In vitro maturation and attempted fertilization of cattle follicular oocytes

1970 ◽  
Vol 75 (3) ◽  
pp. 393-396 ◽  
Author(s):  
J. Sreenan
Keyword(s):  
Growth Medium ◽  
Follicular Fluid ◽  
In Vitro Maturation ◽  
Negative Results ◽  
Final Maturation

SUMMARYMaturation studies were carried out on 241 cattle oocytes. Media used were bovine follicular fluid and growth medium. Both media supported a resumption of meiosis in ca. 80 % of cases, but growth medium proved superior in terms of final maturation.Fertilization attempts were carried out on 564 oocytes following culture (27–32 h) in growth medium. In vitro fertilization attempts (107 oocytes) and in vivo fertilization attempts (82 oocytes) in the rabbit yielded negative results. Following in vivo fertilization attempts in the ewe, ova containing pronuclei as well as apparently normal cleaved ova were recovered.

176 IN VITRO MATURATION OF DOG OOCYTES IN CANINE FOLLICULAR FLUID

2014 ◽  
Vol 26 (1) ◽  
pp. 202
Author(s):  
K. Reynaud ◽  
S. Canguilhem ◽  
S. Thoumire ◽  
S. Chastant-Maillard
Keyword(s):  
Follicular Fluid ◽  
In Vitro Maturation ◽  
Assisted Reproductive Technologies ◽  
Cumulus Cells ◽  
Reproductive Technologies ◽  
Control Group ◽  
Limiting Factor ◽  
Cytoplasmic Maturation

In the canine species, assisted reproductive technologies, especially in vitro maturation (IVM) and IVF, are still ineffective. The main limiting factor remains the immaturity of the oocytes collected from anestrus ovaries. The ability of an oocyte to reach the MII stage in vitro is linked to the diameter of its follicle and anestrus oocytes, collected from small (<1 mm) follicles, are profoundly immature (De Lesegno et al. 2008). The objective of this study was to improve cytoplasmic quality by mimicking in vivo conditions; that is, to test the effect of pure preovulatory follicular fluid (FF) on survival and IVM rates of anestrus dog oocytes, in order to improve the nuclear and cytoplasmic maturation of these immature oocytes. Follicular fluids samples were collected from 54 Beagle bitches at 2 stages: before the LH peak (n = 23 bitches) and after the LH peak (n = 31 bitches). Only follicular fluid samples from large (>4 mm) follicles were collected and pooled by stage. Control oocytes were matured in 20% FCS/M199 medium. Groups of 5 oocytes were in vitro matured in 30 μL of follicular fluid, in half-area 96-well plates (5% CO2, 38°C). After 72 h of IVM, oocytes were denuded, fixed, and stained for DNA and tubulin before observation by confocal microscopy, and nuclear stages were classified as GV-A to GV-E, MI, and MII (Reynaud et al. 2012). A total of 460 oocytes were collected from 13 anestrus bitches and allocated to either the control medium (n = 155), the Pre-LH FF (n = 145) or the Post-LH FF (n = 160) groups. After 72 h of IVM, the morphology of the cumulus–oocyte complexes (COC) in the post-LH group was different from that of the others: cumulus cells appeared more compact and darker. Analysis of the nuclear stages showed that the degeneration rate was significantly higher (P < 0.05) in the post-LH group (58.7%) than in the pre-LH (40.9%) or in the control group (34.4%). No significant differences (P > 0.05) were observed between the 3 groups in the rate of immature GVA-B oocytes (36.4, 28.5, and 25.3% in the control, Pre-LH, and Post-LH groups, respectively), in the rate of meiotic resumption (GV-C/D/E, MI, MII stages, 44.4, 51.9, and 38.7% in the control, Pre-LH, and Post-LH groups, respectively). Metaphase II rates were not significantly different (12.1, 8.6, and 4.8% in the control, Pre-LH, and Post-LH groups, respectively). In conclusion, canine COC may survive when exposed to IVM in pure follicular fluid, but the degeneration rate was higher in the post-LH group. The presence of follicular fluid did not inhibit meiosis resumption, but did not significantly improve IVM rates. To better mimic in vivo conditions, IVM in a sequence of media, such as IVM in follicular fluid followed by IVM in oviducal fluid remains to be tested.

275 TEMPORAL PATTERN OF STEROID HORMONE CONCENTRATIONS DURING IN VIVO AND IN VITRO MATURATION OF BOVINE OOCYTES

2015 ◽  
Vol 27 (1) ◽  
pp. 226 ◽  
Author(s):  
C. Blaschka ◽  
H. Stinshoff ◽  
F. Poppicht ◽  
C. Wrenzycki
Keyword(s):  
Follicular Fluid ◽  
In Vitro Maturation ◽  
Steroid Hormone ◽  
Organic Anion ◽  
Hormone Concentration ◽  
Cholesterol Sulfate ◽  
Anion Transporter ◽  
Sulfated Steroids

Steroid hormone concentration and property can be modulated via different processes. Sulfoconjugation via sulfotransferases (SULT) changes steroids from hydrophobic to hydrophilic, necessitating a transport system such as the sodium-dependent organic anion transporter (SOAT; SLC10A6). Steroid sulfatase (STS) removes the sulfate moiety from conjugated steroids, transforming them to the free active ones. Moreover, present in vitro maturation systems do not completely mimic the in vivo situation resulting in oocytes of reduced quality. The present study investigates the local effects of sulfated steroids during follicular and oocyte development in vivo and in vitro. Follicles of bovine abattoir-derived ovaries were categorized according to their size (3 to 5, 6 to 8, 9 to 14, and >15 mm) after dissection and measurement via a caliper. Only nonatretic follicles were used (Kruip and Dieleman, 1982). Follicular fluid was collected via aspiration and analysed for the presence of steroids and their sulfated counterparts via LC-MS/MS. Moreover, oocytes were in vitro maturated with a standard protocol. The medium was measured via radioimmunoassay after 0, 4, 8, 12, 16, 20, and 24 h to detect 17β-oestradiol (E2) and progesterone (P4). Data were tested using analysis of variance (ANOVA) followed by multiple pairwise comparisons using Tukey's test. A P-value of ≤0.05 was considered significant. It was possible to detect 17β-oestradiol, progesterone, testosterone, 17β-oestradiol sulfate, estrone sulfate, pregnenolone sulfate, cholesterol sulfate (Table 1), and furthermore androstendione, estrone, androsterone, and 17OH-pregnenolone. During IVM, P4 significantly increased in the medium (4 h: 3.3 ± 1.0 ng mL–1; 24 h: 9.8 ± 1.7 ng mL–1), whereas the E2 concentration did not change (4 h: 52.8 ± 12.1 pg mL–1; 12 h: 68.4 ± 3.7 pg mL–1; 24 h: 66.9 ± 19.7 pg mL–1). In addition, preliminary data suggest that transcripts of the steroid metabolizing and transporting enzymes (SULT1E1, STS, SLC10A6) were present in cumulus cells from immature bovine COC. These results indicate for the first time that only small amounts of sulfated steroids are present in bovine follicular fluid. However, the related enzymes are present at the mRNA level. Further studies are underway to analyse the protein level. Table 1.Steroid hormone concentration in follicular fluid We thank Prof. Dr Wudy and A. Sánchez Guijo for the LC-MS/MS analysis. Furthermore, we gratefully acknowledge the financial support of the German Research Foundation (DFG; FOR 1369).

Glucose Metabolism Characterization During Mouse In Vitro Maturation Identifies Alterations In Cumulus Cells†

2021 ◽  
Author(s):  
Nazli Akin ◽  
Lucia von Mengden ◽  
Anamaria-Cristina Herta ◽  
Katy Billooye ◽  
Julia Leersum ◽  
...  
Keyword(s):  
Glucose Metabolism ◽  
In Vitro Maturation ◽  
Culture Media ◽  
Cumulus Cells ◽  
Routine Practice ◽  
Glycolytic Activity ◽  
Direct Measurements ◽  
Final Maturation

Abstract In vitro maturation (IVM) is an assisted reproduction technique with reduced hormone-related side effects. Several attempts to implement IVM in routine practice have failed, primarily due to its relatively low efficiency compared to conventional in vitro fertilization (IVF). Recently, capacitation (CAPA)-IVM, a novel two-step IVM method, has improved the embryology outcomes through synchronizing the oocyte nuclear and cytoplasmic maturation. However, the efficiency gap between CAPA-IVM and conventional IVF is still noticeable especially in the numerical production of good quality embryos. Considering the importance of glucose for oocyte competence, its metabolization is studied within both in vivo and CAPA-IVM matured mouse cumulus-oocyte-complexes (COCs) through direct measurements in both cellular compartments, from transcriptional and translational perspectives, to reveal metabolic shortcomings within the CAPA-IVM COCs. These results confirmed that within in vivo COC, cumulus cells are highly glycolytic, whereas oocytes, with low glycolytic activity, are deviating their glucose towards pentose phosphate pathway. No significant differences were observed in the CAPA-IVM oocytes compared to their in vivo counterparts. However, their cumulus cells exhibited a precocious increase of glycolytic activity during the pre-maturation culture step and activity was decreased during the IVM step. Here, specific alterations in mouse COC glucose metabolism due to CAPA-IVM culture were characterized using direct measurements for the first time. Present data show that, while CAPA-IVM cumulus cells are able to utilize glucose, their ability to support oocytes during final maturation is impaired. Future CAPA-IVM optimization strategies could focus on adjusting culture media energy substrate concentrations and/or implementing co-culture strategies.

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.

179 DIFFERENT GONADOTROPIN SUPPLEMENTATIONS ALTER mRNA EXPRESSION PATTERN IN BOVINE OOCYTES DURING IN VITRO MATURATION

2017 ◽  
Vol 29 (1) ◽  
pp. 198
Author(s):  
C. Blaschka ◽  
B. Zimmer ◽  
C. Wrenzycki
Keyword(s):  
Mrna Expression ◽  
Expression Pattern ◽  
Relative Abundance ◽  
In Vitro Maturation ◽  
Immature Oocytes ◽  
Mrna Expression Pattern ◽  
First Polar Body ◽  
Final Maturation

During final maturation (between LH surge and ovulation) in vivo, a switch from oestradiol to progesterone dominance within the follicle is well described. The aim was to mimic the in vivo situation during in vitro maturation via the supplementation of different gonadotropins. Groups of 30 cumulus-oocyte complex (abattoir-derived ovaries) were matured in TCM 199 plus different gonadotropins (eCG/hCG; FSH/LH, each in 0.05 or 0.01 IU; only FSH 0.05 IU; without gonadotropins) using a standard protocol without oil overlay. In Experiment 1, denuded oocytes were collected at 0 h (immature) and after 24 h of in vitro maturation (IVM; exhibit first polar body). In Experiment 2, oocytes were collected at different time points [0 (immature), 4, 8, 12, 16, 20, and 24 h] after IVM in eCG/hCG-supplemented medium. They were individually stored at −80°C until analyses. Transcripts of developmental competence (BMP15, GDF9, ZAR1), glucose or steroid metabolism (G6PD, STAR), and progesterone receptors (PGR, PGRMC1/2) were examined in individual oocytes via quantitative RT-PCR (n = 5). For statistical analyses, 1-way ANOVA followed by a Tukey test was used. Relative abundance of BMP15 transcripts was significantly lower (P ≤ 0.05) in oocytes of the group matured for 24 h with FSH/LH 0.01 IU, FSH 0.05 IU, and without gonadotropins than in immature oocytes. Relative amount of G6PD and PGRMC2 mRNA was significantly lower (P ≤ 0.05) in mature oocytes of the group with FSH/LH 0.01 IU, FSH 0.05 IU, and without gonadotropins than in immature ones. Relative abundance of GDF9, STAR, and ZAR1 transcripts was significantly lower (P ≤ 0.05) in oocytes of the group with eCG/hCG, FSH/LH 0.01 IU, FSH 0.05 IU, and without gonadotropins compared with immature oocytes. Relative abundance of PGR mRNA was significantly higher (P ≤ 0.05) in mature oocytes of the group with eCG/hCG than in immature oocytes, FSH/LH 0.01IU, FSH 0.05 IU, and without gonadotropins (Experiment 1). Relative amount of GDF9 transcripts was significantly lower (P ≤ 0.05) in mature oocytes collected after 24 h than in immature ones. Relative abundance of PGR mRNA was significantly higher (P ≤ 0.05) in oocytes collected after 20 and 24 h of IVM than in immature ones. Relative amount of ZAR1 transcripts was significantly lower (P ≤ 0.05) in oocytes collected after 16, 20, and 24 h of IVM than in immature oocytes; likewise, they were significantly lower (P ≤ 0.05) in oocytes collected after 12, 16, 20, and 24 h than in oocytes collected after 4 h of IVM. Relative amount of STAR mRNA was significantly lower (P ≤ 0.05) in oocytes collected after 24 h than in immature ones, and significantly lower (P ≤ 0.05) in oocytes collected after 16, 20, and 24 h than in oocytes collected after 4 h of IVM (Experiment 2). The results suggest a down-regulation of most transcripts during the period of IVM with different gonadotropin supplements with exception of PGR. Furthermore, most transcripts follow a timely regulated mRNA expression pattern during the entire IVM period. We gratefully acknowledge the financial support of the German Research Foundation (DFG; FOR 1369, WR 154/3–1).

Pure preovulatory follicular fluid promotes in vitro maturation of in vivo aspirated equine oocytes

2002 ◽  
Vol 57 (7) ◽  
pp. 1765-1779 ◽  
Author(s):  
I.B Bøgh ◽  
J Bézard ◽  
G Duchamp ◽  
M Baltsen ◽  
N Gérard ◽  
...  
Keyword(s):  
Follicular Fluid ◽  
In Vitro Maturation

Aspiration Revisited: Prospective Evaluation of a Physiologically Pressurized Model With Animal Correlation and Broader Applicability to Filler Complications

2021 ◽  
Author(s):  
Hyoung-Jin Moon ◽  
Won Lee ◽  
Ji-Soo Kim ◽  
Eun-Jung Yang ◽  
Hema Sundaram
Keyword(s):  
Normal Saline ◽  
False Negative ◽  
Vascular Complications ◽  
Filler Injection ◽  
Negative Results ◽  
Needle Position ◽  
False Negative Results ◽  
In Vivo Testing

Abstract Background Aspiration testing before filler injection is controversial. Some believe that aspiration can help prevent inadvertent intravascular injection, while others cite false-negative results and question its value given that the needle position always changes somewhat during injection procedures. Objectives To test the relation of false-negative results to the viscosity of the material within the needle lumen and determine whether a less viscous material within the needle lumen could decrease the incidence of false-negative results. Methods In vitro aspiration tests were performed using 30-G and 27-G needle gauges, two cross-linked hyaluronic acid fillers, normal saline bags pressurized at 140 and 10 mmHg to mimic human arterial and venous pressures, and three needle lumen conditions (normal saline, air, and filler). Testing was repeated three times under each study condition (72 tests in total). For in vivo correlation, aspiration tests were performed on femoral arteries and central auricular veins in three rabbits (4–5 aspirations per site, 48 tests in total). Results In vitro and in vivo testing using 30-G needles containing filler both showed false-negative results on aspiration testing. In vitro and in vivo testing using needles containing saline or air showed positive findings. Conclusions False-negative results from aspiration testing may be reduced by pre-filling the needle lumen with saline rather than a filler. The pressurized system may help overcome challenges of animal models with intravascular pressures significantly different from those of humans. The adaptability of this system to mimic various vessel pressures may facilitate physiologically relevant studies of vascular complications.

Temperature gradients in vivo influence maturing male and female gametes in mammals: evidence from the cow

2017 ◽  
Vol 29 (12) ◽  
pp. 2301 ◽  
Author(s):  
R. H. F. Hunter ◽  
F. López-Gatius ◽  
O. López-Albors
Keyword(s):  
Follicular Fluid ◽  
Open Surgery ◽  
Temperature Gradients ◽  
Fluid Temperature ◽  
Preovulatory Follicle ◽  
Male And Female ◽  
Direct Measurements ◽  
Final Maturation ◽  
Preovulatory Follicles

Since 1980 several reports have indicated that temperatures vary between preovulatory follicles and other ovarian tissues in rabbit, cow, pig and human. However, these observations did not achieve prominence; they were regarded as artefacts due to the use of anaesthetics and open surgery (laparotomy). Recently, without resorting to anaesthesia or surgery, direct measurements of temperature in preovulatory follicles have been performed in the cow by means of a thermistor probe introduced into the antrum under ultrasonic guidance. Such follicles revealed a mean antral (follicular fluid) temperature 0.74°C and 1.54°C cooler than uterine surface and rectal temperatures respectively in ovulating cows, whereas no such temperature differences were detected in non-ovulating cows. Cows are predominantly monovular and preovulatory follicles attain a diameter of 15–22 mm or more. These features and the timescale of response to the preovulatory gonadotrophin surge make them a valuable model for the human preovulatory follicle. Temperature gradients are interpreted primarily in a context of final maturation of gametes immediately before the onset of fertilisation. Preovulatory follicular temperature in women could be assessed by a comparable approach and might become a valuable selection guide for oocyte viability.

scholarly journals Biofilm Formation by and Antifungal Susceptibility of Candida Isolates from Urine

2007 ◽  
Vol 73 (6) ◽  
pp. 1697-1703 ◽  
Author(s):  
N. Jain ◽  
R. Kohli ◽  
E. Cook ◽  
P. Gialanella ◽  
T. Chang ◽  
...  
Keyword(s):  
Risk Factors ◽  
Growth Medium ◽  
Biofilm Formation ◽  
Chart Review ◽  
Antifungal Susceptibility ◽  
Retrospective Chart Review ◽  
Urine Samples ◽  
Rpmi Medium

ABSTRACT Biofilm formation (BF) in the setting of candiduria has not been well studied. We determined BF and MIC to antifungals in Candida spp. isolates grown from urine samples of patients and performed a retrospective chart review to examine the correlation with risk factors. A total of 67 Candida spp. isolates were grown from urine samples from 55 patients. The species distribution was C. albicans (54%), C. glabrata (36%), and C. tropicalis (10%). BF varied greatly among individual Candida isolates but was stable in sequential isolates during chronic infection. BF also depended on the growth medium and especially in C. albicans was significantly enhanced in artificial urine (AU) compared to RPMI medium. In nine of the C. albicans strains BF was 4- to 10-fold higher in AU, whereas in three of the C. albicans strains and two of the C. glabrata strains higher BF was measured in RPMI medium than in AU. Determination of the MICs showed that planktonic cells of all strains were susceptible to amphotericin B (AMB) and caspofungin (CASPO) and that three of the C. glabrata strains and two of the C. albicans strains were resistant to fluconazole (FLU). In contrast, all biofilm-associated adherent cells were resistant to CASPO and FLU. The biofilms of 14 strains (28%) were sensitive to AMB (MIC50 of <1 μg/ml). Correlation between degree of BF and MIC of AMB was not seen in RPMI grown biofilms but was present when grown in AU. A retrospective chart review demonstrated no correlation of known risk factors of candiduria with BF in AU or RPMI. We conclude that BF is a stable characteristic of Candida strains that varies greatly among clinical strains and is dependent on the growth medium. Resistance to AMB is associated with higher BF in AU, which may represent the more physiologic medium to test BF. Future studies should address whether in vitro BF can predict treatment failure in vivo.

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