrogênio. Após 165 dias da aplicação do lodo de esgoto, a dose de 31Mg ha-1 permitiu o fechamento completo (100% da taxa de cobertura do solo) e a maior resistência dos tapetes de grama. O mesmo resultado foi encontrado quando utilizou-se a adubação química. A testemunha e as parcelas que receberam 10Mg ha-1 de lodo não formaram tapete. O lodo de esgoto aplicado em superfície promoveu redução da massa dos tapetes de grama esmeralda. O uso do escarificador reduziu a resistência dos tapetes de grama esmeralda. Os valores de metais pesados encontrados no lodo de esgoto estavam abaixo do limite estabelecido pelas normas que regulamenta a utilização do lodo de esgoto na agricultura.

Aiming to evaluate sewage sludge effect in the grass sod qualitiy and formation it was installed in Itapetininha - SP one experiment using Zoysia japonica Steud. The experimental design adopted was randomized blocks with split plot design, with four replications, the main plots consisting of five doses of sludge (0, 10, 20, 30 and 40Mg ha-1, a dry basis), another treatment with NPK, according to the requirement of the culture, and subplots with two management systems (with and without chisel use, to break a compacted surface layer). The sludge used came from "Estação de Tratamento de Esgoto de Jundiaí/SP". The doses of sludge applied correspond to the doses of 100, 200, 300 and 400kg ha-1 of nitrogen. 165 days after sewage sludge application, the dose of 31Mg ha-1 allowed the complete closing (100% of soil cover rate) and greater resistance of the zoysiagrass sod. The same results were found when chemical fertilization was used. The control and the plots that received 10Mg ha-1 of sludge did not formed sod. The sewage sludge applied to surface promoted reduction of zoysiagrass sod mass. The use of surface chisel reduced the strength of zoysiagrass sod. The heavy metals values found in sewage sludge were below the limit set by the rules governing the sewage sludge use in agriculture.

Uso do clorofilômetro no manejo da adubação nitrogenada para milho em sucessão a pastagem de Brachiaria decumbens / Use of chlorophyll meter in nitrogen fertilizing management to corn after Brachiaria decumbens pasture

A resposta do milho à adubação nitrogenada em cultivo após pastagem depende da espécie, da quantidade de palha, da relação C:N, da população microbiana, das condições climáticas e de outros fatores. O objetivo desta pesquisa foi avaliar a utilização da intensidade da cor verde da folha (ICV), medida pelo clorofilômetro, como indicativo para o manejo da adubação nitrogenada em cobertura, na cultura do milho, em sucessão a pastagem de Brachiaria decumbens Stapf. O experimento foi conduzido em casa de vegetação, em vasos com 29 litros de um Latossolo Vermelho distrófico, com três plantas por vaso. Foi utilizado o delineamento de blocos ao acaso, em esquema fatorial 4 x 4, com cinco repetições, constituído por quatro níveis de nitrogênio (0, 50, 100 e 150mg dm-3) e quatro de níveis de palhada de B. decumbens (0, 5, 10 e 15Mg ha-1), aplicados em cobertura. As doses de N foram parceladas em três épocas: a)1/3 na semeadura; b)1/3 no estádio de 4-5 folhas e c)1/3 no estádio de 7-8 folhas. As determinações da ICV da folha foram efetuadas um dia antes e uma semana após a adubação nitrogenada em cobertura. As quantidades de fitomassa seca de braquiária influenciaram a ICV da folha somente no estádio de 7-8 folhas. A manutenção de maiores ICV da folha do estádio de 4-5 (46,6 unidades SPAD) até o estádio de 8-9 folhas (53,0 unidades SPAD) possibilitou o maior acúmulo da fitomassa seca na parte aérea do milho. O monitoramento da ICV da folha pode auxiliar na avaliação da disponibilidade de N para a planta do milho durante o ciclo.

The corn response to the nitrogen fertilizing cultivated after pasture depends on of the species, straw amount, C: N rate, microbial population, climatic conditions and other factors. The study was aimed at evaluating the use of the green color intensity of the leaf (GCI), measured by the chlorophyll meter, as indicative for nitrogen fertilizing management sidedress in the corn crop, after B. decumbens Stapf pasture. The experiment was carried out in greenhouse conditions, in pots with 29 liters of a Red Latosol (Oxisol), with three corn plants per pot. The randomized blocks design was used, in 4x4 factorial arrangement, with five replications, constituted by four nitrogen rates (0, 50, 100 and 150mg dm-3 of soil) and four levels of B. decumbens straw (0, 5, 10 and 15Mg ha-1), applied in sidedressing. The N doses were splitting in three times: a) 1/3 in the seeding; b) 1/3 in the 4-5 leaves stage and c) 1/3 in the 7-8 leaves stage. The determinations of GCI of the leaf were made one day before and one week after the sidedress nitrogen fertilizing. The levels of B. decumbens straw only influenced GCI in the seven-eight leaves stage. The maintenance of larger GCI of the four-five leaves stage (46.6) to the eight-nine leaves (53.0) made possible the largest accumulation of the corn shoot dry matter. The monitoring of GCI can help in the evaluation of the N available for the corn during season.

Doses de nitrogênio e potássio na produção de grama esmeralda / Nitrogen and potassium rates in the zoysia grass sod production

O nitrogênio e potássio são os nutrientes requeridos em maiores quantidades pelas gramas e no Brasil não se tem informação da quantidade a ser aplicada para se obter a formação de tapete em menor tempo possível. Dois experimentos foram instalados em vasos em casa de vegetação, com o objetivo de avaliar o efeito de doses de nitrogênio e de potássio na produção de tapetes de grama esmeralda (Zoysia japonica). O delineamento utilizado para cada experimento foi fatorial com doses de N ou K e épocas de avaliação. Foram aplicadas quatro doses de nitrogênio (0, 200, 400 e 600 kg ha-1) e quatro doses de potássio (0, 100, 200, e 300 kg ha-1). As doses de nitrogênio e potássio foram aplicadas parceladamente em cobertura. O aumento das doses de N influenciou a taxa de cobertura do solo pela grama (TCS) permitindo a formação do tapete com a dose de 408 kg ha-1 de N aos 198 dias após a colheita do tapete anterior, tempo menor quando comparado com as demais doses. A concentração de N na folha e da cor verde da grama foram influenciadas pelas doses de N podendo ser utilizadas para auxiliar na recomendação das doses de N. O aumento das doses de K não influenciou na TCS pela grama, sendo o teor no solo (1,4 mmol c dm-3) suficiente para a produção dos tapetes de grama esmeralda.

The nitrogen and potassium are nutrients required in bigger amounts by grasses, and in Brazil there is no information about the amount that must be applied to get sod formation in the shortest lesser possible time. Two experiments were carried out in a green-house, aiming to evaluate the effect of nitrogen and potassium doses on the Zoysiagrass sod production. The design adopted for each experiment was factorial with doses of N or K and times of evaluation. Four nitrogen doses (0, 200, 400 and 600 kg ha-1) and four K2O doses (0, 100, 200, and 300 kg ha-1) had been applied. Nitrogen and potassium doses were applied split doses and topdressed. The soil cover rate by the grass (TCS) was influenced by the increase of nitrogen doses allowing the sod formation with 408 kg ha-1 of nitrogen 198 days after the harvest of the previous sod, shorter time when compared to other doses. The N concentration in the leaf and green color of the grass were influenced by nitrogen doses and could be used to assist in N doses recommendation. The increase of K doses did not influenced in TCS by the grass, the content in the soil (1,4 mmolc dm-3) was enough for the Zoysiagrass sod production.