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Acta Scientiae Veterinariae, 2011. 39(Suppl 1): Abstracts - 25th Annual Meeting SBTE-Brazil. August 2011. A259 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS” MOURA PIG SPERM VIABILITY AFTER CRYOPRESER OPRESERVATION Mar ariana Grok oke Mar arques ques, Almir lmiro Dahmer ahmer, Vit itor Hugo Grings & Elsio Antonio Per ereia eia de Figueir igueiredo edo EMBRAPA SUÍNOS E AVES, CONCORDIA, SC, BRAZIL. The Moura pig breed was introduced soon after the discovery of Brazil, and possibly descended from Iberian breeds. In the first decades of the 20th century, the breed was widespread in southern Brazil. The main characteristics of this breed are rusticity, disease resistance and meat production with a higher rate of marbling (Favero et al., 2007, Rev Bras Agroecology 2, 1662-65). On the other hand, the breed selection has reduced genetic variability, limiting the Moura pigs to only 29 animals registered in the ABCS (Brazilian association of swine producers). This data demonstrates the importance of the formation of a germplasm bank for this breed. The objective of this study was to evaluate the effect of cryopreservation on the quality of Moura pig semen. The rich fraction of the ejaculate of 9 males was frozen by the method of Hulsenberg (Westendorf et al., 1975, Dtsch Tierärztl Wochenschr 82, 261-67) with 5 x 10 9 spermatozoa/straw. After collection and thawing, semen samples were evaluated for sperm motility, plasma membrane integrity (using the eosin/nigrosine dye test), acrosomal integrity (using Pope dye), and mitochondrial activity (Hrudka 1987, Int J Androl 10, 809-28). The data were compared using a Sign test (Campos 1983, Statistical experimental non parametric, ed. 4, 349). Average losses of 52.91% of motile spermatozoa (78.33 ± 3.22% and 25.42% ± 3.56, collection and thawing, respectively, P < 0.0001), and 40.67% in membrane integrity (74.00 ± 3.81% and 33.33% ± 3.47 collection and thawing, respectively, P < 0.0001) were observed. A milder decrease (12.61%) was observed in acrosomal integrity (63.50 ± 2.64% and 51.33% ± 3.25, collection and thawing, respectively, P= 0.0193). Regarding mitochondrial activity after thawing, 82% of sperm mitochondrial activity in grades 1 or 2 was observed, indicating the predominance of active sperm. Rath et al. (2009, Soc Reprod Fertil Suppl 66, 51-66) describe the successful results of fertility using deep intra-uterine insemination (DUI) with 1-2 x 10 9 viable sperm after thawing. Although semen cryopreservation in pigs is challenging, and ejaculates of Moura pigs were not previously selected due to few available boars, the data shown here demonstrates that the semen of Moura pigs has good quality, and consequently can be used in DUI. Banking of Moura pig semen would serve a useful purpose in expansion of the breed, both genetically and in number. Keywords: cryopreservation, semen, moura. A260 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS” VITRIFICATION TION OF IN VITRO PRODUCED BOVINE EMBRYOS AFTER CHEMICAL LIPOLYSIS D aniela Mar artins Paschoal 1 , Mat eus José Sudano 2 , Tatiana da Silv ilva Rasc ascado ado 1 , Luis Car arlos Oña Magalhães 1 , Letícia Fer err ari i Cro c omo 1 , Joao Ferreira de Lima Neto 1 , Midyan Daroz Guastali 1 , Rosiara Rosária Dias Maziero 1 , Alicio Martins Júnior 2 & Fernanda da Cruz Landim Alvarenga 1 1 FMVZ/UNESP BOTUCATU, BOTUCATU, SP, BRAZIL. 2 FMVA/UNESP ARAÇATUBA, ARAÇATUBA, SP, BRAZIL. The present experiment aimed to induce cytoplasmic lipolysis in IVP bovine embryos using forskolin (Sigma) which is an activator agent of adenylate cyclase and is related to cAMP levels (Seamon et al., 1981; Proc Natl Acad Sc USA 78, 3363-67). Presumptive zygotes were cultured with SOFaa+BSA in the FCS presence or absence until D7. In D6 was added forskolin (F) in the median resulting in 4 groups: FCS (2.5% FCS and without F), 0%FCS (without FCS and F), FCS+F (2.5% FCS + 10 µM F) and 0%FCS+F (without FCS + 10 µM F). The cleavage was analyzed on D3 and the blastocyst rate on D7. On seventh day was performed: viability and ultra structural evaluation and embryos vitrification followed by viability evaluation. The IVP embryos were compared to in vivo-produced embryos. Data were analyzed with ANOVA, using the general linear model (GLM) SAS (SAS Inst Inc, Cary, NC, USA). Sources of variation in the model included FCS concentration, forskolin addition and first order interactions; all factors were considered fixed effects. The arcsine transformation (vy/100) was applied to percentage data. If the ANOVA was significant, means were separated by a Tukey’s test. There were no differences on cleavage, blastocyst production rates and the number of cells/embryo between in vitro and in vivo produced groups (P > 0.05). A higher rate of damaged cells was observed in 0%FCS group (11.3 ± 11.3 b ) comparison with FCS and FCS+F groups (P < 0.01). In vivo-produced embryos (5.1 ± 1.4ab) were similar to IVP embryos (FCS: 1.1 ± 1.3 a , 0%FCS: 11.3 ± 11.3 b ; FCS+F: 1.6 ± 3.2 a , 0%FCS+F: 6.8 ± 6.9 ab ). With the ultrastructural evaluation, the same characteristics were found between IVP and in vivo groups. The FCS group had higher amounts of lipid droplets, which decreased after the F addition. Just as the 0%FCS group had a higher rate of damaged cells, it showed a greater presence of cellular debris (cellular degeneration) being reduced with F addition. After vitrification there was no difference on re-expansion rate between the groups (P > 0.05), but the in vivo group (124.2 ± 12.2 b ) had a greater number of cells/embryo in relation to IVP groups (FCS: 42.6 ± 17.2 a ; 0%FCS: 65.1 ± 34.7 a ; FCS+F: 59.9 ± 46.2 a ; 0%FBS+F: 40.1 ± 12.6 a ; P < 0.05). When the damaged cells rate was assessed, the FCS group showed the highest value (69.7 ± 20.7 a ) compared to 0%FCS (20.3 ± 22.7 b ), FCS+F (39.4 ± 33.0 b ), 0%FCS+F (27.0 ± 26.7 b ) and in vivo (14.1 ± 20.1 b ; P < 0.05). The serum presence was harmful to embryo cryopreservation, but this effect was reversed when forskolin was added. Keywords: ivp, forskolin, vitrification. s466
Acta Scientiae Veterinariae, 2011. 39(Suppl 1): Abstracts - 25th Annual Meeting SBTE-Brazil. August 2011. A261 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS” VITRIFICATION TION OF IN VITRO PRODUCED BOVINE EMBRYOS SUPPLEMENTED WITH TRIIODOTHYR THYRONINE (T3) AND THYROXINE (T4) Maria Clara Costa Mattos, Emerson Roberto Siqueira, Flávia Spessopoto Pavan Rochelle, Ana Carolina Freitas Pereira, Bruno Valente Sanches, José Henrique Fortes Pontes, André Gomiero Rigo, Lucas Lopes Moino, Rodrigo Mendes Untura & Andrea Cristina Basso IN VITRO BRAZIL S/A, MOGI-MIRIM, SP, BRAZIL. The aim of this study was to evaluate the triiodothyronine (T3) and thyroxine (T4) supplementation on in vitro early embryonic development and cryotolerance of these vitrified IVP embryos. Oocytes from ovaries of slaughterhouse were fertilized with conventional sperm from a single bull. After 18 to 22 h, presumptive zygotes were denuded and divided into two groups: In T3T4 Group (n = 322) were added 50 mg/mL of T3 (Sigma 6397) and 50 ng/mL de T4 (Sigma 2502) to the culture media and in the Control Group (n = 392) in vitro embryo culture (IVC) was performed without T3 and T4 addition. Embryos were cultured for seven days in 38.5°C and atmosphere with 5% of CO2, 5% of O2 and 90% of N2. The cleavage and blastocyst production were assessed from the total number of oocytes on Days 3 and 7, respectively. On Day 7, embryos were vitrified at grade 1 blastocyst stage by Cryotop method with cryoprotectants solutions of Ethylene Glycol (EG) + Dimethyl sulfoxide (DMSO). Embryos of both groups were thawed and placed in culture for 48 h to assess hatching rates. The cleavage rate in T3T4 Group (79.8%) was higher than the Control Group (72.3%, P = 0.03). However, the blastocyst rate was not different between groups (27.3% in T3T4 Group and 31.4% in Control Group, P = 0.24). The percentage of vitrified blastocysts did not differ between groups (44.3% in T3T4 Group and 43.3% in Control Group, P = 0.86). Finally, the hatching rates after 48 h of culture were also similar between T3T4 Group (87%, n = 39) and Control Group (85%, n = 53; P = 0.85). Based on these results, we conclude that T3 and T4 supplementation did not increase blastocyst production. However, it seems not affect the quality and cryotolerance of IVP embryos. Therefore, future studies should also consider the mechanisms of action of these hormones during early in vitro embryonic development and its impact on the conception rates of cryopreserved embryos. [Acknowledgements: In vitro Brazil SA.]. Keywords: bovine embryo, vitrification, t3 and t4. A262 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS” BOVINE OOCYTE VITRIFICATION: TION: EFFECTS ON FERTILIZA TILIZATION TION RATES José Felipe Warmling Sprícigo 1 , Rodolfo Rumpf 2 & Margot Alves Nunes Dode 2 1 UNIVERSIDADE DE BRASÍLIA, BRASILIA, DF, BRAZIL. 2 EMBRAPA-CENARGEN, BRASÍLIA, DF, BRAZIL. Although vitrification has been reported as the most promissory method for cryopreservation of oocytes, it is quite inefficient, N resulting in low rates of cleavage and blastocyst formation. This information suggest that vitrification effects can already be observed in the beginning of embryonic development. The aim of this study was to evaluate the fertilization rates of bovine oocytes submitted to vitrification by cryotop at different moments, during in vitro maturation (IVM). Cumulus-oocyte-complexes were obtained from slaughterhouse ovaries, were selected and distributed into four groups: non-vitrified, used as control group (CG), vitrified immediately after selection (V0), vitrified at 8 h (V8) and vitrified at 22 h (V22) of IVM. After warming the four groups were returned to the incubator to complete the 24 h of maturation. At the end of IVM, the four groups were transferred to fertilization drops and incubated for 18 h with sperm selected by Percoll gradient. At the end of the period of fertilization the oocytes of the four groups were denuded, fixed and stained with lacmoid for the assessment of fertilization. Zygote that presented sperm head, decondensed sperm head, male and females pronuclei, cleavage or polyspermy were classified as fertilized. And those with unidentifiable chromatin, were classified as degenerated. Data were compared using Chi-square test (P < 0.05). Oocytes of V0 group (n = 112) showed the lowest percentage of fertilization (44%) among the vitrified groups, while V8 (n = 119) and V22 (n = 106) showed lower percentages compared to CG (n = 143) with rates 63%, 66% and 81% respectively. The rate of unfertilized oocytes was similar among all groups, but groups of vitrified oocytes showed a higher percentage of polyspermy CG (11%, 3%), V0 (9%, 6%), V8 (12%, 7%) and V22 (7%, 12%). The most discrepant difference between GC (7%) and vitrified treatments was observed for the percentages of degenerated oocytes, where V0 (46%) was the most affected (P < 0.05), followed by V8 (24%) and V22 (25%), which were similar. The results suggest that vitrification reduces the fertilization rate and induces an increase in the rate of degenerated oocytes, especially in the stage of germinal vesicle. Therefore, the reduction in fertilization rate is due to loss of oocyte viability that occurs as a result of vitrification procedure, and immature oocytes in GV are the most affected ones.[Financial support: CNPq, CAPES and EMBRAPA]. Keywords: vitrification, bovine, oocyte. s467
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