scholarly journals 101 OVARIAN RESPONSE AND DEVELOPMENTAL COMPETENCE OF OOCYTES COLLECTED BY OPU IN SHEEP TREATED WITH GnRH ANTAGONIST

2005 ◽  
Vol 17 (2) ◽  
pp. 201
Author(s):  
F. Berlinguer ◽  
A. Gonzalez-Bulnes ◽  
S. Succu ◽  
G. Leoni ◽  
I. Rosati ◽  
...  

The use of a single dose of GnRH antagonists during the progestagen treatment prior to superovulatory treatment protocols in sheep increases the number of smaller follicles able to grow and ovulate in response to the exogenous FSH treatment (Lopez-Alonso C et al. 2004 Reprod. Fertil. Dev. 16, 233). The aim of our study was to test if such treatment affects the in vitro developmental competence of oocytes collected by ovum pick up (OPU) from GnRH-antagonist treated sheep during an ovarian by perstimulation protocol. Adult Sarda sheep (n = 18) were synchronized by the insertion of intravaginal sponges (Day 0) which were left in situ for 12 days; on Day 7, group A (n = 10) received a single dose of 3 mg of Antarelix (Teverelix, Europeptides, France) s.c., while group B (n = 8) served as control. All animals received 96 IU of FSH (Ovagen, ICP, New Zealand) administered in 4 equal doses given i.m. every 12 h starting on Day 10. Twelve hours after the last FSH administration oocytes were collected by OPU technique. Follicular growth was monitored by transrectal ultrasonography from Day 7 to Day 11. Collected oocytes were matured, fertilized, and cultured in vitro up to blastocyst stage under standard conditions used in our laboratory (Berlinguer F et al. 2004 Theriogenology 61, 1477–1486). After IVF, uncleaved oocytes were stained with acetolacmoid to evaluate chromatin configuration, while the cleaved ones were cultured in SOF + 0.4% BSA up to the blastocyst stage. Data were analyzed by ANOVA statistical analysis after arcsine transformation of the value percentages. Ultrasonographic monitoring showed a significant increase in the number of follicles (mean ± SEM) present in the ovaries from Day 8 to Day 11 of treatment in group A compared to group B (Day 8: 19 ± 5.1 vs. 13 ± 3.4, P > 0.05; Day 9: 20.1 ± 4.6 vs. 14.1 ± 2.4, P > 0.001; Day 10: 22.5 ± 6.1 vs. 14.7 ± 2.7, P > 0.001; Day 11: 25.3 ± 5.1 vs. 20.5 ± 4.1, P > 0.05), thus confirming that GnRH antagonist administration enhances ovarian response to exogenous FSH stimulation. On the other hand, oocytes collected from untreated sheep lead to a higher blastocyst output (P = 0.014), as illustrated in the table. These results indicated that although GnRH antagonist administration caused a significant increase in the ovarian response to the hormonal treatment, the final blastocyst output was significantly lower compared to that of the control group. This finding seems to suggest an impairment in the developmental competence of treated sheep oocytes. Table 1. In vitro maturation, fertilization, and developmental capacity of oocytes collected from follicles of GnRH antagonist-treated (group A) and untreated (group B) sheep This work was supported by funds from the Spanish MEC (projects SC 00-051-C3.1 and HI2002-0004) and the Italian MIUR (cofin).

2007 ◽  
Vol 19 (1) ◽  
pp. 288
Author(s):  
C. Kubota ◽  
T. Kojima ◽  
T. Nagai ◽  
X. Tian ◽  
X. Yang

The timing of IVM–IVF–IVC is restricted by the onset of oocyte maturation, and sometimes oocytes must be treated at midnight. If we could regulate the timing of IVM of oocytes without decreasing their developmental competence, the IVM–IVF–IVC system could be a more applied technology. The present study was performed to examine the effects of in vitro storage of bovine oocytes in simple media prior to maturation culture to manipulate the start of IVM. Bovine follicular fluid (bFF), Dulbecco's PBS (PBS), M199 Earle salts (M199), and Earle salts supplemented with 5 mM NaHCO3 (M199A) were used as the fundamental media, after an addition of antibiotics, for in vitro storage of bovine cumulus–oocyte complexes (COCs) collected from ovaries obtained at the slaughterhouse. The fundamental media except for bFF were supplemented with 10% fetal bovine serum (FBS) or 1 mg mL−1 polyvinyl alcohol (PVA). COCs were collected from follicles (3–8 mm in diameter) and washed twice in each medium; then approximately 50 COCs were submerged in 1 mL of each medium in cryotubes (Falcon #2812, 2.5 mL; Becton Dickinson Labware, Lincoln, NJ, USA), which were stored in a container kept at 38.5°C for 22 h under air-closed condition (in vitro storage: IVS). Subsequently, the stored COCs were in vitro-matured (IVM) for 22 h in M199 with 10% FBS and 20 µg mL−1 estradiol, fertilized (IVF), and cultured in CR1aa (IVC) for examination of their development to the blastocyst stage (Kubota et al. 1998 Mol. Reprod. Dev. 51, 281–286). Fresh oocytes without IVS were used as controls. The nuclear status of oocytes after IVS–IVM was compared to that of control oocytes by aceto-orcein stain. Their developmental rates to the blastocyst stage after IVM–IVF–IVC were compared between experimental and control groups. The experiment was repeated more than 3 times, and results were statistically analyzed using Student's t-test. When bFF and PBS supplemented with FBS or PVA were used for IVS, the rates of survived COCs after IVS and the development to the blastocyst stage after IVM–IVF–IVC (bFF (n = 87): 0%, 0%; PBS/FBS (n = 72): 84%, 1%; and PBS/PVA (n = 81): 89%, 6%, respectively) were significantly lower than those of the control group (n = 406; 97% and 29%, respectively). On the other hand, when M199A supplemented with FBS or PVA was used for IVS, the survival rate after IVS and the developmental rate to the blastocyst stage after IVS–IVM–IVF (M199A/FBS (n = 97): 82%, 28%; and M199A/PVA (n = 111): 98%, 31%, respectively) did not differ from those of the control group. After IVS, cumulus expansion was not seen and most of the oocyte nuclei reached the GVBD stage. These results suggest that the nuclear maturation progress of bovine oocytes can be regulated for at least 22 h in M199A without any deleterious influence on the number of oocytes surviving at an immature state after the storage and their subsequent development to the blastocyst stage after IVM–IVF–IVC. The delayed maturation allows a flexible fertilization schedule which is advantageous in research and industrial applications.


2018 ◽  
Vol 30 (1) ◽  
pp. 189
Author(s):  
L. Landeo ◽  
R. S. Molina ◽  
M. E. Zuñiga ◽  
T. R. Gastelu ◽  
C. Sotacuro ◽  
...  

The objective of this study was to evaluate the in vitro developmental competence of alpaca embryos bisected at different embryonic stages. Gametes were obtained from ovaries and testes collected from a local abattoir. Cumulus-oocyte complexes (COC) were recovered (n = 120) by aspiration of ovarian follicles using a 5-mL syringe with an 18-gauge needle. Then, COC with at least 3 layers of cumulus cells and a homogeneous cytoplasm were matured in TCM-199 supplemented with 10% FCS, FSH (0.02 IU [JM1] [P2] [P3]), and 0.01 mg mL−1 oestradiol 17β [JM4] for 26 h at 38.5°C and 5% CO2 in air. After in vitro maturation, COC were placed in a 30-mL Petri dish containing FERT-TALP solution for 30 min. Then, epididymal alpaca spermatozoa (3 × 106 mL−1) were added to the dish and co-incubated with the COC for 20 h at 38.5°C and 5% CO2 in air. Motile epididymal sperm were selected by swim-up method centrifuged for 15 min at 350 × g in 2 mL of SPERM-TALP supplemented with 6 mg mL−1 of fatty-acid-free BSA. Sperm pellet was extended and culture in 5% CO2 in air at 38.5°C for 45 min. Thirty-three viable embryos at different stages [2-cells (n = 6), 8-cells (n = 15), and morulae (n = 12)] were bisected into approximately equal halves using a micro-surgical blade. The embryos were previously treated with 2 mg mL−1 of protease from Streptomyces griseus (P 8811, Sigma, St. Louis, MO, USA) for 2 min to remove the zona pellucida. After bisection, the demi-embryos were cultivated in in vitro culture (IVC) medium containing 0.036 mg mL−1 sodium pyruvate, 0.146 mg mL−1 l-glutamine, 1% essential amino acids, 0.5% nonessential amino acids, and supplemented with 10% FCS using the well-of-the-well system. The demi-embryos were incubated for 7 days (changing the media every 48 h) in 5% CO2 in air at 38.5°C. Additional embryos (n = 60) were obtained using the same conditions described above and used as a control group (unmanipulated). We obtained 66 demi-embryos [2-cells (n = 12), 8-cells (n = 30), and morulae (n = 24)] after bisection that were considered for IVC. From 12 demi-embryos bisected at 2-cell and 30 bisected at 8-cell stages, 3 (25%) and 30 (100%) reached the morula stage respectively. However, they did not develop any further. Interestingly, 18 demi-embryos bisected in morula reached the blastocyst stage (80%). For unmanipulated embryos, we obtained 42% (25/60), 35% (21/60), 32% (19/60), and 28% (17/60) of cleavage, morulae, and blastocyst and hatched blastocyst rates, respectively. In conclusion, alpaca embryos bisected at earlier stages (less than 8-cell) are not suitable to produce blastocysts. The earliest stage to produce blastocyst from bisected alpaca embryos is the morula stage.


Author(s):  
Jayendra R. Gohil ◽  
Vishal S. Rathod ◽  
Bhoomika D. Rathod

Objective: To study the effect and safety of Fenofibrate in uncomplicated hyperbilirubinemia in newborn with 6-month follow-up. Materials and Methods: This is a randomized controlled clinical trial conducted in 60 normal term neonates admitted for uncomplicated hyperbilirubinemia in NICU at Sir T G Hospital, Bhavnagar from January 2012 to December 2012. The data included: age, sex, total serum bilirubin (TSB), weight and duration of phototherapy. All neonates enrolled in the study received phototherapy. They were divided in two groups of 30 each: control group A and group B receiving Fenofibrate (100 mg/kg single dose). There was statistically insignificant difference between the parameters of age, sex, weight and TSB between the two groups at hospitalization. Data was analyzed by using appropriate statistical methods. Results: Mean values for total serum bilirubin in Fenofibrate group B at 24 and 48 hours after admission were significantly lower than those for control group A (p<0.0001,  p=0.0001). There was no significant difference in fall of TSB between 24 and 48 hours. The mean duration of phototherapy in Fenofibrate group (44.8h: 24-72h) was significantly shorter than that in control group (55.2 h: 24‐96 h) (P=0.02). There were no side effects of the drug observed during the study and during 6 months follow up period. Conclusion: Fenofibrate as a single 100 mg/kg dose in healthy full term neonates, is effective and a safe drug (till six-month follow-up) for neonatal hyperbilirubinemia, that can decrease the time needed for phototherapy and hence hospitalization. Effect of a single dose seems to wane after 24 hours.


2017 ◽  
Vol 8 (3) ◽  
pp. 83-89
Author(s):  
Johnny Holanda De Gauw ◽  
Lara Maria Melo Costa ◽  
Rodrigo Neves Silva ◽  
Natanael Barbosa Santos ◽  
Maria Dânia Holanda Tenorio

Objective: This study aimed to evaluate the effect of ferrous sulfate (FS) on demineralized and non-demineralized human deciduous teeth. Additionally, it was evaluated the penetration extent of FS and its remineralizing effect on the enamel of deciduous teeth using Polarized Light Microscopy (PLM). Method: The sample comprised 44 human deciduous teeth. The 44 crowns were divided randomly into four groups: group A (FS after demineralization), group B (FS without demineralization), group C (only demineralization), and group D (control group). FS at 0.45 mol/L-1 was used daily (15 days) and demineralization was done by pH cycling (7 days). Then, three longitudinal slices of the crowns were photographed using PLM. The degree of penetration of the lesion or stain was measured in micrometers, as well as the distance between the external enamel surface and the core of lesion. Results: Group A showed a dark stain on the outer surface of enamel larger than the group B. It is suggested, a remineralizing effect when comparing groups, A and C. The mean depth and standard deviation for groups A, B, and C were 4.27µm (±1.49), 3.72 µm (±1.68) and 5.00 µm (±1.84), respectively. No dark stains were observed in group D. Conclusion: FS stained the demineralized and non-demineralized human deciduous teeth. However, dark stains in the non-demineralized teeth were smaller or absent, than in the demineralized teeth. Therefore, FS may have a protective effect against demineralization.


2004 ◽  
Vol 16 (2) ◽  
pp. 231 ◽  
Author(s):  
A. Gonzalez-Bulnes ◽  
J. Santiago Moreno ◽  
R.M. Garcia-Garcia ◽  
C.J.H. Souza ◽  
A. Lopez-Sebastian ◽  
...  

In goats, as in other mammals, the use of treatment with GnRH antagonists (GnRHa) inhibits gonadotrophin secretion, causing a suppression of the growth of large ovarian follicles. Thus, GnRHa treatment could be useful to decrease the effects of dominant follicles prior to ovarian stimulation, increasing the number of gonadotrophin-responsive follicles at the start of FSH treatments and improving the ovarian response in terms of transferable embryos. However, in goats, the beneficial effects of this treatment is annulled by a high number of unfertilised ova and degenerated embryos (2003, Cognie et al., Theriogenology 59, 171–188), which suggests deficiencies in oocyte developmental competence per se or induced by endocrine or follicular alterations during the peri-ovulatory period. We have tested whether these failures can be related to a prolongation of gonadotrophin down-regulation and/or alterations in follicular function after cessation of the antagonist, during the period of administration of the superovulatory treatment, around 4 days after the end of GnRHa treatment. A total of 15 does received 45-mg FGA intravaginal sponges (Chronogest®, Intervet Int, H), the first group of 10 females were treated with daily injections of 0.5mg of the GnRHa Teverelix (Antarelix™, Zentaris, G) for 6 days from Day 5 of sponge insertion, while five does acted as controls receiving saline. Endocrine and ovarian function were monitored daily from Day −5 to Day 4 (Day 0=day of last GnRHa injection). Pituitary activity was determined by measuring plasma FSH and LH, and follicular activity by ultrasonographic monitoring of all &gt;2mm follicles and by assessing plasma inhibin A levels. During GnRH antagonist treatment, the mean plasma LH concentration was lower in treated than control goats (0.5±0.2 v. 0.7±0.5ng/mL, P&lt;0.0005); however, the FSH levels remained unaffected (0.8±0.4 v. 0.8±0.5ng/mL). In this period, treated does also showed an increase in the number of small follicles 2–3mm in size (10.7±0.7 v. 8.4±0.6, P&lt;0.05), and a decrease in both the number of follicles &gt;4mm in size (5.0±0.3 v. 6.8±0.5, P&lt;0.005) and the secretion of inhibin A (120.9±10.7 v. 151.6±12.6pg/mL, P&lt;0.05). After GnRHa treatment, LH levels increased in treated goats from the day after the last Teverelix injection (Day 1), so that LH levels were the same as controls on Day 3 (0.6±0.1 v. 0.6±0.2ng/mL). However, there were even greater numbers of small follicles than during the period of GnRHa treatment (15.4±0.6 in treated v. 8.9±0.7 in control, P&lt;0.0005). Moreover, the number of follicles &gt;4mm in size and the secretion of inhibin A remained lower in treated goats (3.9±0.3 follicles and 84.4±7.0pg/mL v. 5.4±0.5 follicles, P&lt;0.05 and 128.9±14.2pg/mL, P&lt;0.05). These results indicate that pituitary secretion of gonadotrophins is restored shortly after the end of GnRHa treatment, but the number of follicles and the secretion of inhibin A are affected. This may be relevant to the failures in ovulation and/or fertilization reported for superovulatory protocols with GnRHa pre-treatments in goats.


Author(s):  
Hassan Mohammadipour Anvari ◽  
Maarouf Ansari Kazaj ◽  
Khosro Kolahdouzan ◽  
Nasser Ghobanian ◽  
Afsaneh Khobeydeh

Background: Sore throat is one of the major complications of tracheal intubation after general anesthesia. Pregabalin is an analgesic, the anti neuropathic pain and analgesic effects of which have been demonstrated in various studies. This study examined the effects of single dose pregabalin one hour before tracheal intubation, to prevent sore throat after extubation. Methods: In a double-blind, randomized clinical trial, 60 patients who had undergone general and urologic surgeries at Imam Reza hospital in Tabriz, Iran, since March to July 2015 that required tracheal intubation, were included in the study. The patients were randomly divided into two groups (group A, 30 patients and group B, 30 patients). In the group A, an hour before anesthesia, one pregabalin tablet (300mg) was given to the patients. For the patients of the group B, the placebo was given. After awareness of patients, the severity of sore throat was measured and recorded by VAS scale after 2, 6 and 24 hours of the surgery. Results: Severity and incidence of sore throat after tracheal intubation were not significantly different between two groups. Meanwhile, no side effects of pregabalin were observed in the group A. Conclusion: Administration of pregabalin as a single dose of 300 mg one hour prior to anesthesia and intubation decreased the incidence and severity of sore throat in the case group than the control group, although the amount of this reduction was not statistically significant between the two groups.


2007 ◽  
Vol 19 (1) ◽  
pp. 184 ◽  
Author(s):  
T. Somfai ◽  
M. Ozawa ◽  
J. Noguchi ◽  
H. Kaneko ◽  
K. Ohnuma ◽  
...  

The present study investigated the ability of in vitro-matured (IVM) porcine oocytes to be fertilized in vitro after vitrification. Oocytes matured in vitro for 46 h according to Kikuchi et al. (2002 Biol. Reprod. 66, 1033–1041) were cryopreserved by solid surface vitrification (SSV; Dinnyes et al. 2000 Biol. Reprod. 63, 513–518) or subjected to the steps of SSV without cooling (toxicity control, TC). Oocyte viability was assessed 2 h after treatment by morphology and fluorescein diacetate staining. Live oocytes were in vitro-fertilized (IVF) and cultured (IVC) for 6 days according to Kikuchi et al. (2002). Fertilization and pronuclear development of oocytes were assessed 10 h after IVF by aceto-orcein staining. Cleavage and blastocyst rates were recorded during IVC. Glutathione (GSH) and hydrogen peroxide levels in oocytes were analyzed by DTNB-glutathione disulfide reductase recycling assay and 20,70-dichlorofluorescein fluorescence assay, respectively. Data were analyzed by ANOVA and paired t-test. The rate of live oocytes after SSV was lower compared to the control and the TC groups (54.4%, 100%, and 100%, respectively; P &lt; 0.05). Sperm penetration rates of SSV oocytes were lower than those of the control group (51.9% and 67.8%, respectively; P &lt; 0.05). Significantly fewer penetrated oocytes in the SSV group formed male pronuclei than those in the control and the TC groups (66.7%, 96.5%, and 98.5%, respectively; P &lt; 0.05). There were no differences in second polar body extrusion and monospermy rates between the treatment groups. The cleavage rate of SSV oocytes was significantly lower than that of the control and the TC groups (13.3%, 46.6%, and 47.7%, respectively; P &lt; 0.05). Blastocyst rates of control and TC oocytes were similar (20.7% and 23.6%, respectively), whereas only a single embryo developed to the blastocyst stage in the SSV group. GSH content of SSV oocytes was significantly lower than that of the control oocytes (7.3 pM and 10.5 pM, respectively), whereas the peroxide level was higher in SSV oocytes than in the control oocytes (59.0 and 50.5 FIU, respectively; P &lt; 0.05). Our results reveal a cryopreservation-related drop of intracellular GSH level in oocytes, which may cause their decreased ability to form a male pronucleus and their increased sensitivity to oxidative stress. These factors might contribute to the low developmental competence of vitrified oocytes. This work was supported by a grant-in-aid for the Japanese Society for the Promotion of Science Postdoctoral Fellowship for Foreign Researchers (P05648) and the Bilateral Scientific and Technological Collaboration Grant between Hungary and Japan (TET, no. JAP-11/02).


2019 ◽  
Vol 9 (9) ◽  
pp. 1286-1291
Author(s):  
YiPing Tian ◽  
XiaoLing Guo ◽  
RuiJun Cai ◽  
LanXia Li

The in vitro temperature experiment was used to determine the magnetic field strength, and the magnetic bodies were isolated and purified as carriers. In the previous study, pHSP-PLk1-siRNA/DOX complexes were constructed to target loose drugs. The subjects were arranged into 4 groups, namely magnetosome+drug group (group A), magnetosome+ blank control (group B), magnetosome+drug+magnetic field (group C), magnetosome+blank control+magnetic field (D) Group); co-incubated with human osteosarcoma cell line U2OS. The uptake rate and cell morphology of osteosarcoma cells were observed by laser confocal microscopy at 12, 24, 48 and 72 h. The cell cycle was observed by the aid of flow cytometry. The cells were detected by RT-PCR and Western blot. PLk1 mRNA and expression of protein levels, MTT assay for cell proliferation, adhesion assay and Transwell chamber for cell adhesion and invasion, and apoptosis kit for apoptosis.


2015 ◽  
Vol 2015 ◽  
pp. 1-7 ◽  
Author(s):  
Zhe Wang ◽  
Li Wang ◽  
Meng-ming Xia ◽  
Wei Sun ◽  
Cheng-ke Huang ◽  
...  

The objective of this work was to investigate the effect of orally administered genistein on the pharmacokinetics of imatinib and N-desmethyl imatinib in rats. Twenty-five healthy male SD (Sprague-Dawley) rats were randomly divided into five groups: A group (control group), B group (multiple dose of 100 mg/kg genistein for consecutive 15 days), C group (multiple dose of 50 mg/kg genistein for consecutive 15 days), D group (a single dose of 100 mg/kg genistein), and E group (a single dose of 50 mg/kg genistein). A single dose of imatinib is administered orally 30 min after administration of genistein (100 mg/kg or 50 mg/kg). The pharmacokinetic parameters of imatinib and N-desmethyl imatinib were calculated by DAS 3.0 software. The multiple dose of 100 mg/kg or 50 mg/kg genistein significantly (P<0.05) decreased theAUC0-tandCmaxof imatinib.AUC0-tand theCmaxof N-desmethyl imatinib were also increased, but without any significant difference. However, the single dose of 100 mg/kg or 50 mg/kg genistein has no effect on the pharmacokinetics of imatinib and N-desmethyl imatinib. Those results indicated that multiple dose of genistein (100 mg/kg or 50 mg/kg) induces the metabolism of imatinib, while single dose of genistein has no effect.


2013 ◽  
Vol 2013 ◽  
pp. 1-6 ◽  
Author(s):  
Xian-yun Liu ◽  
Tao Xu ◽  
Wan-shu Li ◽  
Jun Luo ◽  
Pei-wu Geng ◽  
...  

The purpose of this study was to determine the effect of apigenin on the pharmacokinetics of imatinib and N-desmethyl imatinib in rats. Healthy male SD rats were randomly divided into four groups: A group (the control group), B group (the long-term administration of 165 mg/kg apigenin for 15 days), C group (a single dose of 165 mg/kg apigenin), and D group (a single dose of 252 mg/kg apigenin). The serum concentrations of imatinib and N-desmethyl imatinib were measured by HPLC, and pharmacokinetic parameters were calculated using DAS 3.0 software. The parameters ofAUC(0-t),AUC(0−∞),Tmax,Vz/F, andCLz/Ffor imatinib in group B were different from those in group A (P<0.05). Besides,MRT(0−t)andMRT(0−∞)in groups C and D differed distinctly from those in group A as well. The parameters ofAUC(0-t)andCmaxfor N-desmethyl imatinib in group C were significantly lower than those in group A (P<0.05); however, compared with groups B and D, the magnitude of effect was modest. Those results indicated that apigenin in the short-term study inhibited the metabolism of imatinib and its metabolite N-desmethyl imatinib, while in the long-term study the metabolism could be accelerated.


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