scholarly journals 319IN VITRO MATURATION OF EQUINE OOCYTES IN A COMPLETELY DEFINED MEDIUM SUPPLEMENTED WITH PROGESTERONE

2004 ◽  
Vol 16 (2) ◽  
pp. 279
Author(s):  
B. Merlo ◽  
E. Iacono ◽  
F. Prati ◽  
G. Mari
Keyword(s):  
Polar Body ◽  
Basal Medium ◽  
Defined Medium ◽  
Chi Square ◽  
Nuclear Maturation ◽  
Maturation Rate ◽  
Chi Square Test ◽  
The Media ◽  
Cytoplasmic Maturation

A completely defined medium for in vitro maturation (IVM) of equine oocytes has not yet been developed, since most of the media used for IVM are supplemented with serum or BSA. Furthermore, in this species there is no report about the influence of progesterone on maturation, although it has already been used as supplement (500ngmL−1) in EMMI (Maclellan LJ et al., 2001, Theriogenolgy 55, 310 abst). The aims of this study were to develop a completely defined medium for equine oocyte maturation and to investigate the effect of progesterone on nuclear maturation. Equine oocytes were collected by follicular scraping of abattoir-derived ovaries between April and June. The basal medium for maturation was SOFaa supplemented with pFSH-LH 0.1IUmL−1 (Pluset, Laboratorios Calier, Barcelona, Spain), EGF* 50ngmL−1, ITS (Insulin, Transferrin, Sodium selenite), L-cysteine 1.2mM, Maturation SOF (MSOF). Compact cumulus-oocyte complexes were selected, washed three times in H-SOF and matured in one of the following media (15–20 oocytesmL−1): (1) MSOF+FCS 10% (MSOF-FCS), (2) MSOF+progesterone 100ngmL−1 (MSOF-P4), (3) MSOF. After 24h of culture in 5% CO2 in air at 38.5°C, the oocytes were denuded by gently pipetting in a 0.25% trypsin solution, washed and stained with Hoechst 33258 (10μgmL−1 in PBS) for 30min at room temperature. Oocytes were examined under a fluorescent microscope to assess nuclear maturation. Only oocytes with an evident polar body and metaphase II plate (MII) were considered mature. The experiment was done in 6 replicates. Chi Square test was used for statistical analysis (Statistica for Windows – Stat Soft Inc., Tusla, OK, USA). Significance was assessed for P<0.05. The results of this study show that MSOF can be considered a suitable completely defined medium for IVM of equine oocytes. Adding progesterone significantly (P<0.05) increases the nuclear maturation rate at 24h of culture. It can be speculated that although cumuls cells produce this hormone, supplementation is useful to reach progesterone concentrations similar to those present in follicular fluid (early dominant 63.4±19.3ngmL−1, healthy preovulatory follicle 1094.3±170.9ngmL−1; Gerard N et al., 2002, Reproduction 124, 241–248). Further studies are needed to investigate the influence of progesterone on cytoplasmic maturation and to test the effect of different progesterone concentrations and time of maturation in a completely defined system.*All chemicals were purchased from Sigma, St. Louis, MO, USA, unless otherwise stated. Table 1 Maturation of equine oocytes in different media

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 >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.

358 MORPHOLOGICAL CHANGES IN VITRIFIED WATER BUFFALO CUMULUS - OOCYTE COMPLEXES AND THEIR IN VITRO NUCLEAR MATURATION

2007 ◽  
Vol 19 (1) ◽  
pp. 294
Author(s):  
R. C. S. Yadav ◽  
A. Sharma ◽  
G. N. Purohit
Keyword(s):  
Normal Form ◽  
Ethylene Glycol ◽  
Morphological Changes ◽  
Lower Percentage ◽  
Good Tool ◽  
Chi Square ◽  
Nuclear Maturation ◽  
Chi Square Test ◽  
Vitrification Solution

Morphological changes and in vitro nuclear maturation of buffalo cumulus–oocyte complexes (COCs) was evaluated subsequent to their cryopreservation by vitrification in solutions containing 4 M, 6 M, 8 M, and 10 M concentrations of glycerol (G) or ethylene glycol (EG) or their combination. COCs collected from buffalo ovaries by aspiration (n = 1342) were equilibrated in 50% of the vitrification solution and then placed in the vitrification solution (Dulbecco's phosphate-buffered saline+0.5M sucrose + 0.5% BSA + cryoprotectant). COCs were transferred to empty semen straws, kept over LN vapor for 2–3 min, and then plunged into LN. After 7–10 days of storage, COCs were warmed and evaluated for morphological damage. Morphologically normal COCs were cultured in vitro (9 replicates each with 5–10 oocytes in 50–100-µL culture drops) in TCM-199 medium supplemented with 5µgmL-1 FSH, 5µgmL-1 LH, and 1 ngmL-1 estradiol with 25mM HEPES, 0.25mM pyruvate, and antibiotics. The COCs were incubated for 24 h at 38±1°C and 5% CO2 in humidified air in a CO2 incubator and evaluated for nuclear maturation at the end of 24 h of culture. Freshly collected COCs were also matured in vitro and kept as controls (n=142). The proportions of COCs retrieved in morphologically normal form were compared by chi-square test; the arcsin transformed data of the proportions of oocytes matured was compared by Duncan's new multiple range test. The proportions of oocytes recovered in a morphologically normal form were highest in the 6M EG group (95.23%), followed by 8M EG (94.0%) and 6M G (90.6%) groups. At 10M concentration, a significantly (P <0.05) lower percentage of oocytes was morphologically normal. The morphological abnormalities recorded were change in shape, rupture of zona pellucida, and leakage of oocyte contents. A significantly higher (65.62%; P <0.05) proportion of fresh oocytes reached metaphase-II compared to oocytes vitrified in all concentrations of G and EG. The proportion of oocytes reaching metaphase-II increased with increasing concentrations of both G and EG, but at 10M concentration the proportion of oocytes reaching metaphase-II decreased. The proportions of COCs reaching metaphase-II in 4M, 6 M, 8M, and 10M glycerol were 6.9%, 21.2%, 25.7%, and 5.5%, respectively. The respective proportions of COCs reaching metaphase-II in 4M, 6 M, 8M, and 10M ethylene glycol were 21.9%, 34.3%, 40.8%, and 7.5%. No significant benefit of in vitro maturation of oocytes was seen for oocytes vitrified in a combination of both G and EG. It was concluded that although vitrification brings about some damage to the oocytes, yet it appears to be a good tool for oocyte cryopreservation, and 8M concentration of either G or EG appears to be optimum for vitrification of buffalo oocytes.

256 LEPTIN AND GLUCOSE INFLUENCE PORCINE NUCLEAR MATURATION

2009 ◽  
Vol 21 (1) ◽  
pp. 226
Author(s):  
E. Silva ◽  
R. L. Krisher
Keyword(s):  
Leptin Receptor ◽  
Oocyte Quality ◽  
Defined Medium ◽  
Chi Square ◽  
Nuclear Maturation ◽  
The Metabolic Syndrome ◽  
Obesity And Diabetes ◽  
Maturation Medium ◽  
Negative Effect

Leptin (LEP), a product of the obese gene, regulates food intake and may be associated with the metabolic syndrome. In addition, leptin receptor has been identified in the luteal and granulosa cells of the pig, suggesting that this protein may play a role in fertility. Research indicates that LEP is associated with insulin sensitivity; thus, the addition of different concentrations of LEP and glucose (GLUC) in maturation media may have an impact on oocyte maturation. The objective of the current experiments was to determine the effect of LEP (Experiment 1), and LEP in combination with GLUC (Experiment 2) on the nuclear maturation of porcine oocytes. This could potentially be a good model system in which to study the effects of obesity and diabetes on oocyte quality. Cumulus–oocyte complexes were cultured in a chemically defined medium, Purdue porcine medium (PPM) for 42 h, in 7% CO2 in air and at 38.7°C. In Experiment 1, medium was supplemented with 5 different concentrations (0, 1, 10, 20, and 100 ng mL–1) of LEP, in the presence of 2 mm GLUC. Experiment 2 was designed as a 3 × 3 factorial, with LEP (0, 1, and 10 ng mL–1) and GLUC (0, 5, and 50 mm). Oocytes were fixed and stained after IVM (136 to 155/treatment, 7 replicates; and 39 to 90/treatment, 5 replicates for Experiment 1 and 2, respectively). Nuclear maturation was scored as 1 (mature; T or MII) or 0 (not mature). Data were analyzed by GLM ANOVA and chi-square. There was no difference in nuclear maturation between 0, 1, 10, 20, and 100 ng mL–1 LEP (79.4, 82.3, 74.5, 77.2, and 83.7% mature, respectively). According to these data, LEP alone did not have an effect on in vitro maturation of porcine oocytes when using a chemically defined maturation medium. Absence of GLUC in the medium had a negative effect on maturation (39.5%; P < 0.01) compared with treatment with 5 mm (88.2%) and 50 mm (74.7%) GLUC, independent of LEP. In the presence of 5 mm GLUC, there was no difference between 0, 1, and 10 ng mL–1 LEP (83.9, 94.9, and 88.3%, respectively). However, LEP did affect nuclear maturation when COC were cultured in the absence of GLUC or with excessive GLUC. Leptin (10 ng mL–1) tended (P = 0.1) to promote nuclear maturation (46.2%) in the absence of GLUC (0 mm), compared with 0 ng mL–1 LEP (32.3%). In high-GLUC (50 mm) medium, 1 ng mL–1 LEP had a positive effect (P < 0.05) on nuclear maturation (87.8%) compared with 0 ng mL–1 (66.7%) and 10 ng mL–1 LEP (72.3%). LEP (0 or 10 ng mL–1) inhibited (P < 0.05) nuclear maturation in the presence of high (50 mm) GLUC compared with that in 5 mm GLUC. These data demonstrate that addition of 10 ng mL–1 LEP in maturation media may mitigate the negative effect of maturation in the absence of GLUC. Moreover, in the presence of excessive GLUC, 1 ng mL–1 LEP stimulates nuclear maturation. These results suggest that LEP and GLUC may interact to regulate oocyte nuclear maturation in obese or diabetic individuals, or both.

scholarly journals Oxygen tension and oocyte density during in vitro maturation affect the in vitro fertilization of bovine oocytes

2015 ◽  
Vol 36 (6Supl2) ◽  
pp. 4277 ◽  
Author(s):  
Angelo Bertani Giotto ◽  
Daniela Dos Santos Brum ◽  
Francielli Weber Santos ◽  
Antonio Carlos Galarça Guimarães ◽  
Cibele Garcia Moreira Gonçalves ◽  
...  
Keyword(s):  
Antioxidant Activity ◽  
Oxygen Tension ◽  
Polar Body ◽  
Ros Production ◽  
Low Oxygen ◽  
Nuclear Maturation ◽  
First Polar Body ◽  
Cytoplasmic Maturation ◽  
Bovine Oocytes

<p>Oocyte maturation is the key factor affecting the fertilization and embryonic development. Factors such as oocyte density and oxygen tension can directly influence the IMV. Thus, the objective of this study was to evaluate the effect of the association of oxygen tensions (5% or 20%) with different oocyte densities (1:10?l or 1:20?l) in the <em>in vitro </em>maturation (IVM) of bovine oocytes on maturation and fertilization rates, ROS production and antioxidant activity. Three experiments were performed with bovine oocytes that were obtained from slaughterhouse ovaries. After selection, the oocytes were randomly distributed in four treatments: 1:10/5%; 1:10/20%; 1:20/5%and 1:20/20% for each experiment. In experiment I, nuclear maturation status and cytoplasmic maturation were evaluated through detection of the first polar body by immunofluorescence and the mitochondrial reorganization assay. In experiment II, ROS production and antioxidant activity were analyzed in oocytes and IVM medium after 24 h of maturation through detection of ROS, reduced glutathione (GSH) and Superoxide dismutase activity by spectrofluorimetric methods. In experiment III, fertilization was evaluated through pronucleus formation, sperm penetration with or without decondensation and polyspermy rates by immunofluorescence. In experiment I, the nuclear maturation and cytoplasmic maturation were similar among treatments (P&gt;0.05). In experiment II, reactive oxygen species in oocytes were elevated in treatments with low oxygen tension which was independent of oocyte density (P&lt;0.05). Additionally, ROS levels in IVM medium were higher in treatments with high oocyte density by volume of medium, which was independent of oxygen tension (P&lt;0.05). In Experiment III, the fertilization and penetration rates were higher in the treatment with 20% oxygen tension and high oocyte density (P&lt;0.05). Furthermore, a high incidence of polyspermy was observed in groups with high oxygen tension and low oocyte density (P&lt;0.05). In conclusion, the results of this study indicate an interaction between oxygen tension and oocyte density, which increases ROS production in certain associations and subsequently influences the rates of <em>in vitro </em>fertilization of bovine oocytes. The improved rates of IVF were obtained when IVM was conducted using 20% oxygen tension and high oocyte density (1:20 ul).</p>

199 PREMATURATION OF BOVINE OOCYTES WITH BUTYROLACTONE I AND/OR CILOSTAMIDE: EFFECTS ON MEIOSIS PROGRESSION, CYTOPLASMIC MATURATION AND GENE EXPRESSION

2012 ◽  
Vol 24 (1) ◽  
pp. 212
Author(s):  
G. Z. Mingoti ◽  
F. Filion ◽  
P. Vincent ◽  
L. C. Smith
Keyword(s):  
Cell Communication ◽  
Phosphodiesterase Inhibitor ◽  
Developmental Competence ◽  
Mitochondrial Activity ◽  
Additional Time ◽  
Chi Square ◽  
Developmental Potential ◽  
Nuclear Maturation ◽  
Cytoplasmic Maturation

Meiotic block during a prematuration culture (pre-IVM) before in vitro maturation (IVM) is suggested as a way to provide additional time to synchronize oocyte-somatic cell communication, leading to improved cytoplasmic maturation and nuclear meiotic competence and the acquisition of critical cellular functions necessary for developmental competence. This study was designed to evaluate the effects of the inhibitors butyrolactone I (Bl-I) and cilostamide on oocyte nuclear and cytoplasmic maturation. For that, 2 well-established methods of pre-IVM and IVM for cilostamide (Albuz et al. 2010 Hum. Reprod. 25, 2999–3011) or Bl-I (Hashimoto et al. 2002 Biol Reprod. 66, 1696–1701) were used. Abattoir-collected oocytes were IVM in maturation medium (MM: mSOF with 0.8% BSA and hormones) for 24 h, without a previous pre-IVM culture (control). Cilostamide-group oocytes were treated for the first 2 h in vitro (pre-IVM) with 100 μM of the adenylate cyclase activator forskolin and 500 μM IBMX (nonspecific phosphodiesterase inhibitor) and then oocytes underwent extended IVM for 30 h in the presence of 20 μM cilostamide (type 3 phosphodiesterase inhibitor; pre-IVM 2 h + IVM 30 h). The Bl-I-group oocytes were pre-IVM for 24 h with 100 μM Bl-I diluted in TCM-199 supplemented with 0.2 mM pyruvate and then were IVM in MM for 20 h (pre-IVM 24 h + IVM 20 h). The Bl-I + Cilost group was a combination of both procedures: oocytes were first pre-IVM with Bl-I for 24 h and then cultured as described for the cilostamide group (pre-IVM 24 h + IVM 30 h). Cultures were carried out at 38.5°C in 5% CO2 in humidified air. After IVM, oocytes were stained with 500 nM mitotracker red to assess the mitochondrial membrane potential (Δψm) and with 10 μg mL–1 of Hoescht 33342 to evaluate the nuclear maturation (n = 207). Images were captured by an Olympus confocal microscope and analyzed with Fluoview software. The TUNEL assay was used to detect oocyte DNA fragmentation (n = 74). Relative amounts of mRNA for apoptotic-related genes were quantified after IVM in individual oocytes using real-time PCR (Kameyama et al. 2007 Reproduction 133, 423–32). Means were compared by ANOVA and Tukey's test or by chi-square (P ≤ 0.05). The percentage of oocytes reaching metaphase II after IVM did not differ among groups (73.8 to 90.4%; P ≥ 0.05), indicating that in vitro meiotic resumption was normal. The Δψm, expressed in arbitrary units of fluorescence, was 1.0 ± 0.1a (control), 2.7 ± 0.4b (Bl-I), 3.2 ± 0.5b (Cilost) and 2.1 ± 0.3ab (Bl-I + Cilost). The percentage of TUNEL-positive oocytes (18.8–41.3%) did not differ among groups (P ≥ 0.05). The relative abundance of BAX (1.0 ± 0.4 to 2.3 ± 0.4) and BCL-XL (1.0 ± 0.3 to 0.3 ± 0.1) transcripts was unaffected by pre-IVM and IVM (P ≥ 0.05). In conclusion, except for an increase in mitochondrial activity, pre-IVM with cilostamide and/or Bl-I did not affect cytoplasmic and nuclear oocyte maturation. However, oocyte developmental potential needs to be better evaluated in a future study through assessment of embryonic development. We acknowledge FAPESP and NSERC.

173 EFFECTS OF GLUCOSE METABOLISM DURING IN VITRO MATURATION ON CYTOPLASMIC MATURATION OF GOAT OOCYTES

2014 ◽  
Vol 26 (1) ◽  
pp. 201
Author(s):  
J.-H. Tan ◽  
Y.-B. Wang ◽  
H.-L. Xie ◽  
Q. Li ◽  
X.-Y. Liu ◽  
...  
Keyword(s):  
Glucose Metabolism ◽  
Oocyte Maturation ◽  
Polar Body ◽  
Pentose Phosphate ◽  
Blastocyst Development ◽  
Nuclear Maturation ◽  
First Polar Body ◽  
Cytoplasmic Maturation ◽  
Effect Of Glucose

It is well known that oocyte maturation consists of 2 processes: nuclear maturation and cytoplasmic maturation. Nuclear maturation refers to resumption of the first meiosis and extrusion of the first polar body (PB1), and cytoplasmic maturation is manifested as acquisition of the ability to complete pre-implantation development. Although it is recognised that energy supply is essential for oocyte maturation and there have been many reports on the effect of glucose metabolism on oocyte nuclear maturation, studies on the effect of glucose metabolism on ooplasmic maturation are limited. In the present study, goat oocytes recovered from slaughterhouse ovaries were cultured for 24 h in a simplified CR1 (sCR1) medium (NaCl, KCl, NaHCO3, CaCl2, BSA, and eCG) supplemented with glucose (10 mM) and/or lactate (3.5 mM) in the presence or absence of pentose phosphate pathway (PPP) inhibitor dehydroepiandrosterone (DHEA, 100 μM) or glycolysis inhibitor iodoacetate (1 μM). At the end of maturation culture, oocytes with PB1 were either activated by treatment with ionomycin plus 6-DMAP to observe embryo development, or assayed for total glutathione concentrations (GSX) and reduced glutathione (GSH)/oxidized glutathione (GSSG) ratios. Embryos were cultured for 9 days in CR1aa medium (NaCl, KCl, NaHCO3, calcium lactate, sodium pyruvate, glutamine, EAA, NEAA, and FCS) at 38.5°C under 5% CO2 in humidified air. In the absence of inhibitors, oocyte maturation rates of 82, 65, and 76%, and blastocyst rates of 7, 0, and 7%, were obtained, respectively, after oocytes were matured in sCR1 supplemented with glucose, lactate, or both. When oocytes were matured in sCR1 containing glucose and lactate in the presence of DHEA or iodoacetate, oocyte maturation rates were 69 and 67%, respectively, with no blastocyst produced in either case. However, whereas the presence of DHEA produced 12% morulae, no morulae were observed in the presence of iodoacetate. Furthermore, GSX concentrations (pmol/oocyte) were 8.5, 6.5, and 7.2, whereas GSH/GSSG ratios were 1.8, 0.3, and 0.5, respectively, after oocyte maturation without inhibitors or with 300 μM DHEA or 3 μM iodoacetate. The difference in GSX concentration was statistically significant (P < 0.05; one-way ANOVA) between DHEA and iodoacetate. In conclusion, using a culture system (sCR1 containing 3.5 mM lactate) that sustained oocyte nuclear maturation but did not support blastocyst development, we have studied the effect of PPP and glycolysis of glucose metabolism on the cytoplasmic maturation of goat oocytes. The results suggest that both PPP and glycolysis are essential for ooplasmic maturation of goat oocytes, and that both promote oocyte cytoplasmic maturation by increasing glutathione synthesis and reduction. This study was supported by grants from the National Basic Research Program of China (Nos. 2012CB944403 and 2014CB138503) and the China National Natural Science Foundation (Nos. 31272444 and 30972096).

scholarly journals Progesterone influences cytoplasmic maturation in porcine oocytes developingin vitro

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e2454 ◽  
Author(s):  
Bao Yuan ◽  
Shuang Liang ◽  
Yong-Xun Jin ◽  
Jeong-Woo Kwon ◽  
Jia-Bao Zhang ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Polar Body ◽  
Specific Inhibitor ◽  
Blastocyst Stage ◽  
Female Reproduction ◽  
Nuclear Maturation ◽  
First Polar Body ◽  
Porcine Oocyte ◽  
Cytoplasmic Maturation

Progesterone (P4), an ovarian steroid hormone, is an important regulator of female reproduction. In this study, we explored the influence of progesterone on porcine oocyte nuclear maturation and cytoplasmic maturation and developmentin vitro. We found that the presence of P4 during oocyte maturation did not inhibit polar body extrusions but significantly increased glutathione and decreased reactive oxygen species (ROS) levels relative to that in control groups. The incidence of parthenogenetically activated oocytes that could develop to the blastocyst stage was higher (p< 0.05) when oocytes were exposed to P4 as compared to that in the controls. Cell numbers were increased in the P4-treated groups. Further, the P4-specific inhibitor mifepristone (RU486) prevented porcine oocyte maturation, as represented by the reduced incidence (p< 0.05) of oocyte first polar body extrusions. RU486 affected maturation promoting factor (MPF) activity and maternal mRNA polyadenylation status. In general, these data show that P4 influences the cytoplasmic maturation of porcine oocytes, at least partially, by decreasing their polyadenylation, thereby altering maternal gene expression.

214 EFFECT OF LONG-DISTANCE TRANSPORTATION OF OVARIES ON THE DEVELOPMENT OF IN VITRO-MATURED - IN VITRO-FERTILIZED - IN VITRO-CULTURED BOVINE EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 206
Author(s):  
M. Kanae ◽  
K. Hisaichi ◽  
S. Jaswant ◽  
D. Osamu
Keyword(s):  
Calf Serum ◽  
Control Group ◽  
Long Distance ◽  
Chi Square ◽  
Maturation Rate ◽  
Long Time ◽  
Chi Square Test ◽  
Thermos Flask ◽  
Vitro Development

Keeping ovaries for a long time before oocyte recovery affects the maturation rate and blastocyst rate (BL rate), but a change in the temperature of the ovary at the time of transportation does not affect the BL rate (Ribeiro et al. 2008 Anim. Reprod. Sci. 108, 171–179). The objective of this study was to investigate the effect of long-distance transportation of ovaries on the in vitro development of bovine oocytes into blastocysts. Ovaries in the transportation group (Group T) were collected from a slaughterhouse, placed inside a Thermos flask, and transported to the laboratory within 17 to 21 h. The Thermos flask was covered with a freezer pack in a foam polystyrene box (Nakatate et al. 2006 Reprod. Fertil. Dev. 18, 193). The temperature of the Thermos flask was measured with a temperature recorder. Cumulus–oocyte complexes (COC) were collected by aspirating 2- to 6-mm follicles from the ovaries. The COC were matured in TCM-199 supplemented with 5% calf serum and 0.02 AU mL–1 of FSH. Groups of 20 COC were incubated in 100-μL drops of IVM media at 38.5°C under an atmosphere of 5% CO2 in air for 20 h. After 18 h of gamete co-culture (3 × 106 sperm mL–1), the presumptive zygotes were cultured in CR1aa medium supplemented with 5% calf serum for 9 days at 38.5°C under an atmosphere of 5% CO2, 5% O2, and 90% N2. Embryonic development was evaluated at 48 h after IVF (total cleavage rates, CL rate) and on Days 7 to 9 (BL rate). The ovaries in the control group (Group C) were collected from a slaughterhouse different from that of the Group T ovaries and transported to the laboratory in saline (23°C) in a Thermos flask within 3 h. These ovaries were then used for the collection of COC in the same way as for Group T. Data were analysed by chi-square test. A total of 25 experiments were performed using the ovaries from Groups C and T (n = 3945 v. n = 7218). The CL rate and the BL rate for Group T were significantly lower than those for Group C (41.8 v. 62.0% and 16.1 v. 29.7%; P < 0.01). In Group T, the duration of ovary transportation did not affect the CL rate (within 19 h, between 19 and 20 h, and more than 20 h), but the BL rate was significantly decreased with an increase in transport time (18.7 v. 16.1 v. 14.8%). The temperature change in the Thermos flask during transportation ranged from 12.0 to 25.1°C; the temperature was maintained virtually constant or decreased. The CL rates of the groups that underwent a temperature (t) change (t ≤ 5.0 and 5.0 < t ≤ 10.0) were not significantly different, but the BL rate in the group with 5.0 < t ≤ 10.0 was significantly lower than that of the other group (16.7 v. 13.5%; P < 0.01). These results suggest that the long-distance transportation of ovaries affects in vitro embryo development. Moreover, a decrease in temperature during transportation may have an adverse effect on blastocyst formation.

272 INTRACELLULAR NITRIC OXIDE LEVEL OF PORCINE OOCYTES IS NEGATIVELY CORRELATED WITH OOCYTE MATURATION RATE AND CUMULUS EXPANSION INDEX IN A CHEMICALLY DEFINED MEDIUM

2011 ◽  
Vol 23 (1) ◽  
pp. 234
Author(s):  
T. Uozumi ◽  
H. Funahashi
Keyword(s):  
Nitric Oxide ◽  
Oocyte Maturation ◽  
Defined Medium ◽  
Chemically Defined Medium ◽  
Dibutyryl Camp ◽  
Cumulus Expansion ◽  
Nuclear Maturation ◽  
Porcine Oocyte ◽  
Maturation Rate

Nitric oxide (NO) has been known to inhibit nuclear maturation in cumulus–enclosed oocytes in rodents. The objective of this study was to examine if meiotic stimulators, such as dibutyryl cAMP and epidermal growth factor (EGF), influence intracellular NO level of oocytes and if the level is correlated with oocyte maturation rate and cumulus expansion in a chemically defined medium. Oocyte–cumulus complexes (OCC) were aspirated from mid-size follicles (3–6 mm in diameter) of prepuberal porcine ovaries. The OCC were cultured in modified porcine oocyte medium with various supplements – gonadotropins plus dibutyryl cAMP (Gn + cAMP), EGF plus dibutyryl cAMP (EGF + cAMP), dibutyryl cAMP alone (cAMP), EGF alone (EGF), and non-supplements (none) – for a first 20-h period and then in fresh porcine oocyte medium (without those supplements) for another 24 h in an atmosphere of 5% CO2 in air at 39°C. Following in vitro maturation culture, OCC were assessed for the degree of cumulus expansion (scored from 0 as cumulus free to 5 as full expansion) and then additionally cultured with DAF2-DA, an indicator of NO, for an additional 1-h period in the same condition. The oocytes were denuded with 0.1% hyaluronidase, and the intensity of fluorescence was measured. The oocytes were also fixed, stained with acetic orcein, and observed for meiotic stage. Statistical analysis was performed by ANOVA with a Bonferroni-Dunn post hoc test (significance, P < 0.05). Maturation rates and cumulus expansion indexes were significantly affected by various supplement conditions (Table 1). The intensity of fluorescence showing intracellular NO level was also different among experimental groups (Table 1). A negative correlation was found between intracellular NO intensity and maturation rate (r2 = 0.71) or cumulus expansion index (r2 = 0.70). From these results, we conclude that there is a synergistic effect of cAMP and EGF on cumulus expansion and oocyte maturation and the reduction of oocyte NO levels in a chemically defined medium. Furthermore, a reduction of oocyte NO level seems to be included in the induction of cumulus expansion and oocyte maturation. Table 1.Effects of supplements on nuclear maturation, cumulus expansion, and intracellular NO level of porcine oocytes1

165 In vitro maturation of ovine and caprine oocytes during breeding and nonbreeding seasons

2019 ◽  
Vol 31 (1) ◽  
pp. 207
Author(s):  
M. Markle ◽  
C. K. Mak ◽  
V. Medina ◽  
C. R. F. Pinto
Keyword(s):  
Breeding Season ◽  
In Vitro Maturation ◽  
Statistical Significance ◽  
Polar Body ◽  
Cumulus Cells ◽  
Nonbreeding Season ◽  
Nuclear Maturation ◽  
Significant Difference ◽  
Maturation Rate

The current study investigated the in vitro meiotic competence of ovine and caprine oocytes that underwent nuclear maturation during the breeding and nonbreeding seasons. We hypothesised that maturation rates of ovine and caprine oocyte would be significantly lower during the nonbreeding season. Ovine (Katahdin crossbred) and caprine (mainly Spanish crossbred) ovaries were collected from a local abattoir in the southern United States. Age of the animals was not determined. Cumulus-oocyte complexes (COC) were harvested by slicing the ovaries and searching using a stereomicroscope. Oocytes with more than 3 layers of unexpanded cumulus cells and with evenly granulated cytoplasm were selected for in vitro maturation (IVM). A commercial bovine IVM media (IVF Bioscience, Falmouth, United Kingdom) was used throughout the study. After 24h of IVM, ovine and caprine oocytes were denuded and oocytes with an extruded polar body (meiotic metaphase II oocytes) were considered to have reached nuclear maturation. The seasons in this study were defined as follows: breeding season=September to April and nonbreeding season=May to July. The presence of corpus hemorrhagicum or corpus luteum in at least 70% of the ovaries indicated the breeding season for the animals. Proportions of oocytes undergoing nuclear maturation were analysed using a two-tailed Chi-squared test. Statistical significance was set at P ≤ 0.05. The ovine maturation rate was 59% (65/111) and 49% (254/519) and the caprine maturation rate was 70% (39/56) and 40% (64/162) during the breeding and nonbreeding seasons, respectively. These results show a significant difference in nuclear maturation for caprine oocytes (P&lt;0.001) during the breeding and nonbreeding seasons; however, there was no significant difference in nuclear maturation for ovine oocytes (P=0.06) during the breeding and nonbreeding seasons. High environmental temperatures during the nonbreeding season may have had detrimental effects on oocyte nuclear maturation in caprine but not in ovine oocytes. Why oocytes from these 2 species differ on how they are adversely affected by season remains to be elucidated.

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