An in vitro method for assessment of amino acid bidirectional transport and intracellular metabolic fluxes in mammary epithelial cells.

Understanding uptake of AA by mammary tissue as supply varies is critical for predicting milk component production. Our objective was to develop an in vitro method to quantify cellular uptake, efflux, and intracellular metabolism of individual AA that could be implemented for evaluating these factors when AA supply and profile are varied. Bovine primary mammary epithelial cells were grown to confluency and exposed to medium with an AA profile and concentration similar to lactating dairy cow plasma for 24 h. Cells were then preloaded in medium enriched with 15N-labeled AA for 24 h followed by removal of the 15N-labeled medium and incubation with medium enriched with 13C-labeled AA for 0, 15, 60, 300, 900, 1,800, and 3,600 s. Extracellular free AA and intracellular free and protein-bound AA were analyzed for concentrations and isotopic enrichment by gas chromatography-mass spectrometry. A dynamic, 12-pool model was constructed representing extracellular and intracellular free and protein-bound pools of an AA, and their respective 15N and 13C isotopes. Markov chain Monte Carlo simulation (n = 5,000) was conducted to evaluate prediction errors by deriving standard errors and posterior distributions for rate constants, fluxes, and pools. Cellular Ala influx and efflux were higher than Leu, reflecting Ala role in driving system L transport and the high capacity of sodium-dependent transport. The Ala and Leu turnover rates were 181 and 95, 580 and 857, and 74 and 157% per hour for extracellular, intracellular, and fast protein-bound pools, respectively. The intracellular and extracellular Ala to Leu ratios were quite different, meaning the blood AA profile is not the AA profile provided for protein translation. The high level of exchange and rapid turnover of pools provide a mechanism for matching the AA supplies to the precision necessary for translation. This also understates the importance of using experimental medium similar to what is observed in vivo given that some AA depend on other AA for influx (exchange driven). The average root mean squared prediction error across the isotope enrichments, pools, and concentrations was 9.7 and 14.1% for Ala and Leu, respectively, and collinearity among parameters was low, indicating adequate fit and identifiability. The described model provides insight on individual AA transport kinetics and a method for future evaluation of AA transport and intracellular metabolism when subjected to varying AA supplies.

Assessment of erythrocyte damage and in-line pressure changes associated with simulated transfusion of canine blood through microaggregate filters.

OBJECTIVE: To determine whether passage of whole blood through a microaggregate filter by use of a syringe pump would damage canine erythrocytes. SAMPLE: Blood samples obtained from 8 healthy client-owned dogs. PROCEDURES: Whole blood was passed through a standard microaggregate filter by use of a syringe pump at 3 standard administration rates (12.5, 25, and 50 mL/h). Prefilter and postfilter blood samples were collected at the beginning and end of a simulated transfusion. Variables measured at each time point included erythrocyte osmotic fragility, mean corpuscular fragility, RBC count, hemoglobin concentration, RBC distribution width, and RBC morphology. In-line pressure when blood passed through the microaggregate filter was measured continuously throughout the simulated transfusion. After the simulated transfusion was completed, filters were visually analyzed by use of scanning electron microscopy. RESULTS: Regardless of administration rate, there was no significant difference in mean corpuscular fragility, RBC count, hemoglobin concentration, or RBC distribution width between prefilter and postfilter samples. Additionally, there were no differences in in-line pressure during the simulated transfusion among administration rates. Echinocytes were the erythrocyte morphological abnormality most commonly observed at the end of the transfusion at administration rates of 12.5 and 25 mL/h. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that regardless of the administration rate, the microaggregate filter did not alter fragility of canine RBCs, but may have altered the morphology. It appeared that the microaggregate filter would not contribute to substantial RBC damage for transfusions performed with a syringe pump.

Assessment of anoxia tolerance and photoperiod dependence of GABAergic polarity in the pond snail Lymnaea stagnalis.

The pond snail Lymnaea stagnalis is reported to be anoxia-tolerant and if the tolerance mechanism is similar to that of the anoxia-tolerant painted turtle, GABA should play an important role. A potentially confounding factor investigating the role of GABA in anoxia tolerance are reports that GABA has both inhibitory and excitatory effects within L. stagnalis central ganglion. We therefore set out to determine if seasonality or photoperiod has an impact on: 1) the anoxia-tolerance of the intact pond snail, and 2) the response of isolated neuroganglia cluster F neurons to exogenous GABA application. L. stagnalis maintained on a natural summer light cycle were unable to survive any period of anoxic exposure, while those maintained on a natural winter light cycle survived a maximum of 4h. Using intracellular sharp electrode recordings from pedal ganglia cluster F neurons we show that there is a photoperiod dependent shift in the response to GABA. Snails exposed to a 16h:8h light:dark cycle in an environmental chamber (induced summer phenotype) exhibited hyperpolarizing inhibitory responses and those exposed to a 8h:16h light:dark cycle (induced winter phenotype) exhibited depolarizing excitatory responses to GABA application. Using gramicidin-perforated patch recordings we also found a photoperiod dependent shift in the reversal potential for GABA. We conclude that the opposing responses of L. stagnalis central neurons to GABA results from a shift in intracellular chloride concentration that is photoperiod dependent and is likely mediated through the relative efficacy of cation chloride co-transporters. Although the physiological ramifications of the photoperiod dependent shift are unknown this work potentially has important implications for the impact of artificial light pollution on animal health.

Comparison of different methods for exogenous DNA uptake by bovine spermatozoa / Comparação de diferentes métodos de incorporação de DNA exógeno pelo espermatozoide bovino

Although genetic manipulation of farm animals is of great interest for animal production and the pharmaceutical industry, its efficiency remains far from satisfactory. Pronuclear injection, which is the most widely used technique for such modification, mainly in mice, remains limited for this species. Some alternatives have been developed such as sperm mediated gene transfer, in which the spermatozoa are used as vectors for DNA delivery during in vitro fertilization. Mature sperm cells are able to spontaneously bind exogenous DNA molecules which may be internalized into sperm nuclei. Given the potential of sperm mediated gene transfer for livestock animals transgenesis, the aim of this study was to evaluate four methods of DNA uptake for sperm mediated gene transfer in bovine: incubation with DNA, plasma membrane alteration induced by calcium ionophore followed by incubation with DNA, electroporation and lipofection. Spermatozoa not exposed to exogenous DNA were used as control group. Cleavage, blastocyst and hatching rates were recorded at 72 hours post insemination (hpi), days 9 and 12 of embryo culture, respectively. Exogenous DNA-positive embryos were evaluated by PCR. No effect of treatment was observed on cleavage, blastocyst and hatching rates. In addition, percentage of DNA positive blastocysts did not differ among experimental groups. In spite of the low number of positive embryos, our results show that all treatments presented similar efficiencies for DNA delivery during in vitro fertilization. In conclusion, although the development rates were similar and constant in all groups, other factors such as exogenous DNA sequence, size and concentration should be considered to improve sperm mediated gene transfer.

Apesar da manipulação genética de animais domésticos ser de grande interesse para a produção animal e para a indústria farmacêutica, a sua eficiência ainda é insatisfatória. A injeção pronuclear, a técnica mais utilizada para tal propósito, principalmente em camundongos, ainda apresenta limitações para esta espécie. Algumas alternativas têm sido desenvolvidas como o uso de espermatozoides como vetores para transferência gênica, na qual a célula espermática tem habilidade espontânea de se ligar à molécula de DNA e internalizá-la. Dado o potencial da transferência gênica mediada por espermatozoide para animais domésticos transgênicos, o objetivo do presente trabalho foi a avaliação de quatro métodos de incorporação de DNA para a transferência gênica mediada por espermatozoides na espécie bovina: incubação com DNA, alteração da membrana plasmática induzida por cálcio ionóforo seguida por incubação com o DNA exógeno, eletroporação e lipofecção. Espermatozoides não expostos ao DNA exógeno foram usados como grupo controle. Os índices de clivagem, blastocisto e eclosão foram avaliados, respectivamente, as 72 horas após a inseminação dos oócitos, bem como, aos 9 e 12 dias de cultivo embrionário. Os embriões positivos para o DNA exógeno foram avaliados por PCR. Nenhum efeito de tratamento foi observado nos índices de clivagem, blastocisto e eclosão. Além disso, a porcentagem de blastocistos positivos para o DNA exógeno não diferiu entre os grupos experimentais. Apesar do baixo número de embriões positivos para DNA exógeno, os resultados obtidos mostram que todos os tratamentos apresentaram eficiências similares. A conclusão obtida foi que, apesar de os índices de desenvolvimento embrionário terem sido similares e constante em todos os grupos experimentais, outros fatores como a sequência, o tamanho e a concentração do DNA exógeno devem ser avaliados para melhorar a transferência gênica mediada por espermatozoides.

Evaluation of in vitro methods for assessment of infection of Australian Culicoides spp. with bluetongue viruses.

Biting midges from the genus Culicoides (Diptera: Ceratopogonidae) are the vectors of several globally important arboviruses that affect livestock. These include orbiviruses from the bluetongue virus (BTV) and African horse sickness virus (AHSV) groups and members of the Simbu serogroup of orthobunyaviruses, such as the recently emerged Schmallenberg virus. In this article, the authors evaluate several methods for feeding wild­caught Australian Culicoides on BTV infected preparations of blood and sucrose. Feeding Culicoides on the membrane of embryonated chicken eggs was identified as the preferred feeding method. Although, cotton wool pads soaked in either virus­infected blood or virus­sucrose mixtures were also successful. A non­destructive nucleic acid extraction technique for the detection of viral RNA in Culicoides was also evaluated as it allows for readily differentiating infected from non­infected Culicoides.