Vitrification of In Vitro Produced Porcine Blastocysts: Influence of Cryoprotectants Toxicity and Embryo Age

Background: Porcine embryos are sensible to all assisted reproduction manipulations, especially the ones that involve cryopreservation. Despite the high cryoprotectant concentrations routinely applied, vitrification is the most effective technique to date. These substances toxicity can also play a negative role in embryo viability. During in vitro porcine embryo production, the speed of development is often unevenly distributed. It is possible that their development speed, affects embryo tolerance to cryoprotectants. This study aimed to evaluate the toxicity of porcine embryos of days 5 or 6 of culture to cryoprotectant agents; as well as to assess embryo survival to vitrification.Material, Methods & Results: Parthenogenetic porcine blastocysts and expanded blastocysts of days 5 and 6 of culture were exposed to toxicity tests (experiments 1 and 2) and vitrification (experiment 3) using different protocols. In the first experiment, three different cryoprotectants were used (Dimethyl sulfoxide - DMSO, Ethylene glycol – EG, and Sucrose - SUC), combined in three different associations (G1: 15% EG + 15% DMSO with 0.5M SUC; G2: 16% EG + 16% DMSO with 0.4M SUC; G3: 18% EG + 18% DMSO with 0.5M SUC). In the fresh Control, embryos of day 6 are more sensible than the ones of day 5, whom showed a lower hatching rate (39.7 vs. 60.8%). After the toxicity (Experiment 1) test, the G1 showed better expansion rates in day 6 (50.0 vs 31.0 and 3.6% for G2 and G3) and higher hatching of day 6 compared to G2 and G3 (23.2, vs. 8.6 and 0.0% for G2 and G3). The fresh non hatched embryos at day 8, derived at day 6, had a lower percentage of cells with cleaved caspase-3 (20.2%) compared with the G1 (30.5%), G2 (31.4%) and G3 (30.5%). The hatched embryos of day 5 from G2 had lower total cell number (TCN) compared with the day 6 hatched embryos, whereas in G1 the TCN was not affected. The second experiment compared EG combined to one of these three extracellular cryoprotectants: Polyvinylpyrrolidone/sucrose/trehalose (respectively groups: PVP, SUC, TRE). The group SUC has raised the best results for day 5 embryos, whereas for day 6 embryos SUC and TRE were both best. The third experiment tested four vitrification protocols, being P1: EG+DMSO+TRE/warming with SUC; P2: EG+DMSO+TRE/warming TRE; P3: EG+TRE/ warming SUC; P4: EG+TRE/warming TRE. The expansion of vitrified day 5 embryos was higher in the P1 (20.0%) in comparison with the other three groups (4.3, 4.3 and 4.4% for P2, P3 and P4, respectively), with no difference for their hatching rates, been it lower comparing to the Control. Day 6 embryos showed no difference in expansion and hatching for the vitrified groups, been them lower than the Control.Discussion: Embryos obtained on day 6 are more sensible than the ones of day 5, fact observed when the embryos were exposed to cryoprotectant solution, as well by the behavior of the no treated Control embryos. The toxicity increases as it does the concentration of intracellular cryoprotectant, where over 16% of the intracellular cryoprotectors already affected the day 6 embryos development. For the day 5 embryos however, 15 or 16% of the intracellular cryoptrotectors, had similar behavior to the embryos. For the extracellular solutions, however, it is variable according the embryos development speed. Indeed, it is necessary to adjust the cryoprotectors to be used to cryopreserve porcine in vitro produced embryos obtained at days 5 and 6 of culture

Intracytoplasmic Sperm Injection after Vitrification of Immature Oocytes in Follicular Fluid Increases Bovine Embryo Production

Background: Despite the low efficiency caused by its harmful effects, vitrification is the technique of choice for oocyte cryopeservation, especially at the germinal vesicle (GV) stage. This enables the banking of female gametes without linkage to the male genotype. Follicular fluid (FF), in vivo, is known to provide an adequate environment to the immature oocyte. The intra-cytoplasmic sperm injection (ICSI), by the other hand, can be used to bypass any sperm penetration disorder, including the ones caused by cryopreservation. This study aimed to evaluate oocyte vitrification in FF based solution, and to asses ICSI efficiency in the fertilization of vitrified/warmed bovine GV oocytes.Material, Methods & Results: Follicles of 2-8 mm in diameter were aspirated from bovine ovaries obtained from a slaughterhouse, selected and maintained into FF from aspiration, until their allocation in the experimental groups. The FF used to prepare the vitrification solution was centrifuged, heat inactivated, filtered through a 0.22 mm pore and stored at -20°C. Oocyte vitrification was done into one of these three solutions: The standard solution TCM-Hepes (TH-Vitri) was compared to a totally FF based solution (FF-Vitri), and to a 50:50 (v/v) mix of both solutions (TH:FF-Vitri). Oocytes were submitted to in vitro embryo production in order to assess embryo production efficiency. A second set of experiments using the FF-Vitri solution compared IVF versus ICSI. With basis on cleaved structures, the morula + blastocyst rate obtained in the Fresh Control (43.9%) was similar to FF-Vitri (31.1%). Conversely, the TH-Vitri (15.7%) and the TH:FF-Vitri (20.4%) rates were significantly lower than the Fresh Control. ICSI showed a positive effect in comparison with IVF. The embryo development rate of Vitri-IVF (18.8%) was the lowest, whereas Vitri-ICSI (37.3%) was similar to the Fresh-IVF (43.9%), but lower than the Fresh-ICSI (57.8%).Discussion: Oocytes cryopreserved in TH based solution are known to show certain rigidity in the zona pellucida, being this event a possible cause to spermatozoa penetration disruption. Our results agree with that, since the fertilization rate for TH-Vitri was significantly lower than for the FF-Vitri. In contrast, GV oocytes vitrified in total versus partial FF based solution showed similar maturation and fertilization rates as the Fresh Control, evidencing the beneficial effect of FF during the course of vitrification. It is possible that FF helped to adjust oocyte maturation, allowing a better nuclear-cytoplasmic synchrony. Also, it might have provided some protection due to its antioxidant properties. The releasing of cortical granules induced by freezing, lead to a zona pellucida hardening and failure in sperm penetration. Factors present in the FF might block this premature releasing of cortical granules, thus ensuring that the egg retains its ability to be fertilized after maturation. The blastocysts produced from the FF-Vitri oocytes were the only ones that had the average ICM similar to the Fresh Control, evidencing that besides the similarity in morula + blastocyst rates, the embryos derived from oocytes vitrified in FF solution have also yielded best quality. When vitrified warmed oocytes were submitted to ICSI, there was an increase in the blastocyst production. This increment of embryo production with ICSI evidences a pathway to overcome the zona pellucida biological barrier. In conclusion, the use of FF as base for vitrification solution improves further embryo development; ICSI increases the embryo production of vitrified/warmed bovine GV stage oocytes

Células fetais bovinas de cultivo primário submetidas a diferentes pressões negativas antes do congelamento em palhetas

O congelamento de células é uma importante ferramenta na preservação de espécies ameaçadas de extinção. Células fetais de cultivo primário obtidas de um bovino clone foram submetidas à pressão negativa (PN) de 200, 500 ou 800 mbar, imediatamente (PN0h) ou três horas antes (PN3h) do congelamento em palhetas finas, com 10% de DMSO como crioprotetor. Células frescas e congeladas sem submissão à PN foram utilizadas como controles. Avaliou-se a viabilidade pós-descongelamento, a curva de proliferação celular, assim como o tempo de duplicação da população (PDT) celular, a cada 24 horas, durante oito dias. Os dados obtidos foram submetidos ao teste de Tukey ou Qui quadrado (P≤0,05). A sobrevivência média dos grupos controle (89,8%) e PN500 0h (88,1%) foi superior aos outros grupos; o tempo de PDT foi semelhante nos grupos fresco (27,5 ± 0,35 h), controle congelado (30,1 ± 2,3 h) e PN500 0h (32,4 ± 1,6 h). O menor tempo foi observado no grupo PN800 0h (21,9 h). O congelamento de células fetais bovinas de cultivo primário, realizado em palhetas de 0,25 mL, com 10% de DMSO, possibilita elevadas taxas de sobrevivência após o descongelamento. A PN modifica a curva de crescimento de células criopreservadas, sendo que as intensidades de 200 ou 500 mbar, aplicadas imediatamente antes do congelamento das células, possibilitam curvas de proliferação semelhantes às obtidas com células frescas.Palavras-chave: células somáticas; criopreservação; estresse controlado; preservação animal

Particularities of sperm and somatic cells in their interactions with the ooplasm: the ivf as a bovine model and the inter-species cloning as a porcine model

The adequate embryo development depends on proper nuclear-cytoplasmic interactions in the embryo. Such interactions are influenced by the donor cell type and quality of recipient oocyte on cloning, as well as by the characteristics of the sperm and the oocyte on in vitro fertilization (IVF). The first study (two experiments) investigated the effect of donor cell type (fibroblastic-like cells - FIB vs. adipocyte-derived mesenchymal stem cells - ADMSC), and host cytoplast (porcine reconstructed with 2 hemi-porcine cytoplasts; mosaic reconstructed with one-hemi-porcine and one hemi-bovine cytoplast; bovine reconstructed using 2 hemi-bovine cytoplasts), on development of porcine somatic cell nuclear transfer (SCNT) embryos. Somatic cell cultures were established from two animals of endangered pig breeds (Mule-foot and Moura) and embryos were reconstructed by hand-made cloning and cultured for 7 days in PZM-3 medium. Mosaic and bovine groups produced lower blastocyst rates than porcine (5.5, 1.9 and 18.0%, respectively). The group ADMSC-mosaic from Moura animal showed an intermediate embryo development on porcine and bovine groups, which is higher than the FIB-mosaic group of the same animal. The percentage of fragmented blastomeres in cleaved embryos and morulas from the mosaic and bovine groups were higher than the porcine. The dynamic of fusion was different according the group of cytoplasm, as observed through mitochondria staining. In the second study (three experiments), we investigated the effect of the histones de-acethylase inhibitor Scriptaid on the in vitro development of the three groups of SCNT reconstructed embryos. Reconstructed embryos were exposed to 500 nM of Scriptaid for 12 h starting just after activation, being then cultured in PZM-3 for 7 days. The blastocyst rates in the porcine (9.2 vs. 17.3%) and mosaic (1.0 vs. 9.2%) groups were increased by Scriptaid treatment (p < 0.05), and the proportion of fragmented morulas was reduced in mosaic (p < 0.05). However, Scriptaid treatment did not increase embryo development in the bovine group. In the second study (three experiments) the influence of distinct oocyte and spermatozoa qualities in their interaction in embryo IVP by IVF was evaluate. On experiment 1 and 3 the blastocyst rates were evaluated (day 7). On experiment 2, two bulls were used for IVF with good or poor oocytes. Bull A did not show difference according the oocyte quality: good (19.8%) and poor (12.7%). Bull B showed higher blastocyst rates in good quality (25.7%) than poor quality oocytes (9.2%). On experiment 2, sperm penetrating capacity was evaluated for both bulls in oocytes of low quality, by sub-zonal sperm injection. The penetration rate observed 3h after the injection from bull A (34.0%) was lower than for bull B (44.3%) (p < 0.05). On experiment 3, both bulls were used for ICSI of good or low quality oocytes and no bull or oocyte quality effect has affected blastocyst rates. In conclusion, the use of reprogramming modulators such as Scriptaid, and alternative technologies such as ICSI are adequate to provide, at least under particular conditions, an increase in embryo developmentO desenvolvimento embrionário depende da adequada interação nucleo-citoplasmática, o que é influenciado pelo tipo de célula doadora e pela qualidade do oócito receptor na clonagem, assim como por características dos espermatozóides e oócitos na fecundação in vitro (FIV). O primeiro estudo foi constituído de dois experimentos. O primeiro experimento avaliou o tipo de célula doadora de núcleo (células fibroblásticas - FIB vs. células mesenquimais derivadas de adipócitos - ADMSC), com diferentes citoplastos receptores (suíno reconstruído com dois hemi-citoplasto suínos; mosaico reconstruído com um citoplasto suíno e um citoplasto bovino; bovino reconstruído com dois hemi-citoplastos bovinos), no desenvolvimento de embriões suínos, clonados por transferência nuclear de células somáticas (TNCS). Os cultivos celulares foram estabelecidos a partir de dois suínos de raças ameaçadas de extinção (casco de mula e moura), sendo os embriões reconstruídos por clonagem manual e cultivados in vitro por 7 dias, em meio PZM-3. Os grupos mosaico e bovino apresentaram produção embrionária menor que o grupo suíno (5,5; 1,9 e 18,0%, respectivamente). O grupo ADMSC-mosaico do animal moura apresentou produção embrionária intermediaria em relação ao controle e ao bovino, e superior ao grupo FIB-mosaico do mesmo animal. A porcentagem de blastômeros fragmentados em embriões clivados e mórulas foi superior nos grupos mosaico e bovino, em relação ao grupo suíno. A dinâmica de fusão, observada conforme a migração mitocondrial entre os citoplastos, foi diferente em função do citoplasto empregado. No segundo experimento foi investigado o efeito do inibidor de desacetilases Scriptaid no desenvolvimento embrionário in vitro dos grupos suíno, mosaico e bovino, utilizando-se células fibroblastos do animal moura. Os embriões reconstruídos foram expostos a 500 nM de Scriptaid por 12 h, iniciando a partir da ativação, sendo então cultivados em PZM-3 por 7 dias. A taxa de produção de blastocistos do grupo controle (9,2 vs. 17,3%) e mosaico (1,0 vs. 9,2%) aumentou com o uso de Scriptaid (p < 0,05), enquanto a proporção de fragmentos em mórulas reduziu no grupo mosaico (9,8 vs. 2,8%) (p < 0,05). No entanto, o uso de Scriptaid não aumentou a produção embrionária no grupo bovino. No segundo estudo, constituído de três experimentos, avaliou-se a influência de distintas qualidades de gametas na produção embrionária por FIV, em bovinos. Nos experimentos 1 e 3, foram avaliadas as taxas de produção embrionária no sétimo dia de cultivo. No experimento 2, dois touros de comprovada eficiência na produção embrionária in vivtro, foram utilizados na FIV de oócitos de qualidade boa e ruim. O touro 1 não mostrou diferença na produção embrionária com oócitos de qualidade boa (19,8%) ou ruim (12,7%). O touro 2 apresentou maior produção embrionária com oócitos bons (25,7%) do que com oócitos ruins (9,2%). No experimento 2, a capacidade penetrante dos dois touros foi avaliada em oócitos de qualidade ruim através da técnica de injeção espermática sub-zonal. A taxa de penetração, observada 3 h apos a injeção, foi menor no touro 1 (34,0%) em comparação ao touro 2 (44,3%) (p < 0,05). No experimento 3, o sêmen de ambos touros foi usado para injeção intra-citplasmática de espermatozóides com oócitos de viii ambas qualidades. Não foi observado nenhum efeito de touro ou oócito na produção embrionária. Os resultados permitem concluir que o uso de moduladores da reprogramação como Scriptaid e tecnologias como a ICSI são alternativas adequadas para incrementar, ao menos em condições particulares, o desenvolvimento embrionárioCoordenação de Aperfeiçoamento de Pessoal de Nível Superio

Vitrification of In Vitro Produced Porcine Blastocysts: Influence of Cryoprotectants Toxicity and Embryo Age

Background: Porcine embryos are sensible to all assisted reproduction manipulations, especially the ones that involve cryopreservation. Despite the high cryoprotectant concentrations routinely applied, vitrification is the most effective technique to date. These substances toxicity can also play a negative role in embryo viability. During in vitro porcine embryo production, the speed of development is often unevenly distributed. It is possible that their development speed, affects embryo tolerance to cryoprotectants. This study aimed to evaluate the toxicity of porcine embryos of days 5 or 6 of culture to cryoprotectant agents; as well as to assess embryo survival to vitrification.Material, Methods &amp; Results: Parthenogenetic porcine blastocysts and expanded blastocysts of days 5 and 6 of culture were exposed to toxicity tests (experiments 1 and 2) and vitrification (experiment 3) using different protocols. In the first experiment, three different cryoprotectants were used (Dimethyl sulfoxide - DMSO, Ethylene glycol – EG, and Sucrose - SUC), combined in three different associations (G1: 15% EG + 15% DMSO with 0.5M SUC; G2: 16% EG + 16% DMSO with 0.4M SUC; G3: 18% EG + 18% DMSO with 0.5M SUC). In the fresh Control, embryos of day 6 are more sensible than the ones of day 5, whom showed a lower hatching rate (39.7 vs. 60.8%). After the toxicity (Experiment 1) test, the G1 showed better expansion rates in day 6 (50.0 vs 31.0 and 3.6% for G2 and G3) and higher hatching of day 6 compared to G2 and G3 (23.2, vs. 8.6 and 0.0% for G2 and G3). The fresh non hatched embryos at day 8, derived at day 6, had a lower percentage of cells with cleaved caspase-3 (20.2%) compared with the G1 (30.5%), G2 (31.4%) and G3 (30.5%). The hatched embryos of day 5 from G2 had lower total cell number (TCN) compared with the day 6 hatched embryos, whereas in G1 the TCN was not affected. The second experiment compared EG combined to one of these three extracellular cryoprotectants: Polyvinylpyrrolidone/sucrose/trehalose (respectively groups: PVP, SUC, TRE). The group SUC has raised the best results for day 5 embryos, whereas for day 6 embryos SUC and TRE were both best. The third experiment tested four vitrification protocols, being P1: EG+DMSO+TRE/warming with SUC; P2: EG+DMSO+TRE/warming TRE; P3: EG+TRE/ warming SUC; P4: EG+TRE/warming TRE. The expansion of vitrified day 5 embryos was higher in the P1 (20.0%) in comparison with the other three groups (4.3, 4.3 and 4.4% for P2, P3 and P4, respectively), with no difference for their hatching rates, been it lower comparing to the Control. Day 6 embryos showed no difference in expansion and hatching for the vitrified groups, been them lower than the Control.Discussion: Embryos obtained on day 6 are more sensible than the ones of day 5, fact observed when the embryos were exposed to cryoprotectant solution, as well by the behavior of the no treated Control embryos. The toxicity increases as it does the concentration of intracellular cryoprotectant, where over 16% of the intracellular cryoprotectors already affected the day 6 embryos development. For the day 5 embryos however, 15 or 16% of the intracellular cryoptrotectors, had similar behavior to the embryos. For the extracellular solutions, however, it is variable according the embryos development speed. Indeed, it is necessary to adjust the cryoprotectors to be used to cryopreserve porcine in vitro produced embryos obtained at days 5 and 6 of culture

Pre-incubation of porcine semen reduces the incidence of polyspermy on embryos derived from low quality oocytes

ABSTRACT: The main cause of low efficiency of in vitro produced porcine embryos is the high polyspermic penetration rates at fertilization, which is aggravated in low quality oocytes. Experiment 1 evaluated the embryo development in high and low quality oocytes. Experiment 2 evaluated the embryo development and quality of low quality oocytes fertilized with sperm pre-incubated during 0h (control), 0.5h, 1h and 1.5h. Experiment 3 investigated fertilization and monospermic rates of the same groups of Experiment 2. Experiment 4 evaluated embryo development, cell density, fertilization and monospermic rates of high quality oocytes using semen pre incubated during the best time observed in the previous experiments. Cleavage and blastocyst rates were analyzed by chi-square test, and remaining data by ANOVA and Tukey test (P≤0.05). The cleavage (74.8 vs 51.7%) and blastocyst (33.7 vs 9.8%) rates were greater in oocytes of high versus low quality, with no differences in cell density. Fertilization rates (65.6 to 79.5%) were not influenced by pre-incubation time. However, semen pre-incubation during 1.5h increased monospermic penetration (53.3%) and cleavage rates (92.5%) in low quality oocytes. Blastocyst rate was improved with 1.5h of semen pre incubation; however they were still lower than that observed with high quality control oocytes. Ultimately, pre-incubation did not influence fertilization, monospermic penetration, embryo development rates, nor cell density in oocytes of high quality. Low-quality porcine oocytes resulted in better rates of embryo development if in vitro fertilized with sperm pre-incubated for 1.5 hour

Intracytoplasmic Sperm Injection after Vitrification of Immature Oocytes in Follicular Fluid Increases Bovine Embryo Production

Background: Despite the low efficiency caused by its harmful effects, vitrification is the technique of choice for oocyte cryopeservation, especially at the germinal vesicle (GV) stage. This enables the banking of female gametes without linkage to the male genotype. Follicular fluid (FF), in vivo, is known to provide an adequate environment to the immature oocyte. The intra-cytoplasmic sperm injection (ICSI), by the other hand, can be used to bypass any sperm penetration disorder, including the ones caused by cryopreservation. This study aimed to evaluate oocyte vitrification in FF based solution, and to asses ICSI efficiency in the fertilization of vitrified/warmed bovine GV oocytes.Material, Methods &amp; Results: Follicles of 2-8 mm in diameter were aspirated from bovine ovaries obtained from a slaughterhouse, selected and maintained into FF from aspiration, until their allocation in the experimental groups. The FF used to prepare the vitrification solution was centrifuged, heat inactivated, filtered through a 0.22 mm pore and stored at -20°C. Oocyte vitrification was done into one of these three solutions: The standard solution TCM-Hepes (TH-Vitri) was compared to a totally FF based solution (FF-Vitri), and to a 50:50 (v/v) mix of both solutions (TH:FF-Vitri). Oocytes were submitted to in vitro embryo production in order to assess embryo production efficiency. A second set of experiments using the FF-Vitri solution compared IVF versus ICSI. With basis on cleaved structures, the morula + blastocyst rate obtained in the Fresh Control (43.9%) was similar to FF-Vitri (31.1%). Conversely, the TH-Vitri (15.7%) and the TH:FF-Vitri (20.4%) rates were significantly lower than the Fresh Control. ICSI showed a positive effect in comparison with IVF. The embryo development rate of Vitri-IVF (18.8%) was the lowest, whereas Vitri-ICSI (37.3%) was similar to the Fresh-IVF (43.9%), but lower than the Fresh-ICSI (57.8%).Discussion: Oocytes cryopreserved in TH based solution are known to show certain rigidity in the zona pellucida, being this event a possible cause to spermatozoa penetration disruption. Our results agree with that, since the fertilization rate for TH-Vitri was significantly lower than for the FF-Vitri. In contrast, GV oocytes vitrified in total versus partial FF based solution showed similar maturation and fertilization rates as the Fresh Control, evidencing the beneficial effect of FF during the course of vitrification. It is possible that FF helped to adjust oocyte maturation, allowing a better nuclear-cytoplasmic synchrony. Also, it might have provided some protection due to its antioxidant properties. The releasing of cortical granules induced by freezing, lead to a zona pellucida hardening and failure in sperm penetration. Factors present in the FF might block this premature releasing of cortical granules, thus ensuring that the egg retains its ability to be fertilized after maturation. The blastocysts produced from the FF-Vitri oocytes were the only ones that had the average ICM similar to the Fresh Control, evidencing that besides the similarity in morula + blastocyst rates, the embryos derived from oocytes vitrified in FF solution have also yielded best quality. When vitrified warmed oocytes were submitted to ICSI, there was an increase in the blastocyst production. This increment of embryo production with ICSI evidences a pathway to overcome the zona pellucida biological barrier. In conclusion, the use of FF as base for vitrification solution improves further embryo development; ICSI increases the embryo production of vitrified/warmed bovine GV stage oocytes

Effect of the cryopreservation method used, the embryonic stage and the use of conjugated linoleic acid isomers on the cryotolerance of in vitro-produced bovine embryos

Conjugated linoleic acid (CLA) might be able to improve the cryotolerance of in vitro-produced (IVP) embryos. The effect of two CLA isomers on the cryotolerance of bovine IVP embryos, as well as that of the stage of embryonic development and the method used for cryopreservation was evaluated by three experiments. In Experiment 1, oocytes (n = 3,917) were fertilized in vitro and cultured with 0, 50, 100, or 200 ?M trans-10, cis-12 (t10, c12 CLA). In Experiment 2, fertilized oocytes (n = 2,131) were cultured with 100 ?M t10, c12 or cis-9, trans-11 (c9, t11 CLA), or a combination of both isomers. The embryos were vitrified at the blastocyst (BL) or the expanded blastocyst (EB) stage. In Experiment 3, oocytes (n = 1,720) were fertilized and cultured with or without 100 ?M t10, c12 CLA, and the blastocysts were vitrified or frozen. Blastocyst development rate as well as the rates of re-expansion and hatching after thawing was recorded. Moreover, the mean cell number and mRNA expression of acetyl-CoA carboxylase (ACC1) and stearoyl-CoA desaturase (SCD1) as well as fatty acid synthase (FASN) multienzyme complex were determined. In Experiment 1, the highest concentration of t10, c12 CLA that did not reduce blastocyst development rate was 100 ?M. In Experiment 2, the rates of re-expansion and hatching among the EBs obtained through IVP after supplementation with t10, c12 CLA (73.1% and 57.7%), with c9, t11 CLA (80.0% and 68.6%), with the combination (78.3% and 52.2%), and with the control group (85.4% and 58.3%) were similar. At the BL stage, the rates of re-expansion and hatching were lower than those at the EB stage, and CLA combination allowed a hatching rate (8.0%) lower than that observed in the control group (40.0%). In Experiment 3, the hatching rates for vitrified EBs (vitrified control; 67.4%) and vitrified CLA EBs (65.8%) were higher than those obtained for frozen EBs, exposed (13.3%) or not exposed (28.6%) to CLA. In addition, in Experiment 3, the hatching rate was higher at the EB stage in vitrified groups, while the rates of BL and EB were similar in frozen groups, thus proving that vitrification was more efficient than freezing for IVP bovine embryos. In Experiment 3, CLA isomer t10, C12 did not influence the embryonic cell number or mRNA expression of ACC1 and SCD1 enzymes, but decreased the mRNA expression of FASN. In conclusion, 100 ?M CLA did not affect subsequent embryonic development. However, neither CLA isomer improved the cryotolerance of IVP bovine embryos.</p