188 HUMAN FOLLICULAR FLUID IN EQUINE IN VITRO MATURATION MEDIUM SUPPORTS IN VITRO MATURATION AND VIABLE INTRACYTOPLASMIC SPERM INJECTION-BLASTOCYST PREGNANCIES

2012 ◽  
Vol 24 (1) ◽  
pp. 206
Author(s):  
C. Makloski ◽  
R. Gotti ◽  
K. Harris ◽  
J. Bottger ◽  
M. Meintjes
Keyword(s):  
Follicular Fluid ◽  
Intracytoplasmic Sperm Injection ◽  
In Vitro Maturation ◽  
Transvaginal Ultrasound ◽  
Human Follicular Fluid ◽  
Immature Oocytes ◽  
Treatment Groups ◽  
Maturation Medium ◽  
And Control

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

scholarly journals LÍQUIDO FOLICULAR EQÜINO NA MATURAÇÃO IN VITRO DE OÓCITOS BOVINOS

2004 ◽  
Vol 9 (1) ◽  
Author(s):  
M.G.L. PINTO ◽  
M.I.B. RUBIN ◽  
C.A.M. SILVA ◽  
T.F. HILGERT ◽  
M.F. SÁ FILHO ◽  
...  
Keyword(s):  
Follicular Fluid ◽  
In Vitro Maturation ◽  
Mineral Oil ◽  
Control Group ◽  
Sodium Pyruvate ◽  
Vitro Fertilization ◽  
Treatment Groups ◽  
Maturation Medium ◽  
Bovine Oocytes

O desenvolvimento embrionário de oócitos bovinos maturados in vitro (MIV) foi avaliado em meio suplementado com líquido folicular eqüino (Lfe). Foram distribuídos 1045 oócitos em 11 repetições formando três grupos tratamentos (T1, T2, T3) e um controle (C). O meio de maturação utilizado foi o TCM-199 acrescido de piruvato de sódio, hormônio folículo estimulante recombinante (rFSHh) e hormônio luteinizante equino (LHe). Suplementou-se esse meio com 10% de soro de égua em estro para o grupo controle e para T1, T2 e T3, o meio foi suplementado com 5, 10, e 20% de LFe, respectivamente. Os oócitos foram maturados in vitro (MIV) por 24h. A fecundação in vitro (FIV) foi realizada em meio Talp-Fert. A MIV e a FIV foram realizadas em estufa a 39ºC com 5% de CO2 em ar e umidade saturada. Os zigotos foram cultivados em meio SOFaaci, sob óleo mineral no interior de bolsas plásticas gaseificadas. As taxas de clivagem e de blastocistos foram observadas diariamente (D), e em D7, foram superiores (P0,05) às do grupo controle. Em D9, a taxa de blastocistos do T2 foi superior (P0,05). O LFe, na concentração de 10% pode ser utilizado, em substituição ao soro de égua em estro para suplementar o meio de MIV de oócitos bovinos. Equine follicular fluid on in vitro maturation of bovine oocytes Abstract Embryo development of bovine oocytes was evaluated using maturation medium supplemented with equine follicular fluid (eFF). One thousand and forty five (1045) oocytes were distributed in 11 replications forming three treatment groups (T1, T2 e T3) and one Control (C). TCM-199 added with sodium pyruvate, rFSHh and LHe was used as maturation medium. This medium was supplemented with 10% estrous mare serum for Control group, and 5, 10, and 20% eFF, respectively, for T1, T2 e T3 groups. In vitro maturation (IVM) of all groups was performed during 24h. In vitro fertilization (IVF) was performed in TALP-FERT medium. IVM and IVF were carried out in an incubator at 39ºC with 5% CO2 in air and saturated humidity. Zygotes were cultured in SOFaaci medium, under mineral oil in gasified bags. Cleavage and blastocyst rates were daily observed (D), and at D7, were higher (P0.05) for those from control group. At D9, blastocyst rate of T2 was higher (P0.05). The eFF, at a 10% concentration, can replace the use of estrous mare serum to supplement the IVM medium of bovine oocytes.

scholarly journals Effects of melatonin and human follicular fluid supplementation of in vitro maturation medium on mouse vitrified germinal vesicle oocytes: A laboratory study

2021 ◽  
pp. 889-898
Author(s):  
Razieh Doroudi ◽  
Zohre Changizi ◽  
Seyed Noureddin Nematollahi-Mahani
Keyword(s):  
Follicular Fluid ◽  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Cell Stage ◽  
Human Follicular Fluid ◽  
Vitro Fertilization ◽  
Treatment Groups ◽  
Ivf Outcomes

Background: Vitrification as the most efficient method of cryopreservation, enables successful storage of oocytes for couples who undergo specific procedures including surgery and chemotherapy. However, the efficacy of in vitro maturation (IVM) methods with vitrified germinal vesicle (GV) oocytes could be improved. Objective: As melatonin and follicular fluid (FF) might enhance IVM conditions, we used these supplements to assess the maturation rate of vitrified GV oocytes and their artificial fertilization rate. Materials and Methods: Four hundred mouse GV oocytes were harvested, vitrified, and assigned into control (C-Vit-GV) and treatment groups of melatonin (M-Vit-GV), human follicular fluid (HFF-Vit-GV), and a combination (M + HFF-Vit-GV). A non-vitrified group of GV oocytes (non-Vit-GV) and a group of in vivo matured metaphase II (Vivo-MII) oocytes served as control groups to evaluate the vitrification and IVM conditions, respectively. Maturation of GV oocytes to MII and further development to two-cell-stage embryos were determined in the different groups. Results: Development to two-cell embryos was comparable between the Vivo-MII and non-Vit-GV groups. IVM and in vitro fertilization (IVF) results in the non-Vit-GV group were also comparable with the C-Vit-GV oocytes. In addition, the IVM and IVF outcomes were similar across the different treatment groups including the M-Vit-GV, HFF-Vit-GV, M + HFF-Vit-GV, and C-Vit-GV oocytes. Conclusion: Employing an appropriate technique of vitrification followed by suitable IVM conditions can lead to reasonable IVF outcomes which may not benefit from extra supplementations. However, whether utilizing other supplementation formulas could improve the outcome requires further investigation. Key words: Vitrification, Germinal vesicle, In vitro oocyte maturation, Melatonin, Follicular fluid.

334 EFFECT OF INSULIN ON IN VITRO MATURATION OF CANINE OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 275
Author(s):  
H. S. Lee ◽  
Y. I. Seo ◽  
X. J. Yin ◽  
S. G. Cho ◽  
I. H. Bae ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Uv Light ◽  
Cumulus Cells ◽  
Oocyte Activation ◽  
Cell Stage ◽  
Oocyte Competence ◽  
Treatment Groups ◽  
Maturation Medium ◽  
Oviduct Fluid

In spite of our increased knowledge of in vitro oocyte maturation techniques, the success rate of obtaining mature canine oocytes in vitro remains very low compared with that for other domestic animals. The inefficient rate of meiotic resumption of canine oocytes is probably due to both the unique reproductive cycle and inappropriate in vitro maturation (IVM) medium. In an unpublished experiment, we found that the concentration of insulin was higher in estrus bitch serum (EBS; 8833 pg/mL) than in dog follicular fluid (DFF; preovulatory follicle, 122 pg/mL), which implies its possible role in the acquisition of oocyte competence. Therefore, in the present study we investigated the effects of supplementing the IVM medium with insulin on the incidence of maturation to metaphase II. Ovaries were collected from various stages of the estrous cycle by ovariohysterectomy, and oocytes with two or more intact cumulus layers and with a diameter >110 �m were selected and used for IVM. Oocytes were cultured in modified synthetic oviduct fluid (2004 Reprod. Nutr. Dev. 44, 105-109) supplemented with 10% EBS, 20 �g/mL estradiol, and different concentrations of insulin (0, 10, 100, or 1000 ng/mL) at 38.5�C, 5% CO2 in air. After 72 h, cumulus cells were removed from around oocytes using a small glass pipette. Denuded oocytes were fixed in 3.7% paraformaldehyde supplemented with 10 �g/mL Hoechst 33342 at room temperature for 40 min. Nuclear status was observed under UV light using a fluorescence microscope. The percentage of oocytes at the metaphase II stage was not different among the four groups 6.8, 1.8, 5.4, and 2.1% in the control, 10, 100, and 1000 ng/mL insulin groups, respectively. The incidence of oocytes with pronuclear-like structures or cleaving beyond the two-cell stage was not significant higher in the 10 and 100 ng/mL insulin treatment groups than in the control and 1000 ng/mL insulin groups 20.0 and 19.6% vs. 6.8 and 6.4%, respectively. These results indicate that the addition of insulin to the in vitro maturation medium of dog oocytes had no effect on the incidence of meiotic maturation to metaphase II, nor did it affect the frequency of occurrence of spontaneous oocyte activation.

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

Zygote ◽  
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.

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

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 &lt; 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 &lt; 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.

scholarly journals O-011. The outcome of intracytoplasmic sperm injection in immature oocytes with or without in-vitro maturation

1999 ◽  
Vol 14 (Suppl_3) ◽  
pp. 6-6 ◽  
Author(s):  
D. Strassburger ◽  
D. Komarovsky ◽  
O. Bern ◽  
E. Kasterstein ◽  
A. Raziel ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
In Vitro Maturation ◽  
Immature Oocytes

191 SUPPLEMENTATION OF MATURATION MEDIUM WITH FOLLICULAR FLUID, EPIDERMAL GROWTH FACTOR AND NEUREGULIN AFFECTS MITOCHONDRIAL DNA REPLICATION, OOCYTE MATURATION AND EMBRYO DEVELOPMENT IN PIGS

2012 ◽  
Vol 24 (1) ◽  
pp. 208
Author(s):  
J. Mao ◽  
K. M. Whitworth ◽  
L. D. Spate ◽  
E. M. Walters ◽  
J. Zhao ◽  
...  
Keyword(s):  
Growth Factor ◽  
Embryo Development ◽  
Oocyte Maturation ◽  
Follicular Fluid ◽  
Copy Number ◽  
In Vitro Maturation ◽  
Meiotic Maturation ◽  
Mtdna Copy Number ◽  
Maturation Medium

Mitochondria supply the majority of ATP in a cell. Mitochondrial DNA (mtDNA) copy number in oocytes might be used as a marker of viability and might be a key determinant of pre-implantation embryo development. However, little is known about mtDNA copy number changes during porcine oocyte maturation and its regulation by extracellular growth factors. The objectives of the current study were to determine the effects of supplementation of in vitro maturation medium with porcine follicular fluid (pFF; 0, 10, 20 and 30%), epidermal growth factor (EGF; 10 ng mL–1), neuregulin 1 (NRG; 20 ng mL–1) and NRG + IGF1 (insulin-like growth factor-1; 100 ng mL–1 + NRG, 20 ng mL–1) during in vitro maturation on mtDNA copy number, oocyte meiotic maturation and subsequent embryo development after parthenogenic activation. Follicular fluid used for the pFF supplementation experiment was prepared from medium-sized (3–6 mm in diameter) healthy follicles. Cumulus–oocyte complexes (COCs) were collected from antral follicles (3–6 mm in diameter), cultured in LH- and FSH-containing maturation medium for 22 h at 38.5°C, transferred into basic maturation medium without FSH and LH and cultured for another 22 h. The basic maturation medium was TCM-199 supplemented with 0.1% polyvinylalcohol (w/v), 3.05 mM D-glucose, 0.91 mM sodium pyruvate, 10 μg mL–1 of gentamicin, 0.57 mM cysteine and without or with different growth factors depending on the experimental design. In total, 177 germinal vesicle (GV) oocytes and 3837 MII oocytes were used for this study. All data were analyzed by the general linear model (GLM) procedure of SAS software (V9.2). The mtDNA copy number in oocytes increased (P < 0.05) from GV to MII stage oocytes (MII oocytes from all treatment groups pooled). Supplementation of IVM media with 10% pFF decreased mtDNA copy number (P < 0.05), whereas 20 and 30% pFF had no major effect on mtDNA copy number, resulting in a quadratic correlation between percentage of pFF and mtDNA copy number. There was a negative linear correlation between percentage of pFF and oocyte meiotic maturation, with a higher percentage of pFF inhibiting meiotic maturation (73.2 ± 5.2, 71.9 ± 4.8, 64.1 ± 8.5 and 65.8 ± 6.4% for 0, 10, 20 and 30% pFF groups, respectively). The mtDNA copy numbers in EGF and NRG-treated MII oocytes were significantly higher than those in GV oocytes, whereas the control was not different (EGF, 237 042.6 ± 22 198.2; NRG, 281 293.4 ± 22 893.5; and control, 231 856.8 ± 21 883.5 in MII oocytes vs 192 288.7 ± 21 675.4 in GV oocytes). The EGF, NRG and NRG+IGF1 treatments enhanced oocyte maturation as well. There was no difference in Day-7 blastocyst formation between EGF, NRG+IGF1 and the control, whereas the NRG treatment enhanced blastocyst formation as compared to the control (23.8 ± 2.4 vs 15.1 ± 2.1%; P < 0.05). This study demonstrated that there was an increase in mtDNA copy number during in vitro maturation. The EGF and NRG treatments stimulated mitochondria biogenesis, which may provide new means to increase oocyte quality and enhance embryonic development.

In Vitro Maturation of Human Immature Oocytes in Culture Medium Supplemented with Patient's Own Serum or Donor Follicular Fluid

2008 ◽  
Vol 25 (3) ◽  
pp. 163-166
Author(s):  
Hiroyuki Kanaya ◽  
Aisaku Fukuda ◽  
Shu Hashimoto ◽  
Yoshihiko Hosoi ◽  
Yoshiharu Morimoto
Keyword(s):  
Follicular Fluid ◽  
In Vitro Maturation ◽  
Culture Medium ◽  
Immature Oocytes
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