Generating bovine embryos through ICSI

Through ICSI, competition between sperms and also sperm-oocyte interaction are avoided thus ICSI proving reliable when sperm is not suitable for IVF. In bovine, the limiting step is represented by low rate of sperm head decondensation subsequent ICSI. Intracytoplasmatic sperm injection allows avoiding many critical moments that may occur during normal or in vitro fertilization. Oocytes were obtained from ovaries from slaughtered cows. These were transported in 0.9% NaCl solution in isothermal bags at a temperature of 25-30 ° C. The ovaries were brought from the slaughterhouse within 2 hours. Harvesting of the oocytes was made through the aspiration method. After maturation, oocytes were fertilized using sperm that was prepared using Percoll method and then treated with TritonX. The volume of the TritonX solution that accompanies the sperma and which remains in the oocyte is extremely important given that by its action, TritonX removes the acrosome, thus releasing a rich enzyme content and facilitating the dehydration of the male pronucleus. Even though the number of 2 nucleus, 2 cells or 4 cells oocytes is inferior to the data found in the literature, compared to the results achieved last year in the assisted reproduction laboratory within CLC-HC Timisoara, it marks significant progress. At the 2 cells stage, there were several oocytes from group 1 (24.39% vs. 12.5%), while at the 4 cells stage there were 14.63% oocytes from group 1 and 25% group 2. The use of TritonX solution for sperm treatment as well as shortening the duration of ICSI execution allowed us to get encouraging results. The results obtained are inferior to those presented in the literature but are far superior to those we obtained last year when the ICSI technique was assembled. Achieving the two- and four-cell embryonic stages justifies us thinking that we are mastering the ICSI technique

Bovine and swine parthenots generating through electrical stimultion of the oocytes

Electrical stimulation is an alternative to chemical activation to induce 2+ influx, responsible for the formation of pores in the cellular membrane. In order to activate the oocytes, electrical stimulation (E.S.) was performed on 30 oocytes derived from gilts (L1), sows (L2), heifers (L3) and cows (L4). We considered that the stage of development of four cells is eloquent for certifying the ES's division triggering and the results we are considering only refer to these parthenots. Following application of ES, oocyte activation occurred as follows: 6.6% at L1, 16.6% at L2, 20% at L3 and 46.6% at L4. It is obvious the higher maturation rate of oocytes from adult females as compared to young females (16.6% in sows versus 6.6% in gilts and 46.6% in cows versus 20% in heifers). The method of electrical stimulation of oocytes in the fusion chamber used in this paper is effective for activating the division in both bovine and swine oocytes. Activation of oocyte division following electrical stimulation is clearly superior when using oocytes from adult females. The electrical stimulation method used generated the upper division activation in cattle compared with the results obtained using swine oocytes

Pronuclei formation subsequent to intracytoplasmic sperm injection in bovine

ICSI represents the top of the range of the assembly protocols of assisted reproduction which presumes the existence of sophisticated equipments, prior acquired routine in preparing the oocytes and sperm and a very good organization and synchronization of the working time. The aim of the present paper is represented by the assembly ICSI technique in the assisted reproduction laboratory within CLC-HC Timisoara . 70% of the oocytes were considered able for ICSI and out of this number 12 (17%) were destroyed meanwhile micromanipulation; the remaining were fertilized using sperm treated with PVP (G1), TritonX (G2) or untreated sperm (control). Taking into account the outcome results the superiority of the method that prepares the sperm with Triton X, even though the fertilization percentage (35%) is clearly inferior toward the ones reported (60-80% by Sekhavati et al., 2012). Using PVP to prepare the sperm has generated a lower percentage of success (30%) besides a overwhelming proportion of oocytes with one pronucleus (1PN-80%) or unfertilized (NE-20%). Previous reports emphasize the fact that the volume of PVP which gets in the oocyte cytoplasm consecutively injecting the sperm can have a harmful effect on the zygote. The efficiency of Triton X to remove the acrosome, in this way dislodging a consistent enzymatic volume and allowing to decondense the male pronucleus it is demonstrated by the 7 fertilized oocytes (35%) by injecting the sperm treated previous with this solution. Untreated sperm it has generated the oocytes fertilization only in proportion of 11% whereas the male pronucleus does not cover in an useful time and requested proportion the transformations needed to support the fecundation. The culture media used for gamete and zygote preparation had generated lower results toward the reports in the literature. The modest outcomes can be explained both through the culture media composition and through the long execution of ICSI. The presence of both pronuclei in some of the oocytes subdued to ICSI (between 30 and 35% depending on the substance used to treat the sperm), proves our capacity to assemble ICSI technique in the assisted reproduction laboratory within CLC-HC

Contrast-Enhanced Ultrasonography with Arrival Time Parametric Imaging as a Non-Invasive Diagnostic Tool for Liver Cirrhosis

Liver biopsy is the gold standard method for staging liver fibrosis, but it is an invasive procedure that is associated with some complications. There are also non-invasive techniques for assessing liver fibrosis, such as elastography and biological tests, but these techniques can fail in detection or generate false measurements depending on the subject’s condition. This study aimed to determine whether liver fibrosis can be evaluated using contrast-enhanced ultrasonography with arrival time parametric imaging using the ultrasound machine’s parametric image software, the method being called (CEUS-PAT). CEUS-PAT was performed on each subject using SonoVue as a contrast agent, and images showing liver parenchyma and the right kidney on a single screen were used for analysis in parametric imaging, which was performed using the proprietary software of the ultrasound system. The ratio between the kidney and liver arrival times was calculated. The study included 64 predominantly male (56.3%) subjects, 37 cirrhotic patients, and 27 healthy volunteers, with a mean age of 58.98 ± 8.90 years. Significant differences were found between the liver cirrhosis and healthy groups regarding CEUS-PAT, 0.83 ± 0.09 vs. 0.49 ± 0.11, p 0.7, with an AUC of 0.98, p < 0.001, Se = 89.19%, Sp = 100%, PPV = 100%, and NPV = 87.1%. We demonstrate that CEUS-PAT achieves excellent performance in diagnosing liver cirrhosis and is a fast method for diagnosing liver cirrhosis that can even be applied in situations where the use of other methods is excluded