92 INSULIN-LIKE GROWTH FACTOR-1 PROTECTS BOVINE PRE-IMPLANTATION EMBRYOS PRODUCED IN VITRO FROM ANTI-DEVELOPMENTAL ACTIONS OF MENADIONE

2016 ◽  
Vol 28 (2) ◽  
pp. 176
Author(s):  
N. A. S. Rocha-Frigoni ◽  
B. C. S. Leão ◽  
P. C. Dall'Acqua ◽  
M. Ambrogi ◽  
G. Z. Mingoti
Keyword(s):  
Oxidative Stress ◽  
Growth Factor ◽  
Molecular Probes ◽  
Insulin Like Growth Factor ◽  
Blastocyst Development ◽  
Chi Square ◽  
Fluorescent Intensity ◽  
Chi Square Test ◽  
Student’S T

The objective of this study was to evaluate the protective effect of insulin-like growth factor (IGF-1) on blastocyst development and cryotolerance of bovine embryos in in vitro culture (IVC) under oxidative stress induced by menadione (MD). Cumulus-oocyte complexes (n = 1421) were matured in TCM-199 with bicarbonate, hormones, and 10% FCS for 22 h. After fertilization, the presumptive zygotes were cultured up to 7 days in SOF medium with 2.5% FCS and 0.5% BSA (control), and also supplemented with 100 μM IGF-1 (IGF). At Day 6, MD was included in the culture medium (0 μM, control; or 5.0 μM, MD) during 24 h. Cultures were conducted at 38.5°C in 5% CO2 in air. The cleavage and blastocysts rates were evaluated, respectively, at Days 3 and 7 (IVF = Day 0). At Day 7, a sample of the blastocysts was stained with 5 μM H2DCFDA (Molecular Probes, Canada) to evaluate the intracellular ROS levels or was stained for TUNEL (In Situ Cell Death Detection Kit, Roche, Indianapolis, IN, USA). Stained embryos were immediately evaluated under an epifluorescence microscope (excitation 495/550 nm and emission 404/590 nm, respectively, for ROS and TUNEL), and the images of embryos stained with H2DCFDA were analysed by Q-Capture Pro image software for determining the fluorescent intensity. Other blastocysts were vitrified (Ingámed®, Maringá-PR, Brazil), and after warming, they were cultured for 24 h to evaluate the re-expansion rates. The results were compared by ANOVA followed by Student’s t-test (mean ± s.e.M) and re-expansion rates by chi-square test (P < 0.05). The cleavage rates did not differ (P > 0.05) among groups (77.1 ± 1.9% to 82.75 ± 2.2%). The blastocyst rates were similar between control (35.4 ± 2.0%) and IGF (34.5 ± 3.7%), and both were higher (P < 0.05) than MD (21.3 ± 2.7%); the IGF+MD group (28.3 ± 1.6%) was similar (P > 0.05) to all groups. The intracellular levels of ROS were higher (P < 0.05) for the MD group (21.7 ± 0.7) than for control (17.0 ± 1.6), and both were similar (P > 0.05) to the IGF (19.2 ± 0.6) and IGF+MD (18.0 ± 1.0) groups. The highest rates of apoptosis were found in the MD group (22.3% ± 2.3) and the smallest in IGF (9.1% ± 0.7), and both differed (P < 0.05) from control (12.8% ± 1.0), and IGF+MD (15.6% ± 1.6). The re-expansion rates were similar between control (77.4%) and IGF (69.2%), and both were higher (P < 0.05) than MD (49.1%); however, the IGF+MD group (57.6%) was similar (P > 0.05) to IGF and MD groups. In conclusion, the supplementation with IGF-1 during IVC reversed the detrimental effects of MD on embryonic levels of ROS and apoptosis, as well as improved the embryo development and cryotolerance of blastocysts under oxidative stress. Financial support was provided by FAPESP (#2012/10083–8 and #2013/07382–6).

scholarly journals Effect of insulin-like growth factor-1 during in vitro oocyte maturation and in vitro culture of bovine embryos

2001 ◽  
Vol 53 (2) ◽  
pp. 207-211 ◽  
Author(s):  
M.D. Quetglas ◽  
L.A. Coelho ◽  
J.M. Garcia ◽  
E.B. Oliveira Filho ◽  
C.R. Esper
Keyword(s):  
Growth Factor ◽  
In Vitro Culture ◽  
Embryo Development ◽  
Calf Serum ◽  
Culture Conditions ◽  
Insulin Like Growth Factor ◽  
Chi Square ◽  
Igf I ◽  
Chi Square Test

The effects of insulin-like growth factor-I (IGF-I) on in vitro maturation (IVM) (experiment I) and on in vitro embryo development (experiment II) of bovine oocytes fertilized in vitro, were evaluated in terms of cleavage (CR), blastocyst (BR) and hatching (HR) rates. For IVM, immature cumulus-oocyte complexes were cultured in TCM-199 medium supplemented with Hepes, sodium bicarbonate, sodium pyruvate, additives, fetal calf serum (B-199 medium) and gonadotropins (14 U/ml PMSG and 7 U/ml hCG). For embryo development, the oocytes/zygotes were cultured in B-199 medium with bovine oviduct epithelial cells in suspension under silicon oil. Treatments for in vitro culture conditions for both experiments were: 1- B-199 + 200 ng/ml IGF-I; 2- B-199 + 100 ng/ml IGF-I; 3- B-199 + 50 ng/ml IGF-I; 4- B-199 + 10 ng/ml IGF-I; 5- B-199 + 0 ng/ml IGF-I. All cultures were performed at 38.5ºC in 5% CO2 in air and the data were analyzed by chi-square test. In experiment I, there were no differences (P>0.05) among treatments for CR, BR or HR. In experiment II, the addition of IGF-I to the embryo culture medium (ECM) resulted in a significant increase in CR while for BR and HR this effect was not observed. The addition of 200 ng/ml IGF-I to ECM increased CR (71.1%) when compared to 100 ng/ml IGF-I (57.6%) or control (56.7%) groups, however, there were no differences when compared to 50 (69.4%) or 10 ng/ml (73.1%) groups. There was no beneficial effect of the addition of IGF-I in the IVM or ECM media on the in vitro development of embryos produced from oocytes matured and fertilized in vitro.

62 EXPOSURE TO ETHYLENE GLYCOL AND DIMETHYL SULFOXIDE CAUSES ACTIVATION AND SPINDLE ANOMALIES IN BUFFALO (BUBALUS BUBALIS) OOCYTES

2009 ◽  
Vol 21 (1) ◽  
pp. 131 ◽  
Author(s):  
M. De Blasi ◽  
E. Mariotti ◽  
M. Rubessa ◽  
S. Di Francesco ◽  
G. Campanile ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Embryo Development ◽  
Sucrose Solution ◽  
Bubalus Bubalis ◽  
Meiotic Spindle ◽  
Blastocyst Development ◽  
Chi Square ◽  
Chi Square Test ◽  
Simple Exposure

Despite the increasing interest, buffalo oocyte cryopreservation is still inefficient, especially in terms of blastocyst development after IVF. The aim of this work was to evaluate chromatin and spindle organization of buffalo in vitro-matured oocytes after vitrification/warming by cryotop and after their simple exposure to cryoprotectants (CP). An overall amount of 251 COC was selected and matured in vitro. In the vitrification group, COC were first exposed to 10% ethylene glycol (EG) + 10% DMSO for 3 min, and then to 20% EG + 20% of DMSO and 0.5 m sucrose, loaded on cryotops, and plunged into liquid nitrogen within 25 s. Oocytes were warmed into a 1.25 m sucrose solution for 1 min and then to decreasing concentrations of sucrose (0.625 m, 0.42 m, and 0.31 m) for 30s each. In order to test CP toxicity, COC were simply exposed to the vitrification and warming solutions. Two hours after warming, oocytes were fixed and immunostained for microtubules using a method previously described (Messinger SM and Albertini DF 1991 J. Cell Sci. 100, 289–298), stained for nuclei with Hoechst, and examined by fluorescence microscopy. Fresh in vitro-matured oocytes were fixed and stained as controls. Data were analyzed by chi-square test; results are shown in Table 1. The percentages of MII oocytes in the control and vitrification groups were greater than in the toxicity group, in which a greater percentage of telophase II stage oocytes were found compared with both the control and vitrification groups, indicating occurrence of activation. Of the MII oocytes, both exposure to CP and vitrification procedures gave greater percentages of oocytes with abnormal spindle and abnormal chromatin configuration compared with the control. An unexpected datum was the evidence of a significant percentage of spontaneously activated oocytes in the toxicity group. We speculate that the lack of activation in the vitrification group may be related to the slowing down of metabolic activity subsequent to thermal shock, and hence, that activation after vitrification may occur later than 2 h post-warming. In conclusion, the simple exposure to CP causes activation of the COC and damage to the cytoskeleton similar to that induced by the whole vitrification protocol. The damages to the meiotic spindle and DNA fragmentation may lead to aneuploidy incompatible with subsequent embryo development and account for the poor embryo development currently recorded in buffalo. Table 1.Chromatin and spindle organization in oocytes vitrified and exposed to cryoprotectants

135 SHORT-TERM CRYOPRESERVATION OF VITRIFIED MOUSE MORULAE AT - 79°C

2007 ◽  
Vol 19 (1) ◽  
pp. 185
Author(s):  
Y. Takagi ◽  
M. Shimizu ◽  
M. Morimura ◽  
S. Yokomizo ◽  
K. Hara ◽  
...  
Keyword(s):  
Liquid Nitrogen ◽  
Blastocyst Stage ◽  
Control Group ◽  
Embryo Viability ◽  
Chi Square ◽  
Significant Difference ◽  
Morula Stage ◽  
Chi Square Test ◽  
Student’S T

Embryos of various species are successfully vitrified and cryopreserved in liquid nitrogen (&lt;−150°C). Like the preservation of frozen somatic cells cooled by dry ice (−79°C), the cryopreservation of embryos at −79°C is useful for a reduction in the shipping costs. The purpose of this study was to evaluate the effect of the cryopreservation period at −79°C on the in vitro embryo viability of vitrified mouse morulae after thawing. Morula-stage mouse embryos were collected from superovulated ICR donors 70 h after hCG injection. The embryos were exposed first to 5% DMSO + 5% ethylene glycol (EG) in Dulbecco's PBS + 20% FCS (mPBS) for 2 min, and then equilibrated for 20–30 s in a vitrification solution composed of 10% DMSO + 10% EG + 0.6 M sucrose in mPBS. The embryos were loaded onto cryoloops (Lane et al. 1999 Nat. Biotech. 17, 1234–1236) and plunged directly into liquid nitrogen. The cryoloops were placed in 1.2-mL cryotubes and stored in a −79°C freezer for 1–7 days. The embryos were warmed by passing through 4 dilution media and rinsed with mWM culture medium. They were then cultured at 37°C in 5% CO2 for 44 h. Non-cryopreserved embryos and embryos cryopreserved in liquid nitrogen served as controls. Data were analyzed by the chi-square test and the Student's t-test. Results are shown in Table 1. There was no significant difference (P &gt; 0.01) in the developmental abilities to the blastocyst stage of the vitrified embryos that were cryopreserved at −79°C for 1 day, 3 days, and 5 days, the embryos cryopreserved in liquid nitrogen, and the non-vitrified control. The blastocyst rate of embryos was significantly lower (P &lt; 0.01) for the Day 7 group than for the control group. The cell numbers of blastocysts were significantly lower (P &lt; 0.01) for the Day 1, Day 3, Day 5, and Day 7 groups than for the control group. This study suggests that vitrified mouse morulae can be successfully cryopreserved at −79°C for 5 days. Table 1. Effect of the cryopreservation period on the viability of vitrified mouse morulae preserved at −79°C

135 LEVELS OF REACTIVE OXYGEN SPECIES (ROS), APOPTOSIS AND CRYORESISTANCE OF BOVINE EMBRYOS PRODUCED IN VITRO IN THE PRESENCE OF ANTIOXIDANTS

2013 ◽  
Vol 25 (1) ◽  
pp. 215
Author(s):  
N. A. S. Rocha ◽  
B. C. S. Leão ◽  
É. Nogueira ◽  
M. F. Accorsi ◽  
G. Z. Mingoti
Keyword(s):  
Oxidative Stress ◽  
Cell Death ◽  
Embryo Development ◽  
Embryo Quality ◽  
Mitochondrial Respiratory Chain ◽  
Molecular Probes ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Fluorescent Intensity

The production of ROS is a normal process that occurs in cellular mitochondrial respiratory chain. However, the increase in ROS due to the high oxygen tension during in vitro production of bovine embryos induces oxidative stress, leading to embryonic development failure. Thus, the aim of this study was to evaluate the effects of supplementation with antioxidants (cysteine – CIST; β-mercaptoethanol – βME and catalase – CAT) during in vitro maturation (IVM) and in vitro culture (IVC) on the embryo development, cryoresistance, and quality, as well as the amounts of intracellular ROS produced during embryo culture. Cumulus–oocyte complexes (n = 565) were IVM in medium TCM-199 supplemented with 0.6 mM CIST, 100 µM βME, 100 UI CAT, or without antioxidants (Contr). After fertilization, zygotes were IVC in SOF medium during the first 72 h (up to Day 3) with or without addition of the same antioxidant used for IVM, and then all the embryos were transferred to SOF medium without antioxidants. All cultures were conducted at 38.5°C in 5% CO2 in air. The cleavage and blastocysts rates were evaluated, respectively, at 72 and 168 h post-insemination. Part of obtained blastocysts (n = 133) was vitrified (Ingámed®, Maringá, PR, Brazil). The remaining was stained (n = 46) with 5 µM of the fluorescent probe 6-carboxy-2′7′-dichlorodihydrofluorescein diacetate (H2DCFDA; Molecular Probes, Invitrogen, Oakville, Canada) or stained (n = 62) for TUNEL (In situ Cell Death Detection Kit, Fluorescein, Roche Applied Science, IN, USA). Stained embryos were immediately evaluated under an epifluorescence inverted microscope and the images of embryos stained with H2DCFDA were analyzed by Q-Capture Pro image software (QImaging, Surrey, BC, Canada) for determining the fluorescent intensity. The vitrified embryos were thawed and cultured for 24 h to evaluate the re-expansion rates. The difference between groups was compared by ANOVA followed by Tukey test, and re-expansion rates by chi-square test (P < 0.05). The cleavage rates were 82.9% ± 3.2a (Contr), 80.4% ± 2.5a (CIST), 87.0% ± 1.8a (βME) and 81.2% ± 3.0a (CAT). The blastocyst rates were 48.7% ± 3.4ab (Contr), 34.4% ± 4.2b (CIST), 36.0% ± 5.6ab (βME), and 56.8% ± 7.5a (CAT). The re-expansion rates were 76.0%ab (Contr), 66.7%b (CIST), 50.0%b (βME), and 66.1%b (CAT). The fluorescent intensity was 1.0 ± 0.07a (Contr), 0.8 ± 0.05bc (CIST), 0.6 ± 0.05c (βME) and 0.7 ± 0.07bc (CAT). The total number of cells was 85.7 ± 3.5a (Contr), 89.6 ± 4.8a (CIST), 99.5 ± 5.4a (βME), and 81.5 ± 4.3a (CAT), and the rate of apoptotic cells was 4.3 ± 1.2a (Contr), 1.5 ± 0.4b (CIST), 1.6 ± 0.4b (βME), and 2.4 ± 0.6ab (CAT). Supplementation with CAT improved embryo development, compared with CIST (P < 0.05). The fluorescent intensity was lower for all embryos produced with antioxidants (P < 0.05) and the rate of apoptosis was reduced in CIST and βME (P < 0.05). In conclusion, supplementation with antioxidants is an interesting strategy to improve the embryo quality since it reduces the cell death caused by oxidative stress. However, such improvement in embryo quality did not affect embryo cryotolerance. Financial support was provided by CNPq and FAPESP 01/18257-2.

scholarly journals Double blinded study on comparison of phosphorylated insulin like growth factor binding protein 1 test and fetal fibronectin test for prediction of preterm delivery

2017 ◽  
Vol 6 (4) ◽  
pp. 1347
Author(s):  
Seema Singhal ◽  
Nivedita Sarda ◽  
Niharika Dhiman ◽  
Kusum Dogra
Keyword(s):  
Growth Factor ◽  
Binding Protein ◽  
Insulin Like Growth Factor ◽  
Chi Square ◽  
Fetal Fibronectin ◽  
Exact Test ◽  
Medical Interventions ◽  
Factor Binding ◽  
Chi Square Test ◽  
Double Blinded

Background: Recently new markers like fetal fibronectin (FFN) and phosphorylated Insulin like growth factor binding protein 1 (phIGFBP-1) have been found useful in diagnosis of pre-term labor. Fetal fibronectin (FFN) and phosphorylated Insulin like growth factor binding protein 1 (phIGFBP-1) were compared to predict the risk of pre- term delivery.Methods: Cervicovaginal samples of 50 symptomatic and 50 asymptomatic pregnant women were tested. Statistical analysis was done using chi square test, fisher’s exact test for qualitative data and for quantitative data unpaired student t test and Mann Whitney test were used.Results: For Symptomatic group phosphorylated Insulin like growth factor binding protein 1 (phIGFBP-1) and fetal fibronectin (FFN) had 100% sensitivity and 100% NPV for predicting delivery within 48 hours, 7days and 14 days. Specificity and PPV of FFN was higher than phIGFBP-1 to predict delivery (Specificity: within 48 hours 63.2% vs 52.6% within 7 days 72.7% vs 60.6%, within 14 days 75% vs 62.5%, ≤37 weeks 76% vs 56%; PPV: within 48 hours 46.2% vs 40%, within 7 days 65.4% vs 56.7%, within 14 days was 69.2% vs 60% and ≤37 weeks 76.9% vs 63%).Conclusions: Rapid bed side dip stick tests for detecting FFN and ph IGFBP-1 in cervicovaginal secretions are useful clinical adjuncts in ruling out delivery within 14 days of test in symptomatic patients, thus avoiding unnecessary hospital stay and medical interventions. However, FFN has slightly higher NPV in predicting risk of pre term delivery.

227 SUPPLEMENTATION WITH CYSTEINE, CYSTEAMINE, AND CATALASE DURING IN VITRO MATURATION PROTECTS BOVINE PRE-IMPLANTATION EMBRYOS FROM ANTI-DEVELOPMENTAL ACTIONS OF MENADIONE

2015 ◽  
Vol 27 (1) ◽  
pp. 203
Author(s):  
G. Z. Mingoti ◽  
N. A. S. Rocha-Frigoni ◽  
B. C. S. Leão ◽  
P. C. Dall'Acqua
Keyword(s):  
Embryonic Development ◽  
In Vitro Maturation ◽  
Animal Feed ◽  
Molecular Probes ◽  
Apoptotic Cells ◽  
Blastocyst Development ◽  
Fluorescent Intensity ◽  
Fluid Medium ◽  
And Control

Menadione (MD), a naphthoquinone used as a vitamin K source in animal feed, can generate reactive oxygen species (ROS) and cause apoptosis. Hence, we examined whether MD reduces development of pre-implantation bovine embryos by inducing ROS production and apoptosis and tested the hypothesis that the anti-developmental actions of MD would be reduced by supplementation of the in vitro maturation (IVM) medium with antioxidants (a mixture of 0.6 mM cysteine + 100 μM cysteamine + 100 UI mL–1 catalase). Cumulus-oocyte complexes (COC; n = 3334; 50 per well in 11 replications) were matured in IVM medium (TCM-199 with bicarbonate, hormones, and 10% FCS) for 22 h. After fertilization, presumptive zygotes were cultured in synthetic oviduct fluid medium (SOF) with 2.5% FCS and 0.5% BSA up to 7 days (Day 0 = IVF). On Day 6, MD was included in the SOF medium (0 μM: Control; or 5.0 μM: MD5) during 24 h. All cultures were conducted at 38.5°C in 5% CO2 in air. In a second experiment, COC were matured in TCM-199 supplemented with or without the mixture of antioxidants (Antiox; only during IVM) and with or without MD at Day 6 in a 2 × 2 factorial design (Control, Control/Antiox, MD5, MD5/Antiox). The cleavage and blastocysts rates were evaluated on Days 3 and 7, respectively. A sample of the blastocysts (n = 1112) was stained for TUNEL (In Situ Cell Death Detection Kit, Roche, Indianapolis, IN, USA) to detect the apoptotic cells or with 5 μM H2DCFDA (Molecular Probes, Canada) to evaluate the ROS levels. Stained embryos were evaluated under an epifluorescence microscope and the images of embryos stained with H2DCFDA were analysed by Q-Capture Pro image software for determining the fluorescent intensity. The means (±s.e.m.) were compared by ANOVA followed by Tukey's test (P < 0.05). In the first experiment, and the cleavage rates were similar (P > 0.05) for Control (80.5% ± 0.9) and MD5 groups (78.9% ± 1.2), but the blastocyst rates were lower (P < 0.05) in the MD5 group (25.9% ± 2.1) when compared with Control (36.4% ± 1.5). The rate of apoptotic cells in blastocysts was higher (P < 0.05) in the MD5 group (19.2% ± 0.7) than in Control (10.0% ± 0.5), as well as the fluorescence intensity for ROS quantification (0.7 ± 0.1 v. 1.1 ± 0.1, respectively, for Control and MD5 groups). In an effort to rescue the impaired embryonic development, a mixture of antioxidants was added to the IVM medium and the results were transitional in response in which MD5/Antiox (28.3% ± 3.5) was intermediate (P > 0.05) between the Controls [Control (38.2% ± 2.5) and Control/Antiox (34.7% ± 1.9) groups] and the MD5 (21.4% ± 2.9) group. Principal effects evaluated were the presence of MD (without: 35.9% ± 2.3 v. with: 24.8% ± 2.3; P < 0.05) or Antiox (without: 28.4% ± 2.3 v. with: 32.3% ± 2.3; P > 0.05). In conclusion, addition of MD to embryonic culture reduces the blastocyst development and increases intracellular levels of ROS and the occurrence of apoptosis. Anti-developmental actions of MD on embryonic development can be partially blocked by treating oocytes with a mixture of antioxidants during IVM.Financial support was provided by FAPESP (2012/10083-8 and 2013/07382-6).

349 EFFECT OF CUMULUS CELLS AND MATURATION CULTURE PERIOD OF OOCYTES ON THE SEX RATIO OF IN VITRO-FERTILIZED BOVINE EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 331 ◽  
Author(s):  
G. Z. Mingoti ◽  
B. C. S. Leão ◽  
F. P. Gottardi ◽  
R.-V. Alonso
Keyword(s):  
Sex Ratio ◽  
Cumulus Cells ◽  
Culture Period ◽  
Blastocyst Development ◽  
Chi Square ◽  
Maturation Medium ◽  
Chi Square Test ◽  
Culture Duration ◽  
The Difference

Studies have reported a high proportion of males from IVP of embryos systems. Factors such as the stage of oocyte maturation at the time of insemination and conditions of the IVC may influence the proportion of sex. Although cumulus cells (CC) improve the fertilization ability of sperm cells, there are some evidence that exposure of sperm to CC increases the proportion of male embryos produced in vitro. The objective of this study was to investigate the effects and interaction between CC and IVM culture period of bovine oocytes on the sex ratio of in vitro-produced embryos. COCs (n = 781) were collected from the ovaries of slaughtered cows, and then matured in vitro for various periods (18, 20, 22, and 24 h). Maturation medium consisted of TCM-199 with 0.2 mM pyruvate, 25 mM sodium bicarbonate, 75 μg mL-1 gentamicin, 0.5 μg mL-1 FSH, 100 IU mL-1 hCG, and 10% FBS. After maturation culture, oocytes were inseminated with frozen-thawed spermatozoa. Oocytes were inseminated included in CC (COC) or denuded of such cells (denuded oocytes, DO). All zygotes were then co-cultured in vitro with CC in SOF medium with 0.5% BSA and 2.5% FBS. Cultures were carried out at 38.5°C and 5% CO2 in air. Cleavage and blastocyst development rates were observed, respectively, at 48 (Day 2) and 168 (Day 7) h post-insemination (hpi). Blastocysts were harvested on Day 7, and the sex of the embryos was examined using the pair of primers S4B (Kageyama S et al. 2004 Theriogenology 66, 509-514). The data (mean ± SEM) were analyzed by ANOVA, in which the effects of the existence of CC (COC v. DO) and IVM culture periods (18 h, 20 h, 22 h v. 24 h), and interaction between these variables were evaluated. Multiple comparisons of means were followed using Tukey’s test (P < 0.05). The chi-square test was utilized to test the difference in the proportion of sex of embryos. Independently of the presence or absence of CC during IVF, there was no effect of IVM culture duration (P > 0.05) on cleavage (81.5 to 94.8% for COC; and 71.7 to 75.8% for DO) and blastocyst rates (20.0 to 35.9% for COC; and 10.0 to 15.5% for DO). But independently of IVM culture duration, the comparison between COC and DO groups revealed that absence of CC adversely affected (P < 0.05) the cleavage (87.0 and 73.3%, respectively; averaged results) and blastocyst rates (24.5 and 13.5%, respectively; averaged results). In COC, the proportion of male blastocysts increased with the progression in maturation culture period from 18 up to 22 h (54.2, 65.9, and 83.3%, respectively). But for DO, the proportion of females was higher, with exception of 22 h IVM group (65.4, 32.3, 53.3, and 60.0%, respectively from 18 to 24 h of IVM). These results indicate that the sex ratio of in vitro-fertilized embryos is apparently influenced by the maturation culture period of the oocytes and by the presence of CC. FAPESP.

145 EFFECT OF RELAXIN ON FERTILIZING ABILITY OF BUFFALO SPERM

2014 ◽  
Vol 26 (1) ◽  
pp. 186 ◽  
Author(s):  
A. R. Elkhawagah ◽  
V. Longobardi ◽  
G. A. Sosa ◽  
G. Albero ◽  
A. Salzano ◽  
...  
Keyword(s):  
Sperm Motility ◽  
Positive Control ◽  
Negative Control ◽  
Cleavage Rate ◽  
Chi Square ◽  
Chi Square Test ◽  
Live Sperm ◽  
Student’S T ◽  
Fertilizing Capability

The aim of this work was to evaluate the effect of relaxin, known to improve fertility parameters of frozen-thawed sperm in other species (Miah et al. 2006 J. Reprod. Dev. 52, 773–779; Miah et al. 2007 Anim. Sci. J. 78, 495–502), on buffalo sperm motility, capacitation, and fertilizing capability. Frozen-thawed sperm from 2 bulls (4 replicates each) were separated by Percoll, diluted to a 20 × 106 mL–1 concentration and incubated in TALP medium in the absence of capacitating agents (negative control), in the presence of 10 μg mL–1 of heparin (positive control) and 100 ng mL–1 of relaxin for 2 h. Following incubation, sperm were exposed for 15 min to 60 mg mL–1 of lysophosphatidylcholine, a fusogenic agent known to induce the acrosome reaction only on capacitated sperm. To evaluate acrosome-reacted (AR) live sperm, cells were fixed and stained with Trypan blue-Giemsa (Kovacs and Foote 1992 Biotech. Histochem. 67, 119–124) and evaluated (800 sperm counted/group). Sperm motility was examined by a phase contrast microscope, whereas the fertilizing capability was evaluated by heterologous IVF. Abattoir-derived bovine oocytes (n = 258, 86 per group) were in vitro matured and fertilized according to standard procedures (Rubessa et al. 2011 Theriogenology 76, 1347–1355) with buffalo sperm in the absence of capacitating agents and in the presence of 10 μg mL–1 of heparin and 100 ng mL–1 of relaxin. Twenty hours after IVF, presumptive zygotes were denuded and cultured in SOF for 24 h, when cleavage rate was evaluated and confirmed by fixation with absolute ethanol overnight and staining with 2.5 μg mL–1 of Hoechst 33342 after zona removal by pronase (2 mg mL–1) digestion. The differences in the percentages of AR sperm and cleavage among groups were analysed by a chi square test and those in sperm motility by Student's t-test. Acrosomal loss was observed in 10.8% of the sperm after thawing, which may indicate freezing-induced capacitation, and, hence, this value was detracted from the percentages of AR recorded following incubation. After 2 h of incubation, 100 ng mL–1 of relaxin significantly (P < 0.05) increased the percentages of live AR sperm (P < 0.05) compared with the negative control (31.3 ± 2.2 and 25.8 ± 2.8, respectively), with intermediate results in the positive control (27.0 ± 2.2). Motility was significantly improved (P < 0.05) when sperm were exposed to 100 ng mL–1 of relaxin compared with both the negative and positive control (73.7 ± 2.4, 60.0 ± 4.1, and 60.0 ± 7.1, respectively). A significant (P < 0.05) improvement of cleavage rate was recorded both in the positive control (71.5 ± 4.8) and in the group treated with 100 ng mL–1 of relaxin (70.7 ± 0.5) compared with negative control (52.1 ± 1.5). In conclusion, these preliminary results indicate that relaxin at the concentration of 100 ng mL–1 improves sperm motility, capacitation, and the IVF capability of buffalo sperm.

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