Vaginal smear, progesterone levels, and ultrasound examination of the ovaries as methods of determining the moment of ovulation in bitches comparative study

Researches in the scientific literature reveal that the study of vaginal citology and interpretation of progesterone values do not represent certain methods to determine the ovulation time. 6 different breed have been investigated in this study (Labrador Retriever, Tibetan Mastiff, Bichon Maltese, West Highlander White Terrier and the Bucovina Shepherd). All es were monitored in terms of cyto-vaginal smear, the P4 level and ultrasound examination to determine the ovulation moment. The rapid disappearance of the follicular antrum cavity, correspondent to ovulation, was detected only with two. Although ultrasound changes during the estrous cycle were well-studied, the exact ovulation moment cannot be predicted accurately. To optimize the results of determining the ovulation moment it is recommended to collate the ultrasound examination with at least one of the other two methods of investigation

Evaluation of sows oocytes viability through Trypan Blue staining after vitrification

Along sperm and embryo cryopreservation that are used routinely also in animal assisted reproduction, studying are done also on animal oocyte cryopreservation in order to find the best conditions to preserve their viability. Vitrification is one of the methods that can be used in order to preserve oocytes. The higher reactive oxygen species that are formed during in vitro conditions can influence the success of assisted reproduction technique. The aim of this study was to evaluate the antioxidant potential of ascorbic acid (0.5mM) and rosmarinic acid (105μM) added in media for in vitro maturation on swine oocyte subjected to vitrification. COC’s viability after vitrification was evaluated by 0.02% Trypan Blue staining. Comparing experimental groups C (vitamin C) and AR (rosmarinic acid) with group M (control), relative to the number of vitrified oocytes, a slight increase in their viability is observed, with 16.67% (C, class I) and 33.33% (AR, class II), respectively. Regardless of the treatment applied, the oocyte class is associated with viability (p = 0.048). Due to low number of oocytes used in each group we can concluded that supplementation of oocyte maturation media before vitrification with rosmarinic acid and ascorbic acid could produce a slight increase in viability

Effect of rozmarinic acid supplementation on in vitro maturation of bovine oocytes

Antioxidants supplementation of in vitro culture media exerts the key role to reduce the effects of reactive oxidative species produced during assisted reproduction technique. The objective of the study was to determine the effect of rosmarinic acid addition to the in vitro culture media on bovine oocytes maturation rate based on morphological changes. Bovine COC’s were matured according to their morphological class (class I, II and III) in two groups: control (M) and supplemented with rosmarinic acid (105 μM, AR) in TCM 199 HEPES modification media at 38.50C in 5% CO2 humidified air atmosphere for 24h. Comparing the groups, relative to the number of COC’s matured, a increase in their maturation features is observed, with 26.81% % (AR1), 21.67% (AR2) and 23.34% (AR3), respectively in groups supplemented with rosmarinic acid. The oocyte class is associated with their capacity to develop in vitro based on their morphological examination

Protocol developing for identification of vegetal matrices used in ammodytes ammodytes freeze-dried venom adulteration

Presence of corn flour adulteration was detected by extracting the DNA from 25mg of freeze dried venom and using it as template in PCR amplification with zein specific primers known to be highly specific for corn species. The obtained amplicon was purified from agarose gel and sequenced in order to further confirm the presence of corn specific DNA sequences. The sequence thus obtained was uploaded in a DNA Data Base, and aligned with the reference zein sequence. The 99% of similarity between the two sequences enables us to confirm the corn flour adulteration in the analyzed venom sample

Comparative boar and bull semen evaluation after percoll treatment

There are numerous methods used in IVF both in bovine and pig for sperm separation and Percoll gradient is one of them. After using Percoll gradient spermatic parameters were as following: motility increased to 78.33% in boar samples and to 75% in bull samples; the number of live spermatozoa increased with 42.91%, 31.46% and 21.88% in boar sample, respectively with 19.37%, 9.34% and 5.85% in bull samples; normal spermatozoa increase to 98.52-98.92% in boar and to 84.22-93.97% in bull samples. Acrosome integrity, another parameter studied, indicate that in bull sample intact acrosome was before 83.97%, 87.58% and 88.1%, respectively 91.39%, 93.11% and 92.56% after using Percoll gradient, similar increase was in boar sample to 95.49%-98.42%. Therefore, Percoll is an easier and fast way to select viable and normal spermatozoa for IVF technique

BCL2 and bax gene expression in cumulus-oocytes complexes in cow

In vitro maturation (IVM) is the first and the most important step for IVF technique because the success of in vitro embryos production highly depends on oocyte quality. In the present study it was aimed to identify the optimal time of maturation of cattle oocytes, in which IVF techniques are very important and frequently applied. One of the indicators of IVF failure is the apoptosis, occurring in the participating cells on the whole cycle of development. Therefore is essential to identify highly accurate the apoptotic moment. In this respect, the expression of two genes: Bax and Bcl2 was assessed in three stages of maturation of oocytes: time 0, 24h and 48h of cultivation on maturation culture media. These genes have been previously shown to play an essential role in apoptotic process of the cells. The genes expression was evaluated by Quantitative Revers-Transcription PCR, using SYBR Green reagents, having as target the total ARN isolated from the complex oocytes - cumulus cells. The value of genes expression was normalized using as control the Actin gene biomarker. Obtained data were interpreted using 2ΔΔC(T) method and the statistical analysis was performed using ANOVA algorithms. In the determinations, it was found that the expression of the two : Bax and Bcl2 genes are somewhat antagonist, so that at 24 hours of maturation time are both over-expressed comparing to the 0 moment. By comparison, Bcl2 expression is low, which indicates that the apoptotic process has not started and that the stress of in vitro cultivation has not reached the maximum level. Bax gene expression indicates the existence of high stress factors, but not the entry of a cell into apoptosis process. Therefore, it can be considered that the optimum time interval for maturation in culture media is 24h. At 48h the Bax gene overexpression compared to the Bcl2 gene expression indicates the entry of cells into apoptotic process, knowing that a very high level of the Bax gene may have an inhibitory effect on Bcl2 gene experssion

Vitrification of germinal vesicle and metaphase II swine oocytes

In vitro maturation of oocytes is a technique that implies extended cultivation of immature oocytes, recovered by aspiring the cumulus-oocytes complexes from follicles, their maturation under adequate conditions for 48 hours, until they reach the metaphase II stage, so they can be in vitro fertilized later on. Oocytes, once recovered from the follicles, remain in the germinal vesicle stage, representing the diplotene of the first meiotic division at 0 hours of maturation. After maturation, swine oocytes evolve, passing through several stages, from germinal vesicle breakdown, to the first meiotic division and then achieving the metaphase II stage. Vitrification is one of the most interesting cryopreservation techniques, more and more used in the past few years, due to the advantages this technique has, compared to freezing. Vitrification prevents formation of ice crystals during the cooling process; the liquid gets a glassy solid structure, that stops molecular mobility, without any structural reorganization of the liquid that contains the oocytes. Freezing is a slow process, that can take hours until completed and it is very difficult to establish a balance between the agents that produce osmotic injury, due to ice crystals formation and due to osmotic stress. Assisted reproduction techniques rely also on using gametes stored at very low temperatures, in order to enable unlimited access to them. Whereas vitrification can be performed in different stages of gamete and zygote development, in this research we wanted to see the effectiveness of applying this process at the beginning or during gamete processing. Cultivation of oocytes to maturation and their potential use in assisted reproduction techniques, enabled the oocyte development to metaphase II for 52,63% of those belonging to Group 1 ( vitrification / cultivation) and for 78,46% of the oocytes belonging to Group 2 (maturation / vitrification / cultivation). Obviously, cultivation of oocytes before vitrification brings more advantages for the meiotic resumption. Regardless the time of vitrification induction, after warming, the oocytes resumed their meiotic process. By using both protocols, we gathered satisfying rates (over 50%) of oocytes ready to be used in assisted reproduction techniques

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