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1.
Antioxidants have been widely used during in vitro production to decrease the negative effect of reactive oxygen species. It was reported that the complex resveratrol–methyl β‐cyclodextrin (RV ‐CD ) improves resveratrol's stability and bioavailability and increases its antioxidant activity. This study evaluates the effect of RV ‐CD during in vitro oocyte maturation (IVM ) or in vitro embryo culture (IVC ) on developmental competence and quantitative changes in gene expression of developmental important genes. In experiment 1, RV ‐CD was added to IVM media and maturation level, embryo development and oocytes, cumulus cells, and blastocysts gene expression by RT ‐qPCR were examined. In experiment 2, presumptive zygotes were cultured in SOF supplemented with RV ‐CD and embryo development and blastocysts gene expression by RT ‐qPCR were studied. A group without RV ‐CD (control?) and a group with cyclodextrin (control+) were included. No differences were found in cleavage rate or blastocyst yield between groups. However, the expression of LIPE was higher in blastocysts derived from oocytes treated with resveratrol compared with control groups (p  <  .05). Blastocysts produced by IVC with resveratrol showed that RV ‐CD could modify the expression of genes related to lipid metabolism (CYP 51A1 , PNPLA 2 and MTORC 1 ) compared with control groups (p  < .05). RV ‐CD in the IVM and IVC media could reduce accumulated fat by increasing lipolysis and suppressing lipogenesis of blastocysts.  相似文献   

2.
Paraoxonase‐1 (PON1) is an enzyme found in serum and follicular fluid that protects cell membrane and circulating lipids against oxidative damage. The aims of this study were to measure the direct effects of recombinant PON1 (rPON1) on bovine oocyte maturation at the molecular level (gene expression) and to measure the carry‐over effects of PON1 on pre‐implantation embryo development in vitro. COCs were submitted to IVM with the addition of 0.0, 0.02, 0.04 and 0.08 mg ml?1 of rPON1, corresponding to an average PON1 arylesterase enzyme activity of 2.2 ± 0.4, 15.5 ± 1.5, 30.2 ± 3.0 and 57.9 ± 5.0 U ml?1, respectively. The results indicated that addition of rPON1 during IVM improved embryo development in a dose‐dependent manner as D7 embryo development was 22.2%, 29.4%, 32.2% and 37.0% for the treatment groups, respectively (p = 0.02). In conclusion, addition of PON1 enzyme during IVM exerted dose‐related positive effects on embryo development rates to blastocysts.  相似文献   

3.
Oocyte maturation depends on the metabolic activity of cumulus–oocyte complex (COC) that performs nutritive and regulatory functions during this process. In this work, the enzymes [phosphofructokinase (PFK) and malate dehydrogenase (MDH)] were tested to elucidate the metabolic profile of porcine COCs during the in vitro maturation (IVM). Enzymatic activity was expressed in U/COC and U/mg protein (specific activity) as mean ± SEM. In vitro maturation was performed with 2‐oxoglutarate (5, 10 and 20 mm ) or hydroxymalonate (30, 60 and 100 mm ) inhibitors of PFK and MDH, respectively. The PFK and MDH activities (U) remained constant during maturation. For PFK, the U were (2.48 ± 0.23) 10?5 and (2.54 ± 0.32) 10?5, and for MDH, the U were (4.72 ± 0.42) 10?5 and (4.38 ± 0.25) 10?5 for immature and in vitro matured COCs, respectively. The specific activities were significantly lower after IVM, for PFK (4.29 ± 0.48) 10?3 and (0.94 ± 0.12) 10?3, and for MDH (9.08 ± 0.93) 10?3 and (1.89 ± 0.10) 10?3 for immature and in vitro matured COCs, respectively. In vitro maturation percentages and enzymatic activity diminished with 20 mm 2‐oxoglutarate or 60 mm hydroxymalonate (p < 0.05). Viability was not affected by any concentration of the inhibitors evaluated. The U remained unchanged during IVM; however, the increase in the total protein content per COC provoked a decrease in the specific activity of both enzymes. Phosphofructokinase and MDH necessary for oocyte IVM would be already present in the immature oocyte. The presence of inhibitors of these enzymes impairs the meiotic maturation. Therefore, the participation of these enzymes in the energy metabolism of the porcine oocyte during IVM is confirmed in this study.  相似文献   

4.
Melatonin may play an important role in protecting gametes and embryos from the potential harmful effects of oxidative stress. In this study, we first examined two different heat stress (HS) treatments for in vitro oocyte maturation (Experiment 1: 38.5 vs 41.0°C, during the first 20 h; Experiment 2: 38.5 vs 41.5°C, during the entire period) on bovine oocyte maturation and embryo development. Second, we tested different melatonin concentrations added to the maturation and culture medium (Experiment 3: 0, 10?12, 10?9, 10?4 m ; Experiment 4: 0, 10?3 m ), both with and without HS (38.5 or 41.5°C, respectively). In Experiment 1, the HS treatment resulted in a lower maturation rate and number of cells/blastocyst (C/B) and a higher blastocyst rate than that in the control group. In Experiment 2, oocytes/embryos from heat‐stressed oocytes (HSO) had a lower maturation, cleavage and blastocyst rates, as well as a lower C/B compared with the control. In Experiment 3, in HSO groups, 10?4 m melatonin resulted in an increased blastocyst rate compared with 0 m melatonin, with a similar blastocyst rate to the non‐HSO without melatonin. Melatonin did not have any effect in embryos from non‐HSO groups compared with the control. In Experiment 4, 10?3 m melatonin produced lower cleavage and blastocyst rates in HSO and lower blastocyst rate in non‐HSO when compared to melatonin‐untreated oocytes/embryos. In conclusion, 10?4 m melatonin was found to alleviate bovine oocytes from the harmful effects of HS.  相似文献   

5.
This work was designed to evaluate the ovarian follicular development, oocytes morphology, methods of oocytes reterival, and the effect of different in vitro maturation (IVM) media on cumulus cell expansion and nuclear maturation of Jennies oocytes. Experiment 1, the number of small (<6 mm), medium (6 to 9 mm) and large size (>10 mm) ovarian follicles was recorded. Cumulus-oocyte-complexes (COCs) were reterived and classified into 4 Grades based on their cumulus-cells investment and the homogenous of the ooplasm. In Experiment 2, COCs were recovered by using 18-G, 20-G needle or slicing and scraping of ovarian follicles to determine the number and morphology of the recovered COCs. In Experiment 3, Grade A and B COCs were IVM in DMEM-HG, DMEM-LG, DMEM-F12, TCM199, TCM199-F12 or CR1aa media supplemented with 10 % FCS?+?10 μg FSH/mL?+?10 IU hCG/mL?+?50 μg/mL gentamicin. Maturation was performed for 36 h at 38.5 °C under 5 % CO2 in humidified air. After IVM, cumulus cell expansion and oocytes nuclear canfiguration were determined. An average of 6.40?±?0.26 follicles was recorded per Jenny ovary, representing 3.37?±?0.46, 1.89?±?0.14 and 1.14?±?0.16, for the small, medium and large size follicles, respectively. Oocyte recovery was higher (P?P?P?P?P?P?P?P?Conclusion: Slicing and scraping or aspiration of follicles using 18-G needle increased the number and percentage of Grade A Jennies oocytes. TCM199-F12, CR1aa and TCM199 medi are more suitable for IVM of Jenny oocytes by promoting cumulus cells expansion and nuclear maturation to M II stage.  相似文献   

6.
The purposes of the present study were to examine the effect of naloxone, a mu‐opioid receptor (MOR) antagonist, on porcine oocyte maturation and embryo development. MOR gene was expressed in germinal vesicle (GV) and metaphase II (M‐II) porcine oocytes, one‐, four‐cell stage embryos and blastocysts. In blastocysts, MOR gene was mainly expressed in inner cell mass (ICM) cells. Supplementation of 10?8 mol/L naloxone in in vitro maturation (IVM) medium increased the maturation rate (P < 0.05). However, 10?4 mol/L naloxone reduced the maturation rate (P < 0.05) compared with the control. The presence of naloxone during IVM had no effects on fertilization status and subsequent embryonic development after in vitro culture (IVC). The addition of 10?3 mol/L dibutyryl cyclic adenosine monophosphate (dbcAMP), and 10?8 mol/L naloxone together into IVM medium increased nuclear maturation (P < 0.05) compared with the addition of either dbcAMP or naloxone alone. Supplementation with naloxone in IVC medium did not improve embryonic development. However, at the concentrations of 10?6 mol/L and 10?8 mol/L, naloxone increased the ratio of ICM to total cells in blastocysts (P < 0.05). In conclusion, at low concentration, naloxone increases maturation rate and the ratio of ICM to total cells in blastocysts. Naloxone and cAMP have a synergistic effect on oocyte maturation.  相似文献   

7.
Ascorbic acid (AC) used as antioxidant in embryo culture is very sensitive and degrades unavoidably in aqueous solution. Methyl‐β‐cyclodextrin (CD) improved the stability of AC in solution to elevated temperature, light, humidity and oxidation. The aim of this study was to evaluate the effect of the complex AC‐CD during in vitro maturation (IVM) or in vitro culture (IVC) on oocyte developmental competence and subsequent embryo development and quality. AC‐CD (100 µM) was added to IVM media, and maturation level and embryo development were examined. Matured oocytes, their cumulus cells and produced blastocysts were snap‐frozen for gene expression analysis by RT‐qPCR. Besides, in vitro‐produced zygotes were cultured with 100 µM of AC‐CD and blastocysts were as well snap‐frozen for gene expression analysis. A group without AC‐CD (control?) and other with CD (control+) were included. No differences were found on maturation, cleavage or blastocyst rates. However, in matured oocytes, AC‐CD downregulated BAX, GPX1 and BMP15. In cumulus cells, AC‐CD downregulated BAX/BCL2 and GSTA4 while upregulated BCL2 and CYP51A1. The expression of SL2A1, FADS1, PNPLA and MTORC1 was downregulated in blastocysts derived from oocytes matured with AC‐CD, while in blastocysts derived from zygote cultured with AC‐CD, CYP51A1 and IGF2R were downregulated and PNPLA2 was upregulated. In conclusion, AC‐CD in both IVM and IVC media may reduce accumulated fat by increasing lipolysis and suppressing lipogenesis in blastocysts derived from both oocytes and zygotes cultured with AC‐CD, suggesting that CD improves the quality of embryos and bioavailability of AC during IVM and IVC.  相似文献   

8.
The objectives of the present study were to determine ionic and organic composition of seminal plasma, sperm concentration and their relationships in the Persian sturgeon (Acipenser persicus). In this regard, ionic content (Na+, K+, Cl?, Ca2+ and Mg2+) and organic content (total protein, glucose, cholesterol and triglyceride) along with sperm concentration were measured in 17 specimens of the Persian sturgeon. The seminal plasma contained 59.53 ± 2.56 mm /l sodium, 9.1 ± 1.42 mm chloride, 4.72 ± 0.3 mm potassium, 1.45 ± 0.075 mm calcium and 0.7 ± 0.072 mm magnesium. The following organic contents were found: total protein 0.11 ± 0.02 g/dl, glucose 22.18 ± 4.16 mg/dl, cholesterol 6.67 ± 1.04 mg/dl and triglyceride 15.2 ± 0.65 mg/dl. The mean sperm concentration was estimated to be 1.6 ± 0.12 (×109 sperm/ml). A significant relationship was found between sperm concentration and K+ of seminal plasma (r = 0.533, p < 0.05). Significant correlations were observed between ionic contents: Na+ vs Cl? (r = ?0.854, p < 0.01) and Mg2+ vs K+ (?0.583, p < 0.05). Also, level of triglyceride was negatively correlated with Mg2+ (r = ?0.503, p < 0.05). Presented data could be considered as a complementary study for developing special extenders and protectant solutions for improving artificial fertilization in this valuable species.  相似文献   

9.
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10.
Melatonin enhances in vitro embryo development in several species by improving the oocyte developmental competence during in vitro maturation (IVM). Melatonin has a wide range of actions, from scavenging reactive oxygen species (ROS) to regulating gene expression, and it can also act by way of melatonin receptors. The aim of this study was to determine the mechanism of action of melatonin during the IVM of juvenile goat oocytes and the role of the membrane receptors. Melatonin receptor 1 was immunolocalized in cumulus cells and oocytes before and after 24 hr of IVM. The effect of melatonin on oocyte developmental competence was tested in three experimental IVM groups: (a) control, (b) 10?7 M melatonin, and (c) 10?7 M melatonin +10?7 M luzindole (an inhibitor of both melatonin receptors). After IVM oocytes were assessed for ROS levels, mitochondrial activity, adenosine 5′‐triphosphate (ATP) concentration and relative gene expression (ACTB, SLC1A1, SOD1, GPx1, BAX, DNMT1, GCLC and GDF9). IVM‐oocytes were in vitro fertilized and cultured under conventional conditions. Blastocyst rate and quality (differential cell count) were assessed at 8 days post‐fertilization. Melatonin decreased ROS levels, increased mitochondrial activity and ATP content and increased blastocyst quality compared to control group (55.8 vs. 30.4 inner cell mass ICM, p < 0.05). There was no effect on the relative gene expression due to treatment with melatonin. In conclusion, we have showed that melatonin improves oocyte developmental competence in juvenile goats by reducing ROS levels and improving mitochondrial activity.  相似文献   

11.
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12.
The quality of porcine blastocysts produced in vitro is poor in comparison with those that develop in vivo. We examined the quality of in vitro‐matured and fertilized (IVM/IVF) oocytes, their abilities to develop to blastocysts under in vivo and in vitro conditions, and the potential of the embryos to develop to term after transfer. IVM/IVF oocytes were either transferred and the embryos recovered on Days 5 and 6 (100% and 87.5%, respectively) (‘ET‐vivo’ embryos), or cultured in vitro for 5 or 6 days (‘IVC’ embryos). The proportion of blastocysts differed significantly between the two groups on Day 5 (20.6% and 8.0%, respectively), but not on Day 6 (23.8% and 21.2%, respectively). The mean number of cells in ET‐vivo blastocysts on Days 5 or 6 was significantly higher (72.8 and 78.7, respectively) than that in IVC blastocysts (22.1 and 39.7, respectively). When IVM/IVF oocytes and IVC blastocysts on Day 6 were transferred, all (three and three, respectively) developed to piglets (16 and 16, respectively), without any difference in the rates of development to term (2.1% and 2.6%, respectively). These data suggest that, although blastocyst production differs between the two culture conditions, IVM/IVF oocytes possess the same ability to develop to term.  相似文献   

13.
The comparative pharmacokinetics of ivermectin (IVM), between healthy and in Escherichia coli lipopolysaccharides (LPS) injected sheep, was investigated after an intravenous (IV) administration of a single dose of 0.2 mg/kg. Ten Suffolk Down sheep, 55 ± 3.3 kg, were distributed in two experimental groups: Group 1 (LPS): treated with three doses of 1 μg LPS/kg bw at ?24, ?16, and ?0.75 hr before IVM; group 2 (Control): treated with saline solution (SS). An IV dose of 0.2 mg IVM/kg was administered 45 min after the last injection of LPS or SS. Plasma concentrations of IVM were determined by liquid chromatography. Pharmacokinetic parameters were calculated based on non‐compartmental modeling. In healthy sheep, the values of the pharmacokinetic parameters were as follows: elimination half‐life (2.85 days), mean residence time (MRT) (2.27 days), area under the plasma concentration curve over time (AUC, 117.4 ng day?1 ml?1), volume of distribution (875.6 ml/kg), and clearance (187.1 ml/day). No statistically significant differences were observed when compared with the results obtained from the group of sheep treated with LPS. It is concluded that the acute inflammatory response (AIR) induced by the intravenous administration of E. coli LPS in adult sheep produced no changes in plasma concentrations or in the pharmacokinetic behavior of IVM, when it is administered intravenously at therapeutic doses.  相似文献   

14.
Comparative studies of ionic composition, osmolality, protein concentration and pH of seminal plasma along with spermatozoa concentrations were carried out in stellate sturgeon, Acipenser stellatus, and Russian sturgeon, Acipenser gueldenstaedtii. Analysis of A. gueldenstaedtii sperm showed significantly higher concentrations of Na+ (34.58 ± 4.61 mm ), Ca2+ (0.35 ± 0.12 mm ), Mg2+ (0.70 ± 0.25 mm ), Cl? (13.50 ± 4.04 mm ) and proteins (0.60 ± 0.29 mg/ml) in the seminal plasma than did seminal plasma of A. stellatus: Na+ (20.08 ± 10.75 mm ), Ca2+ (0.28 ± 0.06 mm ), Mg2+ (0.29 ± 0.05 mm ), Cl? (7.50 ± 3.00 mm ) and 0.30 ± 0.11 mg/ml proteins. Significantly higher concentration of K+ (5.42 ± 1.06 mm ) was observed in A. stellatus compared to A. gueldenstaedtii K+ (2.29 ± 0.50 mm ). Concentration of Na+ was positively correlated with osmolality (r = 0.819), levels of Cl? (r = 0.922) and Mg2+ (r = 0.727) and pH (r = 0.848). The concentration of Mg2+ was positively correlated with protein concentration (r = 0.774), Na+ (r = 0.727), Cl? (r = 0.872) and Ca2+ (r = 0.801). A positive relationship was also found between concentration of K+ and spermatozoa concentration (r = 0.709). Results revealed strong inter‐species differences in several parameters. The data should be useful for artificial fertilization and for cryopreservation of sturgeon sperm.  相似文献   

15.
Melatonin, the major secretory product of the pineal gland, scavenges a variety of reactive oxygen and nitrogen species in vivo and in vitro, indicating that melatonin is a potent function as an antioxidant. The objective of this study was to investigate the effect of melatonin in the presence or absence of hydrogen peroxide (H2O2) on sperm characteristics (motility, viability, survival rate, membrane integrity, lipid peroxidation (LPO) and mitochondria activity) and also to examine the developmental rates to the blastocysts stage of porcine oocytes fertilized in vitro with semen treated with or without melatonin (100 nm ) in the presence or absence of H2O2 (250 μm ). The sperm were treated with melatonin in the presence or absence of H2O2 for 3, 6, 9 and 12 h at 37°C and then analysed for the sperm characteristics. The porcine embryos were produced by in vitro maturation and in vitro fertilization (IVM/IVF) using semen treated with or without melatonin (100 nm ) in the presence or absence of H2O2 (250 μm ) for 6 h. The semen characteristics, including motility, viability, survival rate, membrane integrity and mitochondria activity, were higher in the groups that were treated with melatonin in comparison to other groups, irrespective of incubation periods. Malondialdehyde levels in control, melatonin and melatonin + H2O2 groups were lower than H2O2 only group. A positive correlation was shown among motility, viability, survival rate and membrane integrity, but a negative correlation was observed between LPO and the other evaluation methods. The developmental rates to blastocysts of IVM/IVF porcine oocytes fertilized by semen treated with melatonin were significantly increased compared with any other groups, with the cell number of blastocysts shown to have a similar trend to the developmental rates. These results demonstrate that melatonin can improve the semen characteristics during in vitro storage and support the developmental ability of IVM/IVF embryos in pigs.  相似文献   

16.
Ten gestations in six domestic shorthair cats (Europeans) were monitored daily during the foetal phase of gestation, from the 28th day after the first mating until parturition, using ultrasound with a 12.5‐MHz probe. The development of the various organs over this period was recorded. The diameters of the head (HD) and abdomen (AD) were measured. Skeletal calcification visible on ultrasound occurred in a defined order between the 34th and 40th day of gestation. During the last 30 days of gestation, there was a significant correlation between HD and days before parturition (DBP) (r2 = 0.99) and between AD and DBP (r2 = 0.98). The following equations were obtained: DBP = ?2.10*HD (mm) + 50.74; DBP = ?1.01*AD (mm) + 42.19. The confidence intervals were stable over the last 30 days of gestation. For the HD, the confidence interval was ±1 day in 53% of cases and ±2 days in 85% of cases. For the AD, the confidence interval was ±1 day in 45% of cases and ±2 days in 77% of cases. A table obtained by combining the HD and AD measurements made it possible to estimate the date of parturition within 2 days with a reliability of over 85%.  相似文献   

17.
The traditional stripping procedure for collecting fish semen is associated with the risk of urine contamination, which may significantly affect semen quality and quantity. The use of a catheter as an alternative method for semen collection may overcome this problem. Therefore, this study compared Caspian brown trout (Salmo trutta caspius) semen parameters (i.e. sperm density, seminal plasma osmolality, motility parameters of spermatozoa analysed using computer‐assisted sperm analysis and fertility) between the traditional stripping method and the use of a catheter. All parameter values of the semen collected with a catheter were significantly higher (p < .05; density = 7.67 ± 1.02 × 109 ml?1 and osmolality = 279.28 ± 32.84 mOsm kg?1) than those collected with stripping method (density = 4.85 ± 0.47 × 109 ml?1 and osmolality = 216.42 ± 20.75 mOsm kg?1). Semen collected with a catheter was characterized by higher spermatozoa motility compared with sperm collected via stripping. Similarly, the fertilization ability of sperm collected with a catheter was significantly greater (p < .05) than sperm collected with the traditional stripping method. In conclusion, collection of sperm with a catheter was shown to effectively reduce urine contamination and is therefore recommended for the collection of Caspian brown trout sperm.  相似文献   

18.
Blood and seminal plasma ionic parameters are essential for monitoring health status, detecting illnesses, fish stock conservation and development of artificial propagation methods via extender improvement. In this study, comparison of blood and seminal plasma ionic parameters in beluga, Huso huso (30–45 kg, 1–2 m, n = 10), was made. The results obtained show that Na+ (82.54 ± 5.46), Cl‐ (15.95 ± 0.72) and K+ (3.57 ± 0.15) were predominant ions in the seminal plasma (as mm ). Blood ionic values (as mm ) were determined for Na+ (110.2 ± 1.26), K+ (3.77 ± 0.081), Cl? (60.12 ± 1.5), Ca2+ (2.05 ± 0.35) and Mg2+ (1.9 ± 0.16). Results of the comparison between ionic parameters of seminal and blood plasma indicated that the concentrations of all parameters of blood plasma with the exception of K+ were significantly (p < 0.05) higher than those of seminal plasma.  相似文献   

19.
Seminal plasma (SP) and ovarian fluid (OF) plays an important role as storage media to prevent the activation of gametes both in vivo and under artificial conditions. The objectives of this study were to quantify gamete biochemistry and explore correlations among quantitative characteristics of SP, OF and sperm performance traits of Ide Leuciscus idus and Northern pike Esox lucius. Generally, Na+, K+ and Cl? were found to be the most dominating ions, although concentrations of K+ were higher in SP, while Na+ and Cl? concentrations were higher in OF for both species. Several significant correlations among the biochemical properties such as total protein, glucose, osmolality, cholesterol, K+, Ca2+, Cl? and Mg2+ were observed for SP and OF. Total protein content of Ide SP was positively correlated with sperm activity traits (r ≥ .89, p ≤ .05), while K+ concentration was negatively correlated with sperm traits (r ≥ ?.89, p ≤ .05). Moreover, Ca2+ concentration in Northern pike SP was positively correlated with the percentage of sperm motility (r = . 98, p < .01). In conclusion, these results can be used to better understand the biochemistry of SP and OF, improve methods for short‐ and long‐term storage of gametes and standardize fertilization protocols.  相似文献   

20.
Melatonin has been reported to improve the in vitro development of embryos in some species. This study was conducted to investigate the effect of melatonin supplementation during in vitro maturation (IVM) and development culture on the development and quality of porcine embryos. In the first experiment, when the in vitro fertilized embryos were cultured with different concentrations of melatonin (0, 10, 25 and 50 ng/ml) for 8 days, the blastocyst formation rate of embryos cultured with 25 ng/ml melatonin (10.7%) was significantly increased (p < 0.05) compared to the control embryos cultured without melatonin (4.2%). The proportion of DNA‐fragmented nuclei in blastocysts derived from embryos cultured with 50 ng/ml melatonin was significantly lower (p < 0.05) than that of embryos cultured without melatonin (2.1% vs 7.2%). In the second experiment, when oocytes were cultured in the maturation medium supplemented with different concentrations of melatonin (0, 10, 25 and 50 ng/ml), fertilized and then cultured with 25 ng/ml melatonin for 8 days, there were no significant differences in the rates of cleavage and blastocyst formation among the groups. However, the proportions (2.7–5.4%) of DNA‐fragmented nuclei in blastocysts derived from oocytes matured with melatonin were significantly decreased (p < 0.05) compared to those (8.9%) from oocytes matured without melatonin, irrespective of the concentration of melatonin. Our results suggest that supplementation of the culture media with melatonin (25 ng/ml) during IVM and development has beneficial effects on the developmental competence and quality of porcine embryos.  相似文献   

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