Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

More documents

Recommendations

Info

$(Microsoft PowerPoint - Lecci\363n 2_07.ppt)$

16 t h International Congress on Animal Reproduction 198 Poster Abstracts small volume of adjacent cytoplasm was aspirated into the same pipette. Then, the pipette was withdrawn from the oocyte and the karyoplast was released out. Thereafter the pipette with the remaining donor cells was re-introduced into ooplasm through the same hole of zona pellucida which was made during enucleation, and the donor cell was directly introduced into the cytoplasm of the enucleated oocyte. The reconstructed embryos were activated by electrical stimulation and cultured for 7 days. Results The blastocyst rate of the novel OSNT embryos was 14.5% (16/110) while the same blastocyst rate for standard SCNT embryos was 10.7% (15/140, P < 0.05). In addition, OSNT reduced steps and duration for SCNT program in our laboratory. Conclusion The simple, new OSNT system enables large scale cloning by reduction of procedural steps. Gene expression analysis data of HSP70, Glut-1, poly A, Pou5f, Nanog, Sox9, Cdx2 and Eomes will be presented at the meeting. This study was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MOST) (M10641000001-06N4100-00110 and R01-2007-000-10316-0). P517 Porcine parthenogenote embryo developmental competence under different in vitro maturation systems using Roscovitine Salvador, I 1 *, Alfonso, J 2 , Garcia-Rosello, E 3 , Garcia-Mengual, E 4 , Silvestre, MA 4 1CITA (Centro de Investigación y Tecnología Animal), Instituto Valenciano de Investigaciones Agrarias (CITA-IVIA), Spain; 2 Instituto de Medicina Reproductiva, Spain; 3 Universidad CEU-Cardenal Herrera, Spain; 4 Instituto Valenciano de Investigaciones Agrarias, Spain With the aim of extending the “time frame” to manipulate oocytes for techniques such as ICSI and NT, we adopted a parthenogenetic model to determine the developmental potential of oocytes submitted to different in vitro maturation conditions, involving the use of roscovitine and longer IVM duration. Immature cumulus-oocyte complexes (COC) collected from ovaries of slaughtered gilts were matured in four different maturation systems: 45IVM (control group); 50IVM; corresponding to 45 or 50 hours maturation, respectively, in IVM medium (M199 supplemented with 0.1% PVA, 0.57 mM cysteine,10 ng/mL EGF, antibiotics and hormones for the first 22h maturation period (0.1 IU/ml recombinant human-FSH and -LH); 5R+40IVM and 5R+45IVM, in which COC were cultured in hormone-free IVM medium with 50 µM of roscovitine (Sigma, R7772) for the first 5h, then washed twice and allowed to reach normal maturation in IVM medium for 40 or 45 hours, respectively. Parthenogenote development was induced by stimulating MII oocytes with an electrical set of two DC pulses of 1.2 kV/cm for 30 µsec delivered on an electro-cell porator. After activation, embryos were washed twice and allowed to culture for 7 days in PZM-3 (Yoshioka et al., 2002). When COC were cultured with the 5R+40IVM treatment, nuclear maturation and cleavage rate were significantly lower than with the 45IVM, 50IVM and 5R+45IVM culture treatments (54% vs. 73 -77%, P < 0.05 and 59%; 81-88%, P < 0.05, respectively). However, this difference between groups did not reach statistical significance in blastocyst rates (ranged from 17% to 25%). Regarding embryo quality, blastocysts from 5R+40IVM group presented the lowest average number of cells per blastocyst (P < 0.05). No differences were observed either in MII, cleavage and blastocyst rates or in blastocyst cell number between 45IVM, 50IVM and 5R+45IVM experimental groups. Under our experimental conditions and using parthenogenote embryos as model, we observed that it is feasible to prolong the “time frame” by at least 5 hours to manipulating porcine oocytes in the laboratory without loss of efficiency by using either 5h pre-treatment with roscovitine or prolonging until 50 hours in vitro maturation. This work was supported by INIA and FEDER (RTA2007-0110-00-00). P518 Melatonin supplementation during ovine oocyte IVM enhance blastocyst output and affects protein expression patterns Succu, S 1 *; Satta, V 1 ; Bebbere, D 2 ; Madeddu, M 2 ; Berlinguer, F 2 ; Leoni, G 1 ; Naitana, S 2 1Dept. of Physiological, Biochemical and Cellular Sciences; 2 Dept. Animal Biology; Veterinary Medicine Faculty; Sassari University, v. Vienna 2, 07100 Sassari (Italy) Melatonin exerts a variety of systemic and local functions. Recently evidence of the presence of melatonin receptors in the ovary has been demonstrated. It has been shown to increase the cleavage rates of bovine and porcine preimplantation embryos in vitro, most likely by its anti-apoptotic effect and scavenging activity. The aims of the current study were to test whether melatonin supplementation during ovine oocyte in vitro maturation is able to enhance further developmental rates in vitro, and to verify if its actions at the germ cell level are mediated by a modification in oocyte protein expression pattern. Adult ovine oocytes collected after slicing of abattoir derived ovaries were randomly divided into three experimental groups for in vitro maturation: A) TCM199 plus 10 µg/ml FSH/LH and 100µM cysteamine (MM) supplemented with 10% oestrus sheep serum; B) MM containing 0.4% bovine serum albumin (BSA); C) MM containing 0.4% BSA and 100 μM melatonin. After 24h, oocytes were fertilized and cultured in vitro up to the blastocyst stage. The cleavage rate did not differ between the three groups (85%, 82.9% and 87.8% for A, B and C respectively). Blastocyst output was significantly higher (P < 0.01) in A (47.1%) and B (41.7%) groups when compared to C (23.5%). 2D-electrophoresis was performed on both matured oocytes (10 oocytes/group) and granulosa cells of B and C groups. Silver staining of electrophoresed gels demonstrated a high protein number expressed in C electrophoretic gels compared to B, while two proteins were evidenced in B but not in C gels. Some of these differences may be related to a shift of the isoelectric point, probably due to post-translational modifications. These data provide evidence that melatonin added to oocyte in vitro maturation medium is able to enhance blastocyst output to levels comparable to those obtained routinely using oestrus ovine serum as medium supplement. Moreover, 2D-electrophoresis results revealed that melatonin acts altering oocyte and cumulus cell protein expression patterns, inducing both ex-novo protein synthesis and post-translational modifications. New insights on melatonin actions at the COC level will be drawn after sequencing ex-novo expressed proteins found after in vitro maturation with melatonin (Supported by Fondazione Banco di Sardegna). P519 Establishment of parthenogenetic embryonic stem cell lines in mouse Rungarunlert, S 1 ; Rungsiwiwut, R 1 ; Suphankong, S 2 ; Panasopolkul, S 1 ; Thongphakdee, A 1 ; Dinnyes, A 3,4 ; Tharasinit, T 1 ; Techakumphu, M 1 * 1Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, 10330 Thailand; 2Department of Medical Science, Faculty of Medicine, Chulalongkorn University, Bangkok, 10330 Thailand; 3 Molecular Animal Biotechnology Laboratory, Szent Istvan University, H-2100 Gödöllö, Hungary; 4 BioTalentum Ltd, H-2100 Gödöllö, Hungary Introduction Establishment ES cell from parthenogenetic activated blastocysts would allow creating histocompatible cells for regenerative medicine. Objective The objective of this study to establish embryonic stem (ES) cell lines from parthenogenetically activated of mouse oocytes as a model system for human research. Methods Mature oocytes (n=145) were collected from oviducts at 15 h post-hCG injection and subsequently activated by using 10 mM/ml Sr2+ with 5 µg/ml Cytochalasin B in Ca 2+-free CZB medium for 6 h. Activated oocytes with two pronuclei (2PN) (n=131) were cultured further in vitro in KSOM-AA medium at 37ºC, 5% CO2 in a humidified atmosphere. 96 h after activation expanded blastocysts
16 t h International Congress on Animal Reproduction Poster Abstracts 199 were used for ES cells establishment (n=18) or differential stained for cell number counting. All pathenogenetic embryonic stem (pES) cell lines were characterized by morphology, pluripotency marker of murine ES cells (AP, Oct-4, SSEA-1, Sox2 and Nanog), chromosome number as well as in vitro differentiation. Result The rate of activation and blastocyst formation from MII oocytes were 90±3.13 and 82.5±2.95%, respectively. The total cell number, ICM cell number and ICM: trophectoderm ratios of parthenogenetic blastocysts were 66.8±2.88, 8.1± 0.63 and 1:7.24, respectively. Four pES cell lines were established and the efficiency of ES cell lines establishment were 10-37.5%. All pES cell lines presented a typical morphology of murine ES cell with high nuclear: cytoplasmic ratio, round shape and clear edge colonies. Three of them presented normal chromosome number (2n=40). The pES cell lines possessed high levels of alkaline phosphatase and expressed pluripotency marker including Oct4, Sox2, SSEA1 and Nanog after detecting by immunocytochemistry and RT-PCR. During in vitro differentiation, the pES cell lines formed cystic embryoid bodies in suspension culture and created spontaneous beating clusters in gelatine-coated culture dishes. Conclusion We succeeded to produce ES cell lines from parthenogenetic blastocysts with presenting pluripotency markers and normal chromosome numbers in mouse model. This work was supported by grant from CHE-TRF Senior Scholars, grant No RTA5080010, The National Research Council of Thailand, The Royal Thai Government Scholarship (PhD-SW-INV_20060202/49) and EU FP7 (IAPP 2007 - 218205). P520 Vitrification versus slow-cooling: higher survival rates for vitrified two-cell mouse embryos Temple-Smith, P*; Wang, X; Aguirre Maclennan, I; Momtaz, F; Fage, R; Patel, D; Philips, S; Pangestu, M; Catt, S Education Program in Reproduction and Development, Centre for Reproduction and Development, Monash Institute of Medical Research, Australia Introduction Cryopreservation of cleavage stage embryos by slow cooling is now routine but recently vitrification has gained prominence. Few studies, however, have compared the two techniques using the same cohort of embryos. Here we compare embryo viability in slow-cooled, vitrified or fresh cultured two-cell mouse embryos (2-CEs). Methods Oocytes collected from superovulated female mice [F1 (C57Bl6JxCBA)] were fertilised in vitro with epididymal sperm. Oocytes were washed free of cumulus and sperm and cultured (24h; modified KSOM, 5% CO2 in air). All 2-CEs (cleavage rate, 84%) were distributed randomly into 3 groups (slow-cooling, vitrification and unfrozen controls). For vitrification, 2-CEs were equilibrated (3 min; 37°C) in 10% v/v ethylene glycol, 10% v/v DMSO and placed in vitrification solution (17% v/v ethylene glycol, 17% v/v DMSO, 0.75M sucrose). Each 2-CE was transferred to a fibreplug (2ul), touched on a pre-cooled metal block in liquid N 2 and inserted in a precooled straw (CVM kit, Cryologic). For slow-cooling (SC), up to seven 2-CEs were put in 11% v/v propanediol in KSOM Hepes (10 mins) followed by 10 min in a final freeze solution [11% v/v propanediol, 0.5M sucrose in KSOM Hepes medium; room temp (RT)], and frozen individually in straws using conventional slowcooling protocols in a programmable freezer (Cryologic CL856). Vitrified embryos were warmed at 37°C in 3 solutions (0.3M, 0.2M, 0.1M sucrose, 5min in each); SC embryos were thawed at RT in 3 solutions (0.5, 0.25, 0.125M sucrose). Lysis was assessed 2h after thawing, and all surviving 2-CEs were cultured (72h) to blastocyst and hatching blastocyst stages assessed. Differences between groups were examined using a Chi Square test. Results Lysis rates after thawing were higher for SC embryos (29/113 21.2% cf 4/88 4.5%, P=0.001). Survival and hatching rates after thawing for vitrification (70/84, 82.6%; 42/84, 50% respectively) and SC (67/84, 79.7%; 42/84, 50% respectively) groups were not significantly different from unfrozen controls (73/94, 77.6% and 54/94, 57.7% respectively). However, blastocyst development rate (67/113, 59.2%) of thawed 2-CE in the SC group was significantly lower than in the vitrification group (70/88, 79.5%, P=0.002). Conclusion Comparison of two cryopreservation methods on the same cohort of cleavage stage embryos revealed lower lysis rates after vitrification than after slow cooling, but not higher blastocysts rates from those 2-CE that survived. We suggest that either or both methods could be used for cryopreserving cleavage stage embryos. P521 Flat-headed cat cloned embryos and preliminary embryo transfer Thongphakdee, A 1 *, Manee-In, S 1 , Rungsiwiwut, R 1 , Numchaisrika, P 1 , Siriaroonrat, B 2 , Kamolnorranath, S 2 , Chatdarong, K 1 , Techakumphu, M 1 1Department of Obstetrics Gynaecology and Reproduct, Faculty of Veterinary Science, Chulalongkorn University, Thailand; 2 Zoological Park Organization of H.M. the King, Thailand Introduction Critically endangered flat-headed cat (FC; Prionailurus planiceps) is one of the small wild cats in Thailand’s captive breeding program. Inter-generic nuclear transfer (ig-NT) offers the possibility of FC embryos/offspring production. The purposes of the study were to evaluate (1) in vitro development and quality of ig-NT FC embryos (Study 1) and (2) in vivo developmental competence of their transfer to recipients (Study 2). Methods In Study 1, 145 ig-NT FC couplets were reconstructed by fusion of the enucleated in vitro matured (IVM) domestic cat oocyte together with the starved FC fibroblast cell. The couplets were activated by inducing electrical pulses, with subsequently incubation in activation medium, comprised of cycloheximide and cytoclalasin B, for 4 h. The embryos were cultured in synthetic oviductal fluid medium supplemented with amino acids and fetal bovine serum, at 38.5°C, 5% CO2 in the humidified atmosphere, and monitored for 7 days. The blastocyst quality was evaluated by cell number count. Total of 620 IVM cat oocytes were in vitro fertilized (IVF) and served as control. The cleaved embryos collected at 27 h postinsemination (pi) were divided for their in vitro development observation in Study 1 (n = 171) and in vivo development after transferred to recipients in Study 2. The rest of the cleaved embryos were selected for further study. In Study 2, reconstructed ig-NT FC (n = 135) and IVF cat embryos (n = 75) were transferred to uterine tubes of gonadotrophintreated recipients (n = 3 in each group) on Day 2 after hCG-induced ovulation. Pregnancy was assessed by ultrasonography on Day 30. Results The reconstructed ig-NT FC couplets were 73.8% successfully fused. The fused couplets developed to 88.8% cleavage, 44.8% morula and 8.4% blastocyst stages. Oocytes from control IVF cleaved 54.5% at 27 h pi and those embryos developed to 98% 8-cell, 92% morula and 63% blastocyst stages. The cell number of ig-NT FC (n = 5) and IVF cat blastocysts (n = 22) was not significantly different (69±21 vs. 106.4±43). All (3/3) recipients receiving IVF cat embryos became pregnant and 2 recipients gave to-term kittens, whereas, none (0/3) receiving ig-NT FC embryos was pregnant. Conclusions This study establishes the efficiency of ig-NT FC embryo production. However, developmental ability of ig-NT FC embryos may be one of the limiting factors of pregnancy establishment. P522 Retinol during in vitro fertilization improves development of mouse embryo Towhidi, A 1 *, Farshidpour, MR 2 , Chamani, M 3 , Gerami, A 4 , Nouri, M 1 1Animal Science, Islamic Azad University, Shahre Qods Branch, Islamic Republic of Iran; 2 Animal Science, Islamic Azad University, Varamin Branch, Islamic Republic of Iran; 3 Animal Science, Islamic Azad University, Science and Research branch, Islamic Republic of Iran; 4 Statistics, University of Tehran, Islamic Republic of Iran Introduction Retinoids are recognized as important regulators of vertebrate development, cell differentiation, and tissue function. Pervious studies, were performed both in vivo and in vitro, indicated the influence of retinoids on several reproductive events, including follicular development, oocyte maturation and early embryonic
Page 1 and 2:
REPRODUCTION IN DOMESTIC ANIMALS 43
Page 3 and 4:
16th International Congress on Anim
Page 5 and 6:
16 t h International Congress on An
Page 7 and 8:
Page 9 and 10:
Page 11 and 12:
Page 13 and 14:
Page 15 and 16:
Page 17 and 18:
Page 19 and 20:
Page 21 and 22:
Page 23 and 24:
Page 25 and 26:
Page 27 and 28:
Page 29 and 30:
Page 31 and 32:
Page 33 and 34:
Page 35 and 36:
Page 37 and 38:
Page 39 and 40:
Page 41 and 42:
Page 43 and 44:
Page 45 and 46:
Page 47 and 48:
Page 49 and 50:
Page 51 and 52:
Page 53 and 54:
Page 55 and 56:
Page 57 and 58:
Page 59 and 60:
Page 61 and 62:
Page 63 and 64:
Page 65 and 66:
Page 67 and 68:
Page 69 and 70:
Page 71 and 72:
Page 73 and 74:
Page 75 and 76:
Page 77 and 78:
Page 79 and 80:
Page 81 and 82:
Page 83 and 84:
Page 85 and 86:
Page 87 and 88:
Page 89 and 90:
Page 91 and 92:
Page 93 and 94:
Page 95 and 96:
Page 97 and 98:
Page 99 and 100:
Page 101 and 102:
Page 103 and 104:
Page 105 and 106:
Page 107 and 108:
Page 109 and 110:
Page 111 and 112:
Page 113 and 114:
Page 115 and 116:
Page 117 and 118:
Page 119 and 120:
Page 121 and 122:
Page 123 and 124:
Page 125 and 126:
Page 127 and 128:
Page 129 and 130:
Page 131 and 132:
Page 133 and 134:
Page 135 and 136:
Page 137 and 138:
Page 139 and 140:
Page 141 and 142:
Page 143 and 144:
Page 145 and 146:
Page 147 and 148:
Page 149 and 150: 16 t h International Congress on An
Page 199: 16 t h International Congress on An
show all

Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?