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.

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>0.05 ); but a significant difference was found in blastocyst development rates (P<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.

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.

137 Comparison of two invitro maturation media on polar body extrusion of cattle oocytes

2021 ◽  
Vol 33 (2) ◽  
pp. 176
Author(s):  
S. M. Sithole ◽  
M. L. Mphaphathi ◽  
M. D. Sebopela ◽  
T. L. Nedambale
Keyword(s):  
Imaging System ◽  
Polar Body ◽  
Oocyte Quality ◽  
Developmental Competence ◽  
Significant Difference ◽  
Maturation Rate ◽  
Aspiration Technique ◽  
Level Of Significance ◽  
Polar Body Extrusion ◽  
Quality Type

The invitro embryo production technique is one of the assisted reproduction technologies that has the potential in speeding up genetic improvement in cattle. The developmental competence of invitro-matured oocytes is influenced by several factors during invitro maturation (IVM), such as maturation environment, oocyte quality, type of media, and additives. The objective of the present study was to compare two IVM media (TCM-199 and BO-IVM) on cattle oocyte maturation rate invitro. Cattle ovaries were collected from local slaughterhouse and transported to the laboratory in a thermos flask containing 0.9% saline (Adcock Ingram Critical care) at 37°C. Oocytes were retrieved from the ovaries by the aspiration technique, and then matured invitro in 500µL of TCM-199 (supplemented with 10% fetal bovine serum, FSH, LH, and E2) or in 500µL of commercially available BO- IVM (Bioscience) medium, both covered with mineral oil for 22h at 38.5°C with 5% CO2 and 5% O2. After 22h of IVM, oocyte polar body extrusion was evaluated with the aid of Oosight Imaging System (Hamilton Thorne) connected to an inverted microscope. The total number of oocytes matured in TCM-199 and BO-IVM media were 401 and 396, respectively. The experiment was replicated 19 times. Data were analysed using the GenStat® program (VSN International). Means of different treatments were separated using Fisher’s protected t-test least significant difference at 5% level of significance. No difference was recorded for polar body extrusion rates in TCM-199 (50.6±13.9) and BO-IVM (47.3±13.7) media. In conclusion, TCM-199 and BO-IVM media did not differ in terms of maturation rate; thus, both can be used for successful cattle oocyte IVM.

Maturation promoting factor in ascidian oocytes is regulated by different intracellular signals at meiosis I and II

Development ◽  
1996 ◽  
Vol 122 (7) ◽  
pp. 1995-2003 ◽  
Author(s):  
G.L. Russo ◽  
K. Kyozuka ◽  
L. Antonazzo ◽  
E. Tosti ◽  
B. Dale
Keyword(s):  
Kinase Activity ◽  
Phase 1 ◽  
Polar Body ◽  
Calcium Transients ◽  
Phase 2 ◽  
Phase 3 ◽  
First Polar Body ◽  
Second Polar Body ◽  
Polar Body Extrusion ◽  
Maturation Promoting Factor

Using the fluorescent dye Calcium Green-dextran, we measured intracellular Ca2+ in oocytes of the ascidian Ciona intestinalis at fertilization and during progression through meiosis. The relative fluorescence intensity increased shortly after insemination in a single transient, the activation peak, and this was followed by several smaller oscillations that lasted for approximately 5 minutes (phase 1). The first polar body was extruded after the completion of the phase 1 transients, about 9 minutes after insemination, and then the intracellular calcium level remained at baseline for a period of 5 minutes (phase 2). At 14 minutes postinsemination a second series of oscillations was initiated that lasted 11 minutes (phase 3) and terminated at the time of second polar body extrusion. Phases 1 and 3 were inhibited by preloading oocytes with 5 mM heparin. Simultaneous measurements of membrane currents, in the whole-cell clamp configuration, showed that the 1–2 nA inward fertilization current correlated temporally with the activation peak, while a series of smaller oscillations of 0.1-0.3 nA amplitude were generated at the time of the phase 3 oscillations. Biochemical characterization of Maturation Promoting Factor (MPF) in ascidian oocytes led to the identification of a Cdc2-like kinase activity. Using p13suc1-sepharose as a reagent to precipitate the MPF complex, a 67 kDa (67 × 10(3) Mr) protein was identified as cyclin B. Histone H1 kinase activity was high at metaphase I and decreased within 5 minutes of insemination reaching a minimum level during phase 2, corresponding to telophase I. During phase 3, H1 kinase activity increased and then decayed again during telophase II. Oocytes preloaded with BAPTA and subsequently inseminated did not generate any calcium transients, nonetheless H1 kinase activity decreased 5 minutes after insemination, as in the controls, and remained low for at least 30 minutes. Injection of BAPTA during phase 2 suppressed the phase 3 calcium transients, and inhibited both the increase in H1 kinase activity normally encountered at metaphase II and second polar body extrusion.

scholarly journals Effect of Melatonin on the In Vitro Maturation of Porcine Oocytes, Development of Parthenogenetically Activated Embryos, and Expression of Genes Related to the Oocyte Developmental Capability

Animals ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 209 ◽  
Author(s):  
Ling Yang ◽  
Qingkai Wang ◽  
Maosheng Cui ◽  
Qianjun Li ◽  
Shuqin Mu ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Polar Body ◽  
Melatonin Receptors ◽  
Melatonin Treatment ◽  
In Vitro Development ◽  
First Polar Body ◽  
Mitochondrial Distribution ◽  
Polar Body Extrusion ◽  
Vitro Development

Melatonin treatment can improve quality and in vitro development of porcine oocytes, but the mechanism of improving quality and developmental competence is not fully understood. In this study, porcine cumulus–oocyte complexes were cultured in TCM199 medium with non-treated (control), 10−5 M luzindole (melatonin receptor antagonist), 10−5 M melatonin, and melatonin + luzindole during in vitro maturation, and parthenogenetically activated (PA) embryos were treated with nothing (control), or 10−5 M melatonin. Cumulus oophorus expansion, oocyte survival rate, first polar body extrusion rate, mitochondrial distribution, and intracellular levels of reactive oxygen species (ROS) and glutathione of oocytes, and cleavage rate and blastocyst rate of the PA embryos were assessed. In addition, expression of growth differentiation factor 9 (GDF9), tumor protein p53 (P53), BCL2 associated X protein (BAX), catalase (CAT), and bone morphogenetic protein 15 (BMP15) were analyzed by real-time quantitative PCR. The results revealed that melatonin treatment not only improved the first polar body extrusion rate and cumulus expansion of oocytes via melatonin receptors, but also enhanced the rates of cleavage and blastocyst formation of PA embryos. Additionally, melatonin treatment significantly increased intraooplasmic level of glutathione independently of melatonin receptors. Furthermore, melatonin supplementation not only significantly enhanced mitochondrial distribution and relative abundances of BMP15 and CAT mRNA, but also decreased intracellular level of ROS and relative abundances of P53 and BAX mRNA of the oocytes. In conclusion, melatonin enhanced the quality and in vitro development of porcine oocytes, which may be related to antioxidant and anti-apoptotic mechanisms.

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 Pengaruh urea dalam media maturasi in vitro terhadap tingkat maturasi oosit sapi

2021 ◽  
Vol 10 (2) ◽  
pp. 46
Author(s):  
Sepvian Dewi Kurniawati ◽  
Suryanie Sarudji ◽  
Widjiati Widjiati
Keyword(s):  
Oocyte Maturation ◽  
Polar Body ◽  
Metaphase Plate ◽  
Petri Dish ◽  
Control Group ◽  
Maturation Medium ◽  
First Polar Body ◽  
Maturation Rate ◽  
Follicle Aspiration

This study was aimed to determine the effect of urea in maturation medium on in vitro oocyte maturation rate. The medium used was TCM-199 added with Hepes, NaHCO3, Kanamycin 0.15 IU/mL, PMSG, 0.15 IU/mL hCG, and 10% FBS. Cumulus oocyte complexes (COCs) of cows derived from follicle aspiration were divided into three groups. In control group (P0), the COCs were matured in vitro in a maturation medium without urea addition, meanwhile in the P1 and P2 groups, the medium was added with urea 20 and 40 mg/dL, respectively. Each petri dish contained three drops of maturation medium (300 µl/drops) according to the groups. Microdrops were coated with mineral oil and then incubated in a 5% CO2 incubator, at 39 ˚C with maximum humidity. Aceto-orcein staining was conducted to evaluate the maturation of oocytes based on the achievement of metaphase II phase that is indicated by the presence of metaphase plate and/or first polar body. The result showed that the oocyte maturation rates of P0, P1, and P2 were 51.25, 52.43 (p >0.05), and 46.88 % (p <0.05) respectively. It could be concluded that the presence of urea at 40 mg/dL in maturation medium reduced the percentage of bovine oocyte maturation in vitro.

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.

scholarly journals The Number of LH Receptor could Predict the Success of Oocyte Maturity in the Process of In Vitro Maturation

2016 ◽  
Author(s):  
Adek Amansyah
Keyword(s):  
In Vitro Maturation ◽  
Clinical Laboratory ◽  
Polar Body ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Oocyte Maturity ◽  
Lh Receptor ◽  
First Polar Body ◽  
Significant Difference

Objective: To evaluate the relationship between the number of LH receptor and the success of oocyte maturity in the process of in vitro maturation (IVM). Method: This experimental study was conducted in the Permata Hati Infertility Clinical Laboratory, Dr. Sardjito General Hospital, Yogyakarta, with the samples of 300 oocytes obtained through collecting immature cow’s oocytes from the abattoir and grouped the oocytes into 3 (three) groups based on the pattern of oocyte cumulus cells on the vesicle germinal stage 2 - 8 mm with three layers of cumulus cell. The sample of the cumulus cells from these three groups were taken and the LH receptor examination was done with immunohistochemistry. After that, the IVM process was performed to the three groups and its development for 24 hours was evaluated. Its maturation quality was evaluated with the emergence of the first polar body (1PB) and compared to the other groups and related to the number of LH receptor in the three groups. Result: The result of this study indicated that the oocyte cumulus cells showed a difference of function during IVM process. The maturity rate in this study showed that the number of LH receptor was related to the morphological pattern of oocyte cumulus cells with oocyte maturity. The maturity of the cumulus cells which 100% covered the oocyte was higher than that of the cumulus cells which > 50% and < 30% covered the oocytes, namely, 74% compared to 60% and 12%. The result of this study also showed that the average number of LH receptors in the three groups (A, B, and C) was 183.4, 78.8, and 24.0 respectively. A significant difference was found in the three groups (p < 0.0001). When related to IVM maturity, this difference showed that the bigger number of oocyte cumulus cells influenced the oocyte maturity. Conclusion: The number of LH receptor can be used as a prediction to determine the success of oocyte maturation in the process of in vitro maturation. [Indones J Obstet Gynecol 2013; 1-4:183-7] Keywords: IVM, LH receptor, oocyte cumulus cell

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