scholarly journals L-carnitine Supplementation Enhances Nuclear and Cytoplasmic Maturation Rates of Sheep Oocytes In Vitro

2021 ◽  
Vol 44 (2) ◽  
pp. 131-137
Author(s):  
Z. W. Bhakty ◽  
E. M. Kaiin ◽  
N. W. K. Karja ◽  
M. A. Setiadi
Keyword(s):  
Tissue Culture ◽  
Treatment Group ◽  
Culture Medium ◽  
Fertilization Rate ◽  
Control Group ◽  
Carnitine Supplementation ◽  
Progressive Motility ◽  
Maturation Medium ◽  
Cytoplasmic Maturation

The aim of the present study was to determine the effectiveness of l-carnitine (LC) supplementation on nuclear and cytoplasmic maturation rates of sheep oocytes. In experiment 1, oocytes were maturated for 24 hours in tissue culture medium 199 supplemented with LC at doses of 0.3 mg/mL, 0.6 mg/mL, and 0.9 mg/mL. In experiment 2, oocytes were maturated and fertilized in a media supplemented with LC at a dose of 0.3 mg/mL and incubated with 5x106 sperm/mL for 12 hours. The treatment group consisted of LC supplementation only in maturation medium (P1), only in fertilization medium (P2), and in both maturation and fertilization media (P3). In experiment 3, sperm motility patterns were assessed using CASA after being exposed to fertilization medium supplemented with LC at a dose of 0.3 mg/mL for 0 and 3 hours. Our results showed that supplementation of LC at a dose of 0.3 mg/mL significantly (p<0.05) increased the percentage of oocytes reaching metaphase II (86.7±4.1%) compared to those supplemented with LA at doses of 0, 0.6, and 0.9 mg/mL (73.6±1.2%, 81.4±1.3%, and 70.5±1.6%, respectively). The LC treatment in the fertilization medium only did not influence the number of two pronuclear formations (62.1±2.5%) compared to supplementation either in the maturation medium only (72.0±4.7%) or a combination of both in maturation and fertilization media (68.2±2.7%) (p<0.05). Further results after 3 hours of incubation compared to the control group showed the total motility (24.8±2.04% vs. 17.49±2.37%), progressive motility (14.17±2.03% vs. 6.49±1.64%), and curvilinear velocity (VCL) (119.70±3.73% vs. 71.15±10.59%) (p<0.05) were increased in the fertilization medium containing LC but it did not improve the fertilization rate. It is concluded that supplementation of LC at a dose of 0.3 mg/mL in the maturation medium only could better improve the nuclear and cytoplasmic maturation rates of sheep oocytes.

scholarly journals Penambahan Glutathione pada Medium Fertilisasi Efektif Mendukung Pembentukan Pronukleus dan Perkembangan Awal Embrio Sapi (SUPPLEMENTATION OF GLUTATHIONE IN FERTILIZATION MEDIUM EFFECTIVELY SUPPORT NORMAL PRONUCLEUS FORMATION AND EARLY BOVINE EMBRYONIC DE

2017 ◽  
Vol 18 (3) ◽  
pp. 327
Author(s):  
Aras Prasetiyo Nugroho ◽  
Iman Supriatna ◽  
Mohamad Agus Setiadi
Keyword(s):  
Tissue Culture ◽  
Embryonic Development ◽  
Culture Medium ◽  
Fertilization Rate ◽  
Tissue Culture Medium ◽  
Randomized Design ◽  
Oviduct Fluid ◽  
And Control ◽  
Bovine Oocytes

The objective of this study was to determine fertilization rate effectiveness and early embryonic development competency with glutathione (GSH) supplementation in fertilization medium and culture This study consisted of two experiments comprising each of the four treatment and six repetitions with completely randomized design (CRD) using 651 oocytes. In the first experiment, a total of 317 bovine oocytes were matured in tissue culture medium (TCM) 199 at incubator 5% CO2 with temperature 39 ºC for24 h, then fertilized with sperm separated by swim up technique. Oocyte and sperms were incubated in fertilization medium supplemented with 0.25 mM, 0.50 mM, 1.00 mM GSH. In the second experiment, bovine oocytes were matured in maturation medium and fertilized with same procedure as mentioned before, then cultured in modified synthetic oviduct fluid (mSOF) with the following treatment: supplementation GSH only in fertilization medium (T1), supplementation GSH only in culture medium (T2), and supplementation GSH in both fertilization and culture medium (T3), while control not supplementation GSH (T0). Result of the first experiment showed that supplementation 1.00 mM GSH in fertilization medium can increase higher normal pronucleus (2PN) formation (86,9%) compared to other treatments, 0.50 mM (80.3%), 0.25 mM (73.8%), and control (58.9%) (P<0.05). In the second experiment showed that early bovine embryonic development on 2nd day cultured which reached 5-8 cell on treatment T1 (56.0%) and T3 (53.6%) were higher (P<0.05) compared to treatment T2 (26.2%) and T0 (control) (31.3%). Result of the other were showed that early bovine embryonic development on 4th day cultured which reached 9-16 cell on treatment T1 (26.2%) and T3 (27.4%) were higher (P<0.05) compared to that T2 (11.9%) and T0 (control) (10.8%). In conclusion, 1.00 mM GSH supplementation in medium was more effective in supporting normal pronucleus formation and early fertilization bovine embryonic development compared to in culture medium. ABSTRAK Penelitian ini bertujuan untuk mengetahui tingkat fertilisasi dan kompetensi perkembangan awal embrio sapi dengan penambahan glutathione (GSH) pada medium fertilisasi in vitro (IVF) dan kultur in vitro (IVC). Penelitian ini terdiri atas dua penelitian yang terdiri dari masing-masing empat perlakukan dan enam kali ulangan dengan rancangan acak lengkap (RAL) menggunakan 651 oosit. Penelitian I, sebanyak 317 oosit sapi dalam tissue culture medium (TCM) 199 dimatangkan pada inkubator 5% CO2 dan suhu 39°C selama 24 jam, kemudian difertilisasi dengan spermatozoa yang telah diseleksi menggunakan teknik swim up. Oosit dan spermatozoa diinkubasi pada medium fertilisasi dengan penambahan 0,25 mM, 0,50 mM, dan 1,00 mM GSH. Penelitian II, sebanyak 334 oosit sapi dimatangkan pada medium pematangan dan difertilisasi, kemudian dikultur pada medium modified synthetic oviduct fluid (mSOF), dengan perlakuan: penambahan GSH hanya pada medium fertilisasi (T1), penambahan GSH hanya pada medium kultur (T2), dan kombinasi penambahan GSH pada medium fertilisasi dan kultur (T3). Hasil penelitian I, menunjukkan bahwa penambahan 1,00 mM GSH pada medium fertilisasi dapat meningkatkan pembentukan pronukleus normal (2PN) yang lebih tinggi (86,9%) dibandingkan dengan perlakuan yang lain yaitu 0,50 mM (80,3%), 0,25 mM (73,8%), dan kontrol (58,9%) (P<0,05). Penelitian II menujukkan bahwa perkembangan awal embrio sapi pada hari ke-2 kultur yang mencapai pembelahan 5-8 sel pada perlakukan T1 (56,0%) dan T3 (53,6%) lebih tinggi (P<0,05) dibandingkan dengan perlakuan T2 (26,2%) dan T0 (kontrol) (31,3%). Hasil penelitian lain menunjukkan bahwa perkembangan awal embrio sapi pada hari ke-4 kultur yang mencapai pembelahan 9-16 sel pada perlakuan T1 (26,2%) dan T3 (27,4%) lebih tinggi dibandingkan dengan perlakukan T2 (11,9%) dan T0 (kontrol) (10,8%) (P<0,05). Dapat disimpulkan bahwa penambahan 1,00 mM GSH pada medium fertilisasi lebih efektif dalam mendukung pembentukan pronukleus normal dan perkembangan awal embrio sapi dibandingkan pada medium kultur.

Induction of autophagy during in vitro maturation improves the nuclear and cytoplasmic maturation of porcine oocytes

2014 ◽  
Vol 26 (7) ◽  
pp. 974 ◽  
Author(s):  
Bong-Seok Song ◽  
Ji-Su Kim ◽  
Young-Hyun Kim ◽  
Bo-Woong Sim ◽  
Seung-Bin Yoon ◽  
...  
Keyword(s):  
Treatment Group ◽  
In Vitro Maturation ◽  
Critical Role ◽  
Control Group ◽  
Transcript Levels ◽  
Rapamycin Treatment ◽  
Fertilisation Rate ◽  
Cytoplasmic Maturation

While a critical role of autophagy in mammalian early embryogenesis has been demonstrated, few studies have been conducted regarding the role of autophagy in in vitro maturation (IVM) of immature oocytes. In the present study we investigated the effect of rapamycin, a chemical autophagy inducer, on the nuclear and cytoplasmic maturation of porcine oocytes. Rapamycin treatment led to increased expression of LC3-II, an autophagy marker. Compared with the control group, as well as the 5 and 10 nM rapamycin treatment groups, the rate of MII oocyte production was higher in the 1 nM rapamycin treatment group, indicating improvement in nuclear maturation. In the analyses of cytoplasmic maturation, we found that the level of p34cdc2, a cytoplasmic maturation marker, and the monospermic fertilisation rate were higher in the 1 nM rapamycin treatment group than in the other groups. Moreover, the beneficial effect of 1 nM rapamycin on cytoplasmic maturation of MII oocytes was further evidenced by increases in blastocyst formation rate, total cell number and cell survival. In the blastocyst embryos, anti-apoptotic Bcl-xL transcript levels were elevated in the 1 nM rapamycin-treated group, whereas pro-apoptotic Bax transcript levels were decreased. Collectively, these results suggest that induction of autophagy during IVM contributes to enhancement of the nuclear and cytoplasmic maturation of porcine oocytes.

212 EFFECTS OF VASCULAR ENDOTHELIAL GROWTH FACTOR ON THE EARLY DEVELOPMENT AND POLYSPERMY RATE OF OVINE EMBRYOS PRODUCED IN VITRO

2009 ◽  
Vol 21 (1) ◽  
pp. 204
Author(s):  
H. Luo ◽  
X. Cao ◽  
Y. Zhao ◽  
P. Zhou ◽  
G. Shi
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Treatment Group ◽  
Early Development ◽  
Control Group ◽  
Vascular Endothelial ◽  
Maturation Medium ◽  
Group A ◽  
Group B ◽  
Endothelial Growth Factor

To investigate the effect of vascular endothelial growth factor (VEGF) on the early development and polyspermy rate of ovine embryos in vitro, 2 experiments were conducted with human recombinant VEGF165 supplemented to the media during maturation, fertilization, and culture in vitro, respectively. Ovaries were collected from ewes at a local slaughterhouse. All oocytes surrounded by a multilayer of cumulus cells were collected and rinsed 3 times in maturation medium (control medium and treatment medium, respectively). A total of 100 oocytes in each group were cultured in 4-well plates (Nunc) containing 800 μL of maturation medium at 38.5°C in an atmosphere of 5% CO2 with saturated humidity. Four replicates of each experiment were conducted. Statistical analyses were conducted by ANOVA with SPSS 12.0 software (SPSS Inc., Chicago, IL, USA). Data are expressed as means, and P < 0.05 was considered significant. In Experiment 1, to investigate the effect of VEGF on the early development of ovine embryos in vitro, VEGF was used at 5 ng mL–1 (treatment group A) and 10 ng mL–1 (treatment group B) in maturation medium (TCM-199 + BSA), HSOF fertilization medium, and SOF culture medium. The results showed that the maturation rate was increased significantly (P < 0.01), from 75.76% in the control treatment to 83.98 and 80.23% in treatment group A and treatment group B, respectively. The cleavage rate was increased from 75.85% in the control group to 79.39% in treatment group A (P > 0.05). The development rates of morulae (45.03%) and blastocysts (23.54%) in treatment group A were significantly higher (P < 0.01) than those in the control group (38.94 and 18.09%, respectively). In addition, the development rates of blastocysts in treatment group B (21.05%) were lower than those in treatment group A (P > 0.05) and higher than those in the control group (P > 0.05). In Experiment 2, to investigate the effect of VEGF on the polyspermy rate of ovine embryos in vitro, 5 ng mL–1 of VEGF was used in TCM-199 + BSA maturation medium in this experiment. The results showed that the fertilization rate after 18 h of IVF was increased significantly (P < 0.01), from 75.75% in the control group to 83.86% in the treatment group, and that the polyspermy rate was decreased significantly (P < 0.01), from 12.64% in the control group to 7.68% in the treatment group. These results indicate that VEGF significantly improved the maturation and fertilization rates of ovine oocytes and, consequently, the rate of embryo development in vitro, especially when the medium was supplemented with 5 ng mL–1 of VEGF. The VEGF obviously decreased the polyspermy rate and bated the phenomenon of polyspermy in the process of ovine oocyte IVF. The present study was supported by the National Natural Science Foundation of China (No. 30371035).

scholarly journals Supplementation of Follicle Stimulating Hormon Into In vitro Maturation Medium to Increase Oocytes Maturation and 4 Cell Stadium Embryo Development of Bligon Goat

2018 ◽  
Vol 42 (2) ◽  
Author(s):  
Yanuar Achadri ◽  
Sigit Bintara ◽  
Diah Tri Widayati
Keyword(s):  
Tissue Culture ◽  
Embryo Development ◽  
Embryo Culture ◽  
In Vitro Maturation ◽  
Culture Medium ◽  
Tissue Culture Medium ◽  
Cleavage Rate ◽  
Maturation Medium ◽  
Phosphate Buffered Saline

The study was carried out to investigate the effect of follicle stimulating hormon (FSH) into in vitro maturation medium to increase oocytes maturation and 4 cell stadium embryo development of Bligon goat. Goat ovaries were obtained from a slaughterhouse and transported to the laboratory in a flask of NaCl at temperature of 31 – 34°C. Oocytes were aspirated from 2 – 6 mm of follicles into a 3 mL syringe (23G needle) that contained Dulbecco’s Phosphate-Buffered Saline. Oocytes were divided into three groups, i.e tissue culture medium (TCM) with FSH supplementation 0, 50, and 100 IU/mL. Oocytes were put into those medium and incubated on 39°C, 5% CO2, and 95% humidity for 24 hours. Matured oocytes were fertilized with capacitated frozen thawed-semen and incubated on 39°C, 5% CO2, and 95% humidity for 5 hours. Fertilized oocytes were washed for 3 times in TCM and incubated in the same condition for embryo culture. The data of FSH supplementation and embryo development were analyzed using randomized completely one way classification. The results showed that the percentages of mature oocytes from FSH supplementation 0, 50, and 100 IU/mL were 70,48±23,22, 78,48±15,80, and 80,29±12,86%, respectively. Cleavage rate of the two cells stage were 36,00±14,22, 44,00±33,94, and 57,45±31,78%, respectively, and for the 4 cells stage were 27,33±22,04, 35,33±40,73, and 39,45±20,38%. It is concluded that supplementation of FSH in the maturation medium could not increase the percentages of in vitro maturation and embryo development.

Short-term storage of Chinese sturgeon (Acipenser sinensis) ova in vitro

Zygote ◽  
2021 ◽  
pp. 1-4
Author(s):  
Chun Tan ◽  
Xueqing Liu ◽  
Jing Yang ◽  
Juanjuan Liu ◽  
Hejun Du
Keyword(s):  
Treatment Group ◽  
Fertilization Rate ◽  
Coelomic Fluid ◽  
Control Group ◽  
Hatching Rate ◽  
Chinese Sturgeon ◽  
Artificial Medium ◽  
Storage Duration ◽  
Acipenser Sinensis

Summary In this study, we tried to maintain the vitality of Chinese sturgeon (Acipenser sinensis) ova before fertilization with several treatments in vitro. The ovulated eggs were allocated to groups with different incubation medium (coelomic fluid and artificial media), temperature (4°C and 16°C) and storage duration (2 h and 6 h). The maximum fertilization and hatching rate were observed for the control group in which the ova were fertilized immediately after spawning, with the values of 82.45% and 84.73%, respectively. Compared with the control group, the fertilization and hatching rate of all the treatment groups stored at 4°C or in coelomic fluid decreased significantly (P < 0.05). The fertilization rate of the treatment group stored in artificial medium at 16°C did not change obviously in the first 2 h (P > 0.05), but declined dramatically (P < 0.05) after 6 h. In comparison with the control group, no significant (P > 0.05) reduction was shown in hatching rate of the treatment group stored in artificial medium at 16°C for 6 h. The results showed that the ova of Chinese sturgeon can be stored for at least 6 h at 16°C in artificial medium without weakening; this provides a practical application method for the routine hatchery practice of Chinese sturgeon, as well as certain relevant research.

Mechanism of the adverse effect of hyaluronidase used for oocyte denudation on early development of bovine embryos

Zygote ◽  
2021 ◽  
pp. 1-5
Author(s):  
Shiori Ashibe ◽  
Kanade Irisawa ◽  
Ken Yokawa ◽  
Yoshikazu Nagao
Keyword(s):  
Treatment Group ◽  
Assisted Reproductive Technologies ◽  
Cumulus Cells ◽  
Reproductive Technologies ◽  
Control Group ◽  
Free Calcium ◽  
Parthenogenetic Development ◽  
Early Embryos ◽  
Bovine Oocytes

Summary Hyaluronidase is widely used in animal and human assisted reproductive technologies (ARTs) to remove cumulus cells around oocytes. However, adverse effects of hyaluronidase treatment, such as increased rates of degeneration and parthenogenesis, have been found after treatment of human and mouse oocytes. Currently, the mechanism(s) of the detrimental effects are unclear. The present study was initiated to identify the mechanism of adverse responses to hyaluronidase treatment in bovine oocytes and early embryos. Cumulus cells were removed from cumulus–oocyte complexes (COCs) with or without hyaluronidase and the oocytes were subjected to intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF). Significantly lower rates of blastocyst formation were obtained in the hyaluronidase treatment group after ICSI (22.4%) and IVF (21.2%) compared with the non-hyaluronidase control groups: 36.1% after ICSI and 30.4% after IVF. Next, we examined the effect of hyaluronidase on parthenogenetic development rates and on the cytoplasmic levels of free calcium ions (Ca2+), reactive oxygen species (ROS) and reduced glutathione (GSH). No differences in parthenogenesis rates were found between treated and untreated groups. Ca2+ levels in oocytes from the hyaluronidase treatment group indicated using mean fluorescence intensity were significantly higher (68.8 ± 5.3) compared with in the control group (45.0 ± 2.5). No differences were found in the levels of ROS or GSH between the treated and untreated groups. We conclude that hyaluronidase might trigger an increase in Ca2+ levels in oocytes, resulting in a decreased potential for normal embryonic development.

scholarly journals In Vitro Production of Erythropoietin by Kidneys Perfused With a Serum-free Solution

Blood ◽  
1974 ◽  
Vol 44 (1) ◽  
pp. 77-85 ◽  
Author(s):  
Allan J. Erslev
Keyword(s):  
Tissue Culture ◽  
Atmospheric Pressure ◽  
Culture Medium ◽  
Kidney Tissue ◽  
In Vitro Production ◽  
In Vitro Perfusion ◽  
Serum Free ◽  
Enzymatic Activation ◽  
Vitro Production

Abstract Normal rabbits exposed to 0.4 atmospheric pressure for 3 hr will generate about 40-60 U of erythropoietin during a subsequent 3-hr period. If the kidneys were removed from 3-hr hypoxic animals, washed carefully, and perfused for 3 hr by recirculation with a serum-tissue culture mixture, each kidney generated about 14 U of erythropoietin in vitro. Perfusion of normal kidneys did not result in the production of erythropoietin, and only small amounts were generated if the perfusate contained Puromycin. Three-hour hypoxic kidneys perfused for 3 hr with a serum-free tissue culture medium were found to generate about 8 U of erythropoietin per kidney and similar kidneys perfused with saline about 1 U. These results indicate that erythropoietin is synthesized by kidney tissue and not produced by enzymatic activation of a plasma substrate.

scholarly journals Effect of In-Vitro Alpha Lipoic Acid Addition on Spermatozoa Motility in Sperm Preparation Process

2021 ◽  
Vol 55 (4) ◽  
pp. 246
Author(s):  
Gede Wira Buanayuda ◽  
Hamdani Lunardhi ◽  
Indra Gusti Mansur
Keyword(s):  
Lipoic Acid ◽  
Intrauterine Insemination ◽  
Control Group ◽  
Infertile Couple ◽  
Preparation Process ◽  
Alpha Lipoic Acid ◽  
Sperm Preparation ◽  
Progressive Motility ◽  
Child Hospital

Infertility is a problem for husband and wife, in the last 20 years the number of infertile couples has tended to increase by around 6.5 million pairs. The infertile couple can use the intrauterine insemination method to obtain offspring if a conventional method approach cannot be performed. Insemination requires a sperm preparation stage in which there are centrifugation and resuspension procedures that tend to produce excess reactive oxygen species (ROS). Excessive ROS will damage the motility of the spermatozoa. This study aims to prove the addition of alpha lipoic acid (ALA) as an antioxidant in the process of sperm preparation to improve and maintain better sperm motility. This research is a laboratory study with an experimental research design. The sample consisted of 10 infertile men who visited the Andrology section of the Sayyidah Jakarta Mother and Child Hospital (RSIA), where each ejaculate from the patient would be divided into 3 groups namely (k1) fresh semen as a control group, (k2) sperm preparation group without ALA, (k3) group of sperm preparation with the addition of ALA. The motility of spermatozoa was observed with the WHO 1999 method for 4 hours in units of percent. Progressive motility in k3 (47.95 ± 3.617) was higher than in k2 (38.05 ± 3.278) statistically significantly different after 3 hours of observation (p<0.0001). Progressive motility in k3 (78.8 ± 5.841) was higher than k1 (56.55 ± 7.511) from the initial observation (p <0.0001). The progressive motility of k2 (76.05 ± 6.768) was higher than k1 (56.55 ± 7.511) from the start of the observation (0.0001). It can be concluded that the addition of ALA in the sperm preparation process increases and maintains progressive motility that is better than sperm preparation without ALA addition after 3 hours of observation.

scholarly journals Experimental conversion of virulent RH Toxoplasma gondii tachyzoites in vitro

2001 ◽  
Vol 7 (1-2) ◽  
pp. 181-188
Author(s):  
N. A. Hammouda ◽  
I. R. Ibrahim ◽  
E. D. Elkerdany ◽  
A. Y. Negm ◽  
S. R. Allam
Keyword(s):  
Toxoplasma Gondii ◽  
Culture Medium ◽  
Dna Analysis ◽  
Morphological Changes ◽  
Control Group ◽  
Image Analyser

We aimed to induce conversion of RH-stain tachyzoites to bradyzoites by changing the pH of the culture medium. Alkalization of the medium to pH 8 induced morphological changes in the cultured tachyzoites. The majority of the organism increased in size and changed from a regular crescent shape to a rounded or ovoid shape. Cyst-like structures were formed. Using a computerized image analyser, significant differences in the size of the whole organisms and in their nuclei were observed compared to the control group. The converted organisms also showed significant differences from the control group by quantitative DNA analysis, and did not infect mice.

scholarly journals Indirect Cold Atmospheric Pressure Plasma treatment on Melanoma and Fibroblast Cells

2020 ◽  
Vol 10 (2) ◽  
pp. 2117-2125
Keyword(s):  
Plasma Treatment ◽  
Cancer Cells ◽  
Culture Medium ◽  
Atmospheric Pressure Plasma ◽  
Control Group ◽  
Fibroblast Cells ◽  
Alizarin Red ◽  
Migration Assay ◽  
Melanoma Cancer

Cold atmosphere plasma has been shown as a promising technology for certain cancer treatments. In this paper, we report indirect plasma treatment using CAP discharged in cell culture medium and study the effect of identical plasma stimulated culture medium on melanoma cancer cells and fibroblast cells cultured in vitro. The results of MTT assay, migration assay, ROS detection, and alizarin red assay show that plasma-treated medium can have a strong negative effect on melanoma cancer cells compared with the control group. However, the plasma-treated medium has a less cytotoxic effect on fibroblast cells than that on melanoma cancer cells at the same treatment. This result is attributed to the production of reactive oxygen species in the plasma-treated medium to induce apoptosis and inhibit melanoma cell proliferation and further cell metastasis. According to the results, this study shows the potential of CAP plasma treatment for anti-cancer therapy.

Sign in / Sign up

Export Citation Format

Share Document