Maintaining Plasmodium falciparum gametocyte infectivity during blood collection and transport for mosquito feeding assays in the field

Background Mosquito feeding assays using venous blood are commonly used for evaluating the transmission potential of malaria infected individuals. To improve the accuracy of these assays, care must be taken to prevent premature activation or inactivation of gametocytes before they are fed to mosquitoes. This can be challenging in the field where infected individuals and insectary facilities are sometimes very far apart. In this study, a simple, reliable, field applicable method is presented for storage and transport of gametocyte infected blood using a thermos flask. Methods The optimal storage conditions for maintaining the transmissibility of gametocytes were determined initially using cultured Plasmodium falciparum gametocytes in standard membrane feeding assays (SMFAs). The impact of both the internal thermos water temperature (35.5 to 37.8 °C), and the external environmental temperature (room temperature to 42 °C) during long-term (4 h) storage, and the impact of short-term (15 min) temperature changes (room temp to 40 °C) during membrane feeding assays was assessed. The optimal conditions were then evaluated in direct membrane feeding assays (DMFAs) in Burkina Faso and The Gambia where blood from naturally-infected gametocyte carriers was offered to mosquitoes immediately and after storage in thermos flasks. Results Using cultured gametocytes in SMFAs it was determined that an internal thermos water temperature of 35.5 °C and storage of the thermos flask between RT (~ 21.3 °C) and 32 °C was optimal for maintaining transmissibility of gametocytes for 4 h. Short-term storage of the gametocyte infected blood for 15 min at temperatures up to 40 °C (range: RT, 30 °C, 38 °C and 40 °C) did not negatively affect gametocyte infectivity. Using samples from natural gametocyte carriers (47 from Burkina Faso and 16 from The Gambia), the prevalence of infected mosquitoes and the intensity of oocyst infection was maintained when gametocyte infected blood was stored in a thermos flask in water at 35.5 °C for up to 4 h. Conclusions This study determines the optimal long-term (4 h) storage temperature for gametocyte infected blood and the external environment temperature range within which gametocyte infectivity is unaffected. This will improve the accuracy, reproducibility, and utility of DMFAs in the field, and permit reliable comparative assessments of malaria transmission epidemiology in different settings.

Maintaining Plasmodium Falciparum Gametocyte Infectivity During Blood Collection and Transport for Mosquito Feeding Assays in the Field

BackgroundMosquito feeding assays using venous blood are commonly used for evaluating the transmission potential of malaria infected individuals. To improve the accuracy of these assays, care must be taken to prevent premature activation or inactivation of gametocytes before they are fed to mosquitoes. This can be challenging in the field where infected individuals and insectary facilities are sometimes very far apart. In this study, a simple, reliable, field applicable method is presented for storage and transport of gametocyte infected blood using a thermos flask. MethodsThe optimal storage conditions for maintaining the transmissibility of gametocytes were determined initially using cultured Plasmodium falciparum gametocytes in standard membrane feeding assays (SMFAs). The impact of both the internal thermos water temperature (35.5 – 37.8°C), and the external environmental temperature (room temp – 42°C) during long-term (4hr) storage, and the impact of short-term temperature changes (room temp – 40°C) during membrane feeding assays was assessed. The optimal conditions were then evaluated in direct membrane feeding assays (DMFAs) in Burkina Faso and The Gambia where blood from naturally infected gametocyte carriers was offered to mosquitoes immediately and after storage in thermos flasks. ResultsUsing cultured gametocytes in SMFAs it was determined that an internal thermos water temperature of 35.5°C and storage of the thermos flask between RT (~21.3°C) and 32°C was optimal for maintaining transmissibility of gametocytes for 4 hours. Short-term storage of the gametocyte infected blood at temperatures up to 38°C (range: RT, 30°C and 38°C) did not have a negative effect on gametocyte infectivity. Using samples from natural gametocyte carriers (47 from Burkina Faso and 16 from The Gambia), the prevalence of infected mosquitoes and the intensity of oocyst infection was maintained when gametocyte infected blood was stored in a thermos flask in water at 35.5°C for up to 4 hours.ConclusionsThis study determines the optimal long-term (4 hours) storage temperature for gametocyte infected blood and the external environment temperature range within which gametocyte infectivity is unaffected. This will improve the accuracy, reproducibility, and utility of DMFAs in the field, and permit reliable comparative assessments of malaria transmission epidemiology in different settings.

Thermal properties of wooden based flask from tropical hardwood species

Ogunsanwo OY, Onakpoma I, Korede M. 2020. Thermal properties of wooden based flask from tropical hardwood species. Asian J For 4: 61-64. Most materials used for production of conventional thermos flask (metals and plastics) are not environment friendly with particular concerns about plastic which are not biodegradable. Wood waste management is important in producing environmentally friendly materials and achieving sustainable development in forestry through wood waste utilization as thermos flask. This study was therefore conducted to investigate the thermal properties of wooden thermos flask (WTF) using tropical hardwood species with a view to promoting the use of greener technology. Offcuts of Tectona grandis and Albizia saman were used to produce 12 WTF of two thicknesses (11 mm and 12 mm), with a height of 10.5'' using bamboo as inner lining. The heat loss and heat gain by the wooden flasks and conventional flask were collected using a thermometer which was inserted into the cap of the flasks through a hole drilled in it, the hole was covered at intervals to prevent heat loss and heat gain from the opening. Data was collected every two hours for twelve hours. The study was laid out in a 2×2 factorial experimental in a completely randomized design. Data were analyzed using ANOVA at p=0.05. The highest heat loss (64.00ºC) after 12 hours was observed in Tectona grandis WTF with 11 mm and 12 mm while Albizia saman thermos flask with 12 mm thickness had the least heat loss (62.00ºC). Metallic flask lost only about 30.00 ºC of its heat content after 12 hours. The highest heat gain (28ºC) after 12 hours was observed in Tectona grandis WTF with 11mm and 12mm while Albizia saman WTF with 11 mm thickness had the least heat gain (25.67ºC). Heat gained by WTF was 28.00ºC and 25.83ºC for Tectona grandis and Albizia saman respectively after 12 hours while heat gain by the metallic flask after 12 hours was 18.00ºC. Species and thickness did not significantly affect heat loss and gain of thermos flask. Significant difference was however observed between the heat lost and gained by WTF and metallic flask. Wooden thermos flask still retained heat and prevented loss to a certain degree but technological improvement would perform better.

148 Effect of concentration of methionine in maturation and culture medium on cleavage rate of oocytes from alpaca (Lama pacos)

Maturation time of oocytes from alpacas is around 38 to 40h (Huanca et al. 2009) that would induce an increase in reactive oxygen species during in vitro maturation and IVF and cause cytotoxic damage to gametes. The objective of this study was to determine the optimal concentration of methionine during in vitro maturation on cleavage rate of alpacas oocytes following IVF. Cumulus-oocyte complexes were collected from slaughterhouse ovaries and transported in a thermos flask containing a saline solution 0.9% and antibiotic, antimycotic at 35°C. Cumulus-oocyte complexes were aspirated from follicles >2mm and evaluated with a stereomicroscope for selection. Only cumulus-oocyte complexes with a homogeneous cytoplasm and with 2 or more layers of cumulus cells were selected to be cultured in maturation medium TCM-199 supplemented with 10% FCS (v:v) plus 0.5μg mL−1 FSH, 10μg mL−1 hCG, 0.2mM sodium pyruvate, 50μg mL−1gentamycin and 1μg mL−1 oestradiol under mineral oil by 38h. Testes of mature males were collected from a slaughterhouse and transported to the laboratory. Caudal epididymide was isolated, and fluid, rich in spermatozoa, was aspirated in syringes containing 2mL of Tris-fructose-egg yolk extender. Motile spermatozoa were obtained by centrifugation at 700×g in a Percoll discontinuous gradient (22.5: 45.0%) for 10min. The supernatant was removed by aspiration, and the pellet was resuspended in TL stock and centrifuged again at 700×g for 5min. Spermatozoa and oocytes were co-incubated by 18h at 39°C with 5% CO2. Presumptive zygotes were culture in KSOMaa medium and evaluated at 72h. The treatments include 0, 14 and 21 μM of methionine in maturation and culture medium. Data were analysed by ANOVA, and results are presented in Table 1. The results suggest that addition of methionine in maturation and culture medium improve the cleavage rate in oocytes from alpacas. Table 1.Cleavage rate (%) following in vitro maturation at different concentrations of methionine Proyect 405-PNICP-PIAP-2014, INNOVATE-PERU, is acknowledged.

Determination of Band Gap Energy of Semiconductor in Homojunction Structure Devices by Using Customized Microcontroller Based Apparatus

We have successfully developed a customized apparatus based on microcontroller for simple band gap energy (Eg) measurement of semiconductors in homojunction structure devices. The apparatus consisted of a data acquisition system based on microcontroller AVR ATMega 128 and a thermos flask equipped with temperature controller. It permits recording of current-voltage (I-V) and temperature and subsequently sends data to a computer to enable the computer processing of such data. For samples under tested, we used two types of commercial diode, i.e. Silicon (1N4007) and Germanium (1N60). In this measurement, the voltage across the resistor was used to calculate the current while the voltage across the diode gave the forward bias voltage. The temperature of diode was varied from 5°C to 80°C. During each I-V measurement, the temperature of diode was maintained to be constant by employing a proportional-integral-derivative (PID) controller to the heater. Furthermore, by varying the temperature of diode, we could extract the saturation currents under reverse bias across the diode of each I-V measurement. For the two types of diode, it is found that the Eg of silicon is 1.13 ± 0.03 eV, while that of germanium is 0.71 ± 0.03 eV. This result is closed to the Eg value of each diode indicated in the respective datasheet. Therefore, it suggests for applying this apparatus for measuring Eg of semiconductor in most homojunction structure devices.

214 EFFECT OF LONG-DISTANCE TRANSPORTATION OF OVARIES ON THE DEVELOPMENT OF IN VITRO-MATURED - IN VITRO-FERTILIZED - IN VITRO-CULTURED BOVINE EMBRYOS

Keeping ovaries for a long time before oocyte recovery affects the maturation rate and blastocyst rate (BL rate), but a change in the temperature of the ovary at the time of transportation does not affect the BL rate (Ribeiro et al. 2008 Anim. Reprod. Sci. 108, 171–179). The objective of this study was to investigate the effect of long-distance transportation of ovaries on the in vitro development of bovine oocytes into blastocysts. Ovaries in the transportation group (Group T) were collected from a slaughterhouse, placed inside a Thermos flask, and transported to the laboratory within 17 to 21 h. The Thermos flask was covered with a freezer pack in a foam polystyrene box (Nakatate et al. 2006 Reprod. Fertil. Dev. 18, 193). The temperature of the Thermos flask was measured with a temperature recorder. Cumulus–oocyte complexes (COC) were collected by aspirating 2- to 6-mm follicles from the ovaries. The COC were matured in TCM-199 supplemented with 5% calf serum and 0.02 AU mL–1 of FSH. Groups of 20 COC were incubated in 100-μL drops of IVM media at 38.5°C under an atmosphere of 5% CO2 in air for 20 h. After 18 h of gamete co-culture (3 × 106 sperm mL–1), the presumptive zygotes were cultured in CR1aa medium supplemented with 5% calf serum for 9 days at 38.5°C under an atmosphere of 5% CO2, 5% O2, and 90% N2. Embryonic development was evaluated at 48 h after IVF (total cleavage rates, CL rate) and on Days 7 to 9 (BL rate). The ovaries in the control group (Group C) were collected from a slaughterhouse different from that of the Group T ovaries and transported to the laboratory in saline (23°C) in a Thermos flask within 3 h. These ovaries were then used for the collection of COC in the same way as for Group T. Data were analysed by chi-square test. A total of 25 experiments were performed using the ovaries from Groups C and T (n = 3945 v. n = 7218). The CL rate and the BL rate for Group T were significantly lower than those for Group C (41.8 v. 62.0% and 16.1 v. 29.7%; P < 0.01). In Group T, the duration of ovary transportation did not affect the CL rate (within 19 h, between 19 and 20 h, and more than 20 h), but the BL rate was significantly decreased with an increase in transport time (18.7 v. 16.1 v. 14.8%). The temperature change in the Thermos flask during transportation ranged from 12.0 to 25.1°C; the temperature was maintained virtually constant or decreased. The CL rates of the groups that underwent a temperature (t) change (t ≤ 5.0 and 5.0 < t ≤ 10.0) were not significantly different, but the BL rate in the group with 5.0 < t ≤ 10.0 was significantly lower than that of the other group (16.7 v. 13.5%; P < 0.01). These results suggest that the long-distance transportation of ovaries affects in vitro embryo development. Moreover, a decrease in temperature during transportation may have an adverse effect on blastocyst formation.

157 FIRST IN VITRO EMBRYO PRODUCTION IN ALPACAS (LAMA PACOS)

The objective was to produce alpaca embryos in laboratory due to its potential role for the multiplication of genetically superior animals and for conservation purposes. Ovaries were collected from an alpaca abattoir located in the Central Highlands of Peru and transported in a thermos flask with warm saline and antibiotics to the laboratory located 200 km away on the coast. Alpaca epididymal sperm to be used for fertilization was previously frozen by diluting in a TRIS-Fructose based extender with 10% glycerol and frozen as pellets in liquid nitrogen vapor. From 31 ovaries, 262 cumulus–oocyte complexes (COCs) were collected (mean of 8.5 COCs per ovary) which were matured in TCM-199 supplemented with 10% heat inactivated FCS plus epidermal growth factor (EGF), FSH, LH, estradiol, and cysteamine for 30 h incubation at 38.5°C, 5% CO2 and 90% humidity. The selected oocytes post-maturation were fertilized with the frozen/thawed sperm that was subjected post-thawing to Percoll gradient (90 and 45% Percoll), centrifugation and resuspension in TALP-IVF medium supplemented with 20 μm D-penicillamine, 10 μm hypotaurine, 1 μm epinephrine and 1.1 μg mL–1 of heparin. The oocytes were inseminated with a concentration of 10 × 106 spermatozoa per drop of 100 mL of fertilization medium containing 30 oocytes each and incubated for 24 h at 38.5°C, 5% CO2 and 90% humidity. The presumptive zygotes were transferred to 200 μL drops (30 zygotes per drop) of SOFaa media supplemented with 5% heat-inactivated FCS which was replaced by SOFaa plus 1% heat-inactivated FCS on day 5 after fertilization. The incubation period post-fertilization was up to day 7 at 38.5°C, 5% CO2 and 90% humidity, when the embryos were inspected and graded. The cleavage rate was evaluated at 72 h post-fertilization and embryo development was evaluated on day 5 and 7 post-fertilization. The cleavage rate was 27.1% (71/262) and the percentage of oocytes that reached the stage of morula and blastocyst was 8.0% (21/262). The percentage of blastocyst that hatched when incubated after day 7 was 14.28% (3/21). The in vitro embryo production in alpacas was successful and suggests the possibility for application in intensive reproduction for conservation of South American camelids and for genetic improvement. Research was partially funded by contributions of BIONICHE and SAIS TUPAC AMARU, Junin, Peru.