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The present study evaluated the cryoprotectant efficacy of dimethylacetamide (DMA) and ethylene glycol in a one-step protocol to freeze boar sperm. The sperm-rich portion of the ejaculates from two boars were collected once a week, for 10 weeks. After collection, the ejaculates were diluted (1:1; v/v) in the cooling extender. After determining their spermatozoa concentration, the ejaculates were pooled with the same number of spermatozoa from each boar and stabilized at 20°C for 120 min. Distinct cryoprotectants were added to the cooling extender at 20 °C, at different concentrations, composing six treatments: 1.25% and 2.5% glycerol (control); 1.25% and 2.5% ethylene glycol; 2.5% and 5.0% DMA. The samples were stored in 0.25 mL straws, containing 35 × 106 spermatozoa. After 90 min at 20 °C, the straws were submitted to a cooling curve until 5 °C (0.3 to 0.5 °C/min) and kept at 5°C for 60 min. Freezing was conducted by placing the straws horizontally 5 cm above the liquid nitrogen for 10 min, followed by immersion on liquid nitrogen. After thawing at 37 °C for 30 seconds, sperm quality was evaluated through a computer-assisted semen analysis system and flow cytometry. Sperm motility was greater (P< 0.05) in treatments with 5.0% and 2.5% DMA (22.2 ± 2.6% and 20.0 ± 2.8%, respectively) than in treatment with 2.5% ethylene glycol (8.2 ± 1.0%). The integrity of the plasma membrane (P = 0.08) and mitochondrial membrane potential (P = 0.27) was similar among the treatments. The treatment with 2.5% ethylene glycol was the least efficient to maintain intact acrosome membrane (P< 0.01). Some kinetics parameters (DAP, DCL, DSL, VAP, VCL, VSL e ALH) were positively affected by 5.0% DMA. The one-step freezing protocol resulted in unsatisfactory boar sperm motility after thawing, regardless of the cryoprotectant.

O presente estudo objetivou avaliar a eficácia de um protocolo one-step de congelamento do sêmen suíno utilizando dimetilacetamida (DMA) e etilenoglicol como crioprotetores. Durante 10 semanas, a fração rica dos ejaculados de dois machos suínos foram coletados, uma vez por semana. Após a coleta, os ejaculados foram diluídos (1:1; v/v) no diluidor de resfriamento. Após a avaliação da concentração espermática, os ejaculados foram agrupados em um pool com o mesmo número de espermatozoides de cada macho e estabilizados a 20 °C por 120 min. Os criopropetores foram adicionados ao diluidor de congelamento a 20 °C, em diferentes concentrações, compondo seis tratamentos: glicerol (controle), 1,25% e 2,5%; etilenoglicol, 1,5% e 2,5%; e DMA, 2,5% e 5,0%. As amostras foram armazendadas em palhetas de 0,25 mL contendo 35 x 106 espermatozoides. Após 90 min a 20 °C as palhetas foram submetidas a uma curva de resfriamento até 5 °C (0,3 a 0,5 °C/mim) e mantidas a 5 °C por 60 min. O congelamento foi realizado a partir da colocação das palhetas horizontalmente a 5 cm acima do nitrogênio líquido por 10 min, com sua posterior imersão no nitrogênio líquido. Após o descongelamento a 37 °C por 30 segundos a qualidade espermática foi avaliada através de um sistema computadorizado e por citometria de fluxo. A motilidade espermática foi maior (P < 0,05) nos tratamentos com 5,0% e 2,5% DMA (22,2 ± 2,6% e 20,0 ± 2,8%, respectivamente) do que no tratamento com 2,5% etilenoglicol (8,2 ± 1,0%). A integridade da membrana plasmática (P = 0,08) e potencial de membrana mitocondrial (P = 0,27) foi similar entre os tratamentos. O tratamento com 2,5% de etilenoglicol foi menos eficiente em manter membrana acrossomal intacta (P< 0,01). Alguns parâmetros de cinética espermática (DAP, DCL, DSL, VAP, VCL, VSL e ALH) foram afetados positivamente pelo uso de DMA a 5.0%. O protocolo simplificado para congelamento de sêmen suíno resultou em motilidade espermática insatifatória após o descongelamento, independente do crioprotetor utilizado.

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The cryopreservation of plant germplasm at ultralow temperatures is an alternative technique for the long-term storage of seeds of the genus Coffea sp. However, for this technique to be successful, cell integrity must be maintained at all stages of the process on the basis of scientific research. The present study investigated validated cryopreservation protocols for Coffea arabica L. seeds and evaluate the effects on the physiological and biochemical characteristics of the seeds at each stage of the process. Seeds were dried on silica gel or with saturated saline solution, precooled or not in a biofreezer, immersed in nitrogen, and reheated in a water bath. After each of these steps, the physiological and biochemical quality of the seeds was determined. Pre-cooling is a step that can be dispensed with in the cryopreservation of Coffea arabica seeds, direct immersion in liquid nitrogen being more indicated. Coffea arabica L. seeds tolerate cryopreservation after rapid drying in silica gel up to water contents of 17 or 20% (wb), with greater survival at 17%. The enzyme activities of catalase, polyphenol oxidase and peroxidase are indicators of the quality of C. arabica L. seeds subjected to cryopreservation.

A criopreservação de germoplasma vegetal em temperaturas ultrabaixas é uma alternativa para o armazenamento em longo prazo de sementes do gênero Coffea sp. Entretanto, para que essa técnica apresente sucesso, a realização de pesquisas que garantam a manutenção da integridade celular em todas as etapas do processo é de fundamental importância. O objetivo com o presente trabalho foi investigar protocolos de criopreservação validados para sementes de Coffea arabica L., avaliando separadamente, os efeitos sobre as características fisiológicas e bioquímicas das sementes, em cada etapa do processo. As sementes foram secadas em sílica gel ou em solução salina saturada, submetidas ou não ao pré-resfriamento em biocongelador, em seguida imersas no nitrogênio e reaquecidas em banho-maria. Após cada uma dessas etapas, a qualidade fisiológica e bioquímica das sementes foi determinada. O pré-resfriamento é uma etapa que pode ser dispensada na criopreservação de sementes de Coffea arabica, sendo mais indicada a imersão direta em nitrogênio líquido. Sementes de Coffea arabica L. toleram a criopreservação após secagem rápida em sílica gel até teores de água de 17 ou 20% (wb), com maior sobrevivência a 17%. A atividade das enzimas catalase, polifenoloxidase e peroxidase são indicadoras da qualidade de sementes de Coffea arabica L. submetidas à criopreservação.

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In this study,the causative pathogen was isolated from Eucommia ulmoides,verified using Koch's postulates, identified by morphology and molecular biology, and assessed the biological characteristics. The effects of agar media, pH, temperature, light, carbon sources, and nitrogen sources on hyphal growth of Pestalotiopsistrachicarpicola were under investigation, and the influences of time, liquid matrix, light, temperature on conidial germination were studied. The results showed that P. trachicarpicola was a new pathogen causing black spot disease of E. ulmoides, and the mycelial growth of P.trachicarpicola was the best in the potato dextrose agar (PDA) medium, pH 7.0, the temperature was 20~25 °C, light, and glucose and beef extract were the best for carbon and nitrogen, respectively. The germination rate of the conidia increased from the time, darkness, and a proper supply of sugar. The lethal temperatures of the hyphae and conidia were 52 °C and 72 °C, respectively.We can conclude that appropriate environmental conditions are more conducive to the growth of the pathogen and the germination of spores, which leads to the occurrence of disease. This study constituted the first report on the new causative pathogen in E. ulmoides black spot and the biological characteristics. Theis study provided a theoretical basis to prevent the occurrence of this disease.

Neste estudo, o patógeno causador foi isolado de Eucommia ulmoides, verificado pelos postulados de Koch, identificado por morfologia e biologia molecular, e avaliadas as características biológicas. Os efeitos do meio de ágar, pH, temperatura, luz, fontes de carbono e fontes de nitrogênio no crescimento de hifas de Pestalotiopsis trachicarpicola foram investigados, e as influências do tempo, matriz líquida, luz e temperatura na germinação dos conídios foram estudadas. Os resultados mostraram que P. trachicarpicola é um novo patógeno causador da doença da mancha preta de E. ulmoides, e o crescimento micelial de P. trachicarpicola foi o melhor no meio de ágar batata dextrose (PDA), pH 7.0, a temperatura é de 20~25 °C, luz e extrato de glicose e carne são os melhores para carbono e nitrogênio, respectivamente. A taxa de germinação dos conídios aumentou com o tempo, escuridão e um suprimento adequado de açúcar. As temperaturas letais das hifas e dos conídios foram 52 °C e 72 °C, respectivamente. Podemos concluir que as condições ambientais adequadas são mais propícias ao crescimento do patógeno e à germinação de esporos, o que leva à ocorrência da doença. Este estudo constitui o primeiro relato sobre o novo patógeno causador da mancha negra de E. ulmoides e as características biológicas. O objetivo deste estudo é fornecer uma base teórica para prevenir a ocorrência desta doença.

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Intracytoplasmic Sperm Injection (ICSI) has increased usage in cases of stallion fertility issues, particularly for older stallions, those with reduced sperm numbers or quality, or stallions that have passed away, and only a limited amount of frozen semen is available. By manipulating the frozen semen through thawing, diluting, and refreezing or by cutting the straw under liquid nitrogen, the supply of semen for ICSI can be extended. While ICSI requires a minimal number of spermatozoa per procedure, it is important to consider sperm quality as a crucial factor affecting fertilization and embryo development. Although it is possible to produce healthy embryos and offspring from low quality sperm samples, it is preferable to process and select morphologically and functionally superior sperm to maximize the chances of successful fertilization and embryo development. Several techniques are available for selecting the spermatozoa for ICSI, such as swim-up, washing, density gradient centrifugation, microfluidic sorting, and some combinations. In this review, we will focus on semen type, handling, recent breakthroughs, stallion effects on ICSI efficiency and the prospects of this technology within the equine industry.(AU)

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Date palm (Phoenix dactylifera( cv. Medjool is a significant plant, grown in Jordan. In vitro propagation gives operative resources for the significant propagation of date palms. Maximum callus induction was achieved from MS media supplemented with benzyl amino purine (BA) and naphthalene acetic acid (NAA). The highest plant regeneration was recorded on MS medium supplemented with dichlorophenoxyacetic acid (2,4-D) at 3.0 mg/L, and BA at 2.0 mg/L. A significant positive impact on shoot formation was recorded on MS medium supplemented with 1.0 mg/L BA with 0.5 to 1.5 mg/L NAA in both liquid and solid MS medium. To maintain survival and regrowth capacity, sucrose could be used for medium-term conservation at lower concentrations (0.1 - 0.2 M). In addition, sorbitol might be used at 0.1 M to maintain the quality of explants. The vitrification technique for long-term preservation was experimented. Embryogenic callus was used as explants for conservation. The survival as well as regrowth percentages of non-cryopreserved and cryopreserved tissue cultures were affected by their duration of treatment with the vitrification solution plant vitrification solution 2 (PVS2) and modified plant vitrification solution 2 (MPVS2). Results showed that using PVS2 for 60 minutes for cryopreserved calli was more effective than other treatments. After storage in liquid nitrogen, the highest survival rate (65%) and regrowth rate (40%) were achieved.

A tamareira (Phoenix dactylifera cv. Medjool) é uma planta significativa, cultivada na Jordânia. A propagação in vitro fornece recursos operacionais para a propagação significativa de tamareiras. A indução máxima de calos foi alcançada a partir de meio MS suplementado com benzil amino purina (BA) e naftaleno ácido acético (ANA). A maior regeneração das plantas foi registrada em meio MS suplementado com ácido diclorofenoxiacético (2,4-D) a 3,0 mg/L e BA a 2,0 mg/L. Um impacto positivo significativo na formação de brotos foi registrado em meio MS suplementado com 1,0 mg/L BA com 0,5 a 1,5 mg/L NAA em meio MS líquido e sólido. Para manter a sobrevivência e a capacidade de regeneração, a sacarose pode ser usada para conservação em médio prazo em concentrações mais baixas (0,1 - 0,2 M). Além disso, o sorbitol pode ser utilizado a 0,1 M para manter a qualidade dos explantes. A técnica de vitrificação para preservação em longo prazo foi experimentada. Calos embriogênicos foram utilizados como explantes para conservação. A sobrevivência, bem como as porcentagens de novo crescimento de culturas de tecidos não criopreservadas e criopreservadas, foram afetadas pela duração do tratamento com a solução de vitrificação, a solução de vitrificação de plantas 2 (PVS2) e a solução de vitrificação de plantas modificada 2 (MPVS2). Os resultados mostraram que o uso de PVS2 por 60 minutos para calos criopreservados foi mais eficaz do que outros tratamentos. Após armazenamento em nitrogênio líquido, foram alcançadas as maiores taxas de sobrevivência (65%) e de rebrota (40%).

Resumo

This study was aimed to assess the efficiency of coconut water extender with addition of soy lecithin and sucrose as nonpermeable cryoprotectants for canine semen vitrification, using a simple method that yields a high survival rate of spermatozoa for clinical use. Twelve ejaculates from 12 adult normozoospermic dogs were collected separately by digital manipulation and only the second semen fraction was used in this study. After evaluation of volume, concentration, viability, total and progressive motility, velocity parameters and morphology, semen was diluted with a coconut water extender (50% (v/v(volume per volume)) coconut water, 25% (v/v) distilled water and 25% (v/v) 5% anhydrous monosodium citrate solution) with addition of soy lecithin and fructose at 1% and 0.25M sucrose until final concentration of 100x106 spermatozoa/ml. After equilibration at 5ºC for 60 minutes, semen was vitrified by "direct dropping method" into liquid nitrogen in spheres with a volume of 30 µl. After a week of storage the spheres were devitrified as three of them were dropped into 0.5 mL of CaniPlus AI medium (Minitüb, Germany), which was previously warmed in a water bath at 42ºC for 2 minutes and evaluated about the above mentioned parameters. It was found that vitrification resulted in a lower percentage of viable sperms, normal morphology, total and progressive motilities (p0.05) compared to fresh semen samples. In conclusion, our results demonstrate that vitrification with coconut water extender with addition of 1% soy lecithin and 0.25M sucrose as cryoprotectants, has an excellent potential for routine canine sperm cryopreservation.(AU)

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Due to their tiny size, soil nematodes and plant-parasitic nematodes are challenging to homogenize and collect using traditional mortar and pestle methods. To overcome this, we developed a reliable and efficient nematode preparation and grinding method using the BioPulverizer. The method involves creating a modified sample 'sandwich' with liquid nitrogen-frozen nematode samples placed between two layers of aluminum foil. This 'sandwich' facilitates complete homogenization through cryogenic grinding without any sample loss during transfer. The powerful nematode grinding yields high-quality and high-quantity samples, suitable for nucleic acid release and subsequent molecular identification and other downstream applications.

Resumo

Sperm cryopreservation is an important tool for genetic diversity management programs and the conservation of endangered breeds and species. The most widely used method of sperm conservation is slow freezing, however, during the process, sperm cells suffer from cryoinjury, which reduces their viability and fertility rates. One of the alternatives to slow freezing is vitrification, that consist on rapid freezing, in which viable cells undergo glass-like solidification. This technology requires large concentrations of permeable cryoprotectants (P- CPA's) which increase the viscosity of the medium to prevent intracellular ice formation during cooling and warming, obtaining successful results in vitrification of oocytes and embryos. Unfortunately, this technology failed when applied to vitrification of sperm due to its higher sensitivity to increasing concentrations of P-CPAs. Alternatively, a technique termed 'kinetic sperm vitrification' has been used and consists in a technique of permeant cryoprotectant-free cryopreservation by direct plunging of a sperm suspension into liquid nitrogen. Some of the advantages of kinetic vitrification are the speed of execution and no rate-controlled equipment required. This technique has been used successfully and with better results for motility in human (50-70% motility recovery), dog (42%), fish (82%) and donkey (21.7%). However, more studies are required to improve sperm viability after devitrification, especially when it comes to motility recovery. The objective of this review is to present the principles of kinetic vitrification, the main findings in the literature, and the perspectives for the utilization of this technique as a cryopreservation method.(AU)

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Management is of fundamental importance in increasing the efficiency of liquid hog manure (LHM) when used as a source of nitrogen (N) and minimizing its impact on the environment. This paper evaluates the effect of surface application and injection of LHM and the use of dicyandiamide (DCD) on the dynamics of mineral N in the soil and on the components of yield in wheat crops. An experiment was conducted at the Federal University of Santa Maria, Frederico Westphalen Campus, Rio Grande do Sul state, Brazil, involving the following treatments: T0 - Control; T1 - surface application of LHM (Sup); T2 - subsurface injection of LHM (Inj); T3 - surface application of LHM + DCD (Sup+DCD); T4 - subsurface injection of LHM + DCD (Inj+DCD), and T5 - application of N, phosphorus (P), and potassium (K) in mineral form (NPK) in 2014 and 2015. The mineral N recovered with the injection of LHM was superior to surface application, and DSD reduced the speed with which anionic forms of mineral N appear. There was a greater increase in the number of ears of wheat with LHM injection. In 2014, the number of grains per ear was higher with the injection of LHM + DCD, whereas in 2015 all the treatments were higher than the control. There was no difference in 1,000-grain weight between treatments with LHM. Hectoliter weight was higher with the injection of LHM + DCD and the yields observed in this treatment were also higher, not differing from mineral fertilization. It is concluded that LHM injection provides lower losses of N and DCD and reduces the speed with which anionic forms of mineral N appear. In addition, the final yield of wheat grains does not differ when comparing LHM + DCD with mineral fertilization.(AU)

O manejo do dejeto líquido de suínos (DLS) é de fundamental importância para aumentar sua eficiência como fonte de nitrogênio (N) e minimizar impacto sobre o ambiente. O objetivo deste trabalho foi avaliar o efeito da aplicação em superfície e da injeção de DLS e o uso da dicianodiamida (DCD) sobre a dinâmica de N mineral no solo e nos componentes de produtividade na cultura do trigo. Para isso, um experimento foi conduzido na Universidade Federal de Santa Maria, Campus de Frederico Westphalen, RS com os seguintes tratamentos: T0 - Testemunha, T1 - aplicação em superfície do DLS (Sup), T2 - injeção em subsuperfície do DLS (Inj), T3 - aplicação em superfície do DLS + DCD (Sup+DCD), T4 - injeção em subsuperfície do DLS + DCD (Inj+DCD) e T5 - aplicação de N, fósforo (P) e potássio (K) na forma mineral (NPK) nos anos de 2014 e 2015. O N mineral recuperado com a injeção do DLS foi superior à aplicação superficial e, a DCD reduziu a velocidade com que surgem formas aniônicas de N mineral. Houve maior incremento no número de espigas com a injeção de DLS. No ano 1, o número de grãos por espiga foi superior com a injeção de DLS + DCD, e no ano 2 todos os tratamentos foram superiores à testemunha. Não houve diferença na massa de mil grãos entre os tratamentos com DLS. A massa de hectolitro foi superior com a injeção do DLS + DCD e as produtividades observadas nesse tratamento também foram superiores, não diferindo da adubação mineral. Conclui-se que a injeção de DLS proporciona menores perdas de N mineral, a DCD reduz a velocidade com que surgem formas aniônicas de N mineral e que a injeção do DLS + DCD não difere da adubação mineral quanto à produtividade de grãos.(AU)

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The aim of the present study was to evaluate semen cryopreservation with ACP-Lact® diluent, which consists of coconut water powder (ACP) added to goat milk powder. After thawing, the samples were evaluated for sperm kinetics, membrane evaluation and in vivo insemination. For cryopreservation, a pool was made with the ejaculate of six goats, diluted in four equal aliquots for the respective treatments: T1 (ACP-Lact®); T2 (ACP-Lact® 50%); T3 (ACP + 2.5% egg yolk) and T4 (Tris + 2.5% egg yolk). After dilution of the treatments, the samples were placed in 0.5 ml straws and chilled at a rate of -1.07°C/min. After reaching 4°C and stabilizing for one hour, the straws were placed in nitrogen vapour at -60°C for 15 minutes and then immersed in liquid nitrogen (-196ºC). The straws were thawed in a 37°C water bath and kinetic assessments were performed immediately using a computerized semen analysis program (CSA), viability (EN), membrane functionality (HOST), mitochondrial activity (DAB) and DNA integrity assessment of spermatozoa. For the in vivo experiment, ten goats were inseminated, divided into two groups of five goats each, G1 inseminated with ACP-Lact® and G2 with ACP, by fixed-time artificial insemination (FTAI). Regarding the kinetic parameters, the ACP-Lact® treatment showed higher progressive motility (PM) and sperm velocity than the other treatments (36.77%). In the VSL parameter the ACP-Lact diluent was superior to ACP and Tris. In viability the treatment with ACP-Lact® was superior to the treatment with Tris, 95% and 83% respectively. In FTAI two goats were born out of the 5 goats inseminated with ACP-Lact®. It was concluded that the use of ACP-Lact® for cryopreservation of caprine semen is efficient in maintaining seminal parameters during thawing in vitro and in vivo and proved to be a good alternative extender for the caprine species.(AU)

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The domestic pig breeds are in danger of extinction whereas the erosion of their gene pool is a serious concern because they significantly contribute to the rich biodiversity. Overall aim of this study was to determine the protocol for preserving the semen of the Windsnyer boars for conservation. A total of 18 ejaculates (6 replications/boar) were collected from three Windsnyer boars of proven fertility with the use of hand-gloved approach method, twice per week. Boars semen were pooled and extended with Beltsville Thawing Solution [(BTS) IMV Technologies, France], held at 18°C for 3 hours and centrifuged. The sperm pellet was re-suspended with Fraction A (20% egg yolk + BTS) and cooled at 5°C for 1 hour. Following cooling, semen was divided and diluted into different cryoprotectants (ethylene glycol, glycerol, propanediol, ethylene glycol + glycerol + propanediol) at equal contribution to make the total concentrations [4, 8, 12 and 16% and the 0% (control; without cryoprotectant)] and loaded into 0.25 mL straws. Two cryopreservation methods (liquid nitrogen vapour and controlled rated) were used to cryopreserve the semen straws. Semen straws were thawed at different temperatures (5, 18, 37 and 40°C) and evaluated for sperm motility, viability, and morphology traits. Post-thawed sperm total motility (36.0±5.3) and live normal sperm (49.5±8.3) percentages were recorded to be higher in the treatment supplemented with 16% glycerol (P<0.05). The highest sperm total motility percentage was recorded at 40°C (26.8±3.2) thawing temperature for liquid nitrogen vapour treatment (P<0.05). In conclusion, 16% glycerol was found to be the suitable cryoprotectant concentration for semen cryopreserved with liquid nitrogen vapour method and thawed at 40°C.(AU)

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A total of twentymixtures of weed, B-Lac and molasses were prepared in orderto evaluate an accelerated liquid fertilizer (ALF) based on these plants. A mixture of 85% weed: water (1:1), 10% molasses and 5% B-Lac showed the best characteristics and was reproduced at a pilot scale. ALF was applied to lettuce using the following treatments: one foliar application per week of 10 mL L-1 (FA1), two foliar applications per week of 10 mL L-1 (FA2), one drench application of 50 mL L-1 every week (DA1), a drench application of 50 mL L-1 every two weeks (DA2) and a control without application (CWA). The variables evaluated were total yield, commercial yield, fresh weight, height, head diameter, percentage of dry matter and the concentration of foliar nitrogen, phosphorus, and potassium.The mixtures in the laboratoryand pilot phase were evaluated in a completely randomized design. The field phase was assessed in a completely randomized block design with five treatments and four replications. No significant differences were found between the treatments, except in the percentage of dry matter and potassium content, where FA2 showed the best results (2.35% and 541 mg plant-1, respectively). The highest total yield (26.4 t ha-1) and commercial (24.11 t ha-1) were achieved with DA2; however, the nutritional content was lower than that in the other treatments. Using homolactic fermentation it was possible to recycle weeds and produce ALF, which has potential as a biofertilizer according to its chemical characterization and effects shown on lettuce cultivation.(AU)

Foram preparadas vinte misturas de ervas daninhas, B-Lac e melaço para avaliar um fertilizante líquido acelerado (ALF) baseado nessas plantas. Uma misturade 85% erva:água (1:1), 10% melaço e 5% B-Lac apresentou as melhores características e foi reproduzida em escala piloto. A ALF foi aplicada à alface utilizando os seguintes tratamentos: uma aplicação foliar por semana de 10 mL L-1(AF1), duas aplicações foliares por semana de 10 mL L-1(AF2), uma aplicação via drench de 50 mL L-1a cada semana (AD1), uma aplicação via drench de 50 mL L-1a cada duas semanas (AD2) e um controle sem aplicação (CSA). As variáveis avaliadas foram produtividade total, produtividade comercial, massa fresca, altura, diâmetro da cabeça, porcentagem de matéria seca e concentração foliar de nitrogênio, fósforo e potássio. As misturas na fase de laboratório e piloto foram avaliadas em delineamentos inteiramente casualizados. A fase decampo foi avaliada em blocos casualizados com cinco tratamentos e quatro repetições. Não foram encontradas diferenças significativas entre os tratamentos, exceto no percentual de matéria seca e no teor de potássio, onde o AF2 apresentou os melhores resultados (2,35% e 541 mg planta-1, respectivamente). As maiores produtividades total (26,4 t ha-1) e comercial (24,11 t ha-1) foram obtidas com AD2; no entanto, o teor nutricional foi inferior aos demais tratamentos. Utilizando a fermentação homolática foi possível reciclar as ervas daninhas e produzir FLA, que tem potencial como biofertilizante de acordo com sua caracterização química e efeitos demonstrados no cultivo de alface.(AU)

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ABSTRACT: The heating rate used during semen thawing plays an important role in reducing structural and functional damage to spermatozoa. In this study, we evaluated the influence of thawing temperature on semen quality, reactive oxygen species (ROS) production, and mitochondrial activity of cryopreserved bovine semen. A total of 195 straws of 0.5 mL from five Holstein Friesian bulls were used (39 straws per bull). Samples underwent 8 to 22 years of storage; they were processed under a standard protocol with tris-egg yolk and stored in liquid nitrogen. Samples were thawed for 30 seconds in a water bath at T1: 36 °C, T2: 38 °C or T3: 40 °C. Sperm motility and kinematics, morphology, structural membrane integrity (SMI), functional membrane integrity (FMI), acrosome integrity (AI), ROS, and mitochondrial membrane potential (ΔΨM) of post-thawing bovine sperm were evaluated. Generalized linear models were fitted to the data. Each model included the effects of bull, storage time, and treatment. The Shapiro-Wilk test was used to assess data normality, and means were compared using the Tukey test. T2 and T3 showed better results for sperm motility and kinematic parameters, SMI (%) (T1 41.9 ± 2.3; T2 45.7 ± 1.9; T3 47.4 ± 2.8), ROS (RFU/min) (T1 0.026 ± 0.007; T2 0.032 ± 0.001; T3 0.031 ± 0.001) and high-ΔΨM (RFU x 103) (67.1± 0,4; 71.3 ± 0.4; 74.2 ± 0.4) (P < 0.05). However, T1 had higher FMI (39.3 ± 2.3) than T2 (34.0 ± 1.9) (P < 0.05), though not significantly (P > 0.05) different from T3 (38.4 ± 2.2). Thawing temperatures of 38 °C and 40 °C increases motility, kinetics, membrane integrity, mitochondrial activity and ROS of cryopreserved bovine semen, compared with more conventional thawing at 36 °C.

RESUMO: A taxa de aquecimento usada durante o descongelamento do sêmen desempenha um papel importante na redução dos danos estruturais e funcionais nos espermatozóides. O objetivo desta pesquisa foi avaliar a influência da temperatura de descongelamento na qualidade do sêmen, produção de espécies reativas de oxigênio (ROS) e atividade mitocondrial do sêmen bovino criopreservado. Foram utilizados 195 palhetas de 0,5 mL de cinco touros Holstein Friesian (39 palhetas por touro). As amostras passaram por oito a 22 anos de armazenamento e foram processadas sob protocolo padrão com Tris-gema de ovo e armazenadas em nitrogênio líquido. As temperaturas de descongelamento foram T1: 36 °C, T2: 38 °C, T3: 40 °C, cada uma por 30 segundos em banho-maria. Pós-descongelamento, a motilidade e cinética dos espermatozoides, morfologia, integridade estrutural da membrana (SMI), integridade funcional da membrana (FMI), integridade acrossomal (AI), ROS e potencial de membrana mitocondrial (ΔΨM) foram avaliados. Modelos lineares generalizados foram ajustados. Cada modelo incluiu os efeitos de touro, tempo de armazenamento e tratamento. A normalidade dos dados foi avaliada pelo teste de Shapiro-Wilk e as médias comparadas pelo teste de Tukey. T2 e T3 apresentaram resultados mais elevados para a maioria dos parâmetros de motilidade e cinemática espermática, SMI (%) (T1 41,9 ± 2,3; T2 45,7 ± 1,9; T3 47,4 ± 2,8), ROS (RFU/min) (T1 0,026 ± 0,007; T2 0,032 ± 0,001; T3 0,031 ± 0,001) e alto ΔΨM (RFU x 103) (67,1 ± 0,4; 71,3 ± 0,4; 74,2 ± 0,4) (P < 0,05). No entanto, T1 apresentou maior FMI (%) (39,3 ± 2,3) em comparação a T2 (34,0 ± 1,9) (P < 0,05), mas não foi diferente do T3 (38,4 ± 2,2) (P > 0,05). Conclui-se que as temperaturas de descongelamento de 38 °C e 40 °C produzem um aumento na motilidade, cinética, integridade de membrana, atividade mitocondrial e ROS do sêmen bovino criopreservado, em comparação com o uso mais convencional de uma temperatura de descongelamento de 36 °C.

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A endometrite é uma afecção muito presente na pecuária, sobretudo leiteira, acarretando impactos econômicos para o sistema e para a saúde pública devido ao uso indiscriminado de antimicrobianos no tratamento, principalmente as bases comuns à medicina humana. Nesse relato foi proposto um tratamento alternativo com plasma rico em plaquetas (PRP) em dois animais (01 e 02) da raça Girolando, com 22 e 28 dias em lactação (DEL) e administrado plasma pobre em plaquetas (PPP) em um animal da mesma raça (03) com DEL 25, a fim de se compararem os resultados e a eficácia do PRP. O diagnóstico positivo para endometrite foi obtido através de citologia endometrial, ultrassonografia e cultura bacteriana. Após realização do método de dupla centrifugação do sangue total de um bovino doador, foi obtido um PRP com concentração final de 698 x 10³ plaquetas/ ml que foi ativado por meio de congelamento e descongelamento, em nitrogênio líquido. Cada animal recebeu 10 mL de infusão intrauterina do PRP ativo ou PPP; repetindose as avaliações clínicas após 14 dias da infusão. Observou-se diminuição da secreção intrauterina e do percentual de neutrófilos na citologia endometrial, variação na espessura endometrial e menor crescimento bacteriano. Nesse trabalho, o PRP mostrou-se potencialmente eficaz para controlar inflamações e infecções intrauterinas em vacas com endometrite.(AU)

Endometritis is a prevalent condition in livestock, especially dairy cattle, causing economic impacts on the system and public health due to the indiscriminate use of antimicrobials in treatment, mainly those common to human medicine. In this report, an alternative treatment with platelet-rich plasma (PRP) was proposed in two animals (01 and 02) of the Girolando breed, with 22 and 28 days in lactation (DEL), and platelet-poor plasma (PPP) was administered in one animal of the same breed (03) with DEL 25, in order to compare the results and efficacy of PRP. Positive diagnosis for endometritis was obtained through endometrial cytology, ultrasonography, and bacterial culture. After performing the double centrifugation method of total blood from a donor bovine, a PRP with a final concentration of 698 x 10³ platelets/ml was obtained, which was activated by freezing and thawing in liquid nitrogen. Each animal received 10 mL of intrauterine infusion of active PRP or PPP, with clinical evaluations repeated after 14 days of infusion. A decrease in intrauterine secretion and percentage of neutrophils in endometrial cytology was observed, as well as variation in endometrial thickness and reduced bacterial growth. In this study, PRP proved to be potentially effective in controlling intrauterine inflammation and infections in cows with endometritis.(AU)

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Osmotic dehydration (OD) is a technique used for the partial removal of water from foodstuff, including fruit and vegetables, with the aim of producing a desiccated product. The process involves placing the material in a hypertonic solution for several hours and allowing water to move from the cell compartment into the solution by osmosis. OD is influenced by various factors such as the concentration and composition of the osmotic solution, the solution temperature, the type of agitation and the time of exposure, as well as the size, shape and compactness of the food material. The main advantages of OD over conventional drying processes are the superior quality of the dried products and the minimization of shrinkage. In recent years, research effort has focused on the combination of OD with other technologies, such as ultrasound, cryogenic freezing with liquid nitrogen, pulsed electric field, gamma radiation and high hydrostatic pressure. The application of these methods prior to or concomitant with OD accelerates mass transfer and reduces the drying rate of fruit and vegetables by increasing the permeability of cell membranes. In this manner, combined processes tend to be more efficient and economical in comparison with conventional OD because they reduce operating times and; consequently, energy consumption. In addition, the dried products generated by such coupled processes typically exhibit improved nutritional and physicochemical characteristics. This review summarizes the basic principles and applications of OD in combination with other methods, with particular emphasis on the production of dried fruits.

A desidratação osmótica (DO) é uma técnica utilizada para remover parcialmente a água dos alimentos, incluindo frutas e vegetais, com vistas a produção de alimentos secos. O processo consiste em colocar o material em uma solução hipertônica por várias horas e deixar a água passar do compartimento celular para a solução por osmose. A DO é influenciada por vários fatores como a concentração e composição da solução osmótica, a temperatura da solução, o tipo de agitação e o tempo de exposição, assim como o tamanho, forma e compactação do material alimentar. As principais vantagens da DO em relação aos processos de secagem convencionais são que ela dá origem a produtos secos de qualidade superior e minimiza o encolhimento. Nos últimos anos, tem-se investigado a combinação da DO com outras tecnologias, tais como ultrassom, congelamento criogênico com nitrogênio líquido, campo elétrico pulsado, radiação gama e alta pressão hidrostática. A aplicação desses métodos antes ou simultaneamente com a DO acelera a transferência de massa e reduz a taxa de secagem de frutas e vegetais através do aumento da permeabilidade das membranas celulares. Assim, os processos combinados tendem a ser mais eficientes e econômicos do que a DO convencional, pois reduzem o tempo de operação e, consequentemente, o consumo de energia. Adicionalmente, os produtos desidratados gerados através de processos associados geralmente apresentam melhores características nutricionais e físico-químicas. Esta revisão sumariza os princípios básicos e aplicações da DO em combinação com outros métodos, com ênfase especial dada à produção de frutas secas.

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Global advances in reproductive biotechnology have allowed for the transfer of embryos from donor females with high genetic merit to recipients using the cryopreservation technique, which preserves an embryo of excellent quality and viability, thereby achieving a feasible pregnancy rate. The objective of this study was to evaluate the quality and viability of Holstein embryos that have been cryopreserved for more than 40 years under glycerol freezing. The embryos were transferred to the recipient heifers using a non-surgical method. Two 17-month-old Holstein heifers (360 kg live weights) which were clinically healthy and reproductively active were used as the recipients. Two bovine embryos of Grade 1 quality were thawed and evaluated for their morphology. Of the two embryo transfers, one pregnancy was achieved, resulting in the birth of a calf. Therefore, embryos frozen in liquid nitrogen and glycerol as a cryopreservative for more than 40 years maintained their quality and viability to produce a live calf.

Os avanços globais em biotecnologia reprodutiva permitiram a transferência de embriões de fêmeas doadoras com alto mérito genético para receptoras, usando-se a técnica de criopreservação, que preserva um embrião de excelente qualidade e viabilidade, alcançando, assim, uma taxa de gravidez viável. O objetivo deste estudo foi avaliar a qualidade e a viabilidade de embriões Holstein, criopreservados por mais de 40 anos, sob congelamento de glicerol. Os embriões foram transferidos para as novilhas receptoras, usando-se um método não cirúrgico. Duas novilhas Holstein de 17 meses de idade (360kg de peso vivo), que eram clinicamente saudáveis e reprodutivamente ativas, foram utilizadas como receptoras. Dois embriões bovinos de qualidade Grau 1 foram descongelados e avaliados quanto à sua morfologia. Das duas transferências embrionárias, uma gravidez foi obtida, resultando no nascimento de um bezerro. Portanto, os embriões congelados em nitrogênio líquido e glicerol como criopreservante por mais de 40 anos mantiveram sua qualidade e viabilidade para produzir um bezerro vivo.

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This study aimed to develop a protocol for the cryopreservation of Pseudoplatystoma corruscans semen. For this, mature males were hormonally induced with a single dose of carp pituitary extract (5 mg/kg body weight). Semen was collected and evaluated. Two cryoprotectants were tested to compose the diluents: dimethyl acetamide (DMA) and dimethyl sulfoxide (Me2SO), in two concentrations (8% and 10%), + 5.0% glucose + 10% egg yolk. The semen was diluted in a 1: 4 ratio (semen: extender), packed in 0.5 mL straws and frozen in a dry shipper container in liquid nitrogen vapors. After thawing, sperm kinetics, sperm morphology and DNA integrity of cryopreserved sperm were evaluated. Pseudoplatystoma corruscans males produced semen with sperm motility > 80%. After thawing, all treatments provided semen with total sperm motility > 40%, with no significant difference (P < 0.05) between them, as well as between the other sperm kinetic parameters evaluated. The treatments with DMA provided a smaller fragmentation of the DNA of the gametes. Sperm malformations were identified in both fresh and cryopreserved semen, with a slight increase in these malformations being identified in sperm from thawed P. corruscans semen samples.(AU)

Este estudo teve como objetivo desenvolver um protocolo para a criopreservação do sêmen de Pseudoplatystoma corruscans. Para tal, machos maduros foram induzidos hormonalmente com uma dose única de extrato de hipófise de carpa (5 mg/kg de peso vivo). O sêmen foi coletado e avaliado. Sendo testados para compor os diluentes, dois crioprotetores: dimetil acetamida (DMA) e dimetil sulfóxido (Me2SO), em duas concentrações (8% e 10%), + 5,0% glicose + 10% gema de ovo. O sêmen foi diluído na proporção 1: 4 (sêmen: extensor), embalado em palhetas de 0,5 mL e congelado em container dryshipper em vapores de nitrogênio líquido. Após o descongelamento, foram avaliados os aspectos cinéticos espermáticos, a morfologia espermática e a integridade do DNA dos espermatozoides criopreservados. Os machos de P. corruscans produziram sêmen com motilidade espermática > 80%. Todos os tratamentos proporcionaram após o descongelamento sêmen com motilidade espermática total > 40%, sem diferença significativa (P < 0,05) entre eles, como também entre os demais parâmetros cinéticos espermáticos avaliados. Os tratamentos com DMA proporcionaram uma menor fragmentação do DNA dos gametas. Malformações espermáticas foram identificadas, tanto no sêmen fresco, como no criopreservado, sendo identificado um aumento discreto dessas malformações nos espermatozoides das amostras de sêmen descongeladas de P. corruscans.(AU)

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Sperm motility and kinematics analysis are important to predict bull fertility. However, there are other molecules in the sperm with the ability to improve the pregnancy rate. For example, PLCZ1 is a sperm protein that plays a unique role in the activation of the zygote and is important for the survival of the embryo. The objective of this work was to compare the expression of PLCZ1 mRNA in sperm cells of Chihuahuan Criollo and European bulls in the winter and summer seasons, under a low-input system. Six (3.33 ± 0.43 years old) bulls (three Criollo, three European) were used. Gross and individual motility were measured in semen obtained by electrostimulation. The cell pack was pelletized by centrifugation and stored in liquid nitrogen. The sperm cells were purified and total RNA was extracted. cDNA was synthesized to perform qPCR and measure the relative level of PLCZ1 transcripts in each bull. There were no differences in individual motility, however, gross motility was lower (P < 0.05) in Criollo bulls, both in the winter (71.1 ± 2.8 vs. 76.6 ± 2.8%) and in the summer season (58.9 ± 2.8 vs. 77.7 ± 2.8%). PLCZ1 expression was 5.3 times higher (P < 0.05) in winter than in summer (5.09 ± 1.09 vs 0.959 ± 1.09). No difference (P>0.05) was found in the expression levels of PLCZ1 between both breeds (4.36 ± 1.09 vs 1.69 ± 1.09), for Criollo and European, respectively. Although the animals presented seminal motility within the recommended limits for insemination, the expression levels of PLCZ1 vary depending on the time of the year and this might impact the rate of successful pregnancies. Therefore, it is important to complement conventional analysis of seminal quality with molecular characteristics.(AU)

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The heating rate used during semen thawing plays an important role in reducing structural and functional damage to spermatozoa. In this study, we evaluated the influence of thawing temperature on semen quality, reactive oxygen species (ROS) production, and mitochondrial activity of cryopreserved bovine semen. A total of 195 straws of 0.5 mL from five Holstein Friesian bulls were used (39 straws per bull). Samples underwent 8 to 22 years of storage; they were processed under a standard protocol with tris-egg yolk and stored in liquid nitrogen. Samples were thawed for 30 seconds in a water bath at T1: 36 °C, T2: 38 °C or T3: 40 °C. Sperm motility and kinematics, morphology, structural membrane integrity (SMI), functional membrane integrity (FMI), acrosome integrity (AI), ROS, and mitochondrial membrane potential (ΔΨM) of post-thawing bovine sperm were evaluated. Generalized linear models were fitted to the data. Each model included the effects of bull, storage time, and treatment. The Shapiro-Wilk test was used to assess data normality, and means were compared using the Tukey test. T2 and T3 showed better results for sperm motility and kinematic parameters, SMI (%) (T1 41.9 ± 2.3; T2 45.7 ± 1.9; T3 47.4 ± 2.8), ROS (RFU/min) (T1 0.026 ± 0.007; T2 0.032 ± 0.001; T3 0.031 ± 0.001) and high-ΔΨM (RFU x 103) (67.1± 0,4; 71.3 ± 0.4; 74.2 ± 0.4) (P < 0.05). However, T1 had higher FMI (39.3 ± 2.3) than T2 (34.0 ± 1.9) (P < 0.05), though not significantly (P > 0.05) different from T3 (38.4 ± 2.2). Thawing temperatures of 38 °C and 40 °C increases motility, kinetics, membrane integrity, mitochondrial activity and ROS of cryopreserved bovine semen, compared with more conventional thawing at 36 °C.

A taxa de aquecimento usada durante o descongelamento do sêmen desempenha um papel importante na redução dos danos estruturais e funcionais nos espermatozóides. O objetivo desta pesquisa foi avaliar a influência da temperatura de descongelamento na qualidade do sêmen, produção de espécies reativas de oxigênio (ROS) e atividade mitocondrial do sêmen bovino criopreservado. Foram utilizados 195 palhetas de 0,5 mL de cinco touros Holstein Friesian (39 palhetas por touro). As amostras passaram por oito a 22 anos de armazenamento e foram processadas sob protocolo padrão com Tris-gema de ovo e armazenadas em nitrogênio líquido. As temperaturas de descongelamento foram T1: 36 °C, T2: 38 °C, T3: 40 °C, cada uma por 30 segundos em banho-maria. Pós-descongelamento, a motilidade e cinética dos espermatozoides, morfologia, integridade estrutural da membrana (SMI), integridade funcional da membrana (FMI), integridade acrossomal (AI), ROS e potencial de membrana mitocondrial (ΔΨM) foram avaliados. Modelos lineares generalizados foram ajustados. Cada modelo incluiu os efeitos de touro, tempo de armazenamento e tratamento. A normalidade dos dados foi avaliada pelo teste de Shapiro-Wilk e as médias comparadas pelo teste de Tukey. T2 e T3 apresentaram resultados mais elevados para a maioria dos parâmetros de motilidade e cinemática espermática, SMI (%) (T1 41,9 ± 2,3; T2 45,7 ± 1,9; T3 47,4 ± 2,8), ROS (RFU/min) (T1 0,026 ± 0,007; T2 0,032 ± 0,001; T3 0,031 ± 0,001) e alto ΔΨM (RFU x 103) (67,1 ± 0,4; 71,3 ± 0,4; 74,2 ± 0,4) (P < 0,05). No entanto, T1 apresentou maior FMI (%) (39,3 ± 2,3) em comparação a T2 (34,0 ± 1,9) (P < 0,05), mas não foi diferente do T3 (38,4 ± 2,2) (P > 0,05). Conclui-se que as temperaturas de descongelamento de 38 °C e 40 °C produzem um aumento na motilidade, cinética, integridade de membrana, atividade mitocondrial e ROS do sêmen bovino criopreservado, em comparação com o uso mais convencional de uma temperatura de descongelamento de 36 °C.

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Toxoplasma gondii can be eliminated in bovine semen. Cryopreserved semen is often used due to the fact that artificial insemination in dairy and beef cattle provides benefits in terms of production. However, little is known regarding the viability and infectivity of T. gondii tachyzoites in cryopreserved bovine semen. In the present study, cattle semen negative for T. gondii were contaminated with 1 x 106 tachyzoites (RH strain) and cryopreserved with and without different cryoprotectants, such as DMSO (concentrations of 2.5%, 5.0%, 7.5%, 8.0% and 10.0%) and glycerol (2.25%, 2.5%, 3.0%, 5.0%, 7.5% and 10.0%), followed by freezing in liquid nitrogen (-196°C). After 24 hours, the samples were thawed and inoculated in 10 mice per cryoprotectant concentration. The mice were evaluated for clinical signs of toxoplasmosis (rough coat, diarrhea, hypoactivity and sudden death) as well as serum titers of IgM and IgG and the presence of tachyzoites in the peritoneal lavage. The results revealed that T. gondii remained infective in all samples. Clinical signs of toxoplasmosis were observed in the mice beginning with the 6th day post-inoculation (DPI) and 100% lethality was found between the 7th and 9th DPI. Viable tachyzoites were recovered from peritoneal exudate of dead mice (except for the control group), with higher mean of tachyzoite counts in the intraperitoneal lavage for 5% DMSO (±3.32 x 106), 8% DMSO (±3.53 x 106), 3% glycerol (±4.75 x 106), 7.5% glycerol (±6.26 x 106) and the absence of cryoprotectant (±3.11 x 106). Seroconversion occurred in the treated groups, with titers of IgG from 1:16 to 1:128 and IgM from 1:16 to 1:512. T. gondii viability and infectivity were maintained in cattle semen during 24 hours of cryopreservation at -196°C with and without cryoprotectant. However, further studies are necessary to determine whether cryopreserved semen contributes to the spread of toxoplasmosis through artificial insemination.

Sabe-se que Toxoplasma gondii pode ser eliminado no sêmen bovino. A inseminação artificial em bovinos leiteiros e de corte proporcionou avanços e benefícios nas produções e para isso o sêmen criopreservado é frequentemente utilizado. No entanto, pouco se sabe sobre a viabilidade e infectividade dos taquizoítos de T. gondii em sêmen bovino criopreservado. Para isso o sêmen bovino, negativo para T. gondii, foi contaminado com 1x106 taquizoítos (cepa RH), criopreservados com ou sem diferentes crioprotetores como DMSO (2.5%, 5.0%, 7.5%, 8.0% e 10.0%) e Glicerol (2.25%, 2.5%, 3.0%, 5.0%, 7.5% e 10.0%) e congelados em nitrogênio líquido (-196°C). Após 24 horas, essas amostras foram descongeladas e inoculadas em 10 camundongos por diluente de concentração de crioprotetor. Os camundongos foram avaliados quanto a sinais clínicos de toxoplasmose (pele áspera, diarreia, hipoatividade e morte súbita), títulos séricos de IgM e IgG e presença de taquizoítos no lavado peritoneal. Os resultados mostraram que T. gondii se manteve infectante em todas as amostras, inclusive naquelas sem crioprotetor. Sinais clínicos de toxoplasmose foram observados nos camundongos a partir do 6º dia pós-inoculação (DPI) e 100% de letalidade foi verificada entre o 7º ao 9º DPI. Nos camundongos mortos, exceto no grupo controle, taquizoítos viáveis foram recuperados do exsudato peritoneal, com maior média de taquizoítos quantificados na lavagem intraperitoneal para DMSO a 5% (±3.32x106), 8% (±3.53x106) e glicerol 3% (±4.75x106), 7,5% (±6.26x106) e livre de crioprotetor (±3.11x106). A soroconversão ocorreu nos grupos tratados com títulos de IgG (1:16 a 1:128) e IgM (1:16 a 1:512). A viabilidade e infectividade do T. gondii no sêmen bovino durante as 24 horas de criopreservação a -196°C foram mantidas com ou sem crioprotetor. No entanto, mais estudos são necessários para verificar se o sêmen criopreservado contribui para a disseminação da toxoplasmose na inseminação artificial.