scholarly journals 78CHRONOLOGICAL EVENTS OF IN VITRO MATURATION IN CAMEL (CAMELUS DROMEDARIES) OOCYTES

2004 ◽  
Vol 16 (2) ◽  
pp. 161 ◽  
Author(s):  
N.A. Wani ◽  
U. Wernery ◽  
M.A. Nowshari
Keyword(s):  
In Vitro Maturation ◽  
Assisted Reproductive Technologies ◽  
Germinal Vesicle ◽  
Reproductive Technologies ◽  
Dromedary Camel ◽  
Glass Slides ◽  
Maturation Medium ◽  
Stereo Microscope ◽  
Group 3

Experiments were conducted to investigate the chronological events and optimum time for in vitro oocyte maturation in the dromedary camel. Follicles measuring 3–12mm were isolated from ovaries obtained from an abattoir and the oocytes harvested by teasing apart these follicles under a stereo microscope. Pooled oocytes were randomly distributed to 4-well dishes (20–25per well) (Nunc, Denmark) containing 400μL of the maturation medium (TCM-199 supplemented with 0.6mgmL−1 calcium lactate, 0.1mgmL−1 L-glutamine, 0.8mgmL−1 sodium bicarbonate, 1.4mgmL−1 HEPES, 0.25mgmL−1 pyruvate, 50μgmL−1 gentamicine, 10μgmL−1 FSH, 10μgmL−1 LH, 1μgmL−1 estradiol and 10% heat-inactivated estrous camel serum) and incubated at 38.5°C under 5% CO2 for 4 to 48h. After every 4h (starting from 0 to 48h), oocytes were denuded by treating them with hyaluronidase (1mgmL−1) followed by repeated pipetting. Denuded oocytes were mounted on glass slides and fixed in 3:1 ethanol:acetic acid for 24h. Oocytes were stained with 1% aceto-orcein and examined under a phase contrast microscope at 400times. for each experimental group, 3 to 7 replications were made. Based on the visualization of the chromatin, oocytes were categorized as at germinal vesicle (GV), diakinesis (DK), metaphase-I (M-I), anaphase (Ana), metaphase-II (M-II) stage and those with no visible chromatin as NVC. At the start of maturation, 75.4% (43/57) of oocytes were at GV stage; however, none of the oocytes revealed a GV at 28 h of maturation (0/97). At 8h of maturation 49.3% (34/69) of oocytes were at DK stage, and after 16h of maturation 50% (49/98) of oocytes were at M-I stage. At 24h of maturation the maximum number of oocytes were in Ana (24.7%, 21/85) stage. At 44h the maximum number of oocytes had reached M-II stage (52%, 103/198) whereas, 10.6% (21/198) of the oocytes were at Ana stage. After 48h the proportion of oocytes with NCV increased to 52.9% (45/85) and the proportion of M-II stage oocytes decreased to 37.6% (32/85). It may be concluded that 40–44h of in vitro maturation yields the highest proportion of matured (M-II stage) oocytes suitable for further use in assisted reproductive technologies in camel.

177 IN VITRO MATURATION AND FERTILIZATION OF OVARIAN OOCYTES OF FREE-RANGING GEMSBOK (ORYX GAZELLA)

2012 ◽  
Vol 24 (1) ◽  
pp. 200
Author(s):  
B. Durrant ◽  
N. Ravida ◽  
D. Van Dien ◽  
C. Young ◽  
P. Mathis
Keyword(s):  
New Mexico ◽  
In Vitro Maturation ◽  
Reproductive Technologies ◽  
Maturation Medium ◽  
Reproductive Techniques ◽  
Embryo Medium ◽  
Game Hunting ◽  
White Sands ◽  
Free Ranging

The gemsbok is a large antelope native to arid regions of southern Africa. Listed by the International Union for Conservation of Nature as a species of least concern, the gemsbok is an excellent model for the development of assisted reproductive techniques for the closely related but critically endangered scimitar-horned oryx (Oryx dammah) and addax (Addax nasomaculatus). Gemsbok were introduced to the White Sands Missile Range by the New Mexico Department of Game and Fish to preserve the habitat by providing big game hunting, which ensures the support and lobby of hunters. Gonads were collected from hunted gemsbok and transported in PBS to the laboratory, where gametes were harvested. Testes were stored at 4°C in PBS until sperm were needed for IVF (18 to 28 h). Ovaries were sliced to release follicular oocytes, which were placed in maturation medium (TCM-199 with FCS, pyruvate, gentamicin and LH, FSH and oestradiol) for 20- to 22-h in vitro maturation culture at 38.8°C in 5% CO2 in air. Oocytes were then washed [Tyrode lactate (TL)-HEPES with BSA, pyruvate and gentamicin] and placed in groups of 5 to 10 in 50-μL drops of IVF-TL medium supplemented with pyruvate, gentamicin and BSA. Sperm were allowed to swim out of sliced epididymides into room temperature IVF-TL medium and then equilibrated for 30 min at 38.8°C. Approximately 2 × 103 motile sperm were added to each oocyte drop and incubated under oil for 21 to 23 h at 38.8°C in 5% CO2 in air. Domestic cattle oocytes were matured in maturation medium at 39°C during shipment to the field site, where they were washed and transferred to IVF medium as described for gemsbok oocytes. Approximately 1.7 × 103 motile gemsbok sperm were added to each drop and oocytes were incubated as described for gemsbok oocytes. At the end of IVF culture, oocytes of both species were stripped of granulosa cells and placed in embryo medium (SOF supplemented with BSA, essential and nonessential amino acids, pyruvate and gentamicin; all media formulations from Applied Reproductive Technologies, Madison, WI, USA) at 38.8°C in 5% CO2 in air. Embryo culture medium was refreshed after 48 h. Embryos were removed from culture after 6 days, examined for cleavage and fixed in PBS:formalin for staining and further analysis. Although gemsbok sperm were capable of fertilizing domestic cow oocytes at the same rate (38.3%) as gemsbok oocytes (37.1%), the antelope oocytes cleaved at a higher rate (29% vs cattle at 15.3%). These results indicate that chilled epididymal gemsbok sperm is capable of fertilizing gemsbok and domestic cattle oocytes and that protocols designed for in vitro maturation and fertilization of cattle oocytes may be successfully used in the field to produce gemsbok embryos (Table 1). Table 1.Fertilization of in vitro-matured gemsbok and cattle oocytes by chilled epididymal gemsbok sperm The authors thank the New Mexico Department of Game and Fish, Troylyn Zimmerly, Dana Powers and the Biology Department of New Mexico Institute of Mining and Technology.

scholarly journals Follicle-stimulating hormone mediates the consumption of serum-derived glycogen by bovine cumulus-oocyte complexes during in vitro maturation

2021 ◽  
pp. 2512-2517
Author(s):  
Ludymila F. Cantanhêde ◽  
Cristiane T. Santos-Silva ◽  
Marcelo T. Moura ◽  
José C. Ferreira-Silva ◽  
Júnior M. B. Oliveira ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Assisted Reproductive Technologies ◽  
Follicle Stimulating Hormone ◽  
Cumulus Cell ◽  
Reproductive Technologies ◽  
Developmental Competence ◽  
Independent Manner ◽  
Oocyte Developmental Competence ◽  
Stimulating Hormone

Background and Aim: Oocyte in vitro maturation (IVM) is an appealing approach for several assisted reproductive technologies and dissecting oocyte maturation. Nonetheless, IVM leads to lower developmental competence and usually relies on undefined, serum-containing media. Therefore, biochemical profiling aimed to explore fluctuations in IVM media content during the acquisition of oocyte developmental competence. Materials and Methods: Bovine cumulus-oocyte complexes (COCs) underwent IVM in TCM199 medium with Earle's salts, supplemented with 2.0 mM L-glutamine, 10% fetal bovine serum, antibiotics, and 0.05 IU/mL porcine follicle-stimulating hormone (FSH+) or vehicle control (CTL) medium for 22 h. Results: FSH withdrawal (CTL) diminished several processes associated with the acquisition of oocyte developmental competence, such as reduced cumulus cell expansion, diminished estradiol synthesis (FSH+: 116.0±0.0 pg/mL vs. CTL: 97.6±18.0 pg/mL), and lower oocyte nuclear maturation rate (FSH+: 96.47% vs. CTL: 88.76%). Fresh media formulations (i.e., TCM199 with FSH or vehicle) were indistinguishable under biochemical profiling threshold conditions. Biochemical profiling showed similar total protein and lipid concentrations between groups. Further, total sugar concentrations diminished from fresh media to their post-IVM counterparts, albeit in an FSH-independent manner. Glycogen concentrations remained unaltered after IVM within CTL media, albeit were substantially lower after IVM under FSH+ conditions. Conclusion: FSH mediates the consumption of serum-derived glycogen by bovine COCs during IVM and implies that serum-free media should contain increased glucose concentrations to facilitate the acquisition of oocyte developmental competence.

196 EFFECTS OF DIFFERENT IN VITRO MATURATION SYSTEMS ON BOVINE EMBRYO DEVELOPMENT

2012 ◽  
Vol 24 (1) ◽  
pp. 210
Author(s):  
S. M. Bernal ◽  
J. Heinzmann ◽  
D. Herrmann ◽  
A. Lucas-Hahn ◽  
B. Timmermann ◽  
...  
Keyword(s):  
Mrna Expression ◽  
In Vitro Maturation ◽  
Assisted Reproductive Technologies ◽  
Expression Profiles ◽  
Reproductive Technologies ◽  
Developmental Competence ◽  
Bovine Embryo ◽  
Blastocyst Quality

Bovine oocytes and embryos have been established as a valuable model for studying human early development, specifically after assisted reproductive technologies (ART). Efforts for the improvement of ART in the last years have focused on culture media and conditions. Recently, Albuz et al. (2010) reported that the culture of bovine cumulus–oocyte complexes (COC) with cyclic adenosine 3′, 5′-monophosphate (cAMP) modulators, before and during extended in vitro maturation (IVM), improved blastocyst quality and yields in mice and cattle. In this study, we investigated the influence of an extended IVM phase in combination with cAMP modulators on blastocyst yields and quality, the effects on mRNA expression profiles and epigenetic marks. We compared these results to the standard protocol (Wrenzycki et al., 2001) used in our laboratory with oocytes from different retrieval methods. Oocytes were retrieved from slaughterhouse ovaries either by slicing or follicular aspiration. The COC were either subjected directly to IVM using the standard TCM-based protocol for 24 h (TCM24-slicing and TCM24-aspiration, respectively) or oocytes that were retrieved by aspiration were treated with forskolin and IBMX for a 2-h pre-IVM period, followed by an extended IVM phase of 30 h in TCM, supplemented with cilostamide (cAMP30-aspiration). Statistical analyses were performed using 1-way ANOVA followed by the nonparametrical Kruskal–Wallis test. Maturation rates were 79.3 ± 2.6% in TCM24-aspiration, 74.2 ± 8.8% in cAMP30-aspiration and 70.4 ± 5.1% in TCM24-slicing oocytes. Matured oocytes were fertilized in vitro with semen from a bull previously proven to be suitable for IVF. Blastocyst rates from presumptive zygotes were significantly higher (P = 0.003) in the TCM24-aspiration group (32 ± 7%) compared to TCM24-slicing (23 ± 7%) and cAMP30-aspiration (22 ± 5%). Analysis revealed that cell numbers were rather similar in the 3 experimental groups (125 ± 19, 128 ± 15 and 129 ± 9), while in vivo-produced blastocysts possessed slightly more cells (134 ± 17; P ≥ 0.05). RT-qPCR analysis of mRNA expression for a panel of genes indicative of embryo quality including DNMT3a, SLC2A8, COX2 and PCK2, showed that blastocysts derived from both aspiration protocols were similar to in vivo embryos, but were different from blastocysts resulting from the ovary-slicing protocol. Specifically, the expression profile of COX2, which is involved in pregnancy outcome and in the response to growth factors, indicates an enhanced developmental competence of aspirated oocytes. However, the transcript level of EGR1 (early growth response) was significantly higher (P = 0.009) in in vivo-derived blastocysts in comparison to all in vitro treatments. The investigation of the epigenetic status of the in vitro-derived blastocysts based on bisulfite sequencing of 2 satellite repeat sequences is currently underway. Results so far indicate that the method of obtaining the oocytes (slicing vs aspiration) for in vitro production of bovine embryos is of greater influence on blastocyst quality than IVM conditions.

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.

127 L-Carnitine Supplementation During In Vitro Maturation Improves Developmental Competence of Canine Oocytes

2018 ◽  
Vol 30 (1) ◽  
pp. 203 ◽  
Author(s):  
A. Salama ◽  
M. Fathi ◽  
M. R. Badr ◽  
A. R. Moawad
Keyword(s):  
Embryo Development ◽  
In Vitro Maturation ◽  
Assisted Reproductive Technologies ◽  
Mammalian Species ◽  
Reproductive Technologies ◽  
Developmental Competence ◽  
Carnitine Supplementation ◽  
Cell Stage ◽  
Nuclear Maturation

In vitro embryo production (IVP) in the domestic bitch is important for conservation of endangered canids. Compared with various domestic animals, the development of assisted reproductive technologies (ART) in the dog has lagged behind, mainly due to the low percentage of oocytes that can reach metaphase II (MII) stage after in vitro maturation (IVM). Beneficial effects of l-carnitine (LC) on embryonic development in culture have been reported in many mammalian species; however, no studies have been conducted in dogs. The aim of the present study was to investigate the effect of LC supplementation during IVM of canine oocytes on nuclear maturation, fertilization status, and pre-implantation development following IVM/IVF. Cumulus-oocyte complexes (COC) were collected by slicing ovaries obtained from dogs (n = 20, 1 to 6 years of age) after ovariohysterectomy. The COC were subjected to IVM for 72 h in a medium (TCM-199) supplemented with LC at different concentrations (0.1, 0.3, 0.6, 1.0, or 2.0 mg mL−1) or without LC supplements (0 mg mL−1; control). Matured oocytes were fertilized in vitro with frozen–thawed spermatozoa, and presumptive zygotes were cultured in SOF medium for 7 days. Frequencies of nuclear maturation (72 h post-IVM), fertilization rates (18 h post-insemination), and embryo development (Days 2 to 5 post-insemination) were evaluated. Data were analysed by one-way ANOVA followed by Tukey’s multiple comparisons test. Supplementation of IVM medium with 0.3 or 0.6 mg mL−1 LC significantly improved (P ≤ 0.05) maturation (35.4% and 41.4%) and fertilization (21.3% and 25.8%) rates compared with the controls and with other LC-supplemented groups; values ranged from 20.1% to 25.0% for maturation and from 12.1% to 14.6% for fertilization. Cleavage (2- to 16-cell stages) was significantly higher (P ≤ 0.05) in the 0.6 mg mL−1 LC supplemented group than the 0.3 mg mL−1 supplemented group (16.3% v. 13.3%). These values were significantly higher (P ≤ 0.05) than those in other groups. Interestingly, 4.5% of IVM/IVF oocytes were developed to morula in 0.6 mg mL−1 LC supplemented group which was significantly higher (P ≤ 0.05) than those developed in the 0.3 mg mL−1 supplemented group (1.0%). No embryos developed beyond the 2- to 16-cell stage in the rest of the groups. In conclusion, l-carnitine supplementation during IVM is particularly efficient in improving nuclear maturation and pre-implantation embryo development of canine oocytes after IVF. These outcomes are important for the improvement of IVM conditions that can advance the efficiency of ART in dogs.

scholarly journals Live birth after in vitro maturation versus standard in vitro fertilisation for women with polycystic ovary syndrome: protocol for a non-inferiority randomised clinical trial

BMJ Open ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. e035334
Author(s):  
Xiaoying Zheng ◽  
Wei Guo ◽  
Lin Zeng ◽  
Danni Zheng ◽  
Shuo Yang ◽  
...  
Keyword(s):  
Polycystic Ovary Syndrome ◽  
Live Birth ◽  
In Vitro Maturation ◽  
Assisted Reproductive Technologies ◽  
Polycystic Ovary ◽  
Reproductive Technologies ◽  
In Vitro Fertilisation ◽  
Ovary Syndrome ◽  
Infertile Women

IntroductionPolycystic ovary syndrome (PCOS) is the first common cause of anovulatory infertility. Currently, in vitro fertilisation (IVF) is recommended when conventional attempts have failed. In vitro maturation (IVM) of human oocytes is an emerging treatment option in infertile women with PCOS. It is a patient-friendly intervention, avoiding the risk of ovarian hyperstimulation syndrome, which is a serious complication of controlled ovarian stimulation in the standard IVF procedure. We plan a randomised controlled trial (RCT) to evaluate whether IVM is non-inferior to the standard IVF for live birth in women with PCOS.Methods and analysisThis is a single-centre, open-label, non-inferiority RCT performed in a large reproductive medicine centre in China. Infertile women with PCOS will be randomised to receive either IVM or standard IVF in a 1:1 treatment ratio after informed consent. IVF procedures used in our study are all standard treatments and other standard-assisted reproductive technologies will be similar between the two groups. The primary outcome is ongoing pregnancy leading to live birth within 6 months of the first oocyte retrieval cycle after randomisation. Pregnancy outcome, maternal safety and obstetric and perinatal complications will be secondary outcomes. The planned sample size is 350 (175 per group).Ethics and disseminationEthical permission was acquired from the Ethics Committee of Peking University Third Hospital. The results will be issued to publications through scientific journals and conference reports.Trial registration numberNCT03463772.

scholarly journals Gene Expression and In Vitro Maturation of Sheep Oocytes using Bee Pollen and Bee Honey as Medium Supplements

2021 ◽  
Vol 25 (03) ◽  
pp. 615-622
Author(s):  
Aaishah M. Kaabi
Keyword(s):  
Gene Expression ◽  
Candidate Genes ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Maturation Medium ◽  
Maturation Rate ◽  
Group 3 ◽  
Najdi Sheep ◽  
Group 1

This study was conducted to investigate the effects of raw honey obtained from black seed or Sider and honeybee pollen as an additive in sheep oocyte maturation medium on the oocyte maturation rate, changes in oocyte glutathione (GSH) levels and expression of developmental candidate genes (GDF-9, MPF, C-MOS, IGF-1, BAX). Healthy immature oocytes of Najdi sheep were cultured in a medium supplemented with 5.0% Sider or Nigella sativa (black seed) honey + 1.0 μg/mL honeybee pollen, and after 24 h of incubation, the effects on the improvement of in vitro oocyte maturation were evaluated. Results demonstrated that the mean oocyte maturation rate was the best in group treated with 5% N. sativa (Group 3) compared with group treated with Sider or N. sativa honey (Group 1A and B, respectively). Mean GSH level was higher in Group 3 oocytes (11.09 ± 0.29 nmol) than in Group 2 oocytes (honey alone; 10.93 ± 0.57; P ≤ 0.05). Mean GSH levels were significantly decreased in Group 1. Expression analysis of candidate genes showed significant upregulation of GDF-9, cyclin B, C-MOS and IGF 1 genes in Group 3 and downregulation of BAX compared with control Group 1. In conclusion, addition of 1.0 μg/mL honeybee pollen along with one of two types of bee honey (Sider and N. sativa) at 5% concentration to the in vitro maturation medium of Najdi sheep oocytes has a beneficial effect in improving the maturation rate and gene expression and increasing the glutathione concentration in matured oocytes. © 2020 Friends Science Publishers

scholarly journals Roscovitine use for the delay of meiotic progression in prepubertal sheep oocytes

2020 ◽  
Vol 55 ◽  
Author(s):  
Letícia Ferrari Crocomo ◽  
Federica Ariu ◽  
Luisa Bogliolo ◽  
Daniela Bebbere ◽  
Sergio Ledda ◽  
...  
Keyword(s):  
Inhibitory Action ◽  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Free Medium ◽  
Nuclear Maturation ◽  
Meiotic Progression ◽  
Maturation Medium ◽  
Inverted Microscope ◽  
Kinetics Of

Abstract: The objective of this work was to evaluate the efficiency of roscovitine on reversibly inhibiting oocytes from prepubertal sheep at the germinal vesicle (GV) stage, and to investigate the kinetics of meiosis progression after inhibitor removal. Cumulus-oocyte complexes, recovered from Sarda breed lambs aged 30-40 days, were cultured for 6 hours in a maturation medium (control) containing 75 μmol L-1 roscovitine (Rosco) at 38.5°C and 5% CO2. Then, the complexes were subjected to in vitro maturation (IVM) for 18 or 23 hours, in an inhibitor-free medium supplemented with gonadotropins. The evaluation of nuclear configuration by Hoescht staining, under a fluorescence-inverted microscope, showed that 88.7% of the lamb oocytes treated with roscovitine remained at the GV stage, as observed for the immature ones (97.3%) stained after collection. The inhibitory action was reversible; however, the proportion of oocytes (83.3%) at the metaphase-II stage, after 23 hours of IVM, was significantly higher than that observed after 18 hours (29.5%), in which meiosis was still in progression with 34.2% oocytes at metaphase-I, 11.6% oocytes at anaphase-I, and 18.5% oocytes at telophase-I. Roscovitine is efficient to arrest the nuclear maturation in oocytes from prepubertal sheep; however, despite the reversibility, meiosis progression is delayed, requiring more time to be completed.

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