4 results
Effect of freezing extender composition and male line on semen traits and reproductive performance in rabbits
- M. P. Viudes-de-Castro, R. Lavara, H. M. Safaa, F. Marco-Jiménez, G. M. K. Mehaisen, J. S. Vicente
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This study was conducted to elucidate the effect of different freezing extenders on two lines selected for hyperprolificacy and longevity (H and LP, respectively). In extender A, dimethyl sulphoxide (Me2SO) and sucrose were used as cryoprotectants. In extenders B and C, the sucrose was replaced by 20% egg yolk, and in extender C the Me2SO was substituted by acetamide. Semen was packaged in 0.25 ml plastic straws and cooled at 5°C for 45 min, and then was frozen in liquid nitrogen vapour for 10 min before being plunged into the liquid nitrogen. Thawing was carried out by immersing the straws in a water bath at 50°C for 10 s. Frozen-thawed semen characteristics and reproductive parameters were affected by freezing. Extender C showed significantly lower post-thawing quality traits than any of the three extenders. Acrosome integrity was significantly improved when Me2SO was used as cryoprotectant. Sucrose replacement by 20% egg yolk had no effect on acrosome integrity but provided significantly lower sperm motility and viability. Freezing extender affected fertility rate, total born, number of implantation sites and gestational losses, obtaining better results when extender A was used. The acrosomal integrity after frozen-thawed process showed a significant correlation with fertility at 12th day and also at birth, indicating that an increase in acrosomal integrity leads to an increase in both fertilities (12th day and at birth). A positive correlation between motility of semen and implantation sites was found. The post-thawing quality traits of semen were not affected by the genetic line, although LP line showed higher total born and lower foetal and gestational losses. The findings of this study suggest that freezing extender composition has a significant effect on the success of rabbit sperm for preservation, and when Me2SO was used as permeable cryoprotectant sucrose provided better protection compared with egg yolk and improved reproductive traits, and, on the other hand, the male genotypes used in the present study had no effect on frozen-thawed sperm parameters but negatively affected some of the reproductive parameters.
Porcine oocyte vitrification in optimized low toxicity solution with open pulled straws
- F. Marco-Jiménez, L. Casares-Crespo, J.S. Vicente
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One of the greatest challenges for reproductive cryobiologists today is to develop an efficient cryopreservation method for human and domestic animal oocytes. The objective of the present study was to optimize a low toxicity solution called VM3 to vitrify porcine oocytes using an open pulled straw (OPS) device and to evaluate the effects on viability, chromosomal organization and cortical granules distribution. Two experiments were conducted in this study. Firstly, we determined the minimum concentration of cryoprotectant present in the VM3 solution required (7.6 M) for vitrification using an OPS device. The appearance of opacity was observed when using a cooling solution at –196°C; no observable opacity was noted as vitrification. In addition, the ultrastructure of oocytes in VM3 or VM3 optimized solution was examined using cryo-scanning electron microscopy. The minimum total cryoprotectant concentration present in VM3 solution necessary for apparent vitrification was 5.6 M when combined with use of an OPS device. Use of both vitrification solutions showed a characteristic plasticized surface. In the second experiment, the relative cytotoxicity of vitrification solutions (VM3 and VM3 optimized) was studied. Oocyte viability, chromosomal organization and the cortical granules distribution were assessed by fluorescent stain. After warming, oocyte survival rate was similar to that of fresh oocytes. The vitrification process significantly reduced correct chromosomal organization and cortical granules distribution rates compared with the fresh oocytes group. However, correct chromosomal organization and cortical granules distribution rates did not differ among oocytes placed in different vitrification solutions. In conclusion, our data demonstrated that the VM3 solution can be optimized and that reduction in concentration to 5.6 M enabled vitrification of oocytes with an OPS device, however use of the VM3 optimised solution had no beneficial effect on vitrification of porcine oocytes.
Effect of lanosterol on the in vitro maturation in semi-defined culture system of prepubertal ewe oocytes
- F. Marco-Jiménez, J.S. Vicente, M.P. Viudes-de-Castro
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The choice of medium and supplements can affect meiotic regulation and may have an impact on the regulation of mammalian oocyte growth and embryonic cell function. The aim of the present study was to assess the effects of oxygen concentration and endogenous lanosterol on the in vitro maturation (IVM) media without serum and based on recombinant human chorionic gonadotrophin in prepubertal ewe oocytes. Firstly, the effect of varying oxygen concentrations (5% and 20%) during IVM in TCM-199 supplemented (4 mg/ml bovine serum albumin (BSA), 100 μM cysteamine, 0.3 mM sodium pyruvate, 0.1 UI/ml recombinant follicle-stimulating hormone (r-FSH; Gonal-F® 75 UI, Serono, Italy), 0.1 UI/ml recombinant leuteinizing hormone (r-LH; Lhadi® 75 UI, Serono, Italy) and 1 μg/ml estradiol-17β) on subsequent nuclear maturation of oocytes examined under ultraviolet light following staining with bisbenzimide (Hoechst 33342) was investigated. Secondly, two concentrations of lanosterol (0, 10 and 50 μM) were added to the IVM medium. Nuclear maturation of oocytes was examined as previously. Lipid content in oocytes, an important indicator of cytoplasmic maturity, was also measured using Nile red fluorescent stain. The results showed that low oxygen concentration affected the nuclear maturation. Similarly, a significantly higher rate of meiosis resumption was observed with 10 μM (72.3%) of lanosterol compared with the control (51.8%) or 50 μM of lanosterol (59.4%). A significantly higher content of lipids was also observed with 10 and 50 μM of lanosterol (7.3 ± 0.2 × 106 and 7.4 ± 0.2 × 106 arbitrary units of fluorescence) compared with the control (6.7 ± 0.2 × 106 arbitrary units of fluorescence). The results indicate that 10 μM lanosterol during IVM in medium without serum and based on recombinant human chorionic gonadotrophin has a positive effect on maturation of prepubertal ewe oocytes.
Effect of different culture systems on mRNA expression in developing rabbit embryos
- M.D. Saenz-de-Juano, C. Naturil-Alfonso, J.S. Vicente, F. Marco-Jiménez
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The rate of zygotes in vitro developed to hatched blastocyst stage was evaluated between two different commercial media (TCM-199 and Ham's F10) and two different culture systems (renewal and non-renewal single medium) to determine the effects of culture conditions on rabbit embryo preimplantation development. The relative transcript abundances of OCT4, vascular endothelial growth factor (VEGF) and epidermal growth factor receptor 3 (erbB3) of resultant blastocysts were also analysed and compared with in vivo developed blastocysts. Results showed an important divergence in mRNA expression between embryos developed under in vivo and in vitro conditions despite there being no significant difference in hatching blastocyst rates between different culture systems and different media. For OCT4, transcript abundance of in vitro culture embryos differs from their in vivo chronological counterparts, but, when the medium is renewed, mRNA expression seemed similar to in vivo developed 4-day-old embryos. In addition, VEGF and erbB3 expression showed marked variation between different in vitro conditions. Therefore, the study of specific transcript abundance in rabbit blastocyst provides a more detailed description of which alterations in gene expression occur due in vitro conditions, and further studies should be carried out to reduce current limitations of long-term culture of rabbit pre-implantation embryos.