Berenice de Ávila Rodrigues

Influence of reproductive status on in vitro oocyte maturation in dogs

In the bitch, oocytes need 48-72 h to complete post-ovulatory maturation to the metaphase II stage in the isthmus of the oviduct, an interval similar to that found in in vitro studies. The effect of estrous cycle stage on in vitro meiotic competence of dog oocytes has been described in several studies. However, there are no reports evaluating the possible effects of pyometra or pregnancy on subsequent potential of oocytes recovered from such females to undergo in vitro maturation. In this study, immature cumulus-oocyte complexes (COCs) were recovered from fresh excised domestic dog ovaries in various reproductive states. The donor females were classified into groups based on stage of the estrous cycle: follicular (proestrus or estrus), luteal (diestrus) or anestrus or at the clinical conditions of pregnancy and pyometra. Grades 1 and 2 oocytes were cultured in vitro at 37 degrees C in TCM-199, supplemented with 25 mM Hepes/l (v/v), and with 10% heat inactived estrous cow serum (ECS), 50 microg/ml gentamicin, 2.2 mg/ml sodium carbonate, 22 microg/ml pyruvic acid, 1.0 microg/ml estradiol, 0.5 microg/ml FSH and 0.03 IU/ml hCG. The nuclear maturation rate was evaluated at 72 h of incubation under Hoechst 33342 (10 microg/ml) staining for fluorescence microscopy. There was no statistical difference in nuclear progression to the MII stage among the various reproductive states (follicular phase, 5.4%; diestrus, 4.2%; anestrus, 4.4%; pyometra, 8.1% and pregnancy, 4.7%). Resumption of meiosis was 24.6% at the follicular phase, 19.6% for diestrus, 16.4% for anestrus, 37.1% for pyometra and 29.2% for pregnancy. Positive and higher numbers of residue above the expected value were observed for the pyometra and pregnancy conditions at the metaphase/anaphase I (MI/AI) stages.Our results indicate that in vitro nuclear maturation of dogs oocytes is not influenced by the in vivo reproductive status of the female. The quality of the oocyte is a more reliable indicator of its potential for meiotic maturation in vitro than the hormonal environment of the donor female at the time of oocyte retrieval.

Cumulus cell features and nuclear chromatin configuration of in vitro matured canine COCs and the influence of in vivo serum progesterone concentrations of ovary donors.

Phenotype integrity is viewed as an indicator of cumulus-oocyte complex (COC) viability. The objectives of this study were: (a) to observe the influence of cumulus investment expansion on the nuclear chromatin configuration of canine oocytes matured in vitro; (b) to examine the relationship between cumulus cell (CC) expansion and its morphology after in vitro maturation (IVM); (c) to ascertain the influence of in vivo serum progesterone (SP) concentrations of ovary donors on oocyte nuclear maturation, CC phenotypes and degrees of CC expansion of in vitro matured COCs. After 48 h of IVM in modified TCM 199, CCs from grade 1 and 2 COCs were stained with propidium iodide. Oocyte chromatin configuration was visualized by Hoechst 33342 stain. Results showed that oocyte IVM was not influenced by degree of CC expansion (D1, D2 and D3) in COCs. From the CC types (C1, C2 and C3), number of C1 types was higher at D1 expansion and differed from those observed at D2 and D3 expansions. Additionally, rates of apoptosis in D1 CCs were lower than those observed in D2 CCs (p < 0.05). Oocyte nuclear maturation was not influenced by in vivo SP concentrations of ovary donors. On the other hand, D3 expansion prevailed in COCs from bitches at SP > 2.5 ng/ml (p < 0.001). Moreover, in vitro CC apoptosis was associated both with low (0-1 ng/ml) and with high (>5 ng/ml) in vivo SP levels. These findings indicate that morphology of CCs from in vitro matured dog oocytes gives valuable information on viability of COCs and could possibly be used as a parameter in predicting the quality of oocytes destined for in vitro fertilization (IVF) and their outcomes.

The Influence of Oxygen Tension on Cumulus Cell Viability of Canine COCs Matured in High-Glucose Medium

High in vitro oxygen (O(2)) tensions are associated with enhanced levels of reactive oxygen species and cumulus oocyte complex (COC) apoptosis. The objective of this study was to determine the influence of O(2) tension on cumulus cell (CC) viability from canine oocytes. Cumulus oocyte complexes were distributed into three groups (CG, T20 and T5) and two O(2) tension levels (20% and 5%). The control group (CG) was matured in vitro in a humidified atmosphere with 5% CO(2) in air in TCM199 with 26.19 mM sodium bicarbonate, 10% (v/v) foetal calf serum (FCS), 0.10 mM gentamicin, 0.20 mM pyruvic acid, 20 microg/ml oestradiol, 0.5 microg/ml follicle-stimulating hormone, 0.03 IU/ml human chorionic gonadotropin, and 1.0 microg/ml human somatotropin. Groups T20 and T5 were matured under 20% or 5% O(2) tensions respectively in a high-glucose medium, without FCS. T20 and T5 were as CG, and supplemented with 0.1% Polyvinyl Alcohol, and 5.5 mM glucose. After 48 h of IVM, CCs from COCs were stained with propidium iodide (1.50 mM). The results showed that viability of CCs (cytoplasmic features and nuclear morphological integrity) was different for the three groups. Rates of apoptosis were at 57.9% (521/900) for CG, 54.4% (490/900) for T20 and 38.9% (350/900) for T5 (p < 0.001). Predominant features in apoptotic cells (n = 1361) were DNA nuclear fragments (94.0%). It was concluded that CCs of canine COCs cultured in high-glucose medium showed significantly less apoptosis than those cultured in medium with FCS. Low O(2) tension was efficient in reducing apoptosis in canine CCs.

Survival rates of mouse blastocyst vitrified in dimethylformamide based solutions associated with ethylene glicol or 1-2 propanediol

O objetivo deste estudo foi determinar o efeito da dimetilformamida (DF) associada com etileno glycol (EG) ou 1-2 propanediol (PROH) durante a vitrificacao, no desenvolvimento in vitro de blastocistos murinos. A toxicidade dos crioprotetores foi avaliada ao expor os embrioes as tres solucoes de equilibrio (ES) compostas pelas misturas de DF, EG ou PROH (10% v/v de cada) em mPBS + 0,5% PVA, em diferentes intervalos de tempo (1, 3 e 10min). Em um segundo experimento, os embrioes foram expostos as mesmas ES (durante 1 e 3min), seguido da exposicao as tres respectivas solucoes de vitrificacao (VS) (20% v/v de cada) durante 30seg. Apos 72 horas de cultivo in vitro, as taxas de expansao e eclosao dos embrioes expostos durante os periodos de 1 e 3min as solucoes de equilibrio ES1 e ES2 foram semelhantes. No entanto, os embrioes expostos durante 10min as solucoes de equilibrio com DF apresentaram taxas de sobrevivencia inferiores a solucao de EG-PROH (P<0,01). Alem disso, as taxas de sobrevivencia dos embrioes expostos a DF-PROH (ES+VS) foram menores que as dos embrioes expostos as outras solucoes (P<0,01). A vitrificacao dos blastocistos foi realizada apos a exposicao dos embrioes nas tres ES+VS (por 1min e 30seg, respectivamente), usando micropipetas de vidro (GMP). As taxas de sobrevivencia foram menores nos blastocistos vitrificados nas solucoes compostas por DF (3%-3/108 e 17,1%-19/111), em relacao a solucao EG-PROH (69%-73/105) (P<0,01). Em conclusao, a DF adicionada como crioprotetor as solucoes de vitrificacao apresenta efeitos deleterios na capacidade de desenvolvimento in vitro dos blastocistos murinos vitrificados.

Influência da secreção prostática autóloga sobre a viabilidade de espermatozóides caninos no pós-descongelamento

Investigou-se o efeito da secrecao prostatica autologa na motilidade pos-descongelamento, na integridade de acrossomo e na adesao intercelular de espermatozoides criopreservados em diluidores com TRIS-gema de ovo e com TRIS-BSA. O experimento mostrou que a adicao de secrecao prostatica promoveu um efeito benefico sobre a manutencao da integridade dos acrossomos. Nao foram observadas alteracoes com referencia a motilidade e a exposicao dos espermatozoides frente a secrecao prostatica, tambem nao levou a uma reducao significativa da adesao interespermatica. No entanto, apos 60 minutos de incubacao a 37oC, a presenca da fracao prostatica parece ter favorecido a dissociacao espermatica dos ejaculados diluidos com TRIS-BSA 0,25% (60% das amostras). Ao final do periodo de observacao, foram verificadas diferentes percentagens de sobrevivencia espermatica nas amostras diluidas com TRIS-gema de ovo (40% das amostras), com TRIS-BSA 0,25% (80% das amostras), com TRIS-BSA 0,50% (40% das amostras) e com TRIS-BSA 1,00% (50% das amostras).