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

Antioxidant effect of vitamin C on porcine oocytes maturated in vitro

Cells are vulnerable to oxidative stress during in vitro culture systems. The objective of the present study was to determine the effect of vitamin C addition in in vitro culture media on porcine oocytes maturation rate based on morphological changes. Porcine COC’s were matured according to their morphological class (class I, II and III) in two groups: control (M) and supplemented with vitamin C (0.5 mM, C) in TCM 199 HEPES (M2520) modification media with hormones (0.88UI/ml FSH, F8174) at 38.50C in 5% CO2 humidified air atmosphere for 44h. The rates of oocytes with cumulus cells expansion were higher with addition of vitamin C as compared to control group, with 7.83% (C1), 70.59% (C2) and 6.04% (C3). It could be concluded from this preliminary study that addition of vitamin C in in vitro maturation medium has a beneficial effect on porcine oocytes especially in C2 group

Boar freeze-dried semen evaluation using Spermac staining

Sperm freeze-drying is a new and alternative method to preserve male gametes in refrigeration or at room temperature. In order to protect sperm integrity special protection is required. The aim of our research was to examine the effect of vitamin C and rosmarinic acid on the spermatozoa integrity after freeze-drying the probes. We analyzed the acrosome reaction and the morphological aspects of boar spermatozoa form three different breed (Pietrain, Large White and Landrace) after rehydration. We observed that the highest percent of spermatozoa with intact acrosome and the least spermatozoa anomalies were in samples were rosmarinic acid was added. As preliminary results we could state that adding antioxidants protects spermatozoa from oxidative stress

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

Boar freeze-dried semen in medium with antioxidants evaluated based on DNA integrity after a long-time preservation

Sperm freeze-drying is considered an alternative method to preserve male gametes in refrigeration or at room temperature condition. In order to preserve sperm integrity special protection is required. The aim of our research was to examine the effect of vitamin C (0.5 mM ) and rosmarinic acid (105 μM) on the DNA spermatozoa integrity after freeze-drying and 36 months of preservation at refrigerator temperature. Our results indicates that more than 90% of DNA boar spermatozoa integrity is not affected by long-time preservation with small differences between experimental groups: with +0.59% higher DNA integrity in AR group from Duroc boar, with +2.83% higher DNA integrity in AR group from Landrace boar and with no differences regarding DNA integrity in group supplemented with vitamin C. The main conclusion of these preliminary results is that DNA integrity of boar freeze-dried semen is not affected by longtime preservation and it can be used further for intracytoplasmatic sperm injection 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

In vitro bovine embryos evaluation based on OCT4, SOX2, IGF1R and IGF2R expression level

In vitro production of bovine embryos comprises a lot of factors that can influence the successful of this technique, oxidative stress being one of them. These factors can influence the evolution of important development processes such as the maternal to zygotic transition and the embryonic genome activation. Adding antioxidants to in vitro culture media exerts the key role to reduce the effects of reactive oxidative species produced during assisted reproduction technique, influencing in a positive way also the early embryonic developement. The objective of this study was to determine the effect of antioxidant rosmarinic acid (105 μM), added to in vitro bovine oocytes maturation media, on the quality of embryo produced based on gene expression level of OCT4, SOX2, IGF1R and IGF2R. For this purpose, we used 35 bovine ovaries taken from slougtherhouse from which we obtain 202 cumulus-oocyte-complexes and 127 of them were maturated in vitro based on morphological aspects. The cumulus-oocyte-complexes were divided in two groups: control (M1, M2, M3) and with acid rozmarinic (AR1, AR2 and AR3). The levels of OCT4, SOX2, IGF1R and IGF2R were the highest in group AR1, embryos obtain from oocytes class I supplemented with rozmarinic acid, where OCT4 expression was 4.08, SOX2 was 27.66, IGF1R and IGF2R were 53.44 and 25.10