144 A comparative study using flat, round, and V-shaped 96-well plates during bovine oocyte in vitro maturation

Paracrine factors secreted by the oocyte (oocyte-secreted factors, OSFs) regulate a broad range of cumulus cell functions including proliferation, differentiation, and apoptosis. The capacity of oocytes to regulate their own microenvironment by OSFs may in turn contribute to oocyte developmental competence. The aim of this study was to determine if OSFs have a direct influence on bovine oocyte developmental competence during in vitro maturation (IVM). Cumulus-oocyte complexes (COCs) were obtained by aspiration of >3-mm follicles from abattoir-derived ovaries. IVM was conducted in Bovine VitroMat (Cook Australia, Eight Mile Plains, Brisbane, Australia) supplemented with 0.1 IU/mL rhFSH for 24 h under 6% CO2 in air at 38.5�C. In the first experiment, COCs were co-cultured with denuded oocytes (DOs, 5/COC in 10 �L) beginning at either 0 or 9-h of IVM. To generate the 9-h DO group, COCs were first cultured intact for 9-h and then denuded. In the second experiment, specific OSFs, recombinant bone morphogenetic protein-15 (BMP-15) and growth differentiation factor 9 (GDF-9), were prepared as partially purified supernatants of transfected 293H cells, and used as 10% v/v supplements in Bovine VitroMat. Treatments were: (1) control (no supplement), (2) BMP-15, (3) GDF-9, (4) BMP-15 and GDF-9, and (5) untransfected 293H control. Following maturation, in vitro production of embryos was performed using the Bovine Vitro system (Cook Australia) and blastocysts were examined on Day 8 for development. Developmental data were arcsine-transformed and analyzed by ANOVA, followed by Tukey's test. Cell numbers were analyzed by ANOVA. Co-culturing intact COCs with DOs from 0 or 9 h did not affect cleavage rate, but increased (P < 0.001) the proportion of cleaved embryos that reached the blastocyst stage post-insemination (50.6 � 1.9 and 61.3 � 1.9%, respectively), compared to COCs cultured alone (40.7 � 1.4%). Therefore, paracrine factors secreted by DOs increased the developmental competence of oocytes matured as COCs. OSFs also improved embryo quality, as co-culture of COCs with DOs (0 or 9 h) significantly increased total cell (156.1 � 1.3 and 159.1 � 1.3, respectively) and trophectoderm (105.7 � 1.3 and 109.8 � 0.4, respectively) numbers, compared to control COCs (total = 148 � 1.2, trophectoderm = 98.2 � 0.8, P < 0.001). BMP-15 alone or with GDF-9 also significantly (P < 0.001) increased the proportion of oocytes that reached the blastocyst stage post insemination (57.5 � 2.4% and 55.1 � 4.5%, respectively), compared to control (41.0 � 0.9%) and 293H-treated (27.1 � 3.1%) COCs. GDF-9 also increased blastocyst yield (49.5 � 3.9%) but not significantly. These results are the first to demonstrate that OSFs, and particularly BMP-15 and GDF-9, directly affect bovine oocyte developmental competence. These results have far-reaching implications for improving the efficiency of IVM in domestic species and human infertility treatment, and support the role of OSF production by oocytes as a diagnostic marker for developmental competence.

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167 INHIBITION OF HEAT SHOCK PROTEIN 90 REDUCES DEVELOPMENTAL COMPETENCE OF BOVINE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv26n1ab167 ◽

2014 ◽

Vol 26 (1) ◽

pp. 197

Author(s):

E. D. Souza ◽

F. B. E. Paula ◽

C. C. R. Quintao ◽

J. H. M. Viana ◽

L. T. Iguma ◽

...

Keyword(s):

Heat Shock ◽

Heat Shock Protein ◽

In Vitro Maturation ◽

Hsp90 Inhibitor ◽

Stress Conditions ◽

Developmental Competence ◽

Bovine Oocyte ◽

Cleavage Rate ◽

Bovine Oocytes

The 90-kDa heat shock protein (HSP90) is a chaperone that is important for maintaing protein homeostasis under stress conditions. HSP90 seems also to be required for maturation of Xenopus oocytes (Fisher et al. 2000 EMBO J. 19, 1516) and first cleavage of mouse zygotes (Audouard et al. 2011 PloS One 6, e17109). This study aimed to evaluate the effect of inhibition of HSP90 by 17-(allylamino)-17-demethoxygeldanamycin (17AAG, Sigma St. Louis, MO, USA) during in vitro maturation (IVM) on bovine oocyte developmental competence. Immature cumulus–oocyte complexes (COC) were randomly allocated in 3 treatments during IVM: T0 (control; n = 240), no HSP90 inhibitor; T1: 2 μM HSP90 inhibitor (17AAG; n = 250) for the first 12 h of IVM; and T2: 2 μM HSP90 inhibitor (n = 188) for 24 h of IVM. In vitro maturation was performed in Nunc plates containing 400 μL of TCM-199 medium (Invitrogen, Carlsbad, CA, USA) supplemented with porcine FSH (Hertape Calier, Juatuba, Brazil) and 10% oestrus cow serum under 5% CO2, 95% humidity, and 38.5°C for 24 h. Oocytes were in vitro fertilized for 20 h and incubated under the same IVM conditions. Semen was processed by Percoll gradient (Nutricell, Campinas, Brazil) an IVF performed with 2 × 106 spermatozoa mL–1. Presumptive zygotes were completely denuded in a PBS solution with hyaluronidase and then cultured in wells with 500 μL of modified CR2aa medium supplemented with 2.5% fetal calf serum (Nutricell) in an incubator at 38.5°C under 5% CO2, 5% O2, 90% N2, and saturated humidity. Cleavage rate was evaluated 72 h post-fertilization and blastocyst rates were evaluated at Day 7 and Day 8. Data from 6 repetitions were analysed by generalized linear model procedure of SAS software (version 9.1; SAS Institute Inc., Cary, NC, USA), and means were compared by Student-Newman-Keuls test. Values are shown as mean ± s.e.m. There was a tendency (P = 0.08) for a lower cleavage rate in T2 (52.6 ± 5.8%) than in T0 (control; 74.2 ± 4.1%). Inhibition of HSP90 by 17AAG for 12 h and 24 h of IVM (T1 and T2, respectively) decreased blastocyst rates at Day 7 (20.4 ± 3.0% and 14.3 ± 2.6%, respectively; P < 0.01) and Day 8 (22.6 ± 4.1% and 16.9 ± 2.7%, respectively; P < 0.05) when compared with control (T0 = 31.8 ± 2.5% and 34.1 ± 2.9% for Day 7 and Day 8, respectively). In addition, the inhibition of HSP90 for 24 h decreased (P < 0.05) the proportion of hatched blastocysts at Day 8 (9.5 ± 5.0% for T2, respectively) when compared with control (T0 = 35.8 ± 3.9%), indicating a reduction on embryo quality. In conclusion, inhibition of HSP90 by 17AAG during IVM results in lower developmental competence, suggesting that this protein is also important for bovine oocytes. Further studies are required to investigate if the role of HSP90 on developmental competence of bovine oocyte is affected when under stress conditions. The authors acknowledge CNPq 473484/2011-0, FAPEMIG and FAPES for financial support.

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Effects of chronic exposure to 2G/3G cell phone radiationon in vitro maturation of bovine oocytes

Indian Journal of Animal Research ◽

10.18805/ijar.10769 ◽

2016 ◽

Author(s):

Yusuf Ziya Guzey ◽

Ali Galip Onal

Keyword(s):

Treatment Group ◽

Mobile Phone ◽

Cell Phone ◽

In Vitro Maturation ◽

Bovine Oocyte ◽

Maturation Period ◽

Mobile Phone Use ◽

Fluorescent Stain ◽

Bovine Oocytes

Mobile phone use has arisen and rapidly been increased during last decades and therefore electromagnetic radiation or heat sourcing from mobile phones and its possible damages has become focus of interest. The aim of the present study was to investigate the effects of mobile phone signals on in vitro maturation of bovine oocytes. A total of 224 bovine cumulus oocyte complexes were matured in vitro and separated into two groups, and only treatment group received mobile phone signals for 100 min by calling 5 min/h. Following maturation period, oocytes were stained by fluorescent stain to assess maturation rates. The results showed that oocytes reaching Metaphase I and Metaphase II stage were 35.2±2.24 % and 48.2±2.21 % for treatment group whereas 11.9±1.45 % and 73.8±4.60 % for controls, respectively (P<0.01). In conclusion, bovine oocyte maturation has been significantly reduced by mobile phone signals.

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Antioxidant Enzyme Activity and Oxidative Stress in Bovine Oocyte In Vitro Maturation

IUBMB Life ◽

10.1080/15216540152035073 ◽

2001 ◽

Vol 51 (1) ◽

pp. 57-64 ◽

Cited By ~ 85

Author(s):

P. D. Cetica ◽

L. N. Pintos ◽

G. C. Dalvit ◽

M. T. Beconi

Keyword(s):

Oxidative Stress ◽

Enzyme Activity ◽

Antioxidant Enzyme ◽

In Vitro Maturation ◽

Antioxidant Enzyme Activity ◽

Bovine Oocyte ◽

And Oxidative Stress

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Gonadotropins-mediated bovine oocyte in vitro maturation and its genetic and epigenetic change- ART safety in vitro model

Fertility and Sterility ◽

10.1016/j.fertnstert.2013.07.385 ◽

2013 ◽

Vol 100 (3) ◽

pp. S481

Author(s):

C.-L. Lu ◽

L.-Y. Yan ◽

T.-R. Wang ◽

R. Li ◽

H.-L. Feng ◽

...

Keyword(s):

In Vitro Maturation ◽

In Vitro Model ◽

Bovine Oocyte ◽

Epigenetic Change ◽

Vitro Model

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250 DEHYDRATED COCONUT WATER FOR IN VITRO SPERM CAPACITATION IN SWINE

Reproduction Fertility and Development ◽

10.1071/rdv17n2ab250 ◽

2005 ◽

Vol 17 (2) ◽

pp. 275 ◽

Cited By ~ 1

Author(s):

A.B. Nascimento ◽

V.P. Oliveira ◽

M.G. Marques ◽

R. Toniolli ◽

R.P.C. Gerger ◽

...

Keyword(s):

In Vitro Maturation ◽

Mammalian Species ◽

Staining Technique ◽

Domestic Animals ◽

Coconut Water ◽

Bovine Oocyte ◽

Sperm Capacitation ◽

Coomassie Blue ◽

Semen Extender

Recently, progress has been achieved in reproduction biotechnology with coconut water as semen extender for domestic animals and in bovine oocyte in vitro maturation and embryo culture. The aim of this study was to evaluate the dehydrated coconut water (ACP250) as semen extender and its effect on in vitro sperm capacitation in swine. Eighteen ejaculates were collected from three crossbred boars of Landrace and Duroc. Semen samples were extended in ACP250 solution or Beltsville Thawing Solution (BTS) and submitted to sperm capacitation after extension (EXT) or after cooling (COO) at 16°C for 24 h. The semen was centrifuged at 400g for 8 min. After centrifugation, the samples were standardized at 2 × 107 spermatozoa/mL, subjected to sperm capacitation in TALP medium with 5 mM of caffeine, and incubated at 38.5°C and 5% of CO2 in high humidity for three h. Four treatments were tested, T1 (ACP250 + EXT), T2 (ACP250 + COO), T3 (BTS + EXT), and T4 (BTS + COO). Sperm capacitation was evaluated by Coomassie Blue G stain, as follows: capacitated spermatozoa were stained in tenuous blue and not capacitated in intense blue. The Coomassie Blue G staining technique was described by Larson and Miller (1999 Mol. Reprod. Dev. 52, 445–449), who demonstrated that sperm from a variety of mammalian species can be stained by this technique, and an adequate observation of the acrosomal status was possible as well. This procedure was described as simple, fast, inexpensive and reliable, and it requires no fluorescence or DIC optics. For statistical analysis, SAS (System for Windows; SAS Institute, Inc., Cary, NC, USA) was utilized (P < 0.05). Comparing the sperm capacitation rates in relation to fresh vs. cooled semen, there were significant differences between T1 (22.3%) and T2 (43.7%) and between T3 (23.4%) and T4 (48.1%) (P < 0.05). In relation to BTS or ACP250 extenders, there were no significant differences between T1 and T3 and between T2 and T4 (P > 0.05). In conclusion, the ACP250 solution is an option for in vitro sperm capacitation in swine, specially with chilled extended semen. This work was supported by FAPESP 01/11931-8 and ACP Biotechnology – CE.

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202 CHARACTERIZATION OF BOVINE OOCYTE CYTOPLASMIC MATURATION WITH COMMON IN VITRO MATURATION PROTOCOLS

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab202 ◽

2016 ◽

Vol 28 (2) ◽

pp. 232

Author(s):

B. A. Foster ◽

F. A. Diaz ◽

P. T. Hardin ◽

E. J. Gutierrez ◽

K. R. Bondioli

Keyword(s):

Gap Junctions ◽

Gap Junction ◽

In Vitro Maturation ◽

Transvaginal Ultrasound ◽

Bovine Oocyte ◽

Hoechst 33342 ◽

Nuclear Maturation ◽

Mitochondrial Distribution ◽

Cytoplasmic Maturation

Modulators of 3′-5′-cyclic adenosine monophosphate have been extensively researched to delay nuclear maturation in in vitro maturation (IVM) systems to improve synchronization of nuclear and cytoplasmic maturation. However while normal maturation for many organelles has been characterised, there is a lack of information on how modulators affect cytoplasmic maturation. The goal of this study was to identify the effect of different components of bovine oocyte maturation systems on 3 aspects of cytoplasmic maturation. Bovine oocytes were collected from mixed breed beef cattle using transvaginal ultrasound guided oocyte aspiration. Oocytes were assigned to 1 of 4 treatments; staining immediately after collection (n = 249) or after 24 h of IVM (n = 270), 2 h of pre-IVM in Forskolin and 3-isobutyl-1-methylxanthine (IBMX; n = 254), or 2 h of pre-IVM followed by IVM (n = 259). Following treatment, half of the recovered oocytes were stained with Hoechst 33342 to determine nuclear maturation status, and Calcein AM for gap junction status. The other half were stained with Hoechst 33342, Mitotracker deep red to identify mitochondria distribution patterns and Alexa Fluor 488 conjugated phalloidin for F actin microfilament distribution. Organelle patterns were coded and statistically analysed using linear models to determine if treatment had an effect on the indicators of cytoplasmic maturation or their agreement with nuclear maturation. Results indicated that there was a high degree of variability in both cytoplasmic and nuclear maturation of oocytes irrespective of treatment group, with many oocytes exhibiting aberrant patterns in both mitochondrial and microfilament distribution. Gap junctions were classified as open (immature), partially open or closed (mature), based on the strength of Calcein fluorescence within the ooplasm. Both treatment and nuclear maturation had a significant effect on gap junction status (P < 0.001) with gap junctions tending to close as oocytes matured, while treatment in pre-IVM maintained open gap junctions, even as meiosis progressed. Mitochondria were classified as peripheral (immature), diffuse, central (mature) or too sparse to accurately classify. There was an unexpectedly high proportion of oocytes with few mitochondria (17%), suggesting an incomplete growth phase before collection. There was no correlation between meiotic stage and mitochondrial distribution (P = 0.73), with the majority of oocytes having diffuse mitochondrial distribution. As normal maturation proceeds, microfilaments aggregate and migrate peripherally. However, neither microfilament aggregation nor redistribution were correlated with nuclear maturation (P = 0.6 and P = 0.11 respectively) or mitochondrial distribution (P = 0.33 and P = 0.06 respectively). Overall, results show that while pre-IVM maintains open gap junctions, the system studied here is not sufficient for improving correlation between cytoplasmic and nuclear maturation. Many deviations from normal cytoplasmic maturation are seen with IVM and these irregularities are maintained with prematuration in Forskolin and IBMX.

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176 Effect of epigallocatechin-3-gallate on bovine oocyte in vitro maturation, fertilization, and development

Reproduction Fertility and Development ◽

10.1071/rdv31n1ab176 ◽

2019 ◽

Vol 31 (1) ◽

pp. 212

Author(s):

Y. Honkawa ◽

Y. Gen ◽

S.-H. Hyon ◽

C. Kubota

Keyword(s):

Oocyte Maturation ◽

In Vitro Maturation ◽

Polar Body ◽

Sperm Concentration ◽

P Value ◽

Bovine Oocyte ◽

Vitro Fertilization ◽

Significant Difference ◽

Maturation Rate

Epigallocatechin-3-gallate (EGCG) is a major ingredient of catechin polyphenols, and a strong antioxidant compound. Huang et al. (2018 Asian-australas. J. Anim. Sci.) reported that adding 50μM EGCG can improve the bovine oocyte maturation rate. In this research, we investigated the effect of EGCG supplementation on different periods in bovine IVF. Cumulus-oocyte complex (COC) collected from ovaries of slaughtered cows were cultured in maturation medium (20 to 30 oocytes per 100-µL droplet), which consisted of TCM-199 with Earle’s salts and 25mM HEPES supplemented with 10% (vol/vol) fetal bovine serum (FBS), 1µg mL−1 oestradiol, 0.02mg mL−1 FSH, and antibiotics at 38.5°C in a humidified atmosphere of 5% CO2 in air for 24h (in vitro maturation, IVM). After IVM, COC were fertilized in the fertilization medium (modified Brackett-Oliphant media supplemented with 10 µgmL−1 heparin, 10mM caffeine, and 3mg mL−1 BSA) for 6h using semen of one bull at final sperm concentration of 1×107 mL−1 (IVF). After IVF, COC were denuded and cultured in culture medium [CR1aa supplemented with 10% (vol/vol) FBS and antibiotics] at 38.5°C in a humidified atmosphere of 5% O2, 5% CO2, and 90%N2 for 8 days (in vitro culture, IVC). The EGCG was supplemented at 10, 25, 50, and 100M in IVM medium; 25 and 50 µM in IVF medium; and 50 and 100 µM in IVC medium. After 24h in IVM medium, COC were denuded by pipetting, fixed in 3:1 ethanol:acetic acid for 24h and then checked for nuclear and polar body by using aceto-orcein stain. After 18h in IVF, the pronucleus in zygote was fixed in 3:1 ethanol:acetic acid for 24h and checked by aceto-orcein staining. Embryo development was evaluated by counting the total number of embryos that had reached compacted morula by 6 to 8 days after IVF. Significant differences were analysed by chi-squared test and residual analysis. A P-value<0.05 was considered statistically significant. When EGCG was added to IVM, there was no significant difference of oocyte maturation rate between all concentrations (0v. 10v. 25v. 50v. 100 μM: 73.9% v. 56.7% v. 76.7% v. 72.7% v. 63.5%). When EGCG was added to IVF, there was no significant difference of fertilized rate (0v. 25v. 50 μM: 59.4% v. 73.7% v. 64.9%). When EGCG was added to IVC, there was no significant difference in development rate (0v. 50v. 100 μM: 26.2% v. 15.7% v. 22.0%). In this research, EGCG addition did not affect bovine in vitro fertilization.

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