Effect of Sodium Selenite on the Metabolite Profile of Epichloë sp. Mycelia from Festuca sinensis in Solid Culture.

Selenium (Se) is an essential micronutrient with many beneficial effects for humans and other living organisms. Numerous microorganisms in culture systems enrich and convert inorganic selenium to organic selenium. In this study, Epichloë sp. from Festuca sinensis was exposed to increasing Na2SeO3 concentrations (0, 0.1, 0.2, 0.3, and 0.4 mmol/L) in Petri dishes with potato dextrose agar (PDA) for 8 weeks. Epichloë sp. mycelia were immediately collected after mycelial diameters were measured at 4, 5, 6, 7, and 8 weeks of cultivation, respectively. Gas chromatography-mass spectrometer (GC-MS) analysis was performed on different groups of Epichloë sp. mycelia. Different changes were observed as Epichloë sp. was exposed to different selenite conditions and cultivation time. The colony diameter of Epichloë sp. decreased in response to increased selenite concentrations, whereas the inhibitory effects diminished over time. Seventy-two of the 203 identified metabolites did not differ significantly across selenite treatments within the same time point, while 82 compounds did not differ significantly between multiple time points of the same Se concentration. However, the relative levels of 122 metabolites increased the most under selenite conditions. Specifically, between the 4th and 8th weeks, there were increases in 2-keto-isovaleric acid, uridine, and maltose in selenite treatments compared to controls. Selenium increased glutathione levels and exhibited antioxidant properties in weeks 4, 5, and 7. Additionally, we observed that different doses of selenite could promote the production of carbohydrates such as isomaltose, cellobiose, and sucrose; fatty acids such as palmitoleic acid, palmitic acid, and stearic acid; and amino acids such as lysine and tyrosine in Epichloë sp. mycelia. Therefore, Epichloë sp. exposed to selenite stress may benefit from increased levels of some metabolite compounds.

Optimization of callus cultures at Echinacea purpurea L. for the amount of caffeic acid derivatives

BACKGROUND: In order to produce an effective callus in Echinacea purpurea L.; determination of the explant type and growth regulators that best respond to callus induction and the optimization of the culture conditions to increase the amount of caffeic acid derivatives (CADs) in the obtained callus. CADs contents of callus cultures of E. purpurea were evaluated by establishing an effective callus induction system in vitro. RESULTS: Various medium containing different growth regulators were tested using leaf, petiole, cotyledon and root as the explants. The best callus development was achieved in MS medium with 1.0 mg l 1 2,4- D + 2.0 mg l 1 BAP in leaf, 1.0 mg l 1 NAA + 0.5 mg l 1 TDZ in petiole, 2.0 mg l 1 NAA + 1.0 mg l 1 TDZ in cotyledon and 0.5 mg l 1 NAA + 0.5 mg l 1 BAP in roots. Upon optimisation of callus growth, each type of explant was cultured for 4, 6, 8 and 10 weeks in medium for the analyses of caftaric acid, chlorogenic acid, caffeic acid and chicoric acid contents. The highest amounts of caftaric acid (4.11 mg/g) and chicoric acid (57.89 mg/g) were found from petiole explants and chlorogenic acid (8.83 mg/g) from root explants at the end of the 10-week culture time. CONCLUSIONS: As a result of the present study, the production of caffeic acid derivatives was performed by providing the optimization of E. purpurea L. callus cultures. Effective and repeatable protocols established in this study may offer help for further studies investigating the production of caffeic acid derivatives in vitro.

Anode biofilm influence on the toxic response of microbial fuel cells under different operating conditions.

The response of microorganisms in microbial fuel cells (MFCs) to toxic compounds under different operating conditions, such as flow rate and culture time, was investigated herein. While it has been reported that MFCs can detect some toxic substances, it is unclear if operating conditions affect MFCs toxicity response. In this study, the toxic response time of MFCs decreased when the flow rate increased from 0.5 mL/min to 2 mL/min and then increased with 5 mL/min. The inhibition rates at 0.5 mL/min, 2 mL/min, and 5 mL/min were 8.4% ± 1.6%, 45.1% ± 5.3%, and 4.9% ± 0.3%, respectively. With the increase of culture time from 7 days to 90 days, the toxic response time of MFCs gradually increased. The inhibition rates at culture times of 7 days, 45 days, and 90 days were 45.1% ± 5.3%, 32.6% ± 6.6%, and 23.2% ± 1.3%, respectively. Increasing the culture time will reduce the sensitivity of MFC. The results showed that MFCs can respond quickly at a flow rate of 2 mL/min after cultivation for 7 days. Under these conditions, the power density can reach 1137.0 ± 65.5 mW/m2, the relative content of Geobacter sp. is 57%, and the ORP of the multilayers changed from -159.2 ± 1.6 mV to -269.9 ± 1.7 mV within 200 µm biofilm thickness. These findings show that increasing the flow rate and shortening the culture time are conducive for the toxicity response of MFCs, which will increase the sensitivity of MFCs in practical applications.

Different Culture Times Affect MicroRNA Cargo in Equine Amniotic Mesenchymal Cells and Their Microvesicles.

Conditioned medium (CM) and microvesicles (MVs) are produced using different protocols: CM is collected following 12-96 h of cell culture without renewal of tissue culture medium, while MVs are collected after overnight cell culture. For future comparative studies in regenerative medicine looking at the efficacy of CM and MVs, it is important to understand how the quality of cell secretions is affected by culture. The aim of this study was to evaluate whether the duration of culturing influences the micro-RNAs (miRNAs) cargo of equine amniotic mesenchymal cells (AMCs) and their MVs. The analysis identified 990 miRNAs. After one night, there were 347 differently expressed (DE)-miRNAs between MVs and cells, whereas after four nights there were 359. About 58.3% of the DE-miRNAs were shared between samples produced under the two conditions. The comparison between miRNA content in AMC cells cultured for one night versus four nights showed eight DE-Equus caballus (eca)-miRNAs, which target genes were involved in immune response to external stimulus, inflammatory response, and production of reactive oxygen species. Comparing MVs isolated from one or four nights, four DE-miRNAs that target genes regulating cell cycle progression and production of reactive oxygen species were found, but only eca-miR-214 was enriched in the MVs after four nights. In conclusion, after 4 days of cell culture, the profile of AMC miRNAs was altered, indicating a probable phenotypic transition versus a new cell culture environment and aging. After this time, MVs accumulated eca-miR-214, which may help cells survive or adapt to new culture conditions.

[Effects of sampling time on polysaccharides of Dendrobium catenatum under temperature-controlled condition].

In order to reveal the accumulation trend of polysaccharides in Dendrobium catenatum and determine the effect of sampling time on polysaccharides, D. Catenatum D21 clone was harvested from January to December after culturing for 2 to 5 months in the growth chamber with constant temperature. Polysaccharides were determined by phenol-sulfuric acid method and the monosaccharide compositions were analyzed by pre-column derivative-UPLC. The results showed that the content of polysaccharide and its key component mannose was positively correlated with the culture time, but the contents of polysaccharides in all kinds of culture peaked from 5 to 6 months, which were consistent with the trend of field planting. The results suggested that the trend of polysaccharide accumulation in the plant could be related to the life rhythm of the sensory seasons of D. catenatum, which was significantly affected by the harvesting season, even under the constant condition of the culture chamber.

Size- and time-dependent growth properties of human induced pluripotent stem cells in the culture of single aggregate.

Aggregate culture of human induced pluripotent stem cells (hiPSCs) is a promising method to obtain high number of cells for cell therapy applications. This study quantitatively evaluated the effects of initial cell number and culture time on the growth of hiPSCs in the culture of single aggregate. Small size aggregates ((1.1 ± 0.4) × 101-(2.8 ± 0.5) × 101 cells/aggregate) showed a lower growth rate in comparison to medium size aggregates ((8.8 ± 0.8) × 101-(6.8 ± 1.1) × 102 cells/aggregate) during early-stage of culture (24-72 h). However, when small size aggregates were cultured in conditioned medium, their growth rate increased significantly. On the other hand, large size aggregates ((1.1 ± 0.2) × 103-(3.5 ± 1.1) × 103 cells/aggregate) showed a lower growth rate and lower expression level of proliferation marker (ki-67) in the center region of aggregate in comparison to medium size aggregate during early-stage of culture. Medium size aggregates showed the highest growth rate during early-stage of culture. Furthermore, hiPSCs proliferation was dependent on culture time because the growth rate decreased significantly during late-stage of culture (72-120 h) at which point collagen type I accumulated on the periphery of aggregate, suggesting blockage of diffusive transport of nutrients, oxygen and metabolites into and out of the aggregates. Consideration of initial cell number and culture time are important to maintain balance between autocrine factors secretion and extracellular matrix accumulation on the aggregate periphery to achieve optimal growth of hiPSCs in the culture of single aggregate.

Culture time of vitrified/warmed zygotes before microinjection affects the production efficiency of CRISPR-Cas9-mediated knock-in mice.

Robust reproductive engineering techniques are required for the efficient and rapid production of genetically modified mice. We have reported the efficient production of genome-edited mice using reproductive engineering techniques, such as ultra-superovulation, in vitro fertilization (IVF) and vitrification/warming of zygotes. We usually use vitrified/warmed fertilized oocytes created by IVF for microinjection because of work efficiency and flexible scheduling. Here, we investigated whether the culture time of zygotes before microinjection influences the efficiency of producing knock-in mice. Knock-in mice were generated using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system and single-stranded oligodeoxynucleotide (ssODN) or PITCh (Precise Integration into Target Chromosome) system, a method of integrating a donor vector assisted by microhomology-mediated end-joining. The cryopreserved fertilized oocytes were warmed, cultured for several hours and microinjected at different timings. Microinjection was performed with Cas9 protein, guide RNA(s), and an ssODN or PITCh donor plasmid for the ssODN knock-in and the PITCh knock-in, respectively. Different production efficiencies of knock-in mice were observed by changing the timing of microinjection. Our study provides useful information for the CRISPR-Cas9-based generation of knock-in mice.

Effects of sampling time on polysaccharides of Dendrobium catenatum under temperature-controlled condition / 中国中药杂志

In order to reveal the accumulation trend of polysaccharides in Dendrobium catenatum and determine the effect of sampling time on polysaccharides, D. Catenatum D21 clone was harvested from January to December after culturing for 2 to 5 months in the growth chamber with constant temperature. Polysaccharides were determined by phenol-sulfuric acid method and the monosaccharide compositions were analyzed by pre-column derivative-UPLC. The results showed that the content of polysaccharide and its key component mannose was positively correlated with the culture time, but the contents of polysaccharides in all kinds of culture peaked from 5 to 6 months, which were consistent with the trend of field planting. The results suggested that the trend of polysaccharide accumulation in the plant could be related to the life rhythm of the sensory seasons of D. catenatum, which was significantly affected by the harvesting season, even under the constant condition of the culture chamber.

Low Agrobacterium tumefaciens inoculum levels and a long co-culture period lead to reduced plant defense responses and increase transgenic shoot production of sunflower (Helianthus annuus L.).

Agrobacterium-mediated plant transformation is typically conducted by inoculating plant tissues with an Agrobacterium suspension containing approximately 108-109 bacteria mL-1, followed by a 2-3-d co-culture period. Use of longer co-culture periods could potentially increase transformation efficiencies by allowing more time for Agrobacterium to interact with plant cells, but bacterial overgrowth is likely to occur, leading to severe tissue browning and reduced transformation and regeneration. Low bacterial inoculum levels were therefore evaluated as a means to reduce the negative outcomes associated with long co-culture. The use of low inoculum bacterial suspensions (approximately 6 × 102 bacteria mL-1) followed by long co-culture (15 d) led to the production of an average of three transformed sunflower shoots per explant while the use of high inoculum (approximately 6 × 108 bacteria mL-1) followed by short co-culture (3 d) led to no transformed shoots. Low inoculum and long co-culture acted synergistically, and both were required for the improvement of sunflower transformation. Gene expression analysis via qRT-PCR showed that genes related to plant defense response were generally expressed at lower levels in the explants treated with low inoculum than those treated with high inoculum during 15 d of co-culture, suggesting that low inoculum reduced the induction of plant defense responses. The use of low inoculum with long co-culture (LI/LC) led to large increases in sunflower transformation efficiency. This method has great potential for improving transformation efficiencies and expanding the types of target tissues amenable for transformation of different plant species.

Culture time and reagent minimization in the chemical PCC assay.

The possibility to reduce the culture time and volume of blood and reagents required for the chemical Premature Chromosome Condensation (PCC) assay is demonstrated in this work. Peripheral whole blood was exposed to gamma radiation (1-20 Gy). Lymphocytes were cultured for 40 h, using 50 µl of blood and 450 µl of culture medium. The dose-response curves were adjusted, using length ratio (LR) of the longest to the shortest chromosome piece, and the frequency of rings per cell. No statistical differences were found between the results obtained with this method and those reported with the classical PCC assay culture. The minimization of culture time and reagents in combination with the automatic measurement of the LR of the chromosome pieces, or the scoring of rings, can be a valuable biodosimetry tool in a mass casualty scenario.

How biophysical in vivo testing techniques can be used to characterize full thickness skin equivalents.

BACKGROUND: The reliability of the biophysical properties of skin equivalents (SEs) remains a challenge for medical applications and for product efficacy tests following the European Directive 2003/15/EC2 on the prohibition of animal experiments for cosmetic products. METHODS: We propose to adapt the biophysical in vivo testing techniques to compare full thickness model growth vs. time. The interest in using such techniques lies in possible comparisons between in vivo and in vitro skin as well as monitoring samples over the culture time. RESULTS: High frequency ultrasound technique, optical coherence tomography (OCT), and laser scanning microscopy were used to analyze SEs morphology at days D42 and D60 whereas their microstructure was assessed through transmission electron microscopy and classical histology. A correlation between these observations and mechanical measurements has been proposed so as to underline the consequence of both the development of the dermis elastic fibers and the epidermis differentiation. CONCLUSION: Ultrasounds measurements show a highly homogeneous dermis whereas the OCT technique clearly distinguishes the stratum corneum and the living epidermis. The increase in the thicknesses of these layers as well as the growth in elastin and collagen fibers results in strong modifications of the samples mechanical properties.

A modified culture strategy of human keratinocytes to shorten the primary culture time.

Shortening time from biopsy to graft preparation is crucial for the coverage of extensive burn wound in major burn patients. This study was aimed to shorten the culture time of keratinocytes by means of one modified cell culture method. Three factors (temperature of dispase, area of skin, and age of patient) that might have impacts on cell behavior were evaluated. The results showed that cells isolated with dispase at 37°C exhibited an improved cell activity than being isolated at 4°C. The time that cells reached subconfluence with various skin areas was in order of 3 cm(2) < 2 cm(2) < 1 cm(2) (120.07 ± 5.03 h, 141.33 ± 4.16 h, and 193.33 ± 6.11 h, respectively). Furthermore, compared with the young and middle-aged groups, proliferation of keratinocytes isolated from children skin tissue was better. These results indicated that 3 cm(2) skin biopsy from a young patient, isolated with dispase at 37°C, was a promising strategy to shorten the cultivation of keratinocytes.

In vitro 3-D model based on extending time of culture for studying chronological epidermis aging.

Skin aging is a complex phenomenon in which several mechanisms operate simultaneously. Among them, intrinsic aging is a time-dependent process, which leads to gradual skin changes affecting its structure and function such as thinning down of both epidermal and dermal compartments and a flattening and fragility of the dermo-epidermal junction. Today, several approaches have been proposed for the generation of aged skin in vitro, including skin explants from aged donors and three-dimensional skin equivalent treated by aging-inducing chemical compounds or engineered with human cells isolated from aged donors. The aim of this study was to develop and validate a new in vitro model of aging based on skin equivalent demonstrating the same phenotypic changes that were observed in chronological aging. By using prolonged culture as a proxy for cellular aging, we extended to 120 days the culture time of a skin equivalent model based on collagen-glycosaminoglycan-chitosan porous polymer and engineered with human skin cells from photo-protected sites of young donors. Morphological, immunohistological and ultrastructural analysis at different time points of the culture allowed characterizing the phenotypic changes observed in our model in comparison to samples of non photo-exposed normal human skin from different ages. We firstly confirmed that long-term cultured skin equivalents are still morphologically consistent and functionally active even after 120 days of culture. However, similar to in vivo chronological skin aging a significant decrease of the epidermis thickness as well as the number of keratinocyte expressing proliferation marker Ki67 are observed in extended culture time skin equivalent. Epidermal differentiation markers loricrin, filaggrin, involucrin and transglutaminase, also strongly decreased. Ultrastructural analysis of basement membrane showed typical features of aged skin such as duplication of lamina densa and alterations of hemidesmosomes. Moreover, the expression of hyaluronan and its surface receptor CD44 drastically decreased as observed during chronological skin aging. Finally, we found that the level of p16INK4A expression significantly increased supporting cellular senescence process associated to our model. To conclude, the major morphological and ultrastructural epidermal modifications observed in both our extended culture skin equivalent model and skin biopsies from old donors validate the relevance of our model for studying chronological aging, understanding and elucidating age-related modifications of basic skin biological processes. In addition, our model provides a unique tool for identifying new targeted molecules intended at improving the appearance of aging skin.

The effect of the number of transferred embryos, the interval between nuclear transfer and embryo transfer, and the transfer pattern on pig cloning efficiency.

To improve the efficiency of producing cloned pigs, we investigated the influence of the number of transferred embryos, the culturing interval between nuclear transfer (NT) and embryo transfer, and the transfer pattern (single oviduct or double oviduct) on cloning efficiency. The results demonstrated that transfer of either 150-200 or more than 200NT embryos compared to transfer of 100-150 embryos resulted in a significantly higher pregnancy rate (48 ± 16, 50 ± 16 vs. 29 ± 5%, p<0.05) and average litter size (4.1 ± 2.3, 7 ± 3.6 vs. 2.5 ± 0.5). In vitro culture of reconstructed embryos for a longer time (40 h vs. 20 h) resulted in higher (p<0.05) pregnancy rate (44 ± 9 vs. 31 ± 3%) and delivery rate (44 ± 9 vs. 25 ± 9%). Furthermore, double oviductal transfer dramatically increased pregnancy rate (83 ± 6 vs. 27+8%, p<0.05), delivery rate (75 ± 2 vs. 27+8%, p<0.05) and average litter size (6.5 ± 2.8 vs. 2.6 ± 1.2) compared to single oviductal transfer. Our study demonstrated that an improvement in pig cloning efficiency is achieved by adjusting the number and in vitro culture time of reconstructed embryos as well as the embryo transfer pattern.

Human bone marrow-derived mesenchymal stem cell gene expression patterns vary with culture conditions.

BACKGROUND: Because of the heterogeneity of human mesenchymal stem cells (MSCs), methods for cell expansion in culture and the effects on gene expression are critical factors that need to be standardized for preparing MSCs. We investigated gene expression patterns of MSCs with different seeding densities and culture times. METHODS: Bone marrow-derived MSCs were plated at densities from 200 cells/cm(2) to 5,000 cells/cm(2), and the gene expression patterns were evaluated over time using a reverse-transcription polymerase chain reaction assay. RESULTS: The mRNA levels of factors that play a critical role in cell migration and tissue regeneration, such as podocalyxin-like protein (PODXL), α4-integrin, α6-integrin, and leukemia inhibitory factor (LIF), were higher in MSCs plated at 200 cells/cm(2) than in MSCs plated at 5,000 cells/cm(2). The mRNA levels of these factors gradually increased for 10 days and then decreased by day 15 in culture. MSCs seeded at 200 cells/cm(2) that were cultured for 10 days expressed high levels of Oct-4 and Nanog. Indoleamine 2,3-dioxygenase, cyclooxygenase-1, and hepatocyte growth factor expression were upregulated in the presence of the proinflammatory cytokine interferon-γ in these cells. CONCLUSION: We found differences in the gene expression patterns of MSCs under different culture conditions. MSCs from 10-day cultures seeded at a low density were efficiently expanded, expressed PODXL, α6-integrin, α4-integrin, and LIF, and maintained properties like stemness and immunomodulation. Therefore, ex vivo expansion of MSCs maintained for an adequate culture time after plating at low cell density can provide an effective regenerative medicinal strategy for cell therapies using MSCs.

Human bone marrow-derived mesenchymal stem cell gene expression patterns vary with culture conditions

BACKGROUND: Because of the heterogeneity of human mesenchymal stem cells (MSCs), methods for cell expansion in culture and the effects on gene expression are critical factors that need to be standardized for preparing MSCs. We investigated gene expression patterns of MSCs with different seeding densities and culture times. METHODS: Bone marrow-derived MSCs were plated at densities from 200 cells/cm2 to 5,000 cells/cm2, and the gene expression patterns were evaluated over time using a reverse-transcription polymerase chain reaction assay. RESULTS: The mRNA levels of factors that play a critical role in cell migration and tissue regeneration, such as podocalyxin-like protein (PODXL), alpha4-integrin, alpha6-integrin, and leukemia inhibitory factor (LIF), were higher in MSCs plated at 200 cells/cm2 than in MSCs plated at 5,000 cells/cm2. The mRNA levels of these factors gradually increased for 10 days and then decreased by day 15 in culture. MSCs seeded at 200 cells/cm2 that were cultured for 10 days expressed high levels of Oct-4 and Nanog. Indoleamine 2,3-dioxygenase, cyclooxygenase-1, and hepatocyte growth factor expression were upregulated in the presence of the proinflammatory cytokine interferon-gamma in these cells. CONCLUSION: We found differences in the gene expression patterns of MSCs under different culture conditions. MSCs from 10-day cultures seeded at a low density were efficiently expanded, expressed PODXL, alpha6-integrin, alpha4-integrin, and LIF, and maintained properties like stemness and immunomodulation. Therefore, ex vivo expansion of MSCs maintained for an adequate culture time after plating at low cell density can provide an effective regenerative medicinal strategy for cell therapies using MSCs.

Multiplicación en sistema de inmersión temporal del clon de Malanga Viequera -Xanthosoma spp / Multiplication in temporary immersion system cocoyam clone Viequera - Xanthosoma spp

El Sistema de inmersión temporal (SIT) constituye una alternativa en la micropropagación de plantas. El presente trabajo se realizó con el objetivo de establecer un protocolo para la multiplicación en SIT del clon de malanga “Viequera”. Se evaluó el efecto de tres tiempos de inmersión (7, 14 y 21 minutos), tres frecuencias de inmersión (2, 4 y 6 horas por día), cuatro volúmenes de medio de cultivo (5, 10, 15 y 20 ml por brote inicial) y cuatro tiempos de cultivo (15, 18, 21 y 25 días) en la multiplicación de los brotes de yemas axilares. Con tiempo de inmersión de 14 minutos cada 4 horas, un volumen de 15 ml de medio de cultivo por brote inicial y 18 días de cultivo, se logró el mejor comportamiento en la multiplicación de los brotes de yemas axilares, con un coeficiente de multiplicación de 10,50. El protocolo propuesto aumenta la productividad del material propagado en comparación con los desarrollados en medios de cultivo semisólidos, lo que representa una reducción en los costos de producción al introducir la multiplicación del cultivo en laboratorios comerciales de propagación.

Temporary Immersion System (TIS) is a alternative in the micropropagation of plants. This work was carried out to establish a protocol for the multiplication of clone TIS cocoyam “Viequera”. The effect of three immersion times (7, 14 and 21 minutes), three immersion frequencies (2, 4 and 6 hours per day), four volumes of culture medium (5, 10, 15 and 20 mL per shoot initial) and four times of cultivation (15, 18, 21 and 25 days) in the multiplication of shoots from axillary buds. With immersion time of 14 minutes every 4 hours, a volume of 15 ml of culture medium for initial outbreak and 18 days of culture, achieved the best performance in the multiplication of shoots from axillary buds, with a coefficient of multiplication 10.50. The proposed protocol increases the productivity of propagated material compared to those developed in semisolid culture media, representing a reduction in production costs by introducing the increase in cultivation in commercial laboratories.

Effect of two temperatures on in vitro nuclear maturation of bitch oocytes: relation to time culture intervals

In the present study, two commonly incubation temperatures used on in vitro oocyte maturation in canine species (Canisfamiliaris) were analysed with the aim to verify the rate of nuclear maturation of bitch oocytes after three culture intervals (48, 72and 96 h). The culture medium was TCM 199, supplemented with 25mM Hepes/l (v/v), with 10% heat inactivated estrous cowserum (ECS), 50mg/mL gentamicin, 2.2mg/mL sodium bicarbonate and 22mg/mL pyruvic acid, 1.0mg/mL oestradiol (E 8875Sigma), 0.5mg/mL FSH (Folltropin-V, Vetrepharm Inc. Ont, Canada) and 0.03IU/mL hCG (Profasi HP, Serono, Aubonne,Switzerland). Oocytes were recovered from ovaries of bitches at random estrous cycle stages by routine ovariohysterectomy(n=14) or by therapeutical ovariohysterectomy (n=8) at the veterinary hospital of the Universidade Federal do Rio Grande do Sul(UFRGS), Brazil. There was no statistical difference in the rate of meiosis of oocytes matured at 37oC or at 39oC at any time point.However, the proportion of oocytes that reached the metaphase II (MII) stage at 37o C after 72 h of in vitro culture (16/123; 13%),tended to be higher (P=0.064), when compared to that matured at the 39oC (6/148; 4,1%). The results demonstrate thattemperatures of 37oC and 39oC are equally effective for the in vitro culture of bitch oocytes.