scholarly journals 291 EFFECT OF DIFFERENT TRANSPORT TEMPERATURES (+4°C, +32°C) ON IN VITRO MATURATION OF OOCYTES COLLECTED FROM CATTLE AND SHEEP OVARIES

2005 ◽  
Vol 17 (2) ◽  
pp. 296
Author(s):  
O.B. Ozdas ◽  
M. Tas ◽  
U. Cirit ◽  
M. Evecen ◽  
K. Demir ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Developmental Stages ◽  
Chi Square ◽  
Medium Temperature ◽  
Factors Affecting ◽  
Group I ◽  
Significant Difference ◽  
Temperature Group ◽  
C 32

At present, blastocyst rates in embryos obtained from in vitro maturation of oocytes, and their fertilization and culture, is still not at the desired level. One of the most important problems encountered in in vitro culture studies is seen in the maturation period of oocytes until they reach the fertilizable level. Transport time of the ovaries and, in particular, temperature of the transport medium used are among the factors affecting complete maturation. The aim of this study was to determine the effects of different transport temperatures (4°C, 32°C) of sheep and cattle ovaries on the in vitro maturation of oocytes. Two experimental groups were formed in the study. Sheep and cattle ovaries were put into saline solution at 32°C. The ovaries were transported at the same temperature (Group I) or at 4°C following a 10-min incubation at room temperature (Group II), in 2–4 h to the laboratory (n = 6). For each group, oocytes were collected from ovaries using the dissection method and selected oocytes were matured in their own group in 700 μL TCM-199 (supplemented with pyruvate, LH, FCS) for 23 h at a gas atmosphere of 5% CO2, 5% O2, 90% N2 and at 38.8°C. At the end of maturation, oocytes were cleansed from their cumulus oophorus cells and fixed in acetic acid-ethyl alcohol (1:3) for 48 h. The developmental stages until MII of oocytes stained with aceto-orcein were then examined under the phase contrast microscope. The chi-square test was used for statistical analysis (Table 1). While oocytes obtained from sheep ovaries transported at +32°C reached the MII stage at a faster rate compared to those at +4°C (P < 0.001), no statistically significant difference was observed between the maturation to the MII stage of oocytes obtained from cattle ovaries transported at +4°C and +32°C. As a result of this study, while it was established that cattle ovaries could be transported at both +4°C and +32°C and that there was no difference in oocyte maturation, a medium temperature of +4°C was determined to be unsuitable for transporting sheep ovaries. Table 1. Stages of development in sheep and cattle oocytes after 23 h of culture This work was supported by Istanbul University.

53 EFFECT OF THE TIME INTERVAL BETWEEN OVARY COLLECTION AND OOCYTE IN VITRO MATURATION ON EQUINE CLONED EMBRYO DEVELOPMENT

2010 ◽  
Vol 22 (1) ◽  
pp. 184
Author(s):  
A. Gambini ◽  
J. Jarazo ◽  
R. Olivera ◽  
D. Salamone
Keyword(s):  
Embryo Development ◽  
In Vitro Maturation ◽  
Limiting Factor ◽  
Time Interval ◽  
Activation Process ◽  
Blastocyst Development ◽  
Sodium Pyruvate ◽  
Chi Square ◽  
Group I

The availability of viable equine oocytes is a limiting factor on in vitro embryo production; therefore, it is necessary to assess some of the variables that affect oocyte viability. The aim of our study was to evaluate one of those variables: the effect of time between the collection of the ovary and oocyte in vitro maturation. Ovaries of slaughtered mares were collected during the breeding season (Argentine, Southern hemisphere). They were separated in bags every half hour and treated separately after arriving at the laboratory. COCs were recovered by a combination of scraping and washing of all visible follicles with a syringe filled with DMEM supplemented with 1 mM sodium pyruvate and 15 IU mL-1 heparin. COCs were matured for 24 to 26 h in 3 groups, according to time interval: 4 to 7 (group I), 7 to 10 (II), and 10 to 12 (III) hours. The medium for maturation was TCM-199 supplemented with 10% fetal bovine serum (FBS), 1 μL mL-1 insulin-transferrin-selenium, 1 mM sodium pyruvate, 100 mM cysteamine, and 0.1 mg mL-1 of FSH at 39°C in a humidified atmosphere of 5% CO2 in air. The cumulus was removed by a trypsin treatment and vortexing in hyaluronidase (1 mg mL-1). Cloning and fusion procedures were performed following the zona-free technique described by Lagutina et al. (2007 Theriogenology 67, 90-98). Two experiments were carried out by using different activation protocols. In experiment 1, the activation process was 22 mM ionomycin in H-TALP for 4 min followed by 3h culture in 1.9 mM 6-DMAP in SOF, whereas in experiment 2, we used 8.7 mM ionomycin in H-TALP for 4 min followed by 4 h culture in 1 mM 6-DMAP and 10 mg mL-1 cycloheximide in SOF. Embryos were cultured in wells of well (WOW) system. Half of the medium was renewed on Day 3 with fresh SOF and on Day 5 with DMEM/F12 with 10% FBS. Cleavage was assessed 48 h after activation; the rate of blastocyst formation was recorded at Days 8 and 9. Results were compared using chi-square test (P < 0.05). In experiment 1, maturation rates were significantly different between group I (n = 135, 54.1%) and III (n = 94, 40.4%), group II did not differ from them (n = 138, 53%). Cleavage rates differed statistically between II (n = 44, 75%) and III (n = 27, 40.7%), but not with group I (n = 53, 98%). No significant differences were found in blastocyst development; however, we observed a certain tendency towards an increase in the blastocyst rate as the time interval was lower (I: 3/53, 5.7%; II: 1/44, 2.3%; III: 0/27, 0%). In experiment 2, there were no significant differences between group I and II in rates of maturation (n = 56, 59% v. n = 111, 44.5%), cleavage (n = 22, 91% v. n = 34, 82%) or blastocyst rates (1/22, 4.5% v. 7/34, 20.6%). We conclude that cloned equine embryo development, using the two activation protocols tested, is not affected when the time interval between ovary collection and oocyte IVM is within 4 to 10 h.

scholarly journals 46 Effect of the addition of α-tocopherol to in vitro maturation media of bovine oocytes

2020 ◽  
Vol 98 (Supplement_2) ◽  
pp. 2-3
Author(s):  
Theisy P Acosta Pérez
Keyword(s):  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Control Group ◽  
Chi Square ◽  
Cumulus Expansion ◽  
Minimum Concentration ◽  
Maturation Rate ◽  
Chi Square Test ◽  
System Data

Abstract α-tocopherol is known to be a powerful antioxidant, in this regard, it was added to bovine oocyte in vitro maturation media to evaluate its effect on oocyte maturation. Oocytes (n = 624) aspirated from ovaries of slaughtered cows were classified by quality and divided in four categories according to cytoplasm appearance and cumulus cells layers. Oocytes were washed in TCM-199 supplemented with fetal bovine serum (FBS) and FSH, then distributed in maturation media (TCM-199 supplemented with FBS, FSH and gentamicin). Three experimental groups of α-tocopherol (50, 100 and 200 mM) and a control group without α-tocopherol were used. Maturation was carried 22 h at 38.5°C in a 5% CO2 atmosphere. Oocytes were examined to determine cumulus expansion as categorical data (expansion or no expansion), as well as cumulus expansion Index (CEI). For CEI determination oocytes were graded 0 to 4, being 0 those with null expansion and 4 those with a noticeable cell expansion, then the number of oocytes were multiplied by the grade given and a sum of the totals was obtained, the new total was divided by the total of oocytes in the group and the result obtained corresponded to the CEI of the group. Results were analyzed with Chi Square test (for maturation rates) and an ANOVA (for the CEI) using the SAS system, data are presented as mean ± standard error. There was no statistical difference between control and α-tocopherol groups (P &gt;0.05). Numerically, the control group showed a higher maturation rate (100%) and obtained a higher CEI (2.44±0.20), followed by the 50 mM group (98.16%; 2.39±0.13), the groups 200 mM (97.40%; 2.00±0.14) and 100 mM (96.25%; 2.06±0.24) were the lowest. The addition of the minimum concentration (50 mM) of α-tocopherol to the maturation media could improve maturation rates without exposing oocytes to toxic effects.

scholarly journals Comparative Evaluation of Fracture Resistance of Endodontically Treated Teeth Restored with Resin Fiber Post and Stainless Steel Post: An In Vitro Study

2015 ◽  
Vol 03 (02) ◽  
pp. 080-084
Author(s):  
Vijay Singh ◽  
Poonam Bogra ◽  
Saurabh Gupta ◽  
Navneet Kukreja ◽  
Neha Gupta
Keyword(s):  
Stainless Steel ◽  
Fracture Resistance ◽  
Testing Machine ◽  
Compressive Force ◽  
Control Group ◽  
Fiber Post ◽  
Endodontically Treated Teeth ◽  
Group I ◽  
Significant Difference

AbstractFracture resistance of endodontically treated teeth restored with post. Aims: This study aims to compare the fracture resistance of endodontically treated teeth restored with resin fiber and stainless steel post. Commercially available prefabricated resin fiber post(Dentsply Maillefer Easy Post), prefabricated stainless steel post(Coltene/Whaledent Parapost) were used. Methods and Material: Forty five maxillary central incisors were obturated and divided into 3 groups: Control Group (Group I) without any post (n = 15), Resin Fiber Post Group (Group II) (n = 15) and Stainless Steel Post Group (Group III) (n = 15). In all Groups except control group, post space was prepared; a post was cemented, and a core build-up was provided. All the specimens were subjected to compressive force under a universal testing machine until fracture. Statistical analysis used: The results were analyzed using the variable analysis test (ANOVA). Results: One-way analysis of variance revealed significant difference among test groups. The control group demonstrated highest fracture resistance (925.2183 N), followed by the resin fiber post group (486.7265 N) and stainless steel post group (423.539N). Conclusions: Teeth restored with resin fiber post showed higher fracture resistance values than prefabricated stainless steel post.

P–431 Human oocytes in-vitro maturation efficacy from infancy to adulthood – is there an optimal age?

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
G Karavani ◽  
P Wasserzug-Pash ◽  
T Mordechai-Daniel ◽  
M Klutstein ◽  
T Imbar
Keyword(s):  
Fertility Preservation ◽  
In Vitro Maturation ◽  
Ovarian Tissue ◽  
Age Groups ◽  
Ovarian Tissue Cryopreservation ◽  
Success Rates ◽  
Human Oocytes ◽  
Tertiary Center ◽  
Significant Difference

Abstract Study question Does human oocytes in-vitro maturation (IVM) effectiveness change throughout childhood, adolescence and adulthood in girls and women undergoing fertility preservation via ovarian tissue cryopreservation (OTC) prior to chemo-radiotherapy exposure? Summary answer The optimal age for IVM is from menarche to 25 years, while pre-menarche girls and women older than 30 years have extremely low maturation rates. What is known already In vitro maturation of oocytes from antral follicles seen during tissue harvesting is a fertility preservation technique with potential advantages over OTC, as mature frozen and later thawed oocyte used for fertilization poses decreased risk of malignant cells re-seeding, as compared to ovarian tissue implantation. We previously demonstrated that IVM performed following OTC in fertility preservation patients, even in pre-menarche girls, yields a fair amount of oocytes available for IVM and freezing for future use. Study design, size, duration A retrospective cohort study, evaluating IVM outcomes in chemotherapy naïve patients referred for fertility preservation by OTC that had oocyte collected from the medium with attempted IVM between 2003 and 2020 in a university affiliated tertiary center. Participants/materials, setting, methods A total of 133 chemotherapy naïve patients aged 1–35 years with attempted IVM were included in the study. The primary outcome was IVM rate in the different age groups – pre-menarche (1–5 years and ≥6 years), post-menarche (menarche–17 years), young adults (18–24 years) and adults (25–29 and 30–35 years). Comparison between paired groups for significant difference in the IVM rate parameter was done using the Tukey’s Studentized Range (HSD) Test. Main results and the role of chance A gradual increase in mean IVM rate was demonstrated in the age groups over 1 to 25 years (4.6% (1–5 years), 23.8% (6 years to menarche) and 28.4% (menarche to 17 years), with a peak of 38.3% in the 18–24 years group, followed by a decrease in the 25–29 years group (19.3%), down to a very low IVM rate (8.9%) in the 30–35 years group. A significant difference in IVM rates was noted between the age extremes – the very young (1–5 years) and the oldest (30–35 years) groups, as compared with the 18–24-year group (p &lt; 0.001). Number of oocytes matured, percent of patients with matured oocytes and overall maturation rate differed significantly (p &lt; 0.001). Limitations, reasons for caution Data regarding ovarian reserve evaluation was not available for most of the patients, due to our pre-op OTC procedures protocol. None of our patients have used their frozen in-vitro matured oocytes, as such further implications of age on in-vitro matured oocytes quality and implantation potential has yet to be evaluated. Wider implications of the findings: Our finding of extremely low success rates in those very young (under 6 years) and older (≥30 years) patients suggest that IVM of oocyte retrieved during OTC prior to chemotherapy should not be attempted in these age group. Trial registration number N/A

scholarly journals Results of modern tuboperitoneal infertility treatment

2009 ◽  
Vol 66 (1) ◽  
pp. 57-62 ◽  
Author(s):  
Drenka Turjacanin-Pantelic ◽  
Dragana Bojovic-Jovic ◽  
Biljana Arsic ◽  
Eliana Garalejic
Keyword(s):  
Statistical Analysis ◽  
Surgical Treatment ◽  
Infertility Treatment ◽  
P Value ◽  
Modern Approach ◽  
Vitro Fertilization ◽  
Group I ◽  
Significant Difference ◽  
Group Ii

Background/Aim. A modern approach to surgical treatment of tuboperitoneal infertility is based on laporascopic techniques. The aim of this study was to compare results of tuboperitoneal infertility treatment by the use of laparoscopy and classical laparotomy. Methods. A retrospectiveprospective study on 66 women treated operatively form tuboperitoneal infertility was performed. Data from patient's anamnesis and those related to the surgical treatment results, obtained by the use of an inquiry, were used in retrospective and prospective analysis, respectively. Chi-square test was used in statistical analysis. P value < 0.05 was considered significant. Results. Classical laparotomy was used on 34 women in a period from 1996 to 1997, while 32 women were operated laparoscopically in a period from 1999 to 2000. The results were as follows: a total number of conceived women was 16 (24%), seven in the group I (20.6%) and nine in the group II (28.1%); 13 women were with one pregnancy, six in the group I (17.6%) and seven in the group II (22%). Twice pregnant were three women, one in the group I (2.9%) and two in the group II (6.2%). The resulting pregnancies were: five women with abortion spontaneous, two in the group I (5.9%) and three in the group II (9.4%); two women with extrauterine pregnancy in the group I (5.9%); three with pretemporal birth, one in the group I (2.9%) and two in the group II (6.2%), while six women were with the temporal birth, two in the group I (5.9%) and four in the group II (12.5%). Statistical analysis showed that there was no significant difference in the results between these two groups. Conclusion. Surgical treatment of tubeperitoneal infertility, regardless of the used methods (classical laparotomy or laparoscopy) was successful in a great number of women. These methods have a great advantage over in vitro fertilization, and they should not be ignored.

scholarly journals Impact of L-carnitine supplementation on the in vitro developmental competence and cryotolerance of buffalo embryos

2021 ◽  
pp. 3164-3169
Author(s):  
Mohamed M. M. El-Sokary ◽  
Al-Shimaa Al-H. H. El-Naby ◽  
Amal R. Abd El Hameed ◽  
Karima Gh. M. Mahmoud ◽  
T. H. Scholkamy
Keyword(s):  
Embryo Culture ◽  
In Vitro Maturation ◽  
Culture Media ◽  
Effective Concentration ◽  
Developmental Competence ◽  
Carnitine Supplementation ◽  
Oocyte Developmental Competence ◽  
High Lipid ◽  
Significant Difference

Background and Aim: Despite many trials, buffalo embryos have poor cryosurvivability because of their high lipid content. L-carnitine was found to be a lipid-reducing agent when added to oocyte and embryo culture media. The study aimed to determine the most effective concentration of L-carnitine to improve the oocyte developmental competence and cryotolerance of buffalo embryos. Materials and Methods: In vitro maturation and embryo culture media were supplemented with four concentrations of L-carnitine: 0 (control), 0.25, 0.5, and 1 mM. Good-quality embryos on 7 days were vitrified using mixtures of dimethyl sulfoxide and ethylene glycol at two concentrations (3.5 and 7 M). Results: The result showed that the cleavage and morula rates were significantly (p<0.05) higher in the 0.5 mM group. Blastocyst rates were significantly (p<0.05) higher at both 0.5 and 1 mM. The rates of viable embryos directly after thawing were significantly (p<0.05) increased in the 0.5 mM group. No significant difference was found in embryos cultured for 24 h after warming among all the groups. Conclusion: The addition of L-carnitine at a concentration of 0.5 mM to the culture media improves the oocyte developmental competence and cryotolerance of buffalo embryos directly after warming but not after 24 h of culture. Nevertheless, further studies must identify how L-carnitine exerts its beneficial micromechanisms.

scholarly journals “Ormocer an innovative technology”: A replacement for conventional cements and veneer? A comparative in vitro analysis

2017 ◽  
Vol 11 (01) ◽  
pp. 058-063 ◽  
Author(s):  
Vini Rajeev ◽  
Rajeev Arunachalam ◽  
Sanjna Nayar ◽  
P. R. Arunima ◽  
Sivadas Ganapathy ◽  
...  
Keyword(s):  
Bond Strength ◽  
Testing Machine ◽  
In Vitro Study ◽  
Sem Analysis ◽  
Innovative Technology ◽  
Group I ◽  
Luting Cement ◽  
Significant Difference ◽  
Vitro Analysis

ABSTRACT Objective: This in vitro study was designed to assess shear bond strength (SBS) of ormocer flowable (OF) resin as a luting agent, ormocer as an indirect veneer material with portrayal of modes of failures using scanning electron microscope (SEM). Materials and Methods: Sixty maxillary central incisors were divided into Group I, II, and III with 20 samples each based on luting cement used. They were OF, self-adhesive (SA) cement, and total etch (TE) cement. These groups were subdivided into “a” and “b” of ten each based on the type of veneering materials used. Veneer discs were fabricated using Ormocer restorative (O) and pressable ceramic (C). Specimens were thermocycled and loaded under universal testing machine for SBS. The statistical analysis was done using one-way ANOVA post hoc Tukey honest significant difference method. Results: A significant difference was observed between the Groups I and II (P < 0.05). The highest mean bond strength when using ormocer veneer was obtained with the Group Ia (19.11 ± 1.92 Mpa) and lowest by Group IIa (8.1 ± 1.04 Mpa), whereas the highest mean bond strength while using ceramic veneer was of similar range for Group Ib (18.04 ± 4.08 Mpa) and Group IIIb (18.07 ± 1.40 Mpa). SEM analysis revealed OF and TE presented mixed type of failure when compared with SA where failure mode was totally adhesive. Conclusion: OF was found equally efficient like TE. Bond strength of ormocer as a veneer was not inferior to ceramic making it one of the promising additions in the field of dentistry.

scholarly journals Microleakage analysis of dental caries lesions sealed with flow resin and compared to microhybrid resin restorations in dentin

2018 ◽  
Vol 66 (2) ◽  
pp. 141-146
Author(s):  
Amanda de Albuquerque VASCONCELOS ◽  
Juliana Tietbohl de Almeida REIS ◽  
Bianca Fiorentin MOURA ◽  
Daniela Cavalcante GIRÃO ◽  
José Carlos Pettorossi IMPARATO ◽  
...  
Keyword(s):  
Composite Resin ◽  
Descriptive Analysis ◽  
Resin Flow ◽  
Occlusal Caries ◽  
Dye Penetration ◽  
Group I ◽  
Significant Difference ◽  
Distal Direction ◽  
Caries Lesions

ABSTRACT Objective: To evaluate the sealing of cavities of dentinal occlusal caries lesions, reproduced in vitro, with flow resin compared to cavity restorations presenting healthy dentin using microhybrid composite resin. Methods: The sample consisted of 27 healthy deciduous molars where cavities of approximately 2 mm in the fossa region were performed and occlusal cleft of each tooth were sealed, impermeabilization was performed and the sample was randomly divided into 2 groups: group I underwent cariogenic challenge and occlusal sealing with resin flow. The teeth of group II were restored with microhybrid composite resin. The teeth were immersed in 5% methylene blue for 8 hours at 37° C and washed until all the dye was removed from the surface. The teeth were sectioned in the mesio-distal direction. The penetration of the dye was evaluated: 0- no penetration; 1- dye penetration up to 1/3 of the restoration; 2- dye penetration up to 2/3 of the restoration depth; 3 - penetration of dye into the pulp wall. The results were analyzed by the Biostat 4.0 program. Descriptive analysis and the mode among the examiners submitted to the Mann-Whitney test. Results: There was no significant difference in microleakage between restoration performed in healthy dentin with microhybrid composite resin or maintenance of infected dentin in primary teeth sealed with resin flow (p = 0.6035). Conclusion: It was concluded that the marginal infiltration of primary molars sealed with microhybrid composite resin and resin flow was not influenced by the removal -or not -of the carious tissue or the material used.

scholarly journals Effects of Short-Term Inhibition of Rho Kinase on Dromedary Camel Oocyte In Vitro Maturation

Animals ◽  
2020 ◽  
Vol 10 (5) ◽  
pp. 750
Author(s):  
Hammed A. Tukur ◽  
Riyadh S. Aljumaah ◽  
Ayman Abdel-Aziz Swelum ◽  
Abdullah N. Alowaimer ◽  
Mutassim Abdelrahman ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Rho Kinase ◽  
Positive Outcome ◽  
Dromedary Camel ◽  
Second Phase ◽  
Short Term ◽  
Rock Inhibitor ◽  
Nuclear Maturation ◽  
Significant Difference

This is the first report on a biphasic in vitro maturation (IVM) approach with a meiotic inhibitor to improve dromedary camel IVM. Spontaneous meiotic resumption poses a major setback for in vitro matured oocytes. The overall objective of this study was to improve in vitro maturation of dromedary camel oocytes using ROCK inhibitor (Y-27632) in a biphasic IVM to prevent spontaneous meiotic resumption. In the first experiment, we cultured immature cumulus–oocyte complexes (COCs, n = 375) in a prematuration medium supplemented with ROCK inhibitor (RI) for 2 h, 4 h, 6 h, and 24 h before submission to normal in vitro maturation to complete 28 h. The control was cultured for 28 h in the absence of RI. In the first phase of experiment two, we cultured COCs (n = 480) in the presence or absence (control) of RI for 2 h, 4 h, 6 h, and 24 h, and conducted real-time relative quantitative PCR (qPCR) on selected mRNA transcripts. The same was done in the second phase, but qPCR was done after completion of normal IVM. Assessment of nuclear maturation showed that pre-IVM for 4 h yielded an increase in MII oocyte (54.67% vs. 26.6% of control; p < 0.05). As expected, the same group showed the highest degree (2) of cumulus expansion. In experiment 2, qPCR results showed significantly higher expression of ACTB and BCL2 in the RI group treated for 4 h when compared with the other groups. However, their relative quantification after biphasic IVM did not reveal any significant difference, except for the positive response of BCL2 and BAX/BCL2 ratio after 4 and 6 h biphasic IVM. In conclusion, RI prevents premature oocyte maturation and gave a significantly positive outcome during the 4 h treatment. This finding is a paradigm for future investigation on dromedary camel biphasic IVM and for improving the outcome of IVM in this species.

326 EVALUATION OF CULTURE DURATION AND PARTIAL CUMULUS CELL REMOVAL DURING CANINE OOCYTE MATURATION

2006 ◽  
Vol 18 (2) ◽  
pp. 270
Author(s):  
C. Hanna ◽  
C. Long ◽  
M. Westhusin ◽  
D. Kraemer
Keyword(s):  
In Vitro Maturation ◽  
Calf Serum ◽  
Cumulus Cell ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Culture Conditions ◽  
Germinal Vesicle Breakdown ◽  
Significant Difference ◽  
Culture Duration

The objectives of this study were to determine whether the percentage of canine oocytes that resume meiosis during in vitro maturation could be increased by either increasing culture duration or by removing approximately one-half of the cumulus cells 24 h after oocytes were placed into culture. Canine female reproductive tracts were collected from a local clinic and ovaries were minced in warm TL-HEPES. Oocytes with a consistently dark ooplasm and at least two layers of cumulus cells were selected, cultured in a basic canine oocyte in vitro maturation medium consisting of TCM-199 with Earl's salts, 2.92 mM Ca-lactate, 20 mM pyruvic acid, 4.43 mM HEPES, 10% fetal calf serum, 1% Penicillin/Streptomycin (GibcoBRL, Grand Island, NY, USA), and 5 μg/mL porcine somatotropin, and incubated at 38.5°C in 5% CO2 in humidified air. Treatment groups were randomly assigned and oocytes were cultured for 60, 84, or 132 h (Basic). From each of these groups, one-half of the oocytes were pipetted through a fine bore pipette to partially remove the cumulus cells 24 h after the start of culture (Basic–1/2). At the end of culture, all oocytes were denuded and the nuclear status was observed with Hoechst 33342 under ultraviolet fluorescence. All data were analyzed by ANOVA with P < 0.05. Since the canine oocyte is ovulated at the germinal vesicle (GV) stage of meiosis and requires up to five days to mature in the oviduct, it was hypothesized that an increased culture time would allow for more oocytes to undergo nuclear maturation to metaphase II (MII). It was also hypothesized that partial removal of cumulus cells would decrease the cumulus cell component in the ooplasm that sustains meiotic arrest, allowing for more oocytes to resume meiosis (RM = germinal vesicle breakdown to MII). Results within each treatment group indicate that there is no significant difference between culture duration and the percent of oocytes that mature to MII. Additionally, there was no significance in the percent of oocytes that resumed meiosis after partial cumulus cell removal. Taken together, these data suggest that neither treatment is effective in canine in vitro maturation systems, given the current maturation culture conditions. Table 1. Nuclear status* of oocytes for three time periods with or without partial cumulus cell removal

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