Effects of cryopreservation on the meiotic spindle, cortical granule distribution and development of rabbit oocytes

Cryopreservation of embryos and oocytes in animal species is considered an important tool in reproduction biotechnology. The aim of this study was to evaluate the effect of vitrification and slow-freezing on the meiotic spindle, cortical granule distribution and developmental competence in rabbit oocytes.Jiménez Trigos, ME. (2011). Effects of cryopreservation on the meiotic spindle, cortical granule distribution and development of rabbit oocytes. http://hdl.handle.net/10251/15556Archivo delegad

Generation of offspring from cryopreserved rabbit (Oryctolagus cuniculus) oocytes

The general aim of this thesis was to optimise the current methodologies of oocyte cryopreservation in order to obtain live offspring from cryopreserved rabbit oocytes. In chapter 1, meiotic spindle configuration, cortical granules (CGs) distribution and oocyte developmental competence were evaluated after cryopreservation with the current slow-freezing and vitrification procedures. The meiotic spindle organisation was dramatically impaired regardless of the method used. Nevertheless, altered CG distribution is more evident in vitrified oocytes than in slow-frozen ones and the developmental rate to blastocyst stage after parthenogenetic activation was only obtained using slow-freezing method. From this chapter it may be concluded that both methodologies equally affect oocyte structure. However, slow-freezing method seems to be the recommended option for this species as a consequence of the sensitivity to high levels of cryoprotectants in this species. The aim of the following two chapters was the optimisation of cryopreservation procedures using different strategies to modify the oocytes in order to make them more cryoresistant. In chapter 2, Taxol and Cytochalasin B were employed to stabilise the cytoskeleton system during vitrification. The effect of these two molecules on the meiotic spindle and chromosome configuration and development to blastocyst stage after parthenogenesis activation were also evaluated. There were no significant differences in the structural configuration between vitrified groups. Regarding cleavage and blastocyst developmental rate, no statistical differences were found between vitrified-non-treated and Taxol-treated oocytes, but no oocytes treated with Cytochalasin B reached this stage. Therefore, structural configuration and blastocyst development were not improved by this pre-treatment. Moreover, Cytochalasin B pre-treatment seems to cause a deleterious effect on developmental ability to blastocyst stage of these oocytes. In chapter 3, oocytes were incubated with cholesterol-loaded methyl-ß- cyclodextrin (CLC) to increase the membrane fluidity and stability and improve their developmental ability after parthenogenetic activation or intracytoplasmic sperm injection (ICSI). Cholesterol incorporation and its presence after cryopreservation were evaluated using confocal microscopy. Results showed that cholesterol was incorporated into the oocyte and remained, albeit in a lesser amount after cryopreservation procedures. However, no improvements on developmental competence were obtained after parthenogenetic activation or intracytoplasmic sperm injection. In the last three chapters of this thesis, the main objective was to develop a reliable technique which would allow us to obtain live offspring from cryopreserved oocytes. For that purpose, in vivo fertilisation using intraoviductal oocyte transfer assisted by laparoscopy was considered a good alternative to bypass the inadequacy of conventional in vitro fertilisation in rabbit. In chapter 4, two recipient models (ovariectomised or oviduct ligated immediately after transfer) were used to compare the ability of fresh oocytes to fertilise in vivo. This first work showed that embryo recovery rates in all transferred groups decreased significantly, but ligated oviduct recipients provided significantly higher results compared to ovariectomised ones. For that reason, in the second experiment the ligated oviduct recipient model was used to generate live births. Results obtained in this chapter suggested that it was possible to obtain offspring from cryopreserved oocytes using this technique, but this kind of animal models compromised the use of the reproductive tract in a high percentage of females. For that reason, chapter 5 was focused on the development of another type of animal model as an alternative. First, the ability of cyanoacrylate tissue adhesive to block the oviducts before the ovulation would take place was evaluated. Then, in vivo fertilisation ability of fresh transferred oocytes after blocking the oviduct with the adhesive was also assessed. Finally, slow frozen oocytes were transferred to generate live birth. Results showed that cyanoacrylate tissue adhesive was effective in blocking the oviduct, as no embryos were recovered in the blocked oviduct six days after artificial insemination (AI). Moreover, this method could fertilise fresh and also slowfrozen oocytes with a higher live birth rate than the previous recipient models. This study showed that successful production of live offspring using slow-frozen oocytes in combination with in vivo fertilisation was possible, which suggested that in vivo environment could help improve the results of oocyte cryopreservation. Thus, this method was employed in the last chapter of this thesis to generate live offspring from vitrified rabbit oocytes for the first time. Results obtained revealed that there were no differences in the rate of birth between vitrified and slow-frozen transferred oocytes. Nevertheless, based on the results with fresh oocytes, further experiments are still needed if the efficiency of cryopreservation procedures are to be improved.Jiménez Trigos, ME. (2014). Generation of offspring from cryopreserved rabbit (Oryctolagus cuniculus) oocytes [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/37977TESI

Effect of in vitro and in vivo conditions on development of parthenogenetic rabbit embryos after vitrification

[EN] Parthenote embryos offer multiple opportunities in biotechnological research, so it is important to analyse the possibilities for their cryopreservation in order to establish a biobank. The aim of this experiment was to determine the effect of culture conditions and vitrification on rabbit parthenogenetic embryos. Parthenotes were cultured under in vivo and in vitro conditions until day 3 (late morula/early blastocyst), when they were vitrified. Immediately after warming, they were newly cultured under in vivo and in vitro conditions till day 6 (blastocyst stage). Both culture conditions showed similar late morula/early blastocyst (0.39±0.056 vs. 0.46±0.043, for in vivo and in vitro, respectively) and blastocyst rates (0.12±0.068 vs. 0.13±0.070, for in vivo and in vitro, respectively). However, no parthenote was recovered when a combination of culture conditions was performed. To our best knowledge, this is the first demonstration of the ability of rabbit parthenogenetic embryos to develop after vitrification, with similar embryo development after in vivo or in vitro culture. Nevertheless, our results highlight the importance of culture conditions on the morphology of parthenote embryos. Therefore, we have described that special attention should be paid on culture conditions to generate parthenote embryos, with a view to their subsequent use, for example in embryonic stem cell production.Statement of funding: This work was supported by funds from the Generalitat Valenciana Research Programme (Prometeoll 2014/036).Naturil Alfonso, C.; Jiménez Trigos, ME.; Vicente Antón, JS.; Marco Jiménez, F. (2015). Effect of in vitro and in vivo conditions on development of parthenogenetic rabbit embryos after vitrification. Cryobiology. 71(1):91-96. doi:10.1016/j.cryobiol.2015.05.003S919671

Live birth from slow-frozen rabbit oocytes after in vivo fertilisation

In vivo fertilisation techniques such as intraoviductal oocyte transfer have been considered as alternatives to bypass the inadequacy of conventional in vitro fertilisation in rabbit. There is only one study in the literature, published in 1989, that reports live offspring from cryopreserved rabbit oocytes. The aim of the present study was to establish the in vivo fertilisation procedure to generate live offspring with frozen oocytes. First, the effect of two recipient models (i) ovariectomised or (ii) oviduct ligated immediately after transfer on the ability of fresh oocytes to fertilise were compared. Second, generation of live offspring from slow-frozen oocytes was carried out using the ligated oviduct recipient model. Throughout the experiment, recipients were artificially inseminated 9 hours prior to oocyte transfer. In the first experiment, two days after unilateral transfer of fresh oocytes, oviducts and uterine horns were flushed to assess embryo recovery rates. The embryo recovery rates were low compared to control in both ovariectomised and ligated oviduct groups. However, ligated oviduct recipient showed significantly (P75: : 532,538.Nottola, S., Coticchio, G., De Santis, L., Macchiarelli, G., Maione, M., Bianchi, S., … Borini, A. (2008). Ultrastructure of human mature oocytes after slow cooling cryopreservation with ethylene glycol. Reproductive BioMedicine Online, 17(3), 368-377. doi:10.1016/s1472-6483(10)60220-9Pereira, R. M., & Marques, C. C. (2008). Animal oocyte and embryo cryopreservation. Cell and Tissue Banking, 9(4), 267-277. doi:10.1007/s10561-008-9075-2Noyes, N., Boldt, J., & Nagy, Z. P. (2010). Oocyte cryopreservation: is it time to remove its experimental label? Journal of Assisted Reproduction and Genetics, 27(2-3), 69-74. doi:10.1007/s10815-009-9382-yDIEDRICH, K., AL-HASANI, S., VAN DER VEN, H., & KREBS, D. (1988). Successful in Vitro Fertilization of Frozen?Thawed Rabbit and Human Oocytes. Annals of the New York Academy of Sciences, 541(1 In Vitro Fert), 562-570. doi:10.1111/j.1749-6632.1988.tb22292.xVincent, C., Garnier, V., Heyman, Y., & Renard, J. P. (1989). Solvent effects on cytoskeletal organization and in-vivo survival after freezing of rabbit oocytes. Reproduction, 87(2), 809-820. doi:10.1530/jrf.0.0870809Siebzehnruebl, E. R., Todorow, S., van Uem, J., Koch, R., Wildt, L., & Lang, N. (1989). Cryopreservation of human and rabbit oocytes and one-cell embryos: a comparison of DMSO and propanediol. Human Reproduction, 4(3), 312-317. doi:10.1093/oxfordjournals.humrep.a136895Cai, X. Y., Chen, G. A., Lian, Y., Zheng, X. Y., & Peng, H. M. (2005). Cryoloop vitrification of rabbit oocytes. Human Reproduction, 20(7), 1969-1974. doi:10.1093/humrep/deh805Salvetti, P., Buff, S., Afanassieff, M., Daniel, N., Guérin, P., & Joly, T. (2010). Structural, metabolic and developmental evaluation of ovulated rabbit oocytes before and after cryopreservation by vitrification and slow freezing. Theriogenology, 74(5), 847-855. doi:10.1016/j.theriogenology.2010.04.009Wang, J., Cong, L., Zhang, Z. G., Cao, Y. X., Wei, Z. L., Zhou, P., … He, X. J. (2009). Double activation improves rabbit freeze–thawed oocytes developmental potential. Zygote, 18(1), 27-32. doi:10.1017/s0967199409990098Jiménez-Trigos, E., Naturil-Alfonso, C., Vicente, J., & Marco-Jiménez, F. (2011). Effects of Cryopreservation on the Meiotic Spindle, Cortical Granule Distribution and Development of Rabbit Oocytes. Reproduction in Domestic Animals, 47(3), 472-478. doi:10.1111/j.1439-0531.2011.01906.xJiménez-Trigos, E., Naturil-Alfonso, C., Vicente, J., & Marco-Jiménez, F. (2012). Post-Warming Competence ofIn VivoMatured Rabbit Oocytes Treated with Cytoskeletal Stabilization (Taxol) and Cytoskeletal Relaxant (Cytochalasin B) Before Vitrification. Reproduction in Domestic Animals, 48(1), 15-19. doi:10.1111/j.1439-0531.2012.02018.xJiménez-Trigos, E., Vicente, J. S., Mocé, E., Naturil-Alfonso, C., Fernandez-Gonzalez, R., Gutierrez-Adan, A., & Marco-Jiménez, F. (2013). Treatment with cholesterol-loaded methyl-β-cyclodextrin increased the cholesterol in rabbit oocytes, but did not improve developmental competence of cryopreserved oocytes. Cryobiology, 67(1), 106-108. doi:10.1016/j.cryobiol.2013.05.006III. Preliminary note on the transplantation and growth of mammalian ova within a uterine foster-mother. (1891). Proceedings of the Royal Society of London, 48(292-295), 457-458. doi:10.1098/rspl.1890.0053CHANG, M. C., CASAS, J. H., & HUNT, D. M. (1970). Prevention of Pregnancy in the Rabbit by Subcutaneous Implantation of Silastic Tube containing Oestrogen. Nature, 226(5252), 1262-1263. doi:10.1038/2261262a0BEDFORD, J. M., & CHANG, M. C. (1962). Fertilization of Rabbit Ova in vitro. Nature, 193(4818), 898-899. doi:10.1038/193898a0Brackett, B. G., & Williams, W. L. (1968). Fertilization of Rabbit Ova in a Defined Medium. Fertility and Sterility, 19(1), 144-156. doi:10.1016/s0015-0282(16)36555-4Keefer, C. L. (1989). Fertilization by sperm injection in the rabbit. Gamete Research, 22(1), 59-69. doi:10.1002/mrd.1120220107Deng, M., & Yang, X. (Jerry). (2001). Full term development of rabbit oocytes fertilized by Intracytoplasmic sperm injection. Molecular Reproduction and Development, 59(1), 38-43. doi:10.1002/mrd.1005Li, G.-P., Chen, D.-Y., Lian, L., Sun, Q.-Y., Wang, M.-K., Liu, J.-L., … Han, Z.-M. (2000). Viable rabbits derived from reconstructed Oocytes by germinal vesicle transfer after intracytoplasmic sperm injection (ICSI). Molecular Reproduction and Development, 58(2), 180-185. doi:10.1002/1098-2795(200102)58:23.0.co;2-7Zheng, Y.-L., Jiang, M.-X., Zhang, Y.-L., Sun, Q.-Y., & Chen, D.-Y. (2004). Effects of oocyte age, cumulus cells and injection methods on in vitro development of intracytoplasmic sperm injection rabbit embryos. Zygote, 12(1), 75-80. doi:10.1017/s0967199404002643Garcı́a-Ximénez, F., & Escribá, M. J. (2002). Viable offspring derived from cryopreserved haploid rabbit parthenotes. Theriogenology, 57(4), 1319-1325. doi:10.1016/s0093-691x(01)00718-xNaturil-Alfonso, C., Saenz-de-Juano, M. D., Peñaranda, D. S., Vicente, J. S., & Marco-Jiménez, F. (2011). Parthenogenic blastocysts cultured under in vivo conditions exhibit proliferation and differentiation expression genes similar to those of normal embryos. Animal Reproduction Science, 127(3-4), 222-228. doi:10.1016/j.anireprosci.2011.08.005Curry, M. R., Kleinhans, F. W., & Watson, P. F. (2000). Measurement of the Water Permeability of the Membranes of Boar, Ram, and Rabbit Spermatozoa Using Concentration-Dependent Self-Quenching of an Entrapped Fluorophore. Cryobiology, 41(2), 167-173. doi:10.1006/cryo.2000.2277Carnevale, E. M., Coutinho da Silva, M. A., Panzani, D., Stokes, J. E., & Squires, E. L. (2005). Factors affecting the success of oocyte transfer in a clinical program for subfertile mares. Theriogenology, 64(3), 519-527. doi:10.1016/j.theriogenology.2005.05.008Deleuze, S., Goudet, G., Caillaud, M., Lahuec, C., & Duchamp, G. (2009). Efficiency of embryonic development after intrafollicular and intraoviductal transfer of in vitro and in vivo matured horse oocytes. Theriogenology, 72(2), 203-209. doi:10.1016/j.theriogenology.2009.02.001Vicente, J. S., Lavara, R., Marco-Jiménez, F., & Viudes-de-Castro, M. P. (2011). Detrimental effect on availability of buserelin acetate administered in seminal doses in rabbits. Theriogenology, 76(6), 1120-1125. doi:10.1016/j.theriogenology.2011.05.020Viudes-de-Castro, M. P., & Vicente, J. S. (1997). Effect of sperm count on the fertility and prolificity rates of meat rabbits. Animal Reproduction Science, 46(3-4), 313-319. doi:10.1016/s0378-4320(96)01628-4Besenfelder, U., & Brem, G. (1993). Laparoscopic embryo transfer in rabbits. Reproduction, 99(1), 53-56. doi:10.1530/jrf.0.0990053Mehaisen GM, Vicente JS, Lavara R (2004) In vivo Embryo Recovery Rate by Laparoscopic Technique from Rabbit Does Selected for Growth Rate. Reprod Dom Anim 39: 347–351.Bavister, B. D. (1995). Culture of preimplantation embryos: facts and artifacts. Human Reproduction Update, 1(2), 91-148. doi:10.1093/humupd/1.2.91Rizos, D., Lonergan, P., Boland, M. P., Arroyo-García, R., Pintado, B., Fuente, J. de la, & Gutiérrez-Adán, A. (2002). Analysis of Differential Messenger RNA Expression Between Bovine Blastocysts Produced in Different Culture Systems: Implications for Blastocyst Quality1. Biology of Reproduction, 66(3), 589-595. doi:10.1095/biolreprod66.3.589Fleming, T. P., Kwong, W. Y., Porter, R., Ursell, E., Fesenko, I., Wilkins, A., … Eckert, J. J. (2004). The Embryo and Its Future1. 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IN VITRO CAPACITATION OF HAMSTER SPERMATOZOA BY FOLLICULAR FLUID. Reproduction, 18(2), 275-286. doi:10.1530/jrf.0.0180275Sultan, K. M., & Bedford, J. M. (1996). Two modifiers of sperm transport within the Fallopian tube of the rat. Reproduction, 108(2), 179-184. doi:10.1530/jrf.0.1080179Fatehi, A. ., Zeinstra, E. ., Kooij, R. ., Colenbrander, B., & Bevers, M. . (2002). Effect of cumulus cell removal of in vitro matured bovine oocytes prior to in vitro fertilization on subsequent cleavage rate. Theriogenology, 57(4), 1347-1355. doi:10.1016/s0093-691x(01)00717-8Mullen SF (2007) Advances in Fundamental Cryobiology of Mammalian Oocytes. University of Missouri, Columbia.Ledda S, Bogliolo L, Succu S, Ariu F, Bebbere D, et al.. (2007) Oocyte cryopreservation: oocyte assessment and strategies for improving survival. Reprod Fertil Dev 19: : 13–23. ReviewFadini, R., Brambillasca, F., Renzini, M. M., Merola, M., Comi, R., De Ponti, E., & Dal Canto, M. (2009). 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Campylobacter epidemiology from breeders to their progeny in Eastern Spain

[EN] While horizontal transmission is a route clearly linked to the spread of Campylobacter at the farm level, few studies support the transmission of Campylobacter spp. from breeder flocks to their offspring. Thus, the present study was carried out to investigate the possibility of vertical transmission. Breeders were monitored from the time of housing day-old chicks, then throughout the laying period (0 to 60 wk) and throughout their progeny (broiler fattening, 1 to 42 d) until slaughter. All samples were analyzed according with official method ISO 10272:2006. Results revealed that on breeder farms, Campylobacter isolation started from wk 16 and reached its peak at wk 26, with 57.0% and 93.2% of positive birds, respectively. After this point, the rate of positive birds decreased slightly to 86.0% at 60 wk. However, in broiler production all day-old chicks were found negative for Campylobacter spp, and the bacteria was first isolated at d 14 of age (5.0%), with a significant increase in detection during the fattening period with 62% of Campylobacter positive animals at the end of the production cycle. Moreover, non-positive sample was determined from environmental sources. These results could be explained because Campylobacter may be in a low concentration or in a non-culturable form, as there were several studies that successfully detected Campylobacter DNA, but failed to culture. This form can survive in the environment and infect successive flocks; consequently, further studies are needed to develop more modern, practical, cost-effective and suitable techniques for routine diagnosis.We thank the ASAV (Valencian Poultry Association) and the Santander bank for the financial support of the project and the staff of the CECAV (Center for Poultry and Animal Feed Quality of the Valencian Region) for their assistance. Sofia Ingresa was supported by a research grant from the Spanish Ministry of Education (programme FPU13/03306). English text version was revised by N. Macowan English Language Service.Ingresa-Capaccioni, S.; Jiménez Trigos, ME.; Marco Jiménez, F.; Català, P.; Vega García, S.; Marin Orenga, C. (2016). Campylobacter epidemiology from breeders to their progeny in Eastern Spain. Poultry Science. 95(3):676-683. https://doi.org/10.3382/ps/pev338S67668395

State of actin cytoskeleton and development of slow-frozen and vitrified rabbit pronuclear zygotes

[EN] This study was focused on the effect of cryopreservation on the state of actin cytoskeleton and development of rabbit pronuclear zygotes. Zygotes were collected from superovulated females and immediately used for 1) slow-freezing in a solution containing 1.5 M 1,2-propanediol and 0.2 M sucrose, or 2) vitrification in a solution containing 42.0% (v/v) of ethylene glycol, 18.0% (w/v) of dextran and 0.3 M sucrose as cryoprotectants. After thawing or warming, respectively, zygotes were evaluated for 1) actin distribution, 2) in vitro or 3) in vivo development to blastocyst. Comparing actin filaments distribution, a significantly higher number of vitrified zygotes with actin distributed in cell border was observed (55 ± 7.7 vs. 74 ± 6.1% for slow-frozen vs. vitrified, respectively). After 24 and 72 h of in vitro development, significant differences in the cleavage and morula rate among the groups were observed (9 ± 2.4 and 3 ± 1.3 vs. 44 ± 3.0 and 28 ± 2.7% for slow-frozen vs. vitrified, respectively). None of the slow-frozen zygotes reached the blastocyst stage, in contrast to the vitrified counterparts (11 ± 1.9%). Under in vivo culture conditions, a significant difference in blastocyst rate was observed between vitrified and fresh embryos (6 ± 1.5 vs. 35 ± 4.4% respectively). Our results showed that alterations in actin cytoskeleton and deteriorated development are more evident in slow-frozen than vitrified pronuclear zygotes. Vitrification method seems to be a more effective option for rabbit zygotes cryopreservation, although pronuclear zygotes manipulation per se resulted in a notable decrease in embryo development.This research was supported by the projects: UGAVIII/16/2015, VEGA 1/0611/15, by the Spanish Research project AGL2014-53405-C2-1-PComision Interministerial de Ciencia y Tecnologia (CICYT), Generalitat Valenciana research program (Prometeo II 2014/036), grant of Slovak Research and Development Agency: APVV-14-0043 and by the European Community under project no 26220220180: Building Research Centre "AgroBioTech". B. Kulikova received fellowship from a Collaborative European Network on Rabbit Genome Biology (RGB-Net) (COST-STSM-TD1101)Kulikova, B.; Jiménez-Trigos, ME.; Makarevich, AV.; Chrenek, P.; Vicente Antón, JS.; Marco-Jiménez, F. (2016). State of actin cytoskeleton and development of slow-frozen and vitrified rabbit pronuclear zygotes. Cryobiology. 72(1):14-20. https://doi.org/10.1016/j.cryobiol.2015.11.009142072

A novel technique for oviduct occlusion to generate live births from cryopreserved rabbit oocytes after in vivo fertilisation

[EN] Intraoviductal transfer technique in combination with in vivo fertilisation has arisen as an effective technique to assess live births after transfer of slow-frozen oocytes in the rabbit. Nevertheless, the great disadvantage of this method is the accumulation of tubal fluid in a large number of females after clamping the oviducts. In this study, we develop an alternative method to minimise damage to the oviduct and increase the birth rate. The aims of this study were (1) to evaluate the ability of cyanoacrylate tissue adhesive to occlude the oviduct for female sterilisation; (2) to evaluate the effect of oviduct occlusion immediately after transferring fresh oocytes on in vivo fertilisation; and (3) to assess this technique to generate live births from fresh and slow-frozen oocytes. In all the experiments, recipients were artificially inseminated 9 h prior to occluding the oviducts. In the first experiment, the left oviduct was blocked with cyanoacrylate tissue adhesive, while the right one was used as a control. Six days later, oviducts and uterine horns were flushed to assess embryo recovery rates. While the embryo recovery rate was 79.2% in the intact oviduct, no embryos were recovered in the blocked one. In the second experiment, fresh oocytes were transferred into both oviducts, which were immediately occluded. Six days later, the in vivo fertilisation success rate was 33.7%. Finally, in the last experiment, slow-frozen oocytes were transferred and the rate of live births was 13.2 ± 4.5%. The study shows that when using this method the generation of live births from slow-frozen oocytes increases significantly. In addition, our results suggest that in vivo environment could help improve the results of oocyte cryopreservationThis work was supported by the Spanish Research Project AGL2011-30170-C02-01 (CICYT) and by funds from the Generalitat Valenciana Research Programme (Prometeo 2009/125). Estrella Jimenez was supported by a research grant from the Education Ministry of the Valencian Regional Government (programme VALi+d. ACIF/2010/262).Jiménez Trigos, ME.; Vicente Antón, JS.; Marco Jiménez, F. (2014). A novel technique for oviduct occlusion to generate live births from cryopreserved rabbit oocytes after in vivo fertilisation. Animal Reproduction Science. 148(3-4):197-204. https://doi.org/10.1016/j.anireprosci.2014.06.007S1972041483-

First pregnancy and live birth from vitrified rabbit oocytes after intraoviductal transfer and in vivo fertilization

[EN] Intraoviductal oocyte transfer in combination with in vivo fertilization has arisen as an alternative method to induce pregnancies from cryopreserved oocytes in rabbits. In this study, offspring were obtained for the first time from vitrified rabbit oocytes using this technique. In all the experiments, recipients were artificially inseminated 9 hours before oocyte transfer. Cryopreserved (vitrified and slow-frozen) and noncryopreserved (fresh) oocytes were transferred into both oviducts, which were immediately closed using cyanoacrylate tissue adhesive to block the entry of the recipient's own oocytes. Three transferred group females that received vitrified oocytes became pregnant and delivered a total of nine live young naturally. The results revealed that there were no differences in the live birth rate between vitrified and slow-frozen oocytes (5.5% and 4.4%, respectively). When fresh oocytes were transferred, this rate increased to 19.2%, whereas in the control females (nontransferred) the rate of offspring obtained was 71.4%. This is the first reported result of the development to term of vitrified rabbit oocytes and suggests that an in vivo environment could help improve the results of oocyte cryopreservation. (C) 2014 Elsevier Inc. All rights reserved.This work was supported by the Spanish Research Project AGL2011-30170-C02-01 Comision Interministerial de Ciencia y Tecnologia (CICYT) and by funds from the Generalitat Valenciana Research Programme (Prometeo 2009/125). E. Jimenez-Trigos was supported by a research grant from the Education Ministry of the Valencian Regional Government (programme VALi+d, ACIF/2010/262). English text version was done by N. Macowan English Language Service.Jiménez Trigos, ME.; Vicente Antón, JS.; Marco Jiménez, F. (2014). First pregnancy and live birth from vitrified rabbit oocytes after intraoviductal transfer and in vivo fertilization. Theriogenology. 82(4):599-604. https://doi.org/10.1016/j.theriogenology.2014.05.029S59960482

Generation of live offspring from vitrified embryos with synthetic polymers Supercool X-1000 and Supercool Z-1000

[EN] BACKGROUND: Ice growth and recrystallisation are considered important factors in determining vitrification outcomes. Synthetic polymers inhibit ice formation during cooling or warming of the vitrification process. OBJECTIVE: The aim of this study was to assess the effect of adding commercially available synthetic polymers SuperCool X-1000 and SuperCool Z-1000 to vitrification media on in vivo development competence of rabbit embryos. METHODS: Four hundred and thirty morphologically normal embryos recovered at 72 h of gestation were used. The vitrification media contained 20% dimethyl sulphoxide and 20% ethylene glycol, either alone or in combination with 1% of SuperCool X-1000 and 1% SuperCool. RESULT: Our results show that embryos can be successfully vitrified using SuperCool X-1000 and SuperCool Z-1000 and when embryos are transferred, live offspring can be successfully produced. CONCLUSIONS: In conclusion, our results demonstrated that we succeeded for the first time in obtaining live offspring after vitrification of embryos using SuperCool X-1000 and SuperCool Z-1000 polymers.This work was supported by the Spanish Research Project AGL2011-30170-C02-01 and by funds from the Generalitat Valenciana Research Programme (Prometeo 2009⁄125). Estrella Jiménez was supported by a research grant from the Education Ministry of the Valencian Regional Government (programme VALi+d. ACIF/2010/262).Marco Jiménez, F.; Jiménez Trigos, ME.; Lavara García, R.; Vicente Antón, JS. (2014). Generation of live offspring from vitrified embryos with synthetic polymers Supercool X-1000 and Supercool Z-1000. Cryo Letters. 35(4):286-292. http://hdl.handle.net/10251/65518S28629235

Use of cyclodextrins to increase cytoplasmic cholesterol in rabbit embryos and their impact on live kits derived from vitrified embryos

[EN] BACKGROUND: Low cryotolerance in oocytes and embryos is frequently associated with lipid accumulation in the cytoplasm. OBJECTIVE: This study aimed to evaluate the effect of cyclodextrin used as a cholesterol loader to change cytoplasmic cholesterol content of embryos and raise their tolerance to cryopreservation. METHODS: In the first experiment compact morulae-early blastocysts were exposed to CLC (0.11 mM and 0.23 mM cholesterol) for 1 hour. In the second experiment, embryos were exposed to CLC (0.11 mM and 0.23 mM cholesterol) and then vitrified. RESULT: Using both concentrations, cytoplasmic cholesterol content was increased. Vitrified groups demonstrated a lower capacity for embryonic development (in vitro and in vivo) compared to the control groups. Nevertheless, live young were obtained in all groups. CONCLUSIONS: In conclusion, we have demonstrated the feasibility of using cyclodextrin as a carrier for cholesterol into rabbit embryo cytoplasm, although further studies are required to clarify the usefulness of CLC use in embryo cryopreservationThis work was supported by the Spanish Research Project AGL2011-30170-C02-01 and by funds from the Generalitat Valenciana Research Programme (Prometeo 2009/125). Estrella Jiménez was supported by a research grant from the Education Ministry of the Valencian Regional Government (programme VALi+d. ACIF/2010/262). English text version revised by N. Macowan English Language Service.Marco Jiménez, F.; Jiménez Trigos, ME.; Lavara García, R.; Vicente Antón, JS. (2014). Use of cyclodextrins to increase cytoplasmic cholesterol in rabbit embryos and their impact on live kits derived from vitrified embryos. Cryo Letters. 35(4):320-326. http://hdl.handle.net/10251/65519S32032635