Refinements in embryo manipulation applied to CRISPR technology in livestock.

The implementation of CRISPR technology in large animals requires further improvements in embryo manipulation and transfer to be applied with commercial purposes. In this study we report (a) developmental competence of CRISPR/Cas microinjected zygotes subjected to in vitro culture in large scale programs in sheep; (b) pregnancy outcomes after early-stage (2-8-cell) embryo transfer into the oviduct or the uterine horn; and (c) embryo survival and birth rate after vitrification/warming of CRISPR/Cas microinjected zygotes. Experiment 1 consisted of a retrospective analysis to evaluate embryo developmental rate of in vitro produced zygotes subjected to CRISPR/Cas microinjection (n = 7,819) compared with a subset of non-microinjected zygotes (n = 701). Development rates to blastocyst on Day 6 were 20.0% for microinjected zygotes and 44.9% for non-injected zygotes (P < 0.05). In Experiment 2, CRISPR/Cas microinjected zygotes were transferred on Day 2 after in vitro fertilization (2-8 cell embryos) into the oviductal ampulla (n = 262) or into the uterine horn (n = 276) in synchronized recipient ewes at prefixed time (i.e., approximately two days after ovulation). Pregnant/transferred recipients (24.0% vs. 25.0%), embryo survival/transferred embryos (6.9% vs. 6.2%), and born lambs/pregnant embryos (72.2% vs. 100.0%) did not differ significantly in the two groups. In Experiment 3, CRISPR/Cas microinjected zygotes were maintained under in vitro culture until blastocyst stage (Day 6), and subjected to vitrification/warming via the Cryotop method (n = 474), while a subset of embryos were left fresh as control group (n = 75). Embryos were transferred into the uterine horn of recipient females at prefixed time 8.5 days after the estrous synchronization treatment (i.e., approximately six days after ovulation). Pregnancy rate (30.8% vs. 48.0%), embryo survival rate (14.8% vs. 21.3%), and birth rate (85.7% vs. 75.0%) were not different (PNS) between vitrified and fresh embryos, respectively. In conclusion, the current study in sheep embryos reports (a) suitable developmental rate after CRISPR/Cas microinjection (i.e., 20%), even though it was lower than non-microinjected zygotes; (b) similar outcomes when Day 2-embryos were placed into the uterine horn instead of the oviduct, avoiding both time-consuming and invasive oviduct manipulation, and extended in vitro culture during one week; (c) promising pregnancy and birth rates obtained with vitrification of CRISPR/Cas microinjected embryos. This knowledge on in vitro embryo development, timing of embryo transfer, and cryopreservation of CRISPR/Cas microinjected zygotes have practical implications for the implementation of genome editing technology in large animals.

Could some procedures commonly used in bioassays with the copepod Acartia tonsa Dana 1849 distort results?

Many organizations have suggested the use of the Calanoid copepod Acartia tonsa in protocols for acute toxicity tests. Nevertheless, these protocols present some problems, such as using 60-180µm meshes to separate specific stages of A. tonsa or carrying out the tests using small volumes that reflect high densities of A. tonsa that do not occur in nature, which could lead to distorted results. In addition, ecotoxicological studies may use statistical approaches that are inadequate for the type of data being analysed. For these reasons, some methodological approaches for bioassays using A. tonsa need to be clarified and revised. In this study, we present information about (i) the retention of copepodite stages of A. tonsa on 180, 330 and 500µm net meshes; (ii) tested storage volumes of 1 organism per 5, 10 or 20mL in each test container (TC); and (iii) considerations about the statistics employed. The results demonstrated that a net mesh of 180µm is capable of retaining all copepodite stages (CI to CVI), contrasting with the recommendation of using a 180µm mesh to separate out adults only. Coarser meshes (330 and 500µm) can also retain different proportions of all copepodite stages, but cannot separate out one developmental stage only. Twenty-five millilitres of medium in an open TC, commonly employed in bioassays simulating densities of 1 organism 5mL-1, completely evaporated, and the results showed that the TCs need to be covered (e.g., PVC film) and filled with a minimum of 100mL of culture medium (simulating densities of 1 organism 20mL-1) to avoid evaporation and increases in salinity. The current use of ANOVA in ecotoxicological studies with proportions of surviving organisms should also be reconsidered since the data are discrete and have a binomial distribution; general linear models (GLMs) are considered more adequate. The information presented here suggests some adjustments that hopefully will enable the improvement of the procedures and methods employed in studies of acute toxicity using the copepod A. tonsa.

Determination of the minimum effective volume of 0.5% bupivacaine for ultrasound-guided axillary brachial plexus block.

BACKGROUND AND OBJECTIVE: The use of ultrasound for needle correct placement and local anesthetic spread monitoring helped to reduce the volume of local anesthetic required for peripheral nerve blocks. There are few studies of the minimum effective volume of local anesthetic for axillary brachial plexus block. The aim of this study was to determine the minimum effective volume (VE90) of 0.5% bupivacaine with epinephrine (1:200,000) for ultrasound guided ABPB. METHOD: Massey and Dixon's up-and-down method was used to calculate the minimum effective volume. The initial dose was 5 mL per nerve (radial, median, ulnar, and musculocutaneous). In case of blockade failure, the volume was increased to 0.5 mL per nerve. A successful blockade resulted in decreased volume of 0.5 mL per nerve to the next patient. Successful blockade was defined as a motor block ≤2, according to the modified Bromage scale; lack of thermal sensitivity; and response to pinprick. The achievement of five cases of failure followed by success cases was defined as criterion to complete the study. RESULTS: 19 patients were included in the study. The minimum effective volume (VE90) of 0.5% bupivacaine with 1:200,000 epinephrine was 1.56 mL (95% CI, 0.99-3.5) per nerve. CONCLUSION: This study is in agreement with some other studies, which show that it is possible to achieve surgical anesthesia with low volumes of local anesthetic for ultrasound-guided peripheral nerve blocks.

Determinacao do volume minimo efetivo de bupivacaina 0,5% para bloqueio do plexo braquial por via axilar guiado por ultrassom / Determination of the minimum effective volume of 0.5% bupivacaine for ultrasound-guided axillary brachial plexus block / Determinacion del volumen minimo efectivo de bupivacaina al 0,5% para el bloqueo del plexo braquial por via axilar guiado por ultrasonido

Justificativa e objetivo: o uso do ultrassom para acompanhar o correto posicionamento da agulha e a dispersão do anestésico local permitiu a redução do volume de anestésico local necessário para o bloqueio de nervos periféricos. Existem poucos trabalhos sobre o volume mínimo efetivo de anestésico local para o bloqueio do plexo braquial via axilar (BPVA). Este estudo foi conduzido com o objetivo de determinar o volume mínimo efetivo (VE90) de bupivacaína 0,5% comadrenalina (1:200.000) para o BPVA guiado por ultrassom. Método: o método up-and-down proposto por Massey e Dixon foi usado para o cálculo do volume mínimo efetivo. A dose inicial foi de 5 ml por nervo (radial, mediano, ulnar e musculocutâneo). No caso de falha do bloqueio, o volume era aumentado em 0,5 mL por nervo. O sucesso do bloqueio resultava na diminuição do volume em 0,5 mL por nervo para o paciente subsequente. O sucesso do bloqueio foi definido como bloqueio da função motora ≤ 2 segundo a escala de Bromage modificada, ausência de sensibilidade térmica e de resposta ao pinprick. Foi definido como critério para término do estudo a obtenção de cinco casos de falha seguidos de casos de sucesso. Resultados: foram incluídos 19 pacientes no estudo. O volume mínimo efetivo (VE90) de bupi-vacaína 0,5% com 1:200.000 de adrenalina foi de 1,56 ml (IC 95% 0,99-3,5) por nervo. Conclusão: este trabalho corrobora alguns estudos que mostram que é possível obter anestesia cirúrgica com baixos volumes de anestésico local para bloqueios de nervo periférico guiados por ultrassom. .

Background and objective: The use of ultrasound for needle correct placement and local anesthetic spread monitoring helped to reduce the volume of local anesthetic required for peripheral nerve blocks. There are few studies of the minimum effective volume of local anesthetic for axillary brachial plexus block. The aim of this study was to determine the minimum effective volume (VE90) of 0.5% bupivacaine with epinephrine (1:200,000) for ultrasound guided ABPB. Method: Massey and Dixon's up-and-down method was used to calculate the minimum effective volume. The initial dose was 5 mL per nerve (radial, median, ulnar, and musculocutaneous). In case of blockade failure, the volume was increased to 0.5 mL per nerve. A successful blockade resulted in decreased volume of 0.5 mL per nerve to the next patient. Successful blockade was defined as a motor block ≤2, according to the modified Bromage scale; lack of thermal sensitivity; and response to pinprick. The achievement of five cases of failure followed by success cases was defined as criterion to complete the study. Results: 19 patients were included in the study. The minimum effective volume (VE90) of 0.5% bupivacaine with 1:200,000 epinephrine was 1.56 mL (95% CI, 0.99-3.5) per nerve. Conclusion: This study is in agreement with some other studies, which show that it is possible to achieve surgical anesthesia with low volumes of local anesthetic for ultrasound-guided peripheral nerve blocks. .

Justificación y objetivo: el uso del ultrasonido para acompanar el correcto posicionamiento de la aguja y la dispersión del anestésico local ha permitido la reducción del volumen de anestésico local necesario para el bloqueo de los nervios periféricos. Existen pocos trabajos sobre el volumen mínimo efectivo de anestésico local para el bloqueo del plexo braquial vía axilar. Este estudio fue llevado a cabo con el objetivo de determinar el volumen mínimo efectivo (VE90) de bupivacaína al 0,5% con adrenalina (1:200.000) para el bloqueo del plexo braquial vía axilar guiado por ultrasonido. Método: el método up-and-down propuesto por Massey y Dixon fue usado para el cálculo del volumen mínimo efectivo. La dosis inicial fue de 5mL por nervio (radial, mediano, cubital y musculocutáneo). En el caso de fallo en el bloqueo, el volumen se aumentaba a 0,5 mL por nervio. El éxito del bloqueo traía la disminución del volumen en 0,5 mL por nervio para el paciente posterior. El éxito del bloqueo se definió como bloqueo de la función motora < 2 según la escala de Bromage modificada, ausencia de sensibilidad térmica y de respuesta al pinprick. Quedó definido como criterio para el término del estudio la obtención de 5 casos de fallo seguidos de casos de éxito. Resultado: se incluyeron 19 pacientes en el estudio. El volumen mínimo efectivo (VE90) de bupivacaína al 0,5% con 1:200.000 de adrenalina fue de 1,56 mL (IC 95%: 0,99-3,5) por nervio. Conclusiones: este trabajo corrobora algunos estudios que muestran que se puede obtener la anestesia quirúrgica con bajos volúmenes de anestésico local para bloqueos de nervio periférico guiados por ultrasonido. .