129 Polar body extrusion following exposure of pig oocytes to different invitro maturation media

2021 ◽  
Vol 33 (2) ◽  
pp. 173
Author(s):  
M. L. Mphaphathi ◽  
K. M. Honneysett ◽  
T. T. Maduwa ◽  
K. G. Pohler ◽  
T. L. Nedambale
Keyword(s):  
Granulosa Cells ◽  
Fetal Bovine Serum ◽  
Polar Body ◽  
Maturation Period ◽  
Treatment Groups ◽  
Maturation Medium ◽  
Significant Difference ◽  
Fetal Bovine ◽  
Slicing Method ◽  
Polar Body Extrusion

The invitro maturation (IVM) rate of pig oocytes is a critical step for subsequent invitro embryo production. The objective of the present study was to compare different IVM media (NCSU 23, NCSU 37, TCM-199, pFF, BO-IVM commercial, and Bovine IVM) on polar body extrusion in pig oocytes following 44h of maturation duration. Ovaries from sows were collected from the local slaughterhouse and transported in warmed (38°C) saline to the laboratory, within 2h following slaughter. The slicing method was used to retrieve oocytes from the ovaries. Collected oocytes (n=353) were selected and washed three times in TCM-199+10% fetal bovine serum. The oocytes were then subjected to IVM as per treatment groups for 44h. Following maturation period, oocytes were removed from each maturation medium and denuded of granulosa cells by vortexing. The oocytes polar body extrusion was evaluated with the aid of OosightTM Imaging Sytem connected to an inverted research microscope. Treatment means were compared using Fisher’s protected least significant difference t-test. Polar body extrusion was 30.0, 18.5, 32.8, 22.1, 32.1, and 27.0% for BO IVM commercial, Bovine IVM, NCSU 23, NCSU 37, TCM-199, and pFF media, respectively (P>0.05). The NCSU 23 and TCM-199 had numerically higher percentages of oocytes with extruded polar body compared with other treatments groups (P>0.05). The lowest (18.5%) polar body extrusion was recorded in Bovine IVM medium. In conclusion, all the IVM media tested can be used during IVM of pig oocytes, with NCSU 23 and TCM-199 having numerically higher polar body extrusion percentages.

138 Effect of different temperatures on invitro maturation rates on cattle oocytes

2021 ◽  
Vol 33 (2) ◽  
pp. 177
Author(s):  
M. D. Sebopela ◽  
M. L. Mphaphathi ◽  
S. M. Sithole ◽  
T. L. Nedambale
Keyword(s):  
Polar Body ◽  
International Comparisons ◽  
Treatment Groups ◽  
First Polar Body ◽  
Significant Difference ◽  
Different Temperatures ◽  
Maturation Rate ◽  
Statistical Program ◽  
Polar Body Extrusion ◽  
Incubation Temperatures

Climate change represents a major stressful environmental condition that compromises the reproductive efficiency of animals. The temperature affects various components of the maturation processes in cattle oocytes, this includes disruptions of oocyte development and maturation. Therefore, the study aimed to compare the effects of different incubation temperatures (32.5, 33.5 34.5, 35.5, 36.5, 37.5, 38.5, 39.5, 40.5, 41.5, 42.5, and 43.5°C) on the maturation rate of cattle oocytes. Cattle ovaries were collected from a local abattoir and transported to the laboratory in thermo flask at 37°C. Oocytes were recovered from ovaries using the aspiration method and matured using 500 uL of TCM-199 medium supplemented with 10% fetal bovine serum, FSH, LH, and E2 covered with mineral oil. The maturation rate of oocytes was determined by the extrusion of the first polar body after 22h of IVM period. The total number of 80 oocytes were matured in each group and replicated 4 times. Data were analysed using GenStat® statistical program (VSN International). Comparisons were considered significantly different (P<0.05) using Fisher’s protected least significant difference test. The oocyte polar body extrusion at 32.5 (0±0), 33.5 (4.5±5.2), 34.5 (22.0±1.4), 35.5 (29.5±7.6), 36.5 (41.5±3.1), 37.5 (55.5±4.1), 38.5 (62.2±3.3), 39.5 (49.5±3.3), 40.5 (17.3±6.4), 41.5 (10.3±5.7), 42.5 (8.2±5.9), and 43.5°C (5.7±7.8) (P<0.05) was recorded. The highest percentages of oocytes with polar body extrusion were at 37.5°C (55.5±4.1) and 38.5°C (62.2±3.3) and differed significantly from all other treatment groups (P<0.05). Temperature lower than 37.5°C and higher than 39.5°C did not intensify oocyte polar body extrusion. We concluded that the temperatures of 37.5°C and 38.5°C were suitable for IVM of cattle oocytes with acceptable polar body extrusion rates compared with other temperatures.

МОДЕЛИРОВАНИЕ СИСТЕМ ЭКСТРАКОРПОРАЛЬНОГО СОЗРЕВАНИЯ ООЦИТОВ КРУПНОГО РОГАТОГО СКОТА С ИСПОЛЬЗОВАНИЕМ СТРУКТУРНЫХ КОМПОНЕНТОВ ОВАРИАЛЬНЫХ ФОЛЛИКУЛОВ *

2019 ◽  
pp. 20-22
Author(s):  
T.I. KUZMINA ◽  
I.V. CHISTYAKOVA
Keyword(s):  
Granulosa Cells ◽  
Follicular Fluid ◽  
Bovine Serum ◽  
Egg Quality ◽  
Fetal Bovine Serum ◽  
Reproductive Technologies ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Cultural Systems ◽  
Fetal Bovine

Создание эффективной унифицированной системы дозревания донорских ооцитов обеспечит повышение результативности инновационных клеточных репродуктивных технологий. В исследовании проведен сравнительный мониторинг показателеймейотического созревания ооцитов коров, созревших в различных системах, дополненных структурными компонентами фолликулов (СКФ стенки фолликулов, клетки гранулезы, белки) и фолликулярной жидкостью,а также потенций к развитию из них доимплантационных эмбрионов. Анализу подверглись ооциты, прокультивированные в следующих системах:среда ТС199 с добавлением 10 фетальной бычьей сыворотки (ФБС), 50 мкг/мл эстрадиола, 10 мкг/мл лютеинизирующего гормона (ЛГ), 10 мкг/мл фолликулостимулирующего гормона (ФСГ) среда ТС199 с 10 эстральной сывороткой коров среда ТС199 с 50 жидкости из фолликулов диаметром 9 мм среда ТС199 с добавлением белков фолликулярной жидкости молекулярной массой 65 кДасреда ТС199 с 10 ФБС и 1106 клеток гранулезы среда ТС199 с 10 ФБС и тканью фолликула. В культуральные среды ко всем исследованным группам ооцитов добавляли антибиотики. Использование CКФ обеспечило значительное снижение доли ооцитов с дегенерированным хроматином, что способствовало увеличению уровня доимпланационных эмбрионов на стадии бластоцисты. Так, доля бластоцист, развившихся из ооцитов, созревших в среде со стенками фолликулов,составила43,5. В этой же группе выявлен минимальный уровень дегенерированных зародышей (6,45). Полученные данные предлагается использовать при моделировании систем дозревания ооцитов коров с целью повышения качества яйцеклеток.The creation of an effective unified maturation system of donor oocytes provides an increase in the efficiency of innovative cellular reproductive technologies. The comparative analysis of the meiotic maturation indicators of bovine oocytes, which were matured in different cultural systems modified by follicular structural components (FSC follicular walls, granulosa cells, proteins) and follicular fluid, as well as the potential for preimplantation embryonic development were evaluated in this study. Oocytes matured in following cultural systems: medium TC199 supplemented with 10 fetal bovine serum and 50 g/ml of estradiol, 10 g/ml of luteinizing hormone (LH), 10 g/ml of folliclestimulating hormone (FSH) medium TC199 with 10 estrous cow serum medium TC199 with 50 liquid from follicles with a diameter of 9 mm medium TC199 supplemented with the follicular fluid proteins with molecular weight 65 kDa medium TC199 with 10 fetal bovine serum and 1106 granulosa cells medium TC199 with the addition of 10 fetal bovine serum and follicle tissues were analyzed. Antibiotics were added to cultural media of all experimental groups of oocytes. The usage of FSC ensured the decrease in the proportion of oocytes with degenerated chromatin, which contribute the rise of the level of preimplantation embryos at the blastocyst stage. Thus, the proportion of blastocysts developed from oocytes matured in medium supplemented with follicular walls was 43.5. In the same experimental group, the number of degenerated embryos was 6.45. The obtained data are supposed to be used for modeling the cultural systems of cow oocytes in order to improve the egg quality.

LOCALIZATION OF MITOCHONDRIA IN DONOR OOCYTES OBTAINED FROM COW OVARIES POST MORTEM

2021 ◽  
pp. 21-24
Author(s):  
Т.И. КУЗЬМИНА ◽  
И.В. ЧИСТЯКОВА
Keyword(s):  
Granulosa Cells ◽  
Fetal Bovine Serum ◽  
Post Mortem ◽  
Follicular Growth ◽  
Antral Follicles ◽  
Cluster Distribution ◽  
Maturation Rate ◽  
Fetal Bovine ◽  
Donor Oocytes ◽  
Cytogenetic Method

Проведен сравнительный морфофункциональный мониторинг популяции донорских ооцитов, выделенных из яичников коров post mortem на разных стадиях овариального цикла, с учетом статуса хроматина и интрацитоплазматической локализации митохондрий ооцит-кумулюсных комплексов. Анализу подвергались ооциты, полученные из антральных фолликулов (Ø 2—6 мм), окруженные не менее, чем 5—6 слоями клеток кумулюса. Показано, что 45% и 50% ооцитов, выделенных из яичников на стадии фолликулярного роста и развитого жёлтого тела, соответственно, имеют периферическую локализацию митохондрий в ооплазме. Доля созревших ооцитов с периферической и равномерной локализацией митохондрий на момент аспирации их из яичников составила 88 и 85% соответственно, а уровень созревших ооцитов в группе с кластерным типом локализации достиг лишь 66%. В группе ооцитов с кластерной локализацией митохондрий отмечен высокий уровень ооцитов с дегенерированным хроматином после 24 ч культивирования (90%) по сравнению с группами с периферической и равномерной локализацией митохондрий (2 и 41% соответственно, P < 0,001). Ооциты с периферической локализацией митохондрий характеризовались гомогенной ооплазмой (58%). Гаметы с кластерным распределением митохондрий в основном имели гетерогенную ооплазму (52%), а клетки с равномерным распределением —  гомо- и гетерогенную ооплазму (29 и 36% соответственно). A comparative monitoring of the population of cow donor oocytes isolated from ovaries at different stages of the ovarian cycle was carried out, the chromatin status and the intracytoplasmic localization of mitochondria of oocyte-cumulus complexes were evaluated. Oocytes obtained from antral follicles (Ø 2-6 mm) were analyzed. Gametes were cultured at a temperature of 38.5°C, in an atmosphere with 5% CO2, 90% humidity for 24 hours in T-199 medium supplemented with 10% fetal bovine serum, 106 granulosa cells/ml, 50 ng/ml bovine prolactin. Mitochondria were visualized with using of vital dye rhodamine 123, the chromatin status was assessed by Tarkowski`s cytogenetic method. It was demonstrated that 45% and 50% of oocytes isolated from the ovaries in the stage of follicular growth and developed corpus luteum had a peripheral localization of mitochondria in ooplasm. The proportion of mature oocytes with the peripheral and spread localizations of mitochondria were 88% and 85%, respectively, while the maturation rate of oocytes with the mitochondrial clusters reached only 66%. Among oocytes with cluster distribution of mitochondria a high percent of cells with signs of chromatin degeneration after 24 h culture was noted (90%) compared with groups of oocutes with peripheral and spread distribution (2% and 41%, respectively, P<0,001). Oocytes with the peripheral localization of mitochondria were characterised by homogeneous ooplasm (58%). Gametes with the clusters mostly had a heterogeneous ooplasm (52%), and cells with the spread distribution – homo- and heterogeneous ooplasm (29% and 36%, respectively).

184 EFFECT OF CONCENTRATION AND EXPOSURE DURATION OF FETAL BOVINE SERUM ON PARTHENOGENETIC DEVELOPMENT OF PORCINE FOLLICULAR OOCYTES

2007 ◽  
Vol 19 (1) ◽  
pp. 208
Author(s):  
H. J. Kim ◽  
S. R. Cho ◽  
C. Y. Choe ◽  
S. H. Choi ◽  
D. S. Son ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Follicular Fluid ◽  
Bovine Serum ◽  
Exposure Duration ◽  
Cytochalasin B ◽  
Fetal Bovine Serum ◽  
Hatching Rate ◽  
Parthenogenetic Development ◽  
Maturation Medium ◽  
Fetal Bovine

The aim of the present experiment was to examine hatching rate as a testing tool of porcine embryo viability before early-stage embryo transfer, such as zygotes or 2-cell stage embryos. We evaluated the optimal concentrations and exposure durations of fetal bovine serum (FBS) on porcine parthenotes. Ovaries were obtained from prepubertal gilts at a local abattoir and brought to the laboratory in physiological saline with antibiotics at 30–33°C. The ovaries were washed and wiped, and then cumulus–oocytes complexes (COCs) in the follicular fluid were aspirated from surface-visible follicles (2–6 mm in diameter) with a 10-mL syringe fitted with an 18-gauge needle. After being washed 3 times with modified phosphate-buffered saline (DPBS; GIBCO, Grand Island, NY, USA) containing 0.3% BSA, the COCs were suspended in maturation medium, NCSU-23 containing 10% (v/v) porcine follicular fluid, 10 ng mL−1 epidermal growth factor (EFG; Sigma-Aldrich Corp., St Louis, MO, USA), 10 µg mL−1 follicular stimulating hormone (FSH; Sigma), 35 µg mL−1 luteinizing hormone (LH; Sigma), 1 mg mL−1 cysteine (Sigma, USA), 100 IU mL−1 penicillin G, and 100 µg mL−1 streptomycin sulfate (GIBCO). After 24 h, the COCs were transferred to the same medium without hormones. The oocytes matured for 48 h were denuded. The oocytes with a visible polar body were selected and returned to the maturation medium without hormones. After 65 h of maturation, oocytes were exposed to PBS with 7% ethanol (v/v) for 7 min, and then the oocytes were washed and treated in TCM-199 containing 5 µg mL−1 cytochalasin B (Sigma) for 5 h at 38.5°C in an atmosphere of 5% CO2 and 95% air with high humidity. After cytochalasin B treatment, the presumptive parthenotes were cultured in PZM-5 medium (IFP, Japan) and cleavage of the parthenotes was assessed at 72 h of activation. Normally cleaved parthenotes were cultured for 8 days to evaluate their ability to develop to the blastocyst and hatching stages. The FBS was added at Day 4 or 5 with concentrations of 2.5, 5, or 10%. The blastocyst rates ranged from 39.1 to 70% in each treatment. However, the hatching rate was dramatically decreased in the non-addition group. In this experiment, the developmental potential may be estimated before embryo transfer by an in vitro culture system up to the hatching stage. Table 1. Effect of concentration and exposure duration of FBS on parthenogenetic development of porcine follicular oocytes

56 THE EFFECT OF VITRIFICATION FOR SHEEP OOCYTES VIABILITY

2015 ◽  
Vol 27 (1) ◽  
pp. 121 ◽  
Author(s):  
Y. M. Toishibekov ◽  
R. K. Tursunova ◽  
M. Sh. Yermekova
Keyword(s):  
Polar Body ◽  
Control Group ◽  
Polar Bodies ◽  
Maturation Medium ◽  
Chi Squared ◽  
Fetal Bovine ◽  
Second Polar Body ◽  
Cryopreservation Techniques ◽  
Humidified Air

Advances in reproduction technologies, such as in vitro maturation, IVF, and in vitro culture, stimulated research for efficient cryopreservation techniques for mammalian oocytes. It is well known that the oocyte is the largest cell of an animal's body and as such, is full of water and, in many species, fat, making it difficult to cryopreserve. The objective of this work was to study the effect of vitrification for cryopreservation of the metaphase II plate (MPII) of sheep oocytes. Ovaries from 20 ewes of Kazakh Arkharo-Merino breed were acquired after slaughter and maintained at 37°C in TCM-199. The maturation medium was TCM-199, containing 1 mM of glutamine, 10% FBS, 5 μg mL–1 FSH, 5 μg mL–1 LH, 1 μg mL–1 oestradiol, 0.3 mM sodium pyruvate, and 100 mM cysteamine. The oocytes were incubated in 400 μL of medium in 4-well dishes covered with mineral oil. The IVM conditions were 5% CO2 in humidified air at 39°C for 24 h. Then they were placed for 10 min in a media with Hoechst 33342 (3 μg mL–1) and cytochalasin B (7 μg mL–1) to facilitate the enucleation of the MPII with a minimum volume of ooplasm. The MPII plates were divided into 2 groups: the vitrification group was exposed to vitrification media containing 1.12 M ethylene glycol (ET) + 0.87 M ME2SO for 5 min and was exposed in vitrification media containing 2.24 M ET + 1.75 M ME2SO for 5 min, and then in vitrification solution containing 4.48 M ET + 40% ME2SO + 0.25 M sucrose for 30 s. Oocytes were loaded into cryoloop and plunged into liquid nitrogen (LN2). Oocytes were thawed in a 25°C water bath and then placed in TCM-199 at 20% fetal bovine serum. After 15 min of incubation the oocytes were activated for extrusion of the second polar body in 1 mg mL–1 Ca ionophore for 5 min and washed for 5 min followed by 4 h in 6-DMAP (0.12 mM) + cycloheximide (0.6 μg mL–1). After activation the MPII were washed and cultured for 20 h. The control group received the same treatment, but they were not vitrified. Differences between the experimental groups were tested using Chi-squared test. Our research showed the expulsion of the second polar body after activation was observed in more than 62.2% of the MPII that were not vitrified (control group), whereas 40.5% of vitrified plates had expulsion of polar bodies (P < 0.05). These preliminary studies showed that it is possible to vitrify MPII plates. On the other hand, the drastic reduction of the volume of the sheep oocytes might make cryopreservation possible with greater efficiency.

Is it possible to alter the embryo lipid accumulation with reduction of fetal bovine serum and use of l-carnitine for in vitro maturation of bubaline oocytes?

Zygote ◽  
2020 ◽  
Vol 28 (2) ◽  
pp. 109-115
Author(s):  
Marivaldo Rodrigues Figueiró ◽  
Joaquim Mansano Garcia ◽  
Marina Ragagnin de Lima ◽  
Maite del Collado ◽  
Naiara Zoccal Saraiva
Keyword(s):  
Lipid Accumulation ◽  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Time Frame ◽  
Success Rates ◽  
High Concentration ◽  
Embryo Production ◽  
Maturation Medium ◽  
Fetal Bovine

SummaryIn vitro embryo production (IVEP) is a procedure that can promote genetic improvement in a short time frame. However, the success rates obtained with this biotechnology in water buffaloes are still inconsistent, and can be associated with the high concentration of lipids in the cytoplasm of oocytes and embryos. The objective of this study was to evaluate the effects of reduced concentration of fetal bovine serum (FBS) and/or use of l-carnitine during in vitro maturation (IVM) on the preimplantation development and lipid accumulation in bubaline embryos. In a first experiment, the lowest concentration of FBS in the IVM medium (0%, 2.5%, 5% or 10%) was determined, and the lowest concentration that maintained good embryo development rates was 5%. In a second experiment, the addition of 5 mM of l-carnitine into the maturation medium was evaluated. The blastocysts produced were submitted to lipid evaluation involving staining followed by observation using optical (Oil Red O) and confocal (BODIPY 493/503) microscopy. No difference was observed between the 5% and 10% FBS groups, which were superior to the 0% and 2.5% groups. Furthermore, the performance of the groups treated with 5% and 10% FBS was better than the groups supplemented with l-carnitine. There was no difference regarding embryo lipid accumulation. The results indicated that it is possible to reduce the FBS concentration to 5% in in vitro maturation medium for production of bubaline embryos, and supplementation with 5 mM l-carnitine does not increase embryo production.

scholarly journals Possible mechanism for acceleration of meiotic progression of bovine follicular oocytes by growth factors in vitro

Reproduction ◽  
2002 ◽  
pp. 135-142 ◽  
Author(s):  
M Sakaguchi ◽  
T Dominko ◽  
N Yamauchi ◽  
ML Leibfried-Rutledge ◽  
T Nagai ◽  
...  
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Kinase Activity ◽  
Polar Body ◽  
Control Group ◽  
Meiotic Cell ◽  
Treatment Groups ◽  
Igf I ◽  
Polar Body Extrusion

The mechanism for the accelerating effects of epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) on the meiotic cell cycle of bovine oocytes cultured in vitro was investigated. Cumulus-oocyte complexes (COCs) were obtained from small (< or = 3 mm in diameter), medium (4-6 mm in diameter) or large (7-10 mm in diameter) ovarian follicles and cultured with or without a combination of EGF and IGF-I (growth factors). Growth factors significantly increased the frequency of first polar body extrusion of oocytes derived from small follicles at 16 h of culture (PB16 oocytes; with growth factors: 75%; without growth factors: 55%), but did not increase the frequency in oocytes from medium or large follicles. COCs from small follicles were cultured with individual growth factors and sampled for kinase activity. The frequencies of polar body extrusion in EGF only (67%) and EGF + IGF-I (75%) treatment groups were significantly higher than those in the control (no growth factor) group (49%), but not significantly higher than in the IGF-I only group (63%). The H1 kinase activity at 6-8 h of culture in each group increased significantly from the baseline value at 0 h of culture, and the H1 kinase activities in the EGF only, IGF-I only and EGF + IGF-I treatment groups were significantly higher than those in the control group at 8 h of culture. MAP kinase activity was significantly higher than the baseline value and significantly higher than that in the control group at 6 h of culture in the EGF treatment group only. In conclusion, EGF and IGF-I act on COCs from small follicles to accelerate the meiotic cell cycle of the oocytes. This accelerating effect may be related to increased H1 and MAP kinase activities during the early stages of maturation.

scholarly journals 298RED DEER (CERVUS ELAPHUS) PARTHENOGENETIC BLASTOCYSTS PRODUCED USING IONOMYCIN/6DMAP ACTIVATION

2004 ◽  
Vol 16 (2) ◽  
pp. 268 ◽  
Author(s):  
S.E. Beaumont ◽  
D.K. Berg ◽  
G.W. Asher
Keyword(s):  
Red Deer ◽  
Polar Body ◽  
Negative Control ◽  
Blastocyst Development ◽  
Cell Counts ◽  
First Polar Body ◽  
Significant Difference ◽  
Chi Square Analysis ◽  
Polar Body Extrusion

Successful activation of red deer oocytes is a necessary prerequisite for the cloning of red deer individuals with desirable genetic characteristics. To investigate this, an established biphasic protocol used for oocyte activation in sheep was investigated for suitability. The method chosen was 5μM Ionomycin for 5min followed by 2mM 6DMAP for 3h ( Loi P et al., 1998 Biol. Reprod. 58, 1177–1187). The medium used during activation and subsequent culture was Deer Synthetic Oviduct Fluid, which has been shown to support routine in vitro fertilization and blastocyst development (15%) of in vitro-matured red deer oocytes (DSOF, Berg D et al., 2003 Theriogenology 59, 189–205). Red deer abattoir-derived COCs were matured in vitro for 22h before random allocation across 3 treatment groups comprising a standard IVF group, the activation group and a negative control group exposed to medium only. Activation treatment oocytes were stripped of cumulus by vortexing in 0.1% hyaluronidase before selecting for first polar body extrusion. First-step activation was performed in medium comprising HEPES-buffered IVF-DSOF containing 4mM Ca2+. Second-step activation used 3mM Ca2+ early DSOF under 7% O2, 5% CO2, and 88% N2 at 38.5°C. Standard IVF was conducted at 23h post-IVM using 4mM Ca2+ IVF-DSOF and 0.5×106mL−1 final sperm concentration. Following activation and IVF, oocytes were washed 3 times in HEPES DSOF before culture for 7 days in sequential DSOF with late DSOF on Day 4 containing 1.5mM Ca2+. Cleavage was assessed 24h after activation, and all blastocysts were fixed for cell counts. Four replicates of each treatment were performed. Cleavage and blastocyst rates were examined by chi-square analysis and cell numbers by ANOVA. First polar body extrusion rate was 84%. Cleavage was similar between the activation treatment and IVF (P&gt;0.05 ); but a significant difference was found in blastocyst development rates (P&lt;0.05) with the Ionomycin and 6DMAP protocol being superior to the IVF treatment. Exposure to high Ca2+ media alone resulted in only 5% of the negative control oocytes cleaving to 2 cells. Results show that Ionomycin and 6DMAP are effective in activating red deer oocytes and DSOF is a suitable medium to produce parthenogenetic blastocysts.

scholarly journals The Role of Ca2 + in Maturation and Reprogramming of Bovine Oocytes: A System Study of Low-Calcium Model

2021 ◽  
Vol 9 ◽  
Author(s):  
Lin Meng ◽  
Hongmei Hu ◽  
Zhiqiang Liu ◽  
Luyao Zhang ◽  
Qingrui Zhuan ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Polar Body ◽  
Rna Seq ◽  
Low Calcium ◽  
Maturation Medium ◽  
First Polar Body ◽  
Significant Difference ◽  
Bovine Oocytes

[Ca2+]i is essential for mammalian oocyte maturation and early embryonic development, as those processes are Ca2+ dependent. In the present study, we investigated the effect of [Ca2+]i on in vitro maturation and reprogramming of oocytes in a lower calcium model of oocyte at metaphase II (MII) stage, which was established by adding cell-permeant Ca2+ chelator BAPTA-AM to the maturation medium. Results showed that the extrusion of the first polar body (PB1) was delayed, and oocyte cytoplasmic maturation, including mitochondrial and endoplasmic reticulum distribution, was impaired in lower calcium model. The low-calcium-model oocytes presented a poor developmental phenotype of somatic cell nuclear transfer (SCNT) embryos at the beginning of activation of zygotic genome. At the same time, oxidative stress and apoptosis were observed in the low-calcium-model oocytes; subsequently, an RNA-seq analysis of the lower-calcium-model oocytes screened 24 genes responsible for the poor oocyte reprogramming, and six genes (ID1, SOX2, DPPA3, ASF1A, MSL3, and KDM6B) were identified by quantitative PCR. Analyzing the expression of these genes is helpful to elucidate the mechanisms of [Ca2+]i regulating oocyte reprogramming. The most significant difference gene in this enriched item was ID1. Our results showed that the low calcium might give rise to oxidative stress and apoptosis, resulting in impaired maturation of bovine oocytes and possibly affecting subsequent reprogramming ability through the reduction of ID1.

Flexural Strength and Cytotoxicity of ZCC, a Resin Modified Zinc-Calcium Liner Prototype

2017 ◽  
Vol 751 ◽  
pp. 649-656
Author(s):  
Pasutha Thunyakitpisal ◽  
Nonglax Thunyakitpisal ◽  
Sirithan Jiemsirilers ◽  
Dujreutai Pongkao Kashima
Keyword(s):  
Flexural Strength ◽  
Mtt Assay ◽  
Fetal Bovine Serum ◽  
Growth Media ◽  
Human Dental Pulp Cells ◽  
Significant Difference ◽  
Human Dental Pulp ◽  
Pulp Cells ◽  
Fetal Bovine ◽  
One Way Anova

Objective: To investigate the flexural strength and cytotoxicity of ZCC, a resin modified zinc oxide-calcium carbonate liner prototype, compared with commercial dental liners. Materials and Methods: ZCC, Dycal, Ultra-Blend® plus (UB), and TheraCal LC® (TC) were evaluated for flexural strength. Six samples of each material were incubated in growth medium (10% fetal bovine serum supplemented DMEM) for 24 h. Primary human dental pulp cells were cultured in the conditioned medium from each sample, with growth media used as a control. Cytotoxicity was determined using an MTT assay. Data were analyzed by one-way ANOVA. The value of p<0.05 was considered significant. Results: UB had the highest flexural strength with Dycal presenting the lowest values (p<0.05). ZCC demonstrated significantly higher flexural strength values compared with those of TC and Dycal (p<0.05). The MTT assay indicated that Dycal conditioned media significantly reduced cell viability at 24 and 48 hours (p<0.05). There was no significant difference in viability between the control, ZCC, UB, and TC groups at 24 and 48 hours (p>0.05). Conclusion: ZCC met the requirements for flexural strength per ISO 9917-2:2010(E). There was no significant difference in viability between the control and ZCC group at 24 and 48 hours.

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