2011 (SBTE) 25th Annual Meeting Proceedings - International ...

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B. Gasparrini. 2011. Ovum pick-up and in vitro embryo production in buffalo species: an update................................................................. jjjjjjjjjjj Acta Scientiae Veterinariae. 39(Suppl 1): s317 - s335. signs are even weaker. The embryo-recipient synchrony is known to play a critical role for embryo survival after ET. In fact, it has been shown that buffalo in vivoproduced cryopreserved embryos can only tolerate asynchrony of 12 h [58]. The identification of the optimal time for ET may even be more critical for in vitro produced embryos because they are delayed in development and less viable. A significant improvement of embryo survival rate following IVC of vitrified-warmed buffalo IVP embryos has been recently obtained by using minimum volume vitrification methods, such as Open Pulled Straw [31] and Cryotop [30] vitrification. The first time OPS was used, the vitrification and warming solutions previously utilized to vitrify buffalo embryos in French traditional straws [50] were compared with those previously utilized for OPS vitrification of cattle embryos [113], and the overall embryo survival rate recorded at 24 h was not significantly different between the two methods (70% vs 62% respectively). Subsequently, the cryotop was utilized for the vitrification of IVP buffalo embryos in 16.5 % EG and 16.5 % DMSO and 0.5 M sucrose, with very promising results [30]. In these studies it was clearly demonstrated that the stage of development affects freezability of IVP embryos, with increased in vitro survival for the advanced embryo stages [30,31], such as the expanded blastocysts and hatched blastocysts (approximately 75%), which are, in any case, better quality embryos since they develop faster in vitro. Unfortunately, only few IVP embryos vitrified by CTV have been transferred so far into recipients (n =10; unpublished data) and, despite a high pregnancy rate (50%) diagnosed on day 30, the development to term was still poor (10%). In a recent study IVP buffalo embryos have been cryopreserved by a two-step vitrification method in straws, to examine the effects of different vitrification medium compositions (40% EG; 25% glycerol + 25% EG, and 25% EG + 25% DMSO) and exposure times (2, 4 and 6 min in the equilibration solution) on the post-thaw development of buffalo IVP embryos [67]. The higher expansion (72%) and hatching rates (46%) after postwarming culture were found for blastocysts vitrified in 25% EG + 25% DMSO with an exposure time of 4 min compared with the other combinations. It is known that cryoprotectants might cause irreparable damage to cytoskeletal components. Therefore, maintaining the integrity of the cytoarchitecture within an embryo during cryopreservation is of utmost importance. In porcines, treatment of embryos with cytochalasin-B prior cryopreservation was found to improve development [36]. In buffalo, cytoskeletal stabilization with cytochalasin-B did not improve the postwarming in vitro developmental competence of vitrified buffalo blastocysts [69] but favoured that of morulae. We may conclude that up to know there is not an overwhelming evidence of the superiority of a method vs the others for buffalo embryo cryopreservation. Different labs use different tools, and combinations of cryoprotectants/exposure times and hence it is also difficult to compare the results. The scenario is also complicated by the utilization of different culture systems, known to affect embryo viability. Regarding the tool, the choice may depend also on the future destination of the cryopreserved embryos, as according to the rules for transportation of embryos among different countries, it is not allowed to use methods based on the direct contact of the embryos with the liquid nitrogen because of the potential sanitary risks. However, in case the superiority of the minimum volume vitrification methods is clearly demonstrated also in terms of development to term after transfer, strategies may be developed to reduce these risks, that should take into account utilization of sterile filtered liquid nitrogen and a safe sealing system of the tools before storage in the tank. It is worth pointing out that the main factor affecting the efficiency of cryopreservation of mammalian embryos is undoubtedly the quality of the embryos and that it is current practice in cattle and other domestic species to operate a strict selection of the embryos prior to cryopreservation. This is not the case in buffalo because the low number of oocytes and, hence, of the embryos produced imposes the cryopreservation of all the embryos obtained, regardless of their quality. A strict selection of only Grade 1 embryos would result in improved cryopreservation efficiency further limiting, though, the number of embryos and, hence rendering the cost-benefit ratio of IVEP more unfavorable. Furthermore, as the embryo quality is in part due to the oocyte quality but is also affected by the culture conditions, the optimization of the culture system is compulsory to improve the efficiency of cryopreservation. VII. CONCLUSIONS The acquisition of more specific information on buffalo oocytes and embryo requirements in vitro, has led to a significant improvement of the IVEP efficiency in this species. However, although blastocyst yield has s328
B. Gasparrini. 2011. Ovum pick-up and in vitro embryo production in buffalo species: an update................................................................. jjjjjjjjjjj Acta Scientiae Veterinariae. 39(Suppl 1): s317 - s335. greatly increased, pregnancy rates are still low and only few calves have been produced after transfer of cryopreserved embryos from live animals. In addition to the low number of oocytes recruitable, that seems to be a feature intrinsic to the species, an important limiting factor is the low resistance to cryopreservation of buffalo in vitro derived embryos, that can be also considered a result of poor viability due to suboptimal culture conditions. In fact, in contrast to the significant improvements of both the IVM and IVF systems for buffalo achieved throughout the years, leading to high blastocyst yields, the progress in the optimization of the IVC system is still far behind. Currently, the IVEP efficiency is highly competitive, with 90% maturation rate, 75-80% cleavage and 30-35% blastocyst rates. Operating a preliminary selection of the donors so that 3 viable oocytes are recovered on average per animal, it can be predicted that in 6 months approximately 50 embryos are produced/ buffalo. This yield is much higher than with MOET programs. However, pregnancy rate after ET of cryopreserved embryos is still poor, ranging from 8 to 25 %. If we consider an average pregnancy rate of 15%, 7 calves can be obtained on average per buffalo after 6 months OPU. It follows that, to make cost-benefits ratio more favourable, the only suitable options at present are to transfer either fresh embryos or frozen embryos previously cultured in vivo, i.e. in an intermediate host. It is likely that the poor pregnancy rate is mainly due to the scarce viability of IVP buffalo embryos, determined by suboptimal culture conditions. This can in part be also related to an inappropriate cryopreservation method. Furthermore, it is possible that the status of the recipient and the perfect synchrony between the age of the embryo and that of the recipient uterus play a role. With this regard, it is worth reminding that the percentage of buffaloes responding to synchronization treatments is low and inconsistent and that a high incidence of embryonic mortality is recorded in the unfavourable season also after natural mating. In conclusion, the optimization of culture conditions is fundamental to increase the viability of IVEP embryos for advanced reproductive strategies to become a routine procedure in buffalo breeding. At the same time efforts should be addressed to identify the best management of the recipients, in order to increase pregnancy maintenance after ET of cryopreserved IVP embryos. N REFERENCES 1 Aitken J.R. 1994. A free radical theory of male infertility. Reproduction, Fertility and Development. 6 (1): 19-24. 2 Baruselli P.S., Gimenes L.U., Carvalho N.A.T., Sá Filho M.F. & Ferraz M.L. 2010. Control of ovarian function for assisted reproductive technologies in buffalo. In: Proceedings of the 9th World Buffalo Congress (Buenos Aires, Argentina). Revista Veterinaria. 21(1): 93-103. 3 Blondin P., Coenen K., Guibault L.A. & Sirard M.A. 1997. In vitro production of bovine embryos: developmental competence is acquired before maturation. Theriogenology. 47(5): 1061-1075. 4 Boccia L., Attanasio L., De Rosa A., Pellerano G., Di Palo R. & Gasparrini B. 2007. Effect of sodium nitroprusside on buffalo sperm capacitation in vitro. Reproduction, Fertility and Development. 19(1): 276 5 Boccia L., Attanasio L., Monaco E., De Rosa A., Di Palo R. & Gasparrini B. 2006. Effect of progesterone on capacitation of buffalo (Bubalus bubalis) spermatozoa in vitro. Reproduction in Domestic Animals. 41(4): 311. 6 Boccia L., De Rosa A., Attanasio L., Di Palo R., Zicarelli L. & Gasparrini B. 2005. Capacitation of buffalo spermatozoa in vitro. Reproduction, Fertility and Development. 17 (2): 270. 7 Boccia L., Di Palo R., De Rosa A., Attanasio L., Mariotti E. & Gasparrini B. 2007. Evaluation of buffalo semen by Trypan blue/ Giemsa staining and related fertility in vitro. In: Proceedings of the 8th World Buffalo Congress (Caserta, Italy). Italian Journal of Animal Science. 6 (Suppl 2): 739-742. 8 Boccia L., Monaco E., Attanasio L., De Rosa A. & Gasparrini B. 2006. Influenza del cambio di coltura (doppio vs singolo) sullo sviluppo di embrioni bufalini in vitro. In: Proceedings of the “XII Giornate scientifiche” (Napoli, Italy). p.171. 9 Bols P.E.J., Ysebaert M.T., Van Soom A. & De Kruif A. 1998. Effects of long term treatment with bovine somatotropin on follicular dynamics and subsequent oocyte and blastocyst yield during an OPU-IVF program. Theriogenology. 49(5): 983- 995. 10 Boni R. 1994. In vitro production in bovine and buffalo species. Buffalo Journal. 2: 147-160. s329
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