Supplementing in-vitro maturation media with human follicular fluid improves both the maturation rate of mouse immature oocytes and the subsequent embryonic development

The aim of this study was 2-fold: (1) To determine if altrenogest-treated mares will yield higher numbers of quality in vitro-matured (IVM) oocytes than early pregnant mares and cycling/control mares and (2) if the addition of human follicular fluid (HFF) to IVM medium can support IVM and viable pregnancies from in vitro-produced blastocysts. In this study, 18 mares were assigned to 3 equally sized treatment groups and each mare was subjected to follicle aspiration every 10 to 11 days without monitoring follicular growth. The 3 treatment groups were altrenogest-treated mares (0.044 mg kg–1 of PO daily), early pregnant mares (30–110 days) and control/cycling mares. Using transvaginal ultrasound guidance, all visible follicles were aspirated. Altrenogest-treated mares each yielded more follicles (8.75) per aspiration session when compared with the control mare group (5.75) and the pregnant mare group (3.72), but there was no difference in oocyte recovery rates among the groups (Table 1). A limited number of these oocytes were subjected to in vitro maturation. After heated (38.5°C) transport of oocytes to an off-site laboratory, the oocytes were placed in maturation medium containing 10% HFF obtained from preovulatory follicles after ovulation induction, 20% serum substitute supplement and no hormones for 36 h. This approach yielded a maturation rate of 61.8, 68.8 and 82.0% for the altrenogest, pregnant and control treatment groups, respectively (not significant). Mature oocytes (n = 65) were injected with frozen-thawed sperm using a standard intracytoplasmic sperm injection (ICSI) technique. Four expanding blastocysts (Table 1) were selectively transported back to the embryo transfer facility and transcervically transferred into recipient mares on Day 6 post-ICSI. These 4 transfers resulted in 2 viable, normally progressing pregnancies, ongoing beyond 60 days of gestation. Both pregnancies resulted from the altrenogest-treated aspiration group. In this study we concluded that (1) altrenogest-treated mares provide more follicles and may be a better source of viable immature oocytes for the production of ICSI embryos and foals, but their overall advantage is unclear; (2) addition of HFF to IVM media, in the absence of added gonadotropins, can support oocyte maturation, blastocyst production and viable pregnancies; (3) an aspiration schedule of every 10 to 11 days without ultrasonic monitoring can yield viable immature oocytes, capable of producing ICSI blastocysts, resulting in viable pregnancies. Table 1.Altrenogest-treated mares compared to early pregnant mares and control mares

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208 EFFECT OF GDF8 AND SB-431542 ON PORCINE OOCYTE DURING IN VITRO MATURATION AND SUBSEQUENT EMBRYONIC DEVELOPMENT

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab208 ◽

2016 ◽

Vol 28 (2) ◽

pp. 235

Author(s):

J. D. Yoon ◽

E. Lee ◽

S.-H. Hyun

Keyword(s):

Treatment Group ◽

Embryonic Development ◽

Follicular Fluid ◽

In Vitro Maturation ◽

Transforming Growth Factor ◽

Formation Rate ◽

Transforming Growth Factor Β ◽

Nuclear Maturation ◽

Porcine Oocyte

Growth differentiation factor-8 (GDF8) is a member of the transforming growth factor-β that has been identified as a strong physiological regulator. SB-431542 (SB) is a specific inhibitor of transforming growth factor-β superfamily type I activin receptor-like kinase (ALK) receptors such as ALK4, ALK5, and ALK7. The purpose of this study is investigation of the effects of GDF8 and SB on porcine oocytes during in vitro maturation and subsequent embryonic development. We first performed ELISA to detect GDF8 concentrations in follicular fluid for each size of follicle; sizes were as follows: small (<3 mm), medium (>3 mm and <6 mm), and large (>6 mm) follicle. After detection of the GDF8 concentration in follicular fluid, we investigated the effect of GDF8 and SB treatment during in vitro maturation (IVM) on nuclear maturation, intracellular glutathione (GSH), and reactive oxygen species (ROS) levels, and embryonic development after IVF and parthenogenetic activation (PA). Data were analysed by ANOVA followed by Duncan using SPSS (Statistical Package for Social Science, IBM, New York, NY, USA) mean ± SEM. The ELISA result showed different concentrations of GDF8 for each grade of follicular fluid: small, 0.479 ng mL–1; medium, 0.668 ng mL–1; and large, 1.318 ng mL–1. During the IVM process, 1.318 ng mL–1 of GDF8 and 5 ng mL–1 of SB were added to the maturation medium as control, SB, SB+GDF8, and GDF8 treatment groups. After 44 h of IVM, GDF8 group (90.4%) showed a significantly higher nuclear maturation rate than control and SB+GDF8 groups (85.4 and 81.7%). The SB group (78.9%) showed significantly reduced nuclear maturation rate compared with control (P < 0.05). The GDF8 treatment group showed a significant decreased intracellular ROS and increased GSH levels compared with other groups (P < 0.05). The SB+GBF8 treatment group showed a significantly better cytoplasmic maturation than the SB treatment group. In the PA embryonic development analysis, the GDF8 treatment group showed a significantly higher blastocyst formation rate compared with other groups (47.9, 37.2, 46.4, and 58.7% respectively; P < 0.05). In the IVF embryonic development analysis, the GDF8 treatment groups showed significantly higher blastocyst formation rate compared with the SB group (28.2 and 42.2%, respectively; P < 0.05). In conclusion, treatment with GDF8 during porcine oocyte IVM improved the embryonic developmental competence via increased cytoplasmic maturation and led to better oocyte maturation from the ALK receptor inhibition by SB.

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Porcine follicular fluid derived from > 8 mm sized follicles improves oocyte maturation and embryo development during in vitro maturation of pigs

Zygote ◽

10.1017/s0967199420000398 ◽

2020 ◽

pp. 1-6

Author(s):

Ji-Eun Park ◽

Sang-Hee Lee ◽

Yong Hwangbo ◽

Choon-Keun Park

Keyword(s):

Embryonic Development ◽

Follicular Fluid ◽

In Vitro Maturation ◽

Developmental Competence ◽

Control Group ◽

Cumulus Expansion ◽

Treatment Groups ◽

Nuclear Maturation ◽

Cytoplasmic Maturation

Summary The aim of the present study was to investigate the effects of porcine follicular fluid (pFF) from large-sized (LFF; >8 mm in diameter) and medium-sized (MFF; 3–6 mm in diameter) follicles on the maturation and developmental competence of porcine oocytes. Cumulus–oocyte complexes (COCs) were collected from follicles 3–6 mm in diameter. The collected COCs were incubated for 22 h with LFF or MFF (in vitro maturation (IVM)-I stage) and were incubated subsequently for 22 h with LFF or MFF (IVM-II stage). Cumulus expansion was confirmed after the IVM-I stage and nuclear maturation was evaluated after the IVM-II stage. Intracellular glutathione (GSH) and reactive oxygen species (ROS) levels were measured and embryonic development was evaluated. Relative cumulus expansion and GSH levels were higher in the LFF group compared with in the MFF group after the IVM-I stage (P < 0.05). After the IVM-II stage, the numbers of oocytes in metaphase-II were increased in the LFF group and GSH content was higher in all of the LFF treatment groups compared with in the MFF treatment groups during both IVM stages (P < 0.05). ROS levels were reduced by LFF treatment regardless of IVM stage (P < 0.05). Blastocyst formation and the total numbers of cells in blastocysts were increased in all LFF treatment groups compared with the control group (P < 0.05). These results suggested that pFF from large follicles at the IVM stage could improve nucleic and cytoplasmic maturation status and further embryonic development through reducing ROS levels and enhancing responsiveness to gonadotropins.

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Supplementing ±-Linolenic acid in the in vitro maturation media improves nuclear maturation rate of oocytes and early embryonic development in the Nili Ravi buffalo

Animal Reproduction ◽

10.21451/1984-3143-ar859 ◽

2017 ◽

Vol 14 (4) ◽

pp. 1161-1169

Author(s):

Asima Azam ◽

Qaisar Shahzad ◽

Asma Ul-Husna ◽

Saima Qadeer ◽

Rabea Ejaz ◽

...

Keyword(s):

Embryonic Development ◽

Linolenic Acid ◽

In Vitro Maturation ◽

Early Embryonic Development ◽

Nuclear Maturation ◽

Maturation Rate

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Effect of follicle-stimulating hormone on Bligon goat oocyte maturation and embryonic development post in vitro fertilization

Veterinary World ◽

10.14202/vetworld.2020.2443-2446 ◽

2020 ◽

Vol 13 (11) ◽

pp. 2443-2446

Author(s):

Diah Tri Widayati ◽

Mulyoto Pangestu

Keyword(s):

In Vitro Fertilization ◽

Embryonic Development ◽

Oocyte Maturation ◽

In Vitro Maturation ◽

Follicle Stimulating Hormone ◽

Fertilization In Vitro ◽

Vitro Fertilization ◽

Maturation Rate ◽

Stimulating Hormone

Background and Aim: Bligon goat is a crossbreed between Etawah and Kacang goat. This crossbreed goat is mostly reared by small farmers. In vitro maturation allows female goat (does) contributes toward reproduction despite the fact that the animal has been slaughtered. The aim of this study was to determine the in vitro maturation rate of Bligon goat oocytes supplemented with follicle-stimulating hormone (FSH), and their ability for further embryonic development after in vitro fertilization. Materials and Methods: Experiment was conducted at the Laboratory of Animal Physiology and Reproduction, Faculty of Animal Science, Universitas Gadjah Mada, Yogyakarta, using Bligon goat ovaries obtained from local slaughterhouse around Yogyakarta. One thousand five hundred cumulus-oocyte complexes were matured for 24 h in tissue culture medium 199 supplemented with 50 IU/L FSH or without FSH (control). First, matured oocytes were evaluated its morphology based on the expansion of cumulus cells and PB1 extrusion. Next, 600 oocytes were then stained with 1% aceto-orcein to examine maturation based on changes in the configuration of chromosomes and nuclear membrane breakdown. Oocytes were considered mature when they reached metaphase II. To prove the ability of mature oocytes to develop into embryos, 900 oocytes were processed for fertilization in vitro. The data were analyzed using analysis of variance. Results: The results indicated that FSH supplementation significantly increased oocyte maturation rate (65.21±7.26 vs. 43.25±6.23%) as indicated by extrusion of PB1 and homologous chromosome pairing and lined in the equator. The rate of degeneration was lower in the FSH-supplemented medium (3.21±0.25 vs. 10.17±3.15%). The blastocyst stage of oocyte developed embryos was reached by 12.43±2.15% and 22.28±4.86% of the control and treatment groups, respectively. Conclusion: FSH supplementation significantly improves oocyte maturation and yields mature oocytes for future embryo development in vitro.

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286 EFFECTS OF PORCINE FOLLICULAR FLUID DURING IN VITRO MATURATION OF OOCYTES ON IN VITRO FERTILIZATION AND EMBRYONIC DEVELOPMENT

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab286 ◽

2007 ◽

Vol 19 (1) ◽

pp. 258

Author(s):

B. Agung ◽

T. Otoi ◽

D. Fuchimoto ◽

S. Senbon ◽

A. Onishi ◽

...

Keyword(s):

In Vitro Fertilization ◽

Embryonic Development ◽

Follicular Fluid ◽

Culture System ◽

In Vitro Maturation ◽

Control Group ◽

Rotating System ◽

Vitro Fertilization ◽

Maturation Medium

When used as a solo maturation medium for oocytes, porcine follicular fluid (pFF) promoted male pronucleus formation (MPF) of oocytes after in vitro maturation (IVM), using a static system, and in vitro fertilization (IVF) in pigs (Naito et al. 1988 Gamete Res. 21, 289–295). However, the developmental competence of oocytes matured in pFF after IVM/IVF has not been reported. This study was conducted to assess the ability of pFF as a maturation medium to support IVM/IVF of porcine oocytes and their subsequent in vitro development. pFF, including cumulus–oocyte complexes (COCs), was aspirated from follicles (2–5 mm in diameter) of prepubertal crossbred gilt ovaries, and large clusters of follicular cells (FC) were removed from pFF by filtration through 212 �m of mesh. All of the COCs in filtered pFF were collected, and COCs with compact cumulus cells were selected for IVM. Also, small clusters of FC were collected by centrifugation of the filtered pFF, and pFF without any cells was prepared by centrifugation and used as a maturation medium (MpFF) after supplementation with FSH and antibiotics. COCs were transferred to 3.5 mL (in a 15-mL test tube) of MpFF with FC (5.2 � 106 cells mL-1) and cultured for 44–48 h at 38.5�C in 5% O2 and 5% CO2 using the rotating culture system. As a control group, COCs were cultured in 2 mL of MpFF without FC in a 35-mm Petri dish by the standard static culture system. After maturation, culture oocytes were co-incubated (IVF) for 5 h with frozen–thawed sperm in vitro, as reported previously (Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041), and then some of them were fixed 10 h after IVF to assess the fertilization status; the rest of them were cultured in PZM (Yoshioka et al. 2002 Biol. Reprod. 60, 112–119) for 7 days to assess their early embryonic development. All of the data were analyzed by ANOVA. Oocytes cultured with FC in the rotating system (R group) showed significantly higher sperm penetration (71.0%), MPF formation (70.5%), and normal fertilization (monospermic fertilization with female and male pronuclei; 31.5%) rates than those in the control group (56.0%, 56.9%, and 17.6%, respectively; P < 0.05). Also, the R group showed significantly higher rates of 8-cell embryos at 2 days after IVF and blastocyst formation at 7 days after IVF than those of the control group (17.2% vs. 8.3% and 10.9% vs. 4.5%, respectively; P < 0.05). These results indicate that porcine oocytes matured in pFF supplemented with FC using the rotating system have the ability to be penetrated by sperm and form MPF, and to develop to the blastocyst stage at higher rates, than oocytes cultured in the standard static maturation culture system. In conclusion, the pFF can be a sole and simple maturation culture medium useful for the in vitro production of blastocysts in pigs.

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In vitro maturation of buffalo oocytes and fertilization by cattle spermatozoa

Bangladesh Journal of Animal Science ◽

10.3329/bjas.v41i1.11969 ◽

2012 ◽

Vol 41 (1) ◽

pp. 6-12

Author(s):

MAMY Khandoker ◽

MMT Reza ◽

LY Asad ◽

S Saha ◽

AS Apu ◽

...

Keyword(s):

Bovine Serum Albumin ◽

Serum Albumin ◽

Follicular Fluid ◽

Bovine Serum ◽

In Vitro Maturation ◽

Fertilization Rate ◽

Maturation Rate ◽

Normal Fertilization

The present research was undertaken to explore the maturation of buffalo oocytes using bovine follicular fluid (BFF) and bovine serum albumin (BSA), as well as subsequent fertilization using cattle spermatozoa. The cumulus oocyte complexes (COCs) were collected by aspiration of slaughterhouse buffalo ovaries. Maturation was performed in TCM 199 supplemented with 10% BFF, 5% BSA or without supplementation (control). The COCs were fertilized in Brackett and Oliphant (BO) medium using capacitated fresh cattle spermatozoa. It was observed that the percentage of COCs reached to M-II stages were 40.78±3.84, 65.74±2.39 and 67.52±0.85; normal fertilization (formation of 2 pronuclei) were 23.28±3.00, 29.30±0.73 and 30.52±1.21 for control, 10% BFF and 5% BSA supplementation, respectively. The supplementation of BFF (10%) and BSA (5%) were given similar results on maturation and increased significantly (p<0.05) than that of the control. It was observed that cattle spermatozoa were fertilized by the buffalo oocytes and the fertilization rate was 23.28% to 30.52% in BFA and BSA supplemented media, respectively. It can be concluded that buffalo oocytes might be fertilized using capacitated cattle spermatozoa and both 10% BFF and 5% BSA could be supplemented in maturation media to enhance the maturation rate as well as fertilization of buffalo oocytes.http://dx.doi.org/10.3329/bjas.v41i1.11969

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Does the Apoptosis Value of Cumulus Cells Play a Role in Rescue Oocyte in Vitro Maturation?

Gynecology Obstetrics and Reproductive Medicine ◽

10.21613/gorm.2020.1087 ◽

2020 ◽

pp. 1

Author(s):

Tulay Irez ◽

Sinem Ercan Dogan ◽

Enver Ciraci ◽

Saadet Busra Aksoyer ◽

Muhammet Sait Toprak ◽

...

Keyword(s):

Experimental Study ◽

Luteinizing Hormone ◽

Embryo Development ◽

In Vitro Maturation ◽

Germinal Vesicle ◽

Cumulus Cells ◽

Immature Oocytes ◽

Maturation Rate

OBJECTIVE: In this study, we aimed to investigate the role of the cumulus cell’s apoptosis parameter in the maturation of immature rescue oocytes. STUDY DESIGN: In this experimental study, donated immature germinal vesicle oocytes were cultured for, in vitro maturation, embryo development in matured germinal vesicle oocytes were compared with apoptotic properties of cumulus cells. RESULTS: In all of the immature oocytes after oocyte in vitro maturation, the maturation rate has been observed as 56.1% and 2PN rate as 63.0%. Afterin vitro maturation of germinal vesicle oocytes, there was no difference in apoptosis rates of the cumulus cells between mature and immature oocytes (p> 0.05). The ratio of 2PN in matured germinal vesicle oocytes showing embryo development was 35.4%. A positive correlation was found between luteinizing hormone values on day 3 and E2 values during HCG days during oocyte maturation and embryo development (p=0.021, p=0.020). In addition, it has been observed that the germinal vesicle oocytes, which have completed their maturation and developed into embryos, have high E2 values during HCG days (p=0.020).CONCLUSION: In our study, it has been demonstrated that in vitro maturation in rescue oocytes from stimulated cycles, embryo development potential could not be explained by the apoptosis parameter.

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303 EFFECT OF SERUM SUPPLEMENTATION AND ESTRUS CYCLE STAGE ON IN VITRO NUCLEAR MATURATION OF CANINE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv17n2ab303 ◽

2005 ◽

Vol 17 (2) ◽

pp. 302

Author(s):

F.Y. Heru ◽

H.J. Oh ◽

M.K. Kim ◽

J. Goo ◽

M.S. Hossein ◽

...

Keyword(s):

Luteal Phase ◽

In Vitro Maturation ◽

Germinal Vesicle ◽

Follicular Phase ◽

Estrus Cycle ◽

Immature Oocytes ◽

Nuclear Maturation ◽

Serum Supplementation ◽

Maturation Rate

The present study investigated the effects of the estrus cycle stage and serum supplementation on nuclear maturation of canine oocytes. Ovaries were collected from a private clinic after ovariohysterectomy and classified into follicular, luteal, or anestrus stages through a combination of ovarian morphology and vaginal cytology. A total of 2214 oocytes from 196 ovaries (903 oocytes from 96 anestrus ovaries, 609 oocytes from 36 follicular ovaries, and 702 oocytes from 64 luteal ovaries) were used for experiments. The oocyte retrieval per ovary was 10, 19, and 12 for anestrus, follicular and luteal-phase ovaries, respectively. In Exp. 1, immature oocytes were cultured for 72 h in TCM-199 alone or TCM-199 supplemented with 10% canine anestrus (CAS), estrus (CES), or diestrus (CDS) serum or fetal bovine serum (FBS). In Exp. 2, immature oocytes were cultured for 72 h in TCM-199 supplemented with 0, 5, 10, or 20% CES. After staining with Hoechst 33342, chromatin state and position as well as spindle formation were evaluated to determine the stage of meiosis: germinal vesicle (GV) stage, germinal vesicle breakdown (GVBD), metaphase I (MI) stage, metaphase II (MII) stage. The experiments with anestrus and luteal-phase oocytes were repeated eight times and follicular-phase oocytes were repeated six times. Data were subjected to analysis of variance (ANOVA) and protected least significant difference (LSD) test to determine differences among experimental groups by using the Statistical Analysis System (SAS, SAS Institute, Inc., Cary, NC, USA) program. Statistical significance was determined where P value was less than 0.05. In Exp. 1, the in vitro maturation of oocytes up to MII stage was higher when oocytes were collected from ovaries in follicular phase. The maturation rate up to MII stage was 0.0 to 1.7%, 1.3 to 10.2%, and 1.0 to 3.2% for the oocytes collected from the anestrus, follicular, and luteal-phase ovaries, respectively, depending on the culture media used. In basic TCM media only, 0.0, 1.3, and 2.3% oocytes reached the MII stage for anestrus, follicular, and luteal-phase oocytes, respectively. A significantly higher rate of maturation was obtained when oocytes collected from follicular phase were cultured in TCM-199 supplemented with 10% CES (10.2%), compared to 10% CAS (4.0%), CDS (2.7%), FBS (1.3%), or the control (1.3%). In Exp. 2, supplementing with 10% CES induced the highest (P < 0.05) maturation rate to the MII stage in oocytes collected from follicular-stage ovaries (11.5%) compared to supplementing with 0% (1.0%), 5% (1.3%), or 20% CES (5.1%). Supplementing with CES (5, 10, or 20%) did not have a significant effect on nuclear maturation of canine oocytes collected from anestrus or luteal-stage ovaries. In conclusion, supplementing in vitro maturation medium with 10% CES increased nuclear maturation of canine oocytes, and canine oocytes collected from follicular-stage ovaries are the most suitable to complete nuclear maturation in vitro. This study was supported by grants from the Biogreen 21-1000520030100000.

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