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Abstracts. II International Symposium on Animal Biology of Reproduction, Nov. 19-22, 2008, São Paulo, SP, Brazil. In vitro culture of caprine preantral follicles enclosed in ovarian tissues: main results from LAMOFOPA-UECE (Fortaleza, Ceará, Brazil) J.R. Figueiredo 1 , M.H.T. Matos 1 , J.R.V. Silva 2 1 Faculty of Veterinary Medicine, LAMOFOPA, PPGCV, State University of Ceara. 2 Biotecnology Nucleus of Sobral (NUBIS), Federal University of Ceara, Sobral-CE, Brazil. The main aim of the researches developed in the Laboratory of Manipulation of Oocytes and Preantral Follicles (LAMOFOPA) is to develop a culture system which ensures high rates of survival, activation and in vitro growth of caprine primordial follicles. Such studies are very important to understand the factors that control the early folliculogenesis. The ovaries used in our study were obtained at local slaughterhouse from adult cyclic goats and transported to the laboratory in Minimal Essential Medium (MEM) supplemented with antibiotics. At the laboratory, each pair of ovary was divided into several cortical fragments and fixed in Carnoy (fresh control) or cultured in 1 ml of control medium. This medium was composed of MEM supplemented with Bovine Serum Albumine (BSA), ITS (Insulin, Transferrin and Selenium), Pyruvate, Glutamine and Hypoxantine (MEM + ) or MEM + plus different concentrations of one of the following substances: Follicle Stimulating Hormone (FSH), Estradiol, Progesterone, Growth and Differentiation Factor-9 (GDF-9), Insulin like Growth Factor-1 (IGF-1), Growth Hormone, Vasoactive Intestinal Peptide (VIP), Kit Ligand (KL), Bone Morphogenetic Protein-6 (BMP-6), -7 (BMP-7) and -15 (BMP-15), Angiotensin (ANG), Vascular Endothelial Growth Factor (VEGF), Epidermal Growth Factor (EGF), Fibroblast Growth Factor-2 (FGF-2). The cultures were performed at 39ºC in 5% CO2 in air for 7 days. To evaluate the efficiency of the media, parameters such as the percentage of follicle survival (by histological and transmission electron microscopy analysis), activation of primordial follicles and follicular and oocyte growth were take into consideration. The results showed that after 7 days of culture, some of the factors (FSH, Progesterone, GDF-9, IGF- 1, VIP, EGF) maintained preantral follicles survival similar to fresh control. In addition, other factors increase follicular activation (FSH, Estradiol, Progesterone, GDF-9, IGF-1, FGF-2, VIP, BMP-7, EGF) or follicular diameter (FSH, Progesterone, GDF-9, IGF-1, FGF-2, VIP) when compared with fresh control. Furthermore, FSH, Estradiol and GDF-9 were more efficient to promote both primordial follicles activation and an increase in follicular diameter than MEM+ alone. Overall, the results demonstrated that all substances tested are important in the control of early folliculogenesis depending on the concentration used. This work is supported by CNPq-Brazil E-mail: jrf@pesquisador.cnpq.br Anim. Reprod., v.6, n.1, p.197, Jan./Mar. 2009 197
198 Abstracts. II International Symposium on Animal Biology of Reproduction, Nov. 19-22, 2008, São Paulo, SP, Brazil. What advances are needed before grafting of cryopreserved ovarian tissues can reach full potential in animal conservation M.C.J. Paris 1,2 , J.M. Shaw 3 1,2 Institute for Breeding Rare and Endangered African Mammals (IBREAM), Edinburgh, Scotland. 1,2 Department of Equine Sciences Faculty of Veterinary Medicine, University of Utrecht, Yalelaan 114, 3584CM, Utrecht, The Netherlands. 3 Melbourne School of Land and Environment, The University of Melbourne, Parkville, 3010, Australia. Grafting of cryopreserved ovarian tissue commenced over 50 years ago and the first report of mouse pups being born came in 1961. Current cryopreservation and grafting protocols make germline preservation and propagation a useful tool that has resulted in fertility restoration (offspring) in moths, mice, rats, sheep and humans. In mice, the most well studied of all species, a wide range of collection, processing, cryopreservation and grafting strategies (including xenografting), have successfully resulted in normal, healthy offspring. Although there is considerable interest in, and a need for, cryobanking of ovarian tissue to preserve the female germline for threatened wildlife species, its usefulness is currently moderated by various practical issues, including how to obtain, process and replace and monitor the ovarian tissue. Our interest is in how to optimize the replacement step. In human medicine, ovarian tissue banking is primarily aimed to help young women who are at risk of early ovarian failure, by storing their tissue and autografting it back to them when they are ready to have children. This approach has limited or no value for animal conservation as there are essentially no instances in which the same individual animal would benefit from having its own ovarian tissue collected, stored and then returned to it. In small animal models all grafting to another individual of the same species is widely used, but to be highly effective the recipient has to be immunologically compromised and/ or fully histocompatible with the donor to avoid rejection. In the case of threatened wildlife species all grafting is graft rejection and disease transmission are both major problems (Shaw et al., 1996; Gosden, 2007; Wei et al., 2007; Petroianu et al., 2007). In our opinion the most valuable advance would therefore be the development of strategies to prevent immune rejection without, aggressive, immunosuppressive drugs, as this would permit allografting. Allografting has several advantages over in vitro culture and ensures that follicles and oocytes are grown in a physiologically compatible environment. If allografting became feasible then ovarian tissue from a genetically valuable individual could be placed in several different (less valuable) recipients and thereby greatly boost the donors reproductive potential; comparable to propagating the male germline via artificial insemination. Tissue collection from a living endangered animal is problematical, but an advantage of ovarian tissue is that functional material is recoverable even for a few hours after death. Another approach that has received attention is the use of xenotransplantation (the transplantation from one species to another) of ovarian tissue to an immunologically compromised recipient; to date rodents have been the most commonly used recipients. This approach can support antral follicle development in a wide range of species including marmoset, elephant, hamster, dog, wallaby, wombat, cat, macaque, cow, pig, rabbit, goat (Paris et al., 2004). The current practice of using immunocompromised mice and rats as xenografting recipients does have limitations including a) the small size of the recipient animals and b) the physiological discrepancies between the donor and recipient which can have consequences for the development of healthy, fertilizable oocytes. On a positive note, live offspring has been achieved in a xenogeneic setting (mouse to rat), but further work is needed to determine the potential of this approach for grafting ovarian tissue over greater xenogeneic barriers. Thus, in summary our presentation will focus on the identification of research needs to address whether and how the tools of ovarian cryopreservation combined with ovarian grafting can aid to the propagation of threatened wildlife species. References Gosden RG. 2007. Survival of ovarian allografts in an experimental animal model. Pediatr Transplant, 11:628-633. Paris MCJ, Snow M, Cox S-L, Shaw JM. 2004. Xenotransplantation: a tool for reproductive biology and animal conservation? Theriogenology, 61:277-291; Petroianu A, Alberti LR, Vasconcellos LS. 2007. Morphologic, endocrinologic and natural pregnancy assessment of allogeneic ovarian orthotopic transplantation without a vascular pedicle in rabbits. Eur J Obstet Gynecol Reprod Biol, 133:70-75; Shaw JM, Bowles J, Koopman P, Wood C, Trounson AO. 1996. Fresh and cryopreserved ovarian tissue samples from donors with lymphoma transmit the cancer to graft recipients. Hum Reprod, 11, 1668-1673; Wei F, Yunze W, Changye S, Hongsheng H, Xiaoli W, Qing L, Wenhao C, Kequan G, Masahiko M, Hiroko H, Takahide M, Susumu I. 2007. Prevention of osteoporosis and hypogonadism by allogeneic ovarian transplantation in conjunction with intra-bone marrow-bone marrow transplantation. Transplantation, 84:1459-1466. Email: m.paris@uu.nl Anim. Reprod., v.6, n.1, p.198, Jan./Mar. 2009
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