Cryopreservation and Culture of Testicular Tissues: An Essential Tool for Biodiversity Preservation.

Systematic cryo-banking of reproductive tissues could enhance reproductive management and ensure sustainability of rare mammalian genotypes. Testicular tissues contain a vast number of germ cells, including at early stages (spermatogonia and spermatocytes), that can potentially develop into viable spermatozoa after grafting or culture in vitro, and the resulting sperm cells then can be used for assisted reproductive techniques. The objective of this review was to describe current advances, limitations, and perspectives related to the use of testicular tissue preservation as a strategy for the conservation of male fertility. Testes can be obtained from mature or prepubertal individuals, immediately postmortem or by orchiectomy, but testicular biopsies could also be an alternative to collect samples from living individuals. Testicular fragments can be then cryopreserved by using slow or ultra-rapid freezing, or even vitrification methods. The composition of cryopreservation media can vary according to species-specific characteristics, especially regarding the cryoprotectant type and concentration. Finally, spermatozoa have been usually obtained after xenografting of testicular fragments into severely immunodeficient mice, while this method still has to be optimized after in vitro culture conditions.

The challenges and perspectives of the selection of starter cultures for fermented cocoa beans.

Fermentation is an essential process step to develop precursor compounds for aroma and flavour characteristics of chocolate, as well as preventing germination of the cocoa bean. Despite the importance of the role of microorganisms during the chocolate production, to date, there are some discrepancies of the "cocobiota" community found during fermentation and the impact of starter culture in fermented cocoa beans. This review provides both a detailed overview of the starter cultures used in fermented cocoa beans and the microbial diversity involved during this process, and an in-depth discussion of the methods used to identify these microorganisms. In this review, we included only published articles from 2008 to 2018 in English language. A total of forty-seven studies contributed to the description of the cocobiota from 13 different countries. In detail, we observed that the most common fermentation method used is the wooden box, followed by heap. Interestingly, 37% of the studies cited in this review did not mention the type of cocoa variety studied. Most of the techniques used to identify the microbiota are fingerprinting based (DGGE); however, few studies have been using next-generation technologies to elucidate the possible functions and interactions among microbes. Our results showed a greater diversity of yeasts if compared with bacterial involved in the fermentation. This review will help researchers seeking to design starter cultures to drive cocoa bean fermentation, and thus achieve a homogenous mass of fermented cocoa beans as well as serve as a guide for assessing methodologies for the identification of microorganisms.

Methodological aspects for the culture and quantification of heterotrophic sulfate-reducing bacteria / Aspectos metodológicos para el cultivo y la cuantificación de bacterias heterotróficas sulfato-reductoras

This paper presents a comparison of various methods for culture, quantification, and maintenance of sulfate-reducing bacteria (SRB) under laboratory conditions, using liquid and semisolid media for water and soil samples. Starkey, Postgate B, API and modified Baars media were used with an incubation time of 21 days in a GasPack™ anaerobic jars type. The modified Baars medium was more efficient for the quantification of SRB in both liquid and semisolid media when compared with other culture media tested, detecting differences of three orders of magnitude in soil samples and in two orders for water samples at 8 days of incubation. The semisolid modified Baars medium in Petri dishes allowed the isolation of pure cultures of SRB by the streak plate method. It was found that strains in liquid modified Baars medium remain viable for up to three months, while in the same semisolid medium were kept only one month.

Este trabajo presenta una comparación de diversos métodos para el cultivo, cuantificación, y mantenimiento de bacterias sulfato-reductoras (BSR) en condiciones de laboratorio, utilizando medios líquidos y semisólidos para muestras de agua y suelo. Se utilizaron los medios de cultivo de Starkey, Postgate B, API y Baars modificado con un tiempo de incubación de 21 días en jarras de anaerobiosis tipo GasPack™. Se determinó que para la cuantificación de SRB, tanto en medio líquido como en semisólido, el medio Baars modificado es más eficiente comparado con los demás medios de cultivo probados, detectando diferencias de tres órdenes de magnitud en muestras de suelo y de dos órdenes de magnitud en muestras de agua a los 8 días de incubación. El medio Baars modificado semisólido servido en placas de Petri permitió el aislamiento de cultivos puros de BSR mediante siembra por agotamiento. Se encontró que en el medio modificado de Baars líquido las cepas se mantienen viables hasta por tres meses mientras que en el mismo medio semisólido sólo se mantienen durante un mes.

Cytotoxicity of dental alginates / Citotoxicidade de alginatos odontológicos

Purpose: To evaluate the cytotoxicity of dental alginate (irreversible hydrocolloid), which is widely used as an impression material in Dentistry. Methods: Four dental products were assessed: J (Jeltrate Traditional), ALG (Alga Gel), PG (Printer Gel), and AVG (Ava Gel). Three control groups were used: positive (C+) cell detergent Tween 80, negative (C-) PBS, and control of cells (CC - no exposure of cells to any substance). Disk-shaped specimens were immersed in Eagle minimum essential. The supernatants were collected after 24, 48, 72, and 168 hours (7 days) for analysis of the toxicity to L929 fibroblast cells after 24-h incubation. Viable cells stained with 0.01% neutral red dye were counted using a spectrophotometer. Data were analyzed by ANOVA and Tukey's test (α=0.05).Results: Significant differences in number of viable cells were found between the alginate groups and C- or CC (P<0.05). The group J showed the highest cytotoxicity level followed by PG, ALG, and AVG. Conclusion: All dental alginates tested showed some cytotoxic response from fibroblasts.

Objetivo: Avaliar a citotoxicidade de alginatos (hidrocolóide irreversível) de uso odontológico, os quais são a categoria de material de moldagem mais utilizada em Odontologia. Metodologia: Foram avaliadas quatro marcas de alginato: grupo J (Jeltrate Tradicional), ALG (Alga Gel), PG (Printer Gel) e AVG (Ava Gel). Utilizaram-se 3 grupos controle: positivo (C+) com detergente celular Tween 80, negativo (C-) com PBS, e controle de célula (CC), onde as células não foram expostas a nenhum material. Espécimes em forma de disco foram imersos em meio mínimo essencial Eagle. O sobrenadante foi coletado 24, 48, 72 e 168 horas (7 dias) para análise de toxicidade para fibroblastos L929 após 24 h de incubação. As células viáveis foram coradas com corante vermelho neutro a 0,01%, fixadas e contadas em espectrofotômetro. Os dados foram analisados por ANOVA e teste de Tukey (α=0,05). Resultados: Houve diferença significativa do número de células viáveis entre os alginatos e os grupos C- ou CC (P<0,05). O grupo J apresentou a maior citotoxicidade, sendo seguido por PG, ALG e AVG. Conclusões: Pode-se concluir que todos os alginatos testados mostraram resposta citotóxica para fibroblastos.

Diets for feed training peacock bass Cichla sp.

The use of prized, carnivorous fish species such as the peacock bass Cichla sp. in either intensive farming or sport fishing demand specific knowledge on feed conditioning strategies for those species. One thousand and fifteen 0.5-g fingerlings were trained for 7 days to feed on ground fish flesh (GF). Seven hundred and seventy six (76%) fish (0.63 ± 0.03 g) feeding on GF were stocked into twelve 0.03-m³ net cages (63 fish/cage) and submitted to gradual feed ingredient transition (GFIT) weaning technique. Moist pellets with 90, 80, 70 or 60% GF (GF-90, GF-80, GF-70 or GF-60, respectively) were offered during the first 4 days of GFIT. No fish accepted GF-00 at the end of GFIT. Fish started on GF-90 or GF-80 fed well until GF dietary levels dropped below 40%. To improve acceptance of pellets containing 30% or less GF, a second trial with four 0.03-m³ net cages stocked with 120, 0.5-g fish feeding on GF was designed. Fish fed on a sequence of moist pellets containing 90, 80, 70, 60, 50 or 40% GF for 3 days. Approximately 81% of the fish accepted GF-40; they were pooled and restocked into nine 0.03-m³ net cages and weaned to GF-00 with a sequence of diets containing 30, 20 and 10% GF plus: 1) a meat-flavored dry, commercial feed (MEAT); 2) a fish-flavored dry, commercial feed (FISH); or 3) MEAT plus 10% krill meal (KM). Fish accepted fish-flavored pellets better than meat-flavored pellets. Addition of 10% krill meal to a meat-flavored feed improved pellet acceptance, even when ground fish flesh comprised only 10% of the feed. However, no fish accepted GF-00 pellets at the end of this study.

A produção comercial de peixes carnívoros como o tucunaré Cichla sp. depende do desenvolvimento de técnicas de condicionamento alimentar para cada espécie. Um mil e quinze alevinos de tucunaré Cichla sp. (peso médio 0,5 g) foram condicionados a aceitar alimento inerte na forma de filé de peixe moído (FP) por um período de 7 dias, com um sucesso de treinamento de 76% (771 peixes). Estes 771 peixes (peso médio 0,63 ± 0,03 g) foram estocados em 12 gaiolas de volume 0,03 m³ (63 peixes/gaiola) e condicionados a aceitar uma dieta seca (FP-00) pelo método da transição gradual dos ingredientes da ração (TGIR) recebendo, durante 4 dias, grânulos úmidos com 90, 80, 70 ou 60% de FP (FP-90, FP-80, FP-70 ou FP-60, respectivamente). Nenhum peixe aceitou FP-00 no final da TGIR. Os peixes alimentados com FP-90 e FP-80 aceitaram bem as dietas enquanto a quantidade de FP não caiu para níveis inferiores a 40%. Um segundo ensaio foi realizado visando melhorar a aceitação das dietas contendo 30% ou menos FP. Para tanto, quatro gaiolas de 0,03 m³ foram estocadas com 120 peixes cada (peso médio 0,5 g), os quais foram inicialmente alimentados com FP e submetidos a TGIR com dietas FP-90, FP-80, FP-70, FP-60, FP-50 e FP-40 por períodos subsequentes de 3 dias. Os peixes que aceitaram GF-40 (81% do total) foram agrupados e estocados em 9 gaiolas de 0,03 m³ e treinados por 3 dias a aceitar FP-00 pelo uso de dietas contendo 30, 20 ou 10% de FP e flavorizadas com: (1) alimento comercial seco sabor carne (ACSC); (2) alimento comercial seco sabor peixe (ACSP); ou (3) ACSC mais 10% de farinha de krill (ACSP+FK). A aceitação de ACSP foi melhor que ACSC. A adição de KM ao ACSC melhorou a aceitabilidade da dieta em comparação com outros aditivos alimentares, mesmo quando a quantidade de FP nas dietas foi reduzida para 10%. Entretanto, ao final dos ensaios, nenhum peixe aceitou grânulos alimentares isentos de FP (FP-00).

Diets for feed training peacock bass Cichla sp

The use of prized, carnivorous fish species such as the peacock bass Cichla sp. in either intensive farming or sport fishing demand specific knowledge on feed conditioning strategies for those species. One thousand and fifteen 0.5-g fingerlings were trained for 7 days to feed on ground fish flesh (GF). Seven hundred and seventy six (76%) fish (0.63 ± 0.03 g) feeding on GF were stocked into twelve 0.03-m³ net cages (63 fish/cage) and submitted to gradual feed ingredient transition (GFIT) weaning technique. Moist pellets with 90, 80, 70 or 60% GF (GF-90, GF-80, GF-70 or GF-60, respectively) were offered during the first 4 days of GFIT. No fish accepted GF-00 at the end of GFIT. Fish started on GF-90 or GF-80 fed well until GF dietary levels dropped below 40%. To improve acceptance of pellets containing 30% or less GF, a second trial with four 0.03-m³ net cages stocked with 120, 0.5-g fish feeding on GF was designed. Fish fed on a sequence of moist pellets containing 90, 80, 70, 60, 50 or 40% GF for 3 days. Approximately 81% of the fish accepted GF-40; they were pooled and restocked into nine 0.03-m³ net cages and weaned to GF-00 with a sequence of diets containing 30, 20 and 10% GF plus: 1) a meat-flavored dry, commercial feed (MEAT); 2) a fish-flavored dry, commercial feed (FISH); or 3) MEAT plus 10% krill meal (KM). Fish accepted fish-flavored pellets better than meat-flavored pellets. Addition of 10% krill meal to a meat-flavored feed improved pellet acceptance, even when ground fish flesh comprised only 10% of the feed. However, no fish accepted GF-00 pellets at the end of this study.

A produção comercial de peixes carnívoros como o tucunaré Cichla sp. depende do desenvolvimento de técnicas de condicionamento alimentar para cada espécie. Um mil e quinze alevinos de tucunaré Cichla sp. (peso médio 0,5 g) foram condicionados a aceitar alimento inerte na forma de filé de peixe moído (FP) por um período de 7 dias, com um sucesso de treinamento de 76% (771 peixes). Estes 771 peixes (peso médio 0,63 ± 0,03 g) foram estocados em 12 gaiolas de volume 0,03 m³ (63 peixes/gaiola) e condicionados a aceitar uma dieta seca (FP-00) pelo método da transição gradual dos ingredientes da ração (TGIR) recebendo, durante 4 dias, grânulos úmidos com 90, 80, 70 ou 60% de FP (FP-90, FP-80, FP-70 ou FP-60, respectivamente). Nenhum peixe aceitou FP-00 no final da TGIR. Os peixes alimentados com FP-90 e FP-80 aceitaram bem as dietas enquanto a quantidade de FP não caiu para níveis inferiores a 40%. Um segundo ensaio foi realizado visando melhorar a aceitação das dietas contendo 30% ou menos FP. Para tanto, quatro gaiolas de 0,03 m³ foram estocadas com 120 peixes cada (peso médio 0,5 g), os quais foram inicialmente alimentados com FP e submetidos a TGIR com dietas FP-90, FP-80, FP-70, FP-60, FP-50 e FP-40 por períodos subsequentes de 3 dias. Os peixes que aceitaram GF-40 (81% do total) foram agrupados e estocados em 9 gaiolas de 0,03 m³ e treinados por 3 dias a aceitar FP-00 pelo uso de dietas contendo 30, 20 ou 10% de FP e flavorizadas com: (1) alimento comercial seco sabor carne (ACSC); (2) alimento comercial seco sabor peixe (ACSP); ou (3) ACSC mais 10% de farinha de krill (ACSP+FK). A aceitação de ACSP foi melhor que ACSC. A adição de KM ao ACSC melhorou a aceitabilidade da dieta em comparação com outros aditivos alimentares, mesmo quando a quantidade de FP nas dietas foi reduzida para 10%. Entretanto, ao final dos ensaios, nenhum peixe aceitou grânulos alimentares isentos de FP (FP-00).