scholarly journals Meiotic Status Does Not Affect the Vitrification Effectiveness of Domestic Cat Oocytes

Animals ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 1371
Author(s):  
Natalia Sowińska ◽  
Jennifer Zahmel ◽  
Wojciech Niżański ◽  
Romy Hribal ◽  
Lorena Fernandez-Gonzalez ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Toxicity Assessment ◽  
Domestic Cat ◽  
Developmental Potential ◽  
Vitro Fertilization ◽  
Negative Effect ◽  
Meiotic Competence

Cryopreservation is important for animal fertility and biodiversity. Unfortunately, cryopreservation of feline oocytes is still an experimental technique. The aims of this study were to analyze the potential toxicity of the cryoprotectants in the vitrification solution (VS) on cat oocytes and to investigate whether the meiotic status of oocytes influences their developmental potential after vitrification. Two experiments were conducted with the VS composed of 20% ethylene glycol, 20% dimethyl sulfoxide, 20% fetal calf serum, 1.5 M trehalose, and 10% Ficoll PM-70: (1) toxicity assessment of the VS on immature cumulus oocyte complexes (COCs), and subsequently in vitro maturation (IVM) and in vitro fertilization; (2) assessment of the influence of the meiotic status on vitrification effectiveness, where immature and in vitro matured COCs were vitrified on the Cryotop. After rewarming, vitrified oocytes were subjected to IVM (immature) and intracytoplasmic sperm injection (ICSI) with fresh epididymal sperm. The toxicity test revealed no negative effect of oocyte exposure to the applied VS on their developmental potential (p > 0.05). Although the vitrification procedure itself significantly reduced the meiotic competence of oocytes, their meiotic status before vitrification (immature vs. in vitro matured) did not influence fertilization and morula rates. The only parameter affected by vitrification was the rate of oocytes suitable for ICSI, which was significantly lower for immature oocytes. Regardless of the meiotic status of vitrified oocytes, morphologically normal morulae were obtained. Moreover, the two meiotic stages examined are suitable for vitrification, with mature oocytes being a better choice when a well-equipped laboratory is available.

Effect of culture conditions on artificial activation of porcine oocytes matured in vitro

1996 ◽  
Vol 8 (8) ◽  
pp. 1153 ◽  
Author(s):  
N Yamauchi ◽  
H Sasada ◽  
S Sugawara ◽  
T Nagai
Keyword(s):  
In Vitro Maturation ◽  
Fetal Calf Serum ◽  
Culture Media ◽  
Calf Serum ◽  
Treated Group ◽  
Culture Conditions ◽  
Culture Period ◽  
Subsequent Response ◽  
Artificial Activation

The effects of culture media used and culture period for in vitro maturation of porcine oocytes on their subsequent response to chemical and electrical activation, were investigated. Activated oocytes were identified by the presence of a pronucleus(ei) or cleavage. Porcine oocytes were cultured for 24, 30, 36, 42 and 48 h in TCM199 with Earle's salts (199) supplemented with 10% fetal calf serum (199-FCS) before electrical stimulation. Although few oocytes were activated after 24 h and 30 h of culture (5.4% and 6.1% respectively), the percentage of activated oocytes increased significantly to 93.2% after 42 h in culture (P < 0.05); however, when the culture period was extended to 48 h, there was a significant decrease to 56.7% (P < 0.05). Oocytes were also cultured in four types of media: (1) 199-FCS; (2) 199 supplemented with 5 mg mL-1 bovine serum albumin (199-BSA); (3) Kreb's-Ringer bicarbonate solution supplemented with 10% FCS (KRB-FCS); and (4) KRB supplemented with BSA (KRB-BSA). After 42 h of culture in each medium, the oocytes were electrically activated. Although rates of maturation of oocytes cultured in the four media were similar (74.0-80.8%), all oocytes except those cultured in 199-FCS failed to be activated. In addition, oocytes were cultured for 36, 42 and 48 h in 199-FCS and then stimulated by treatment with ethanol. Significantly fewer oocytes were activated in the chemically-treated group than in the electrically-treated group. These results indicate that culture conditions used for the culture of porcine oocytes in vitro are important with respect to their subsequent response to artificial activation.

71 VITRIFICATION OF BOVINE OOCYTES IN MEDIA WITH GLYCEROL + ETHYLENE GLYCOL BEFORE AND AFTER IN VITRO MATURATION

2008 ◽  
Vol 20 (1) ◽  
pp. 116
Author(s):  
L. G. Devito ◽  
C. B. Fernandes ◽  
H. N. Ferreira ◽  
F. C. Landim-Alvarenga
Keyword(s):  
Ethylene Glycol ◽  
Liquid Nitrogen ◽  
In Vitro Maturation ◽  
Calf Serum ◽  
Quiescent State ◽  
Developmental Potential ◽  
Immature Oocytes ◽  
Nitrogen Vapor ◽  
Bovine Oocytes

The cryopreservation process aims to keep the cellular metabolism in a quiescent state for an indeterminate length of time. In mammals, oocyte cryopreservation success is important for the establishment of genetic banks. The objective of the present experiment was to evaluate the effect of vitrification on oocyte meiotic ability and the integrity of the metaphase plate in immature and in vitro-matured bovine oocytes. Bovine cumulus–oocytes complexes (COCs) were harvested from slaughterhouse ovaries and randomly divided into 3 groups: (G1) non-vitrified oocytes subjected to in vitro maturation, (G2) immature oocytes vitrified and then subjected to in vitro maturation after warming, and (G3) in vitro-matured oocytes subjected to vitrification. For in vitro maturation, oocytes were incubated for 22 h in 5% CO2 in air in TCM-199 with fetal calf serum, estradiol, LH, FSH, pyruvate, and gentamicin. For vitrification, the oocytes were exposed to the cryoprotectors in three steps: solution 1 containing 1.4 m glycerol in PBS for five min, and then solution 2 containing 1.4 m glycerol and 3.6 m ethylene glycol in PBS for another five min. After exposure to the second solution, the oocytes were transferred to 30-µL drops of solution 3 containing 3.4 m glycerol and 4.6 m ethylene glycol, loaded (5 oocytes per straw) in less than 1 min into 0.25-mL straws between two columns of 0.5 m galactose in PBS separated by two air bubbles, and immediately set in liquid nitrogen vapor. After 1 min of equilibration in liquid nitrogen vapor, the straws were immersed in liquid nitrogen. Warming was performed by holding the straws for 10 s in air, followed by 10 more s in a water bath at 20–22�C. The straws were then shaken 5 to 8 times to mix the bubbles (movement similar to that for a thermometer) and left horizontally for 6 to 8 min at room temperature. The rates of metaphase II and degeneration were analyzed by ANOVA followed by the Student t-test. The oocytes were stained with 100 µg mL–1 Hoechst 33342 and examined in an inverted microscope equipped with fluorescent light (UV filters 535 and 617 mm). Three different routines were realized with a total of 90 oocytes per group. The metaphase II rates in G1 (48/90, 53.3%) and G3 (42/90, 46.6%) were statistically the same (P e 0.05), but were higher (P d 0.05) than in G2 (0/90, 0%). The degeneration rates were: G1 (18/90, 20%), G2 (77/90, 85.6%), and G3 (7/90, 7.8%). The vitrification procedure damaged mainly the immature oocytes, since in the G2 the degeneration rate was higher and the oocytes were not able to resume meiosis. Meanwhile, when oocytes were vitrified after in vitro maturation, the metaphase II rate was similar to the one observed in IVM oocytes not subjected to vitrification. This indicates that the vitrification procedure performed in this experiment did not damage the structure of the metaphase II plate. However, more studies are necessary to predict the developmental potential of these in vitro-matured oocytes.

154 THE EFFECT OF ZINC ON PORCINE IN VITRO MATURATION AND SUBSEQUENT EMBRYONIC DEVELOPMENT AFTER IN VITRO FERTILIZATION

2014 ◽  
Vol 26 (1) ◽  
pp. 191
Author(s):  
Y. Jeon ◽  
J. D. Yoon ◽  
L. Cai ◽  
S. U. Hwang ◽  
E. Kim ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Inductively Coupled Plasma ◽  
In Vitro Maturation ◽  
Formation Rate ◽  
Developmental Competence ◽  
Developmental Potential ◽  
Vitro Fertilization ◽  
Nuclear Maturation ◽  
Significant Difference

Zinc (Zn) is one of the abundant transition metals in biology and is an essential component of most cells. However, there are few reports about the effect of Zn in porcine oocytes. The objective was to investigate the effects of supplementary Zn during in vitro maturation (IVM) of porcine oocytes. We investigated nuclear maturation, intracellular glutathione (GSH) levels, reactive oxygen species (ROS) levels, and subsequent embryonic development after IVF. Before the experiment, Zn concentrations in IVM medium and body fluids were measured using inductively coupled plasma spectrophotometer (sensitivity: 1 μM) and treatment concentrations were determined. Zinc concentration was 12.6 μM in porcine plasma and 12.9 μM in porcine follicular fluid. We confirmed that Zn was not detected in IVM medium. A total of 541 cumulus–oocyte complexes (COC) were used for the evaluation of nuclear maturation. The COC were matured in TCM-199 medium supplemented with various concentrations of Zn (0, 6, 12, 18, and 24 μM). After 44 h of IVM, no significant difference was observed in all groups (metaphase II rate: 85.7, 88.7, 90.4, 90.3, and 87.2%, respectively). A total of 100 matured oocytes were examined for the effects of different Zn concentrations (0, 6, 12, 18, and 24 μM) on porcine oocyte intracellular GSH and ROS levels, which were measured through fluorescent staining and image analysis program. The groups of 12, 18, and 24 μM showed a significant (P < 0.05) increase in intracellular GSH levels (1.45, 1.67, and 1.78, respectively) compared with the control and 6 μM group (1.00 and 1.08, respectively). The intracellular ROS level of oocytes matured with 12, 18, and 24 μM (0.82, 0.68, and 0.55) were significantly (P < 0.05) decreased compared with the control and 6 μM groups (1.00 and 1.03, respectively). Finally, the developmental competence of oocytes matured with different concentrations of Zn (0, 6, 12, 18, and 24 μM) was evaluated after IVF. There were no significantly different in cleavage rates. However, cleavage patterns and blastocyst (BL) formation were different. Fragmented embryo ratio of the 12 μM group (14.9%) was significantly lower than that of the other groups (control, 6, 18, and 24 μM: 26.4, 17.8, 18.4, and 18.0%, respectively). Oocytes treated with 12 μM Zn during IVM had a significantly higher BL formation rate (28.2%) after IVF compared with the control (19.8%). In conclusion, these results indicate that Zn treatment as body fluid concentration during IVM improved the developmental potential of IVF in porcine embryos by increasing the intracellular GSH concentration and decreasing the ROS level. This work was supported, in part, by a grant from the Next-Generation Bio Green 21 Program (No. PJ00956901), Rural Development Administration, and the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2012R1A1A4A01004885, NRF-2013R1A2A2A04008751), Republic of Korea.

scholarly journals 318EFFECT OF PIG FOLLICLE FLUID AND FETAL CALF SERUM ON PORCINE OOCYTE MATURATION AND SUBSEQUENT DEVELOPMENT AFTER ACTIVATION AND SOMATIC CELL NUCLEAR TRANSFER

2004 ◽  
Vol 16 (2) ◽  
pp. 278
Author(s):  
Z. Liu ◽  
L. Lai ◽  
G. Im ◽  
M. Samuel ◽  
D. Wax ◽  
...  
Keyword(s):  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
In Vitro Maturation ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Defined Medium ◽  
Chemically Defined Medium ◽  
Maturation Rate ◽  
Blastocyst Rate

In vitro maturation of porcine oocytes is very important for understanding porcine somatic cell nuclear transfer (SCNT). In order to develop an in vitro maturation system that can provide more high quality oocytes, the effect of porcine follicle fluid (pFF) (gathered from 3–5-mm porcine follicles) and fetal calf serum (FCS: Sigma, St. Louis, MO, USA), as an important additional component of a chemically-defined medium was studied. Cumulus-oocyte complexes (COC) derived from follicles 3–5mm in diameter were cultured in three different media: a chemically-defined medium (CDM: TCM-199 with 0.1mgmL−1 cysteine, 10ngmL−1 EGF, 0.5μgmL−1 LH and 0.5μgmL−1 FSH); CDM with 10% pFF (CDM+p); and CDM with 10% FCS (CDM+F). After 42–44h of maturation, oocytes with a clear polar body were classified as matured oocytes. Matured oocytes stimulated by electric pulse (120v, 30μs, 2 pulse), or enucleated and fused with fibroblasts to construct SCNT embryos by using the same electrical parameters. All of these parthenogenetic and SCNT embryos were cultured in Porcine Zygote Medium-3. The blastocyst rate was assessed under a stereomicroscope on Day 6, and the number of nuclei in the blastocysts was counted under a fluorescent microscope after staining with 5μgmL−1 of Hoechst 33342. All data were subjected to a Generalized Linear Model Procedure (PROC-GLM) of Statistical Analysis System (SAS). The maturation rates of porcine oocytes in CDM and CDM+p were 53.2±3.8% (539/1050) and 69.7±3.8% (587/847), respectively;; in CDM and CDM+F, 61.1±3.1% (471/776) and 70.2±3.7% (577/844), respectively. Oocytes matured in CDM+p and CDM+F showed a higher (P&lt;0.05) maturation rate than those in CDM. The percentages of parthenogenetic blastocysts of oocytes matured in CDM and CDM+p were 13.9±2.1% (35/250) and 20.2±5.3% (64/300), and the numbers of nuclei in these blastocysts were 25.8±2.3 and 25.8±1.4, respectively. The blastocyst rate from CDM- and CDM+F-matured oocytes were 20.1±2.0% (53/272) and 22.2±4.7%(71/298), and the numbers of nuclei in these blastocysts were 24.7±1.5 and 25.3±1.5, respectively. There were no significant (P&gt;0.05) differences in the percentages of parthenogenetic blastocysts and nuclei numbers between CDM and CDM+p, or CDM and CDM+F. The percentages of blastocysts in SCNT embryos derived from CDM and CDM+p were 8.1±1.5% (14/192) and 12.3±1.9% (24/192), while the nuclei numbers in these blastocysts were 26.6±1.2 and 34.5±2.2, respectively. The percentages of blastocysts after SCNT from oocytes matured in CDM and CDM+F were 24.3±4.9% (35/139) and 27.1±5.5% (45/176), while the numbers of nuclei were 29.8±2.5 and 32.2±1.9, respectively. There were no significant (P&gt;0.05) differences between CDM and CDM+p, or CDM and CDM+F in SCNT embryo blastocyst rate, but the SCNT embryos derived from CDM+p showed a higher (P&lt;0.05) nuclear number. In conclusion, these results indicate that 10% pFF or FCS in CDM can promote a higher maturation rate of porcine oocytes. As recipient cytoplasm for SCNT, oocytes matured in CDM+p can support development of blastocysts that contain more nuclei than those matured in CDM alone. Supported in part by Food for the 21st Century and RR13438.

361 EFFECT OF HORMONES, EGF AND β-MERCAPTOETHANOL ON IN VITRO MATURATION OF CAPRINE OOCYTES

2010 ◽  
Vol 22 (1) ◽  
pp. 337 ◽  
Author(s):  
P. Yadav ◽  
S. D. Kharche ◽  
A. K. Goel ◽  
S. K. Jindal ◽  
M. C. Sharma
Keyword(s):  
In Vitro Maturation ◽  
Calf Serum ◽  
Basic Medium ◽  
Control Group ◽  
Vitro Fertilization ◽  
Base Medium ◽  
Maturation Medium ◽  
Maturation Rate ◽  
Group 5

In vitro maturation of oocytes is an integral part of in vitro culture system. In almost all studies of mammalian in vitro maturation, the basic medium is supplemented with serum and hormones. The maturation medium and selection of protein supplements, growth factors, antioxidants and hormones for IVM play an important role in subsequent in vitro fertilization and in vitro embryo development. The objective of the present experiment was to study the effect of exogenous addition of hormones, epidermal growth factor, insulin and β-mercaptoethanol to the maturation medium for in vitro maturation of caprine oocytes. A total of 1540 oocytes were collected from slaughtered goat of 1.5 to 2.5 year of age and randomly divided in to treatment groups. Group 1; COCs were matured in TCM-199 medium containing 10% calf serum (NCS) and 3 mg mL-1 BSA (used as a base medium) for control, Group 2; COCs were matured in a base medium supplemented with hormones (5 μg mL-1 FSH, 5 μg mL-1 LH and 1 μg mL-1 estradiol-17β), Group 3β COCs were matured in a base medium supplemented with 50 ng mL-1 insulin, Group 4β COCs were matured in a base medium supplemented with hormones and 10 ng mL-1 EGF, Group 5; COCs were matured in a base medium supplemented with hormones and 50 mM β-mercaptoethanol and Group 6; COCs were matured in a base medium supplemented with hormones and 50 ng mL-1 insulin. These COCs were matured at 38.5°C in 5% CO2 in air for 27 h. After the maturation, oocytes were separated from cumulus and corona cells by treatment with 0.1% hyaluronidase and by passing through a fine pipette. They are them fixed in 2.5% glutaraldehyde, stained with DAPI and observed under fluorescent microscope for evidence of nuclear maturation. The maturation rates in groups 1 to 6 were 33.6%, 38.0%, 39.7%, 60.0%, 37.4%, and 44%, respectively. Statistical analysis (ANOVA) after arcsin transformation revealed that the maturation rate in group 4 was statistically significant (P < 0.05) as compared to those in groups 1, 2, 3, 5, and 6. The results suggest that the supplementation of EGF in maturation medium significantly enhances the in vitro maturation rate of caprine oocytes.

183 CANINE EMBRYOS OBTAINED BY INTRACYTOPLASMIC SPERM INJECTION

2014 ◽  
Vol 26 (1) ◽  
pp. 206 ◽  
Author(s):  
S. Chastant-Maillard ◽  
K. Reynaud ◽  
S. Thoumire ◽  
M. Chebrout
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Fetal Calf Serum ◽  
Selection Process ◽  
Calf Serum ◽  
Germinal Vesicle ◽  
Sperm Head ◽  
Cell Stage ◽  
Vitro Fertilization

In vitro fertilization encounters 2 specific difficulties in the canine species, with no puppies born to date: low penetration rates (10–50%) and high polyspermia (around 50% of fertilized oocytes; Saint-Dizier et al. 2001 J. Reprod. Fert. Suppl. 57, 147–150). The objectives of the study were to test whether intracytoplasmic sperm injection (ICSI), which overcomes these 2 obstacles, could allow production of canine embryos, using in vivo- or in vitro-matured oocytes. The time of ovulation was determined on 8 Beagle bitches from our experimental kennel by blood progesterone assay and transabdominal ultrasound examination. After ovariohysterectomy 82 to 100 h after ovulation, 58 metaphase II (MII) oocytes were collected by tubal flushing. In parallel, 88 oocytes from 6 anoestrus bitches were matured in vitro (M199 + 20% fetal calf serum for 72 h in 5% CO2 at 38°C). Sperm was collected from 1 Beagle dog with excellent fertility record at natural mating. The sperm was diluted 1 : 100 in PBS/BSA without any selection process. Intracytoplasmic sperm injection was performed at 38°C in M199 HEPES + 20% BSA (4-μm injection pipette; 120-μm holding pipette). One motile spermatozoon of normal morphology was injected per oocyte. Injected oocytes were cultured in vitro for 48 h after injection (M199 + 20% fetal calf serum in 5% CO2 at 38°C) in 4-well open dishes. Oocytes were then fixed and DNA and tubulin were stained for observation by confocal microscopy (Chebrout et al. 2012 Microsc. Microanal. 18, 483–492). Among the 58 MII oocytes recovered in vivo, 7.4% lysed at injection and 20% degenerated during the 48 h after injection. Among the 40 injected oocytes still alive, 6 fragmented (15%) and 4 developed as embryos [10%; 2-pronuclei (n = 2), 2-cell and 6-cell). None of the other oocytes showed decondensed female chromatin. Among the 88 oocytes incubated for in vitro maturation, 13 (14.8%) reached MII. These were successfully injected; 48 h after injection, 3 were embryos at the 2-cell stage and 10 were at the MII stage with a condensed sperm head. Fifty-one non-mature oocytes were injected; 31 were at the germinal vesicle (GV) stage and the stage of others was not determined. Of the GV oocytes, 71% degenerated during culture after injection. The 9 surviving oocytes were still at the GV stage with condensed sperm head 48 h after injection. In conclusion, canine embryos can be obtained through ICSI. Nevertheless, this procedure induced low activation rates. Development at later stages, especially after transfer into a recipient female, is to be evaluated, in particular for in vitro-produced MII oocytes, of lower cytoplasmic competence (Viaris et al. 2008 Reprod. Fert. Dev. 20, 626–639).

Effects of gonadotrophins, growth hormone and prolactin on developmental competence of domestic cat oocytes matured in vitro

1995 ◽  
Vol 7 (5) ◽  
pp. 1061 ◽  
Author(s):  
RD Schramm ◽  
BD Bavister
Keyword(s):  
Growth Hormone ◽  
Calf Serum ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Domestic Cat ◽  
Developmental Competence ◽  
Developmental Potential ◽  
Nuclear Maturation ◽  
Morula Stage

Specific aims were to (1) examine the developmental capacity of felid oocytes matured in vitro and (2) determine the effects of gonadotrophins, growth hormone and prolactin on nuclear and cytoplasmic maturation oocytes in vitro. Oocytes were obtained from excised ovaries of 21 cats, and were matured for 45-46 h in modified CMRL-1066 culture medium (1 mM glutamine, 1 mM pyruvate and 20% bovine calf serum), with one of the following: (1) gonadotrophins (1.0 micrograms mL-1 hFSH+10 micrograms mL-1 hLH), (2) gonadotrophins+10 micrograms mL-1 growth hormone, (3) gonadotrophins+10 micrograms mL-1 prolactin, or (4) no hormones. Oocytes were inseminated with ejaculated cat sperm capacitated in TALP medium. Embryos were cultured in modified CMRL-1066 medium until developmental arrest, then stained with Hoechst 33342 to assess nuclear status or cell number. Gonadotrophins enhanced (P < or = 0.05) the incidence of nuclear maturation, but neither gonadotrophins, growth hormone nor prolactin improved fertilization or developmental potential of oocytes matured in vitro. Mean percentages of mature oocytes that were fertilized and cleaved to or beyond the 2, 4, 8 and 16-cell stages were 80, 77, 66, 42 and 24%, respectively. Three embryos progressed to 40-60 cells, but none developed a blastocoel. Thus, although gonadotrophins enhance nuclear maturation of oocytes in vitro, and mature oocytes are capable of fertilization and development to the morula stage, culture with growth hormone, prolactin or gonadotrophins during maturation in vitro does not enhance developmental competence or overcome the morula-to-blastocyst-stage block in development of domestic-cat oocytes matured in vitro.

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