R. I. T. P. Batista

Freezing goat embryos at different developmental stages and quality using ethylene glycol and a slow cooling rate.

Abstract: The efficiency of an alternative freezing protocol for goat embryos of different morphology and quality was tested. Fifty-eight embryos on Day 6-7 stage were transferred as fresh or after freeze-thawing (n=29/group). For freezing, embryos were placed into 1.5M ethylene-glycol solution for 10min. During this time, they were loaded in the central part of 0.25mL straw, separated by air bubble from columns containing PBS/BSA 0.4% plus 20% BFS. Straws were then frozen using a freezing machine from 20oC to ?6oC at a cooling rate of 3oC/min, stabilization for 15min (seeding after 5min), from ?6 C to ?32oC at 0.6 C/min,and plunged into liquid nitrogen. Frozen embryos were thawed for 30s at 37oC in a water bath. Embryos subjected to fresh transfer were maintained in holding medium (37oC). Fresh and frozen-thawed embryos were transferred at day 7 post-estrus to 30 recipients. Kidding and kid born rates were similar (P> 0.05), respectively, for recipients receiving fresh (66.7% or 10/15; 55.2% or 16/29) or frozen-thawed (60% or 9/15; 51.7% or 15/29) embryos. The cryopreservation of goat embryos using slow-freezing protocol and 1.5MEG resulted in similar efficiency rates of fresh embryos. [Congelacao de embrioes caprinos em diferentes estadios de desenvolvimento e qualidade utilizando etileno glicol e taxa de resfriamento lenta]. Resumo: Este estudo testou a eficiencia de protocolo alternativo de criopreservacao de embrioes caprinos de diferentes qualidades morfologicas. Foram utilizados 58 embrioes, coletados entre o sexto e o setimo dia do ciclo estral (n=29/grupo). Embrioes congelados passaram por solucao 1,5M etilenoglicol por 10min e foram aspirados durante esse tempo para parte central de palheta 0,25mL, separada por bolhas de ar de colunas contendo PBS 0,4% BSA e 20% SFB. As palhetas foram congeladas em maquina de congelacao de 20oC a -6oC, com taxa de resfriamento de 3oC/min, estabilizacao por 15min (seeding apos 5min), -6oC a -32oC a 0,6oC/min, e imersas em nitrogenio liquido. Os embrioes foram descongelados por 30s a 37oC, em agua. Embrioes frescos foram mantidos em solucao de manutencao (37oC). Embrioes frescos e congelados/descongelados foram transferidos para 30 receptoras no setimo dia do ciclo estral. A taxa de partos e a de crias nascidas (respectivamente) foram similares (P>0,05) para receptoras recebendo embrioes frescos (66,7% ou 10/15; 55,2% ou 16/29) ou congelados/descongelados (60,0% ou 9/15; 51,7% ou 15/29). O protocolo de criopreservacao de embrioes utilizado no presente estudo resultou em indices de eficiencia semelhantes aos de embrioes frescos.

Dose and administration protocol for FSH used for ovarian stimulation affect gene expression in sheep cumulus–oocyte complexes

The present study evaluated the effect of four ovarian stimulation protocols on the follicular population and molecular status of cumulus-oocyte complexes (COCs). Twelve Santa Inês ewes (in a cross-over design) received 80 or 120mg FSH alone in a multiple-dose (MD80 and MD120) regimen or in combination with 300IU equine chorionic gonadotrophin (eCG) in a one-shot (OS80 and OS120) protocol. The follicular population, COC recovery rate, mean COCs per ewe and the rate of brilliant Cresyl blue-positive (BCB+) COCs were similar among treatments (P>0.05). The expression of markers of oocyte competence (ZAR1, zygote arrest 1; MATER, maternal antigen that embryo requires; GDF9, growth differentiation factor 9; BMP15, bone morphogenetic protein 15; Bcl-2, B-cell lymphoma 2; BAX, Bcl-2 associated X protein) and the steroidogenic pathway (ERα, oestrogen receptor α; LHr, LH receptor; FSHr, FSH receptor; STAR, steroidogenic acute regulatory protein) was affected by stimulation. Specifically, the expression of markers of the steroidogenic pathway was reduced with increasing FSH dose in the OS protocol. FSH at a dose of 80mg reduced the expression of FSHr and ERα in the OS versus MD protocol. Conversely, in MD protocol, only LHr was affected by increasing FSH dose. In conclusion, 80mg FSH in the MD or OS protocol was sufficient to promote the development of multiple follicles and obtain fully grown (BCB+) oocytes. The MD protocol may be more appropriate for the production of better-quality oocytes.

184 EFFECT OF HISTONE DEACETYLASE INHIBITOR ON DEVELOPMENT OF EMBRYOS DERIVED FROM HEAT-SHOCKED BOVINE OOCYTES

Heat shock affects the oocyte developmental competence and embryonic gene expression. We found that the chromatin organisation of embryos derived from heat-shocked oocytes can also be affected (Camargo et al. 2015 Reprod. Fert. Dev. 27, 132). This study aimed to evaluate whether Scriptaid, a histone deacetylase inhibitor able to modulate the chromatin structure, could influence the development of embryos derived from heat-shocked oocytes. Bovine oocytes were in vitro-matured under conventional temperature (38.5°C) for 24h (non-heat-shock; NHS group) or under 41.5°C for 12h followed by 38.5°C for 12h (heat-shock; HS group). In vitro fertilization was performed under 38.5°C with 5% CO2 in air for 20h. Right after the end of fertilization the presumptive zygotes from the NHS or HS groups were denuded and randomly exposed to 500 nM Scriptaid for 0, 12, or 24h, comprising 6 treatments as followa: NHS-0h (NHS without Scriptaid, n=185); NHS-12h (NHS plus Scriptaid for 12h, n=178); NHS-24h (NHS plus Scriptaid for 24h, n=177); HS-0h (HS without Scriptaid, n=187); HS-12h (HS plus Scriptaid for 12h, n=180); and HS-24h (HS plus Scriptaid for 24h, n=183). After Scriptaid exposure, zygotes were cultured in CR2aa plus 2.5% FCS at 38.5°C with 5% CO2, 5% O2, and 90% N2. Cleavage rate was calculated on Day 2 (44h post-fertilization) and blastocyst rates on Day 7 and 8 post-fertilization. Six replicates were carried out and data was analysed by logistic regression (Proc Logistic, SAS 9.2, SAS Institute Inc., Cary, NC). Significant data were interpreted as odd ratios considering the 95% confidence interval. Values are shown as mean±standard error of the mean. There was no difference (P>0.05) among NHS treatments (NHS-0h, NHS-12h, and NHS-24h) as well as among HS treatments (HS-0h, HS-12h, and HS-24h) for cleavage and blastocyst rates at Day 7. At Day 8, however, the blastocyst rate in the NHS group decreased (P<0.05) as the time of zygote exposure to Scriptaid increased to 24h (33.9±2.8 and 24.2±1.6% for NHS-0h and NHS-24h, respectively), whereas no difference (P>0.05) was found in the HS group (20.7±1.5, 21.2±1.6, and 20.5±2.4% for HS-0h, HS-12h, and HS-24h, respectively). Comparison between NHS and HS treatments showed that cleavage and blastocyst rates at Day 7 and 8 of NHS-0h (88.7±2.8, 30.1±1.5, and 33.9±2.8%, respectively) were superior (P<0.05) to HS-0h (79.3±3.2, 16.9±1.0, and 20.7±1.5%, respectively). Differences (P<0.05) between NHS-12h and HS-12h on blastocyst rates at Day 7 (32.8±3.8 v. 20.6±1.7%, respectively) and at Day 8 (31.7±2.7 v. 21.2±1.6%, respectively) were also found. However, no difference (P>0.05) between NHS-24h and HS-24h was found. We showed that Scriptaid for 24h right after IVF has a negative impact on further development of zygotes derived from oocytes matured under conventional temperature (NHS group), in contrast to zygotes derived from oocytes matured under high temperature (HS group). We concluded that the effect of Scriptaid on embryo development is influenced by the temperature during oocyte maturation.