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.

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.

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

183 CANINE EMBRYOS OBTAINED BY INTRACYTOPLASMIC SPERM INJECTION

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

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

292. Influence of the in vitro environment on rat gametes and pre-implantation embryos

2005 ◽  
Vol 17 (9) ◽  
pp. 123
Author(s):  
N. R. Borg ◽  
M. K. Holland
Keyword(s):  
Protein Profile ◽  
Germinal Vesicle ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
Vitro Fertilization ◽  
Nuclear Maturation ◽  
In Vitro Systems ◽  
Interaction Research

Rat in vitro fertilization (IVF) and culture (IVC) is attempted by few because of its reputation for difficulty. Currently very few functional rat in vitro systems (IVS) exist for sperm–oocyte interaction research. Successful fertilization of rat metaphase II (MII) oocytes was achieved with two different media, Enriched Krebs Ringer Bicarbonate (EKRB) (70.2%) and M16 (57.4%). Using this IVS we have shown that the rat germinal vesicle-intact (GV-i) oocyte lacks the necessary maturity to interact with capacitated caudal epididymal spermatozoa, whether zona pellucida intact (ZP-i) or free (ZP-f). Proteomic analysis of the protein profile of the oolemma from the GV-i stage through to the MII stage in oocytes is being conducted to characterize any maturational changes that may occur. In addition we provide initial evidence to suggest that an acrosome-intact spermatozoa can fuse with the oolemma of a ZP-f MII oocyte during IVF. Although high percentages of polyspermic embryos in ZP-f IVF (64.8–100%) were observed, the possibility that the rat oolemma may undergo a post-fusion block to polyspermy was implied by a small proportion of normally fertilized embryos (3.8–17.0%) in M16 supplemented with different ratios of hyperactived spermatozoa. Despite successful culture to the blastocyst stage for in vivo fertilized zygotes (33.73%) and 2-cell stage embryos (79.3%), IVF embryos have repeatedly failed to develop in culture. Two dimensional analyses of the protein profile of oocytes/embryos immediately prior to fertilization (MII oocyte–101 spots) and the maternal to zygotic transition (MZT) (zygotes–59 spots and 2-cell embryos–84 spots) has shown a difference in patterns of protein expressed. Comparison of IVF zygotes (41 spots) obtained from EKRB displayed reduced protein expression suggesting that nuclear maturation and/or MZT is not being adequately supported. These data illustrate that rat IVF and IVC require suitable media if its problematic reputation is finally to be shed.

282 IMPROVED QUALITY OF PORCINE BLASTOCYSTS BY AGGREGATION OF EMBRYOS AT THE 4 - 8-CELL STAGE

2006 ◽  
Vol 18 (2) ◽  
pp. 248
Author(s):  
S.-G. Lee ◽  
C.-H. Park ◽  
D.-H. Choi ◽  
H.-Y. Son ◽  
C.-K. Lee
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Transgenic Animals ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
Vitro Fertilization

Use of blastocysts produced in vitro would be an efficient way to generate embryonic stem (ES) cells for the production of transgenic animals and the study of developmental gene regulation. In pigs, the morphology and cell number of in vitro-produced blastocysts are inferior to these parameters in their in vivo counterparts. Therefore, establishment of ES cells from blastocysts produced in vitro might be hindered by poor embryo quality. The objective of this study was to increase the cell number of blastocysts derived by aggregating 4–8-cell stage porcine embryos produced in vitro. Cumulus–oocyte complexes were collected from prepubertal gilt ovaries, and matured in vitro. Embryos at the 4–8-cell stage were produced by culturing embryos for two days after in vitro fertilization (IVF). After removal of the zona pellucida with acid Tyrode’s solution, one (1X), two (2X), and three (3X) 4–8-cell stage embryos were aggregated by co-culturing them in aggregation plates followed by culturing to the blastocyst stage. After 7 days, the developmental ability and the number of cells in aggregated embryos were determined by staining with Hoechst 33342 and propidium iodide. The percentage of blastocysts was higher in both 2X and 3X aggregated embryos compared to that of 1X and that of intact controls (Table 1). The cell number of blastocysts also increased in aggregated embryos compared to that of non-aggregated (1X) embryos and controls. This result suggests that aggregation might improve the quality of in vitro-fertilized porcine blastocysts by increasing cell numbers, thus becoming a useful resource for isolation and establishment of porcine ES cells. Further studies are required to investigate the quality of the aggregated embryos in terms of increasing the pluripotent cell population by staining for Oct-4 and to apply improved aggregation methods in nuclear-transferred (NT) porcine embryos. Table 1. Development, cell number, and ICM ratio of aggregated porcine embryos

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.

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