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Articles by Andrea HETTIG
Total Records ( 8 ) for Andrea HETTIG
  Ileana MICLEA , Marius ZAHAN , Vasile MICLEA , Andrea HETTIG , Iulian ROMAN and Florin GHIURU
  As defence against oxidative stress, living systems employ antioxidants that they either produce or take up from the environment. Among these α-tocopherol has been shown to improve swine oocyte maturation and the development of bovine and swine embryos. The goal of this study was to establish the influence of several α-tocopherol concentrations on swine embryo development in vitro, in order to improve culture media. Pig oocytes were cultured for 45 hours at 37°C in 5% CO2 atmosphere; in M199 containing several α-tocopherol (5, 10, 20, 40 and 80 μM) concentrations. Then, they were fertilized in TALP medium using spermatozoa capacitated by centrifugation and incubated for 16-18 hours. Afterwards, the presumed zygotes were cultured in NCSU-23 droplets supplemented with α-tocopherol (5, 10, 20, 40 and 80 μM) in the same conditions. Their development was assessed at 48 and 120 hours. The number of embryos that had developed to the 2 cells, 4-8 cells and morula stages was compared to the control, and the differences analyzed using the analysis of variance and interpreted using the LSD and Duncan tests. At 48 hours after fertilization it was apparent that α-tocopherol supplementation increased embryo development to the 4-8 cell (10, 20, 40, 80 μM) and morula (5, 40, 80 μM) stages. At 120 hours even more embryos had reached the morula stage.
  Ileana MICLEA , Marius ZAHAN , Vasile MICLEA , Andrea HETTIG , Iulian ROMAN , Florin GHIURU and Elena ILISIU
  As defence against oxidative stress, living systems employ antioxidants that they either produce or take up from the environment. Among these lutein has been shown to be a powerful antioxidant that also functions as a signal molecule. The goal of this study was to establish the influence of several lutein concentrations on swine embryo development in vitro, in order to improve culture media. Pig oocytes were cultured for 45 hours at 37°C in 5% CO2 atmosphere; in M199 containing several lutein (2.5, 4, 5, 8, and 10 μM) concentrations. Then, they were fertilized in TALP medium using spermatozoa capacitated by centrifugation and incubated for 16-18 hours. Afterwards, the presumed zygotes were cultured in NCSU-23 droplets supplemented with lutein (2.5, 4, 5, 8, and 10 μM) in the same conditions. Their development was assessed at 48 and 120 hours. The number of embryos that had developed to the 2 cells, 4-8 cells and morula stages was compared to the control, and the differences analyzed using the analysis of variance and interpreted using the LSD and Duncan tests. Lutein (2.5 μM) supplementation has a beneficial effect on embryo development to the morula stage. This is apparent at 48 hours as well as 120 hours.
  Iulian ROMAN , Vasile MICLEA , Andrea HETTIG , Marius ZAHAN , Ileana MICLEA , Florin VARO-GHIURU and Alexandru RUSU
  Supplementation of a chemical defined in vitro maturation (IVM) media with some amino acids was examined in the terms of oocyte maturation and fecundation rate following intracytoplasmic sperm injection (ICSI). Addition of glutamine resulted in a higher (p<0.05) in vitro maturation rate, compared to the total absence of amino acids in media, but in terms of fecundation rate, none of the amino acids used increased the fecundation rate in our research.
  Florin VARO GHIURU , Ioan LADOSI , Iulian ROMAN , Andrea HETTIG , Marius ZAHAN and Vasile MICLEA
  The technical establishment of boar sperm cryopreservation is indispensable for effective breeding of the Mangalita native pig. The objective of the present study was to determine whether ascorbic acid is capable of improving the quality of cryopreserved Mangalita spermatozoa. Ejaculated Mangalita and PIC (as comparison) boar sperm frozen in an extender supplemented with 200μM ascorbic acid (vitamin C), 400μM Trolox® (a vitamin E hydrosoluble analogue), 200+400μM vit.C+vit.E, 200+200μM vit.C+vit.E, or without supplementation was thawed and then evaluated the sperm motility. Treatment with 200+400μM vit.C + vit.E has the most beneficial effect on the Mangalita sperm motility after frozen-thawing among the concentrations tested (P<0.05).
  Marius ZAHAN , Vasile MICLEA , Andrea HETTIG , Ileana MICLEA , Paul RAICA and Iulian ROMAN
  The aim of this research was to characterize Red Mangalitsa breed population from Romania in order to include into the germoplasm preservation program. For this purpose we have chosen genetic characterization by individual fingerprinting using 14 microsatellite markers and also biochemical characterization of carcass quality by fatty acid level determination. Microsatellite markers helped us to characterize the population and give scientific basis for management practices. The fatty acid determination has indicated a higher level of polyunsaturated fatty acids, especially linoleic acid and a higher ratio P/S (polyunsaturated fatty acids / saturated fatty acids). These results confirm than population analyzed has characteristics of Red Mangalitsa and could be use to long germoplasm preservation.
  Andrea HETTIG , Vasile MICLEA , Marius ZAHAN , Iulian ROMAN and Ileana MICLEA
  Porcine oocytes are particularly difficult to cryopreserv, and there are no reports of live offspring production after thawing via in vitro fertilization and embryo transfer. This may be partially due to the large size of the oocyte which has a low surface to volume ratio, making it more difficult for water and cryoprotectants to move across the cell plasma membrane. (Guang-Bin Zhou, 2009). This study was design to investigate the effect of the exposure time of the immature swine oocytes to the vitrification media. Vitrification procedure was performed stepwise: preequilibration medium (PM) with 15% ethylene glycol (EG) and 0.25M trehalose in basic medium (DPBS, supplemented with FCS 20%), and vitrification medium (VM) containing 45% EG and 0.5 M trehalose in basic medium. A total number of 1138 were vitrified in two experimental designs. In the first experiment (n=586) the cells were kept 4 min. in PM and 40 seconds in the VM. In the second experiment (n=552) the oocytes were kept 4 minute in the PM and 1 min in the VM. The eggs were loaded with the vitrification medium in the super fine open pull straws and plunged into the liquid nitrogen. After thawing the oocytes were stained with florescent dyes FDA (fluorescent diacetate) and PI, two complementary dyes and examined at the microscope’s UV light to test their viability. Each experimental design had been done in 5 repetitions. The results shows very significant differences (p< 0.0001) between the 2 experiments. Oocytes exposed to cryoprotectants in the VM for 40 sec presents a higher viability (average: 46,30%) vs. oocytes exposed to VM for 1 min (average: 7,19%). These differences show the importance of the exposure time to high concentrations of cryoprotectors. It is worldwide accepted that the higher the cryoprotectant concentration is the better the result is, but a high concentration also could lead to toxicity and cell death if the exposure time exceed the critical point. It means that 1min exposure to a 45% EG in BM is critical to swine oocytes. An acceptable protocol in vitrification can be made only with repeated exploratory regarding to both exposure time to cryoprotectants and their concentration.
  Andrea HETTIG , Vasile MICLEA , Marius ZAHAN , Iulian ROMAN , Florin VARO-GHIURU and Alexandru RUSU
  The aim of this study was to establish a protocol with a higher number of viable oocytes after the vitrification procedure. The oocytes were collected by the follicular punction method from swine prepuberal ovaries. A total of 1792 immature oocytes were cryopreserved stepwise in previtrification (PV) and vitrification (VM) media, containing ethylene glycol (EG) permeating agent, combined with trehalose a non-permeating agent. The cells were preserved in three experimental ways, with three different concentration of cryoprotectant in the VM respectively 30, 40 and 45% EG. The exposure time of the cells to the cryoprotectants was the same in each experimental design, 4 minutes in the PV and 40 seconds in the VM. The concentration of the trehalose and the EG in the PM were remained the same. The vitrification procedure was performed by the superfine open pull straw method. For each experiment, there were 5 repetitions. After tawing the cells were stained with fluoresceine diacetate (FDA) and propidium iodide (PI), and there were exposed to the microscope UV light to test their viability. The viable oocytes had a florescent green color because of the FDA staining, which binds the esterase present only in the viable cells. The PI penetrates only dead cells, with destroyed membrane, binding the DNA, and giving them a red color. The results show an increase of percentage of viable cells once the concentration of the cryoprotectant is increased. With a 30% of EG in the VM, the medium percentage of viable cells is 10,26%, with a standard deviation of 1, 06. For 40% EG in the VM, the median is 33, 81%, with a standard deviation of 0,96, and 46,30% for a concentration of 45% EG in the VM with a standard deviation of 0,50. The statistical analysis shows significant differences (p< o, ooo1) between the experiments. Vitrification is the solidification of a solution, glass formation, at low temperatures without ice crystal formation. The phenomenon can be regarded as an extreme increase of viscosity and requires either rapid cooling rates or the use of cryoprotectant solutions, which depress ice crystal formation and increase viscosity at low temperatures. (Vajta G., 2000). These results sustain the theory according to which the higher the cryoprotectant is, the better the result is but limited by the exposure time.
  Ileana MICLEA , Nicolae PACALA , Marius ZAHAN , Andrea HETTIG , Iulian ROMAN and Vasile MICLEA
  As defense against oxidative stress, living systems employ antioxidants that they either produce or take up from the environment. Among these α-tocopherol and ascorbic acid are some of the most important and have been shown to protect cell components against oxidation. The goal of this research was to establish whether supplementation with α-tocopherol or/and ascorbic acid could improve viability and maturation of porcine oocytes. Pig oocytes were cultured for 44-45 hours at 37°C in 5% CO2 atmosphere in M199 containing 20 μM α-tocopherol or/and 750 μM ascorbic acid. Afterwards, cumulus oophorus expansion was assessed and oocytes were denuded. Viability and the presence of the first polar body were assessed by fluorescent staining with 3’, 6’ fluorescein diacetate and Hoechst 33258. Differences between treatments were analyzed by the analysis of variance and interpreted using the LSD test. Supplementation with 750 μM ascorbic acid and the combination of α- tocopherol and ascorbic acid resulted in significantly greater (p<0.05) percentages of COCs that were scored as 3. Overall, cumulus expansion was promoted by the 750 μM ascorbic acid treatment. The percentages of oocytes with a visible first polar body were highest when ascorbic acid and the antioxidant mix were added to the maturation media. However, these supplementations had a negative effect on oocyte viability. This was maximal if 20 μM α-tocopherol was present the medium. The ch indicates that 20 μM α-tocopherol has a positive influence on preserving oocyte viability while 750 μM ascorbic acid and the antioxidant combination promote cumulus oophorus expansion and the formation of the first polar body.
 
 
 
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