scholarly journals Effects of disulfide bond reducing agents on sperm chromatin structural integrity and developmental competence of in vitro matured oocytes after intracytoplasmic sperm injection in pigs

Reproduction ◽  
2009 ◽  
Vol 137 (4) ◽  
pp. 633-643 ◽  
Author(s):  
Wen-Min Cheng ◽  
Lei An ◽  
Zhong-Hong Wu ◽  
Yu-Bo Zhu ◽  
Jing-Hao Liu ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Intracytoplasmic Sperm Injection ◽  
Chemical Activation ◽  
Reducing Agents ◽  
Developmental Competence ◽  
Sperm Chromatin ◽  
Dna Integrity ◽  
Acridine Orange Staining ◽  
Sperm Dna

We recently reported that electrical activation followed by secondary chemical activation greatly enhanced the developmental competence ofin vitromatured porcine oocytes fertilized by intracytoplasmic sperm injection (ICSI). We hypothesized that sperm treatment with disulfide bond reducing agents will enhance the development competence of porcine embryos produced by this ICSI procedure. We examined the effects of glutathione (GSH), dithiothreitol (DTT), GSH or DTT in combination with heparin on sperm DNA structure, paternal chromosomal integrity, pronuclear formation, and developmental competence ofin vitromatured porcine oocytes after ICSI. Acridine orange staining and flow cytometry based sperm chromatin structure assay were used to determine sperm DNA integrity by calculating the cells outside the main population (COMP αT). No differences were observed in COMP αT values among GSH-treated and control groups. COMP αT values in GSH-treated groups were significantly lower than that in DTT-treated groups. Following ICSI, GSH treatments did not significantly alter paternal chromosomal integrity. Paternal chromosomal integrity in sperm treated with DTT plus or minus heparin was also the lowest among all groups. GSH-treated sperm yielded the highest rates of normal fertilization and blastocyst formation, which were significantly higher than that of control and DTT-treated groups. The majority of blastocysts derived from control and GSH-treated spermatozoa were diploid, whereas blastocysts derived from DTT-treated spermatozoa were haploid. In conclusion, sperm treatment with GSH enhanced the developmental capacity of porcine embryos produced by our optimized ICSI procedure.

320 EFFECTS OF DIFFERENT ACTIVATION REGIMENS ON PRONUCLEAR FORMATION AND PRE-IMPLANTATION DEVELOPMENTAL COMPETENCE OF IN VITRO-MATURED BOVINE OOCYTES AFTER INTRACYTOPLASMIC SPERM INJECTION

2015 ◽  
Vol 27 (1) ◽  
pp. 249
Author(s):  
M. E. Arias ◽  
R. Sanchez ◽  
R. Felmer
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Standard Procedure ◽  
Chemical Activation ◽  
Developmental Rate ◽  
Fertilization Rate ◽  
Developmental Competence ◽  
Assisted Reproductive Technique ◽  
Pronuclear Formation ◽  
Bovine Oocytes

Intracytoplasmic sperm injection (ICSI) is an assisted reproductive technique that has been used with considerable success in humans; however, in the bovine species the efficiency of this technique is far from optimal. The objective of the present study was to evaluate the effect of 4 chemical activation treatments, 6-dimethylaminopurine (DMAP), cycloheximide (CHX), anisomycin (ANY), and ethanol (EtOH) on the pronuclear formation and embryo development of bovine embryos generated by ICSI. Cumulus-oocyte complexes were aspirated from abattoir ovaries, selected, and matured in 400-µL drops of standard TCM-199 maturation medium for 22 h at 38.5°C and 5% CO2. The ICSI was performed by a standard procedure. Injected oocytes were randomly distributed and activated by 5 µM ionomycin for 5 min (Io) followed by i) 5 µg mL–1 CHX for 5 h (Io/CHX), ii) 3 h window followed by a second Io treatment plus 1.9 mM DMAP for 4 h (2Io/DMAP), iii) 1 µg mL–1 ANY for 5 h (Io/ANY), and iv) 3 h window followed by 7% ethanol (Io/EtoH). Embryos were cultured in 50-µL drops of KSOM medium under mineral oil at 38.5°C and 5% CO2, 5% O2, and 90% N2. Cleavage was recorded at 72 h and blastocyst rate at 192 h. Pronuclear formation analysis was carried out at 18 hpa with Hoechst staining. An oocyte was considered fertilized when 2 polar bodies and 1 female and 1 male pronucleus (or a decondensed sperm head) could be observed. The data were transformed to arcsine, analysed by ANOVA, and means were compared using Tukey's test with Statgraphics Plus 2 Software. Results with a total of 431 injected oocytes (114, 104, 101, and 112 for DMAP, CHX, ANY, and EtOH, respectively) showed differences in cleavage (P < 0.01) in DMAP, CHX, and ANY treatments (86, 72, and 78%, respectively), relative to EtOH (12%). Similarly, the rate of blastocysts/injected oocyte at 192 h was higher with DMAP, CHX, and ANY (41, 20, and 32%, respectively), relative to EtOH (4%). Sham-injected oocytes showed cleavage and blastocyst rates of 67, 43, 68, and 12% and 32, 11, 19, and 5%, for DMAP, CHX, ANY, and EtOH, respectively. Despite the higher developmental rate observed with DMAP, pronuclear formation assessment revealed that fertilization rate was higher in CHX (87%) and ANY (75%) treatments relative to DMAP (35%). In conclusion, the results of the present study show that activation of bovine oocytes after ICSI is more efficient with DMAP and ANY, compared with CHX and EtOH.Provision of ovaries by our local slaughterhouse (Frigorifico Temuco, Chile) and funding support from FONDECYT 1120241 CONICYT, Chile, are gratefully acknowledged.

Influence of bovine sperm DNA fragmentation and oxidative stress on early embryo in vitro development outcome

Reproduction ◽  
2013 ◽  
Vol 146 (5) ◽  
pp. 433-441 ◽  
Author(s):  
Renata Simões ◽  
Weber Beringui Feitosa ◽  
Adriano Felipe Perez Siqueira ◽  
Marcilio Nichi ◽  
Fabíola Freitas Paula-Lopes ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Dna Fragmentation ◽  
Sperm Chromatin ◽  
Dna Integrity ◽  
Sperm Dna Fragmentation ◽  
Cleavage Rate ◽  
Sperm Dna Integrity ◽  
Sperm Dna ◽  
Significant Difference

Sperm chromatin fragmentation may be caused by a number of factors, the most significant of which is reactive oxygen species. However, little is known about the effect of sperm oxidative stress (OS) on DNA integrity, fertilization, and embryonic development in cattle. Therefore, the goal of this study was to evaluate the influence of sperm OS susceptibility on the DNA fragmentation rate and in vitro embryo production (IVP) in a population of bulls. Groups of cryopreserved sperm samples were divided into four groups, based on their susceptibility to OS (G1, low OS; G2, average OS; G3, high OS; and G4, highest OS). Our results demonstrated that the sperm DNA integrity was compromised in response to increased OS susceptibility. Furthermore, semen samples with lower susceptibility to OS were also less susceptible to DNA damage (G1, 4.06%; G2, 6.09%; G3, 6.19%; and G4, 6.20%). In addition, embryo IVP provided evidence that the embryo cleavage rate decreased as the OS increased (G1, 70.18%; G2, 62.24%; G3, 55.85%; and G4, 50.93%), but no significant difference in the blastocyst rate or the number of blastomeres was observed among the groups. The groups with greater sensitivity to OS were also associated with a greater percentage of apoptotic cells (G1, 2.6%; G2, 2.76%; G3, 5.59%; and G4, 4.49%). In conclusion, we demonstrated that an increased susceptibility to OS compromises sperm DNA integrity and consequently reduces embryo quality.

scholarly journals Impaired protamination and sperm DNA damage in a Nellore bull with high percentages of morphological sperm defects in comparison to normospermic bulls

2015 ◽  
Vol 67 (2) ◽  
pp. 417-423 ◽  
Author(s):  
J.T. Carreira ◽  
J.T. Trevizan ◽  
B.H. Kipper ◽  
S.H.V. Perri ◽  
I.R. Carvalho ◽  
...  
Keyword(s):  
Plasma Membranes ◽  
Cell Damage ◽  
Sperm Concentration ◽  
Sperm Chromatin ◽  
Dna Integrity ◽  
Dna Breaks ◽  
Mitochondrial Potential ◽  
Sperm Dna Damage ◽  
Sperm Dna

The routine semen evaluation assessing sperm concentration, motility and morphology, does not identify subtle defects in sperm chromatin architecture. Bulls appear to have stable chromatin, with low levels of DNA fragmentation. However, the nature of fragmentation and its impact on fertility remain unclear and there are no detailed reports characterizing the DNA organization and damage in this species. The intensive genetic selection, the use of artificial insemination and in vitro embryo production associated to the cryopreservation process can contribute to the chromatin damage and highlights the importance of sperm DNA integrity for the success of these technologies. Frozen-thawed semen samples from three ejaculates from a Nellore bull showed high levels of morphological sperm abnormalities (55.8±5.1%), and were selected for complementary tests. Damage of acrosomal (76.9±8.9%) and plasma membranes (75.7±9.3%) as well as sperm DNA strand breaks (13.8±9.5%) and protamination deficiency (3.7±0.6%) were significantly higher compared to the values measured in the semen of five Nellore bulls with normospermia (24.3±3.3%; 24.5±6.1%; 0.6±0.5%; 0.4±0.6% for acrosome, plasma membrane, DNA breaks and protamine deficiency, respectively) (P<0.05). Motility and percentage of spermatozoa with low mitochondrial potential showed no differences between groups. This study shows how routine semen analyses (in this case morphology) may point to the length and complexity of sperm cell damage emphasizing the importance of sperm function testing.

scholarly journals Sperm DNA Integrity Assessment: A New Tool in Diagnosis and Treatment of Fertility

2012 ◽  
Vol 2012 ◽  
pp. 1-6 ◽  
Author(s):  
Mona Bungum
Keyword(s):  
Semen Quality ◽  
Significant Proportion ◽  
Semen Parameters ◽  
Sperm Chromatin ◽  
Dna Integrity ◽  
Sperm Dna Fragmentation ◽  
Integrity Assessment ◽  
Sperm Dna Integrity ◽  
Sperm Dna

Infertility affects 15% of all couples. Although male infertility factors with reduced semen quality are contributing to about half of all involuntary childlessness, the value of standard semen parameters in prediction of fertilityin vivoand choice of proper method for assisted reproduction is limited. In the search for better markers of male fertility, during the last 10 years, assessment of sperm DNA integrity has emerged as a strong new biomarker of semen quality that may have the potential to discriminate between infertile and fertile men. Sperm DNA Fragmentation Index (DFI) as assessed by the flow cytometric Sperm Chromatin Structure Assay (SCSA) can be used for evaluation of sperm chromatin integrity. The biological background for abnormal DFI is not completely known, but clinical data show that DFI above 30% is associated with very low chance for achieving pregnancy in natural way or by insemination, but notin vitro. Already when the DFI is above 20%, the chance of natural pregnancy may be reduced, despite other sperm parameters being normal. Thus this method may explain a significant proportion of cases of unexplained infertility and can be beneficial in counselling involuntary childless couples need ofin vitrofertilisation.

scholarly journals Factors affecting developmental competence of equine oocytes after intracytoplasmic sperm injection

Reproduction ◽  
2004 ◽  
Vol 127 (2) ◽  
pp. 187-194 ◽  
Author(s):  
Y H Choi ◽  
L B Love ◽  
D D Varner ◽  
K Hinrichs
Keyword(s):  
Embryo Culture ◽  
Intracytoplasmic Sperm Injection ◽  
Room Temperature ◽  
Culture Media ◽  
Developmental Competence ◽  
Glucose Medium ◽  
Factors Affecting ◽  
Nuclear Maturation ◽  
High Glucose Medium

This study was conducted to evaluate the effect of initial cumulus morphology (expanded or compact) and duration of in vitro maturation (24, 30 or 42 h) on the developmental competence of equine oocytes after intracytoplasmic sperm injection (ICSI). The effect of manipulation temperature (room temperature vs 37 °C) at the time of ICSI and concentration of glucose (0.55 vs 5.5 mM) during embryo culture was also investigated. The nuclear maturation rates of expanded (Ex) oocytes were significantly (P < 0.001) higher than those of compact (Cp) oocytes at all maturation times (61–72 vs 23–25% respectively). Forty-eight hours after ICSI of mature Ex oocytes, the rate of cleavage with normal nuclei was significantly (P < 0.05) higher for oocytes matured for 24 h than for those matured for 30 or 42 h (73 vs 57–59% respectively). For Cp oocytes, the morphologic cleavage rates for oocytes matured for 30 h were significantly higher (P < 0.05) than for those matured for 24 or 42 h (86 vs 55–61% respectively). The overall proportion of embryos having more than four normal nuclei at 48 h culture was significantly higher (P < 0.05) for Cp than for Ex oocytes. Manipulation temperature did not affect development of embryos from Ex or Cp oocytes at 96 h after ICSI. Culture in high-glucose medium significantly increased morphologic cleavage of Cp, but not Ex, oocytes (P < 0.05). Embryos from Cp oocytes had a significantly higher average nucleus number after 96-h culture than did embryos from Ex oocytes. These data indicate that developmental competence differs between Ex and Cp equine oocytes, and is differentially affected by the duration of maturation and by composition of embryo culture media.

41 STUDY ON THE ESTABLISHMENT OF SOMATIC CELL NUCLEAR TRANSFER USING IN VIVO-MATURED OOCYTES IN DOGS

2006 ◽  
Vol 18 (2) ◽  
pp. 129 ◽  
Author(s):  
G. Jang ◽  
M. Kim ◽  
H. J. Oh ◽  
F. Y. Heru ◽  
M. S. Hossein ◽  
...  
Keyword(s):  
Electrical Stimulation ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Chemical Activation ◽  
Developmental Competence ◽  
Perivitelline Space ◽  
Parthenogenetic Activation ◽  
Vaginal Cytology

The present study was performed to collect in vivo matured canine oocytes for somatic cell nuclear transfer (SCNT) and to investigate the developmental competence of canine parthenogenetic and SCNT embryos as the preliminary research for producing cloned dog. The day of ovulation as described by Hase et al. (2000 J. Vet. Med. Sci. 62, 243-248) was determined by serum progesterone levels and at that time vaginal cytology was performed to assess the cornified index. In vivo-matured oocytes were recovered by retrograde flushing of the oviducts at around 48 h (n = 20) or 72 h (n = 25) after the estimated time of ovulation. Overall size of each oocyte, as well as ooplasmic diameter, zona pellucida thickness, and perivitelline space width, was determined after removing the cumulus cells by pipetting (Exp. 1). To determine activation protocols, two treatments, (1) chemical activation (10 �M Ca ionophore for 4 min, followed by incubation for 4 h with 1.9 mM 6-dimethylaminopurine) and (2) electrical stimulation (3.1?3.4 kV/cm in 0.25M mannitol solution), were evaluated to induce parthenogenetic activation of oocytes (Exp. 2). Donor cells were obtained from the primary cell culture of a canine ear skin biopsy, and SCNT was performed according to our laboratory procedures (Jang et al. 2004 Theriogenology 62, 512-521). Three voltages (1.7?2.0 kV/cm, 2.1-2.4 kV/cm, and 3.1-3.4 kV/cm) were tested for fusion. The fused couplets were subjected to chemical or electrical stimulation as in parthenogenetic activation and in vitro developmental competence was monitored (Exp. 3). As a result, more in vivo-matured canine oocytes were obtained at 72 h (92%) than at 48 h (15%) after ovulation; the 72-h occytes had progesterone concentrations of 4-8 ng/mL and a cornified index (vaginal cytology) of 83.34. The average number of oocytes recovered was 12 and sizes of ooplasmic diameter, cytoplasm, zona pellucida, and perivitelline space in in vivo canine-matured oocytes (n = 120) were 178.8 � 9.3 �m, 125.0 � 8.2 �m, 21.7 � 3.7 �m, and 12.7 � 3.5 �m, respectively. Parthenogenetically activated oocytes developed to the 16-cell and morula stages, but failed to develop to the blastocyst stage. Among the three voltages, in the highest voltage (75.2%) the number of fused couplets was increased compared to either of the other voltages (33.3% and 44.0%). Cleavage rates (60.9% vs. 58.0%) of cloned embryos were not significantly affected by method of activation. In terms of in vitro developmental competence, cloned embryos developed to the 16-cell or morula stage in vitro after electrical or chemical activation, respectively. In conclusion, in the present study we demonstrated that measurement of progesterone levels, in combination with evaluation of vaginal cytology, can be used to determine the estimated time of ovulation in bitches. In addition, we determined fusion/activation protocols that resulted in in vitro development of a portion of parthenogenetically activated and cloned embryos to the 16-cell and morula stages. This study was supported by grants from the Biogreen 21-1000520030100000.

225 DEVELOPMENTAL COMPETENCE AND QUALITY OF PIG EMBRYOS OBTAINED FROM STANDARD IN VITRO FERTILIZATION OR INTRACYTOPLASMIC SPERM INJECTION

2013 ◽  
Vol 25 (1) ◽  
pp. 260 ◽  
Author(s):  
I. Grad-Mandryk ◽  
J. Kosenyuk ◽  
B. Gajda
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Quality Criteria ◽  
Cell Number ◽  
Total Cell ◽  
Developmental Competence ◽  
Tunel Staining ◽  
In Vitro Production ◽  
Total Cell Number ◽  
Vitro Fertilization

In vitro production of porcine embryos is still relatively inefficient. The main reasons for this limited performance are polyspermy after IVF and the poor developmental ability of obtained zygotes. Intracytoplasmic sperm injection (ICSI) is one possible solution to eliminate polyspermy. The aim of this study was to compare the developmental competence of pig zygotes, total cell number, and DNA fragmentation of pig blastocysts derived from IVF or ICSI. Cumulus–oocyte complexes were obtained by aspiration from antral follicles of ovaries collected from slaughtered gilts. The oocytes were then cultured in modified TC-199 medium to metaphase II for 42 h. Semen for IVF was incubated in modified capacitation medium (M199) for 1 h. The sperm fraction (1 × 106 cells mL–1) was introduced into droplets containing oocytes, and then gametes were co-incubated for 4 h in modified TC-199 medium. Intracytoplasmic sperm injection was performed using a mechanical micromanipulator (Research Instruments Limited, Cornwall, UK). Micromanipulation was carried out in modified NCSU-37 medium. The tails of spermatozoa were broken, and then single spermatozoa were aspirated into the injection pipette. The oocyte was fixed by a holding pipette, and the sperm head was then introduced into the oocyte cytoplasm. Presumptive zygotes were cultured in vitro for 144 h in NCSU-23 medium. The embryo quality criteria were developmental competence (morula and blastocyst rates), total cell number per blastocyst, and degree of apoptosis assessed by TUNEL staining. Data were analysed by chi-squared test. The experiment was performed on 136 zygotes (6 replicates) obtained after IVF and 83 zygotes (4 replicates) obtained after ICSI. Percentages of embryos developed to the morula and blastocyst stages were 42.3 ± 6.1 and 28.8 ± 4.7 after IVF, respectively, and 51.7 ± 15.4 and 34.5 ± 18.9 after ICSI, respectively (no differences were observed). Significant differences were noticed in total number of cells per blastocyst between embryos after IVF and ICSI (33.7 ± 5.39 v. 22.8 ± 3.22; P < 0.01). However, there was no difference in the degree of apoptosis between IVF and ICSI embryos (5.14 ± 3.49 and 6.14 ± 4.88, respectively). Our preliminary studies demonstrated a higher proportion of cell numbers in IVF-derived embryos compared with those produced by ICSI, but the developmental competence and degree of apoptosis, as evaluated by the TUNEL method, in both groups were comparable. This study was funded by project N N311 516140 by the NCN, Poland.

scholarly journals Beneficial effect of antioxidant therapy on sperm DNA integrity is not associated with a similar effect on sperm chromatin integrity

2019 ◽  
Vol 4 ◽  
pp. 31-31 ◽  
Author(s):  
Cécile Le Saint ◽  
Isaac-Jacques Kadoch ◽  
François Bissonnette ◽  
Julie Choi ◽  
Jonathan Zini ◽  
...  
Keyword(s):  
Beneficial Effect ◽  
Antioxidant Therapy ◽  
Sperm Chromatin ◽  
Dna Integrity ◽  
Sperm Dna Integrity ◽  
Sperm Dna ◽  
Chromatin Integrity
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