318 EFFECT OF SPERM PRETREATMENT WITH ISOBUTYLMETHYLXANTHINE AND METHYL-β-CYCLODEXTRIN ON THE EFFICIENCY OF BOVINE INTRACYTOPLASMIC SPERM INJECTION

2015 ◽  
Vol 27 (1) ◽  
pp. 248
Author(s):  
L. M. Aguila ◽  
M. E. Arias ◽  
R. S. Sanchez ◽  
T. C. Vargas ◽  
F. A. Zambrano ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Defined Medium ◽  
Fertilization Rate ◽  
Vehicle Group ◽  
Control Group ◽  
Sperm Capacitation ◽  
Chi Square ◽  
Developmental Potential ◽  
Pronuclear Formation

The efficiency of intracytoplasmic sperm injection (ICSI) in bovines is lower than in other species. We propose that in vitro sperm capacitation could optimize the ICSI in cattle. The aim was to evaluate the effects of isobutylmethylxanthine (IBMX) and methyl-β-cyclodextrin (MβCD) on the sperm capacitation and in vitro development of embryos generated by ICSI. Frozen-thawed spermatozoa (3–5 × 106 cells mL–1) were pre-incubated for 2 h at 38.5°C, 5% CO2 in defined medium (Sp-TLP/PVA) supplemented with MβCD (1 mM) or IBMX (0.4 mM) (capacitating conditions). The untreated control group (UTG; not supplemented) and vehicle group (VG) were incubated for 2 h. The non-capacitating control group (NCG) was not supplemented (neither vehicle nor IBMX or MβCD) and not incubated. The sperm viability and capacitation {intracellular calcium [Ca2+]i, plasma membrane fluidity (PMF), and acrosomal reaction} were evaluated by flow cytometry (n = 3 biological replicates). For the ICSI procedure, only motile spermatozoa were selected. After ICSI, oocytes were activated with ionomycin + cycloheximide. Culture was performed at 38.5°C, 5% CO2, 5% O2, 90% N2, saturation humidity in KSOM base medium. Data were analysed by ANOVA and Scheffe's test. Pronuclear formation was evaluated by a chi-square test with Bonferroni's correction. Significance was set at P < 0.05. Pretreated spermatozoa showed lower (P < 0.05) viability (49 and 67% for IBMX and MβCD, respectively) compared with the NCG (89%), UTG (80%), and VG (78%). The [Ca2+]I analysed by median fluorescence intensity (MFI) was lower (P < 0.05) in NCG (117 MFI) with respect to UTG (127 MFI), VG (124 MFI), IBMX (126 MFI), and MβCD (131 MFI). The PMF increased (P < 0.05) with IBMX (115 MFI) and MβCD (106 MFI) compared with NCG (70 MFI), UTG (89 MFI), and VG (65 MFI). Acrosome reaction improved with capacitating treatments with respect to both control groups (16, 23, 8, 4, and 3% for IBMX, MβCD, UTG, VG, and NCG, respectively). Analysis of capacitating v. non-capacitating conditions on ICSI efficiency revealed that the fertilization rate, assessed by pronuclear formation, was higher (P < 0.05) in ICSI-MβCD (76%; n = 46) compared with ICSI-IBMX (55%; n = 53) and ICSI-NCG (50%; n = 44). Nevertheless, there were no differences among groups in cleavage (Day 3): 85, 86, and 84% and blastocyst rates (Day 8): 19, 25, and 18% for ICSI-IBMX (n = 8), ICSI-MβCD (n = 7), and ICSI-NCG (n = 7), respectively. The parthenogenetic and sham injection groups yielded a lower rate of cleavage (73 and 53%, respectively) and blastocyst (13% and 10%, respectively). The results demonstrated an improvement of the fertilization rate of bovine embryos generated by ICSI using sperm capacitated by MβCD pretreatment. However, more studies are necessary to improve in vitro developmental potential of these embryos to the blastocyst stage.Frigorífico Temuco and funding support from FONDECYT 1120241 CONICYT-Chile are gratefully acknowledged.

Effect of sperm pretreatment with sodium hydroxide and dithiothreitol on the efficiency of bovine intracytoplasmic sperm injection

2014 ◽  
Vol 26 (6) ◽  
pp. 847 ◽  
Author(s):  
M. E. Arias ◽  
R. Sánchez ◽  
J. Risopatrón ◽  
L. Pérez ◽  
R. Felmer
Keyword(s):  
Embryonic Development ◽  
Intracytoplasmic Sperm Injection ◽  
Membrane Integrity ◽  
Control Group ◽  
Developmental Potential ◽  
Bovine Spermatozoa ◽  
Pronuclear Formation ◽  
First Time ◽  
In Vitro Embryonic Development

The efficiency of intracytoplasmic sperm injection (ICSI) in bovines is lower than in other species due, in part, to a lack of optimal conditions for its implementation; this has hindered the achievement of high rates of embryonic development and the birth of live offspring. The aim of the present study was to evaluate the effects of pretreatment of bovine spermatozoa with NaOH and dithiothreitol (DTT) on the viability, plasma membrane integrity, DNA fragmentation and in vitro developmental potential of embryos generated by ICSI. Following pretreatment of spermatozoa with 5 mM DTT for 20 min and a low concentration of NaOH (1 mM for 60 min), there were fewer live and acrosome reacted spermatozoa (44% and 34%, respectively) than in the control group without treatment (82%). Spermatozoa subjected to higher alkali concentrations (10–50 mM) were mostly dead and reacted. However, pronuclear formation, cleavage, blastocyst rate and embryo quality did not differ between these pretreatment groups and the untreated control group. In conclusion, we have described, for the first time, the effects of NaOH treatment on bovine spermatozoa and subsequent in vitro embryonic development after ICSI, and have demonstrated that pretreatment of bovine spermatozoa with NaOH or DTT is not necessary for an appropriate in vitro embryo development in this species.

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.

scholarly journals Graft site and gonadotrophin stimulation influences the number and quality of oocytes from murine ovarian tissue grafts

Reproduction ◽  
2006 ◽  
Vol 131 (5) ◽  
pp. 851-859 ◽  
Author(s):  
Hsiao Yun Yang ◽  
Shae-Lee Cox ◽  
Graham Jenkin ◽  
Jock Findlay ◽  
Alan Trounson ◽  
...  
Keyword(s):  
Ovarian Tissue ◽  
Germinal Vesicle ◽  
Fertilization Rate ◽  
Ovarian Tissue Cryopreservation ◽  
Control Group ◽  
Developmental Potential ◽  
Oocyte Yield ◽  
Tissue Grafts

Ovarian tissue cryopreservation and subsequent transplantation can restore fertility in cancer patients. This study used a mouse ovarian grafting model to investigate whether the graft site (bursal cavity, the kidney capsule or subcutaneous) influences the number, fertilization rate and developmental potential of oocytes recovered from grafts and whether using a standard gonadotrophin stimulation protocol would increase oocyte yield from the grafts. Mouse ovarian tissue was grafted into four week old mice and collected three weeks later. Graft recipients were treated either with or without exogenous gonadotrophin stimulation prior to graft collection. Grafted ovaries yielded oocytes that were either at the germinal vesicle (GV) stage or mature metaphase II (MII) stage at collection. These GV oocytes were matured beforein vitrofertilization (IVF), while the MII oocytes underwent IVF immediately. Oocytes collected from the oviducts of non-grafted superovulated mice of the same age served as controls. Two-cell embryos were transferred to pseudopregnant recipients and recovered at day 15 of gestation or left to go to term. Graft retrieval and the number of oocytes from each graft were lowest from the subcutaneous graft site. The number of two-cell embryos produced was significantly higher for oocytes from the grafts to the bursa as compared with the other sites. All graft sites gave rise to embryos with comparable implantation rates and developmental potential to fetuses and offspring following transfer. However, the oocytes from grafted ovaries had a significantly lower developmental potential when compared with the control group. Stimulation with exogenous gonadotrophins did not significantly increase oocyte yield from grafted ovaries but did enhance oocyte maturation and development. In conclusion, graft site affects the number and quality of oocytes produced from ovarian grafts.

scholarly journals Hubungan Anti-Mullerian Hormone (AMH) pada Long dan Short Protocol Terhadap Fertilization Rate Pasien In Vitro Fertilization (IVF)

2021 ◽  
Vol 4 (2) ◽  
pp. 151-161
Author(s):  
Rizki Amalia Wahid ◽  
◽  
Edwin Armawan ◽  
ono Djuwantono
Keyword(s):  
In Vitro Fertilization ◽  
Intracytoplasmic Sperm Injection ◽  
Fertilization Rate ◽  
Chi Square ◽  
Vitro Fertilization ◽  
Fertility Clinic ◽  
Long Protocol

Abstrak Tujuan: Untuk mengevaluasi pengaruh kadar anti-mullerian hormone (AMH) dengan fertilization rate (FR) dan menilai perbedaan pengaruh jenis protokol (long protocol (LP) dan short protocol (SP)) pada tiap tingkat cadangan ovarium terhadap FR pada pasien in vitro fertilization (IVF) dengan Intracytoplasmic Sperm Injection. Metode: Data sekunder dari rekam medis pasien yang menjalani IVF di Aster Fertility Clinic Rumah Sakit Umum Pendidikan dr. Hasan Sadikin pada tahun 2016-2020 dan Bandung Fertility Centre Rumah Sakit Ibu Anak Limijati pada tahun 2018-2019. Penelitian ini analitik observational dengan metode Cohort retrospektif. Hubungan antara dua data kategorik diuji dengan uji chi-square dan uji Kruskal-Wallis digunakan pada data numerik dengan distribusi yang tidak rata pada lebih dari 2 kelompok, Hasil: Hasil data diperoleh nilai rerata kadar AMH secara keseluruhan adalah 3.30 ng/ml dengan rerata capaian FR sebesar 71.97%. Berdasarkan metode IVF yang dipilih, mayoritas pasien menjalani pengobatan SP 54.4% (rerata FR 72.80%) dibandingkan dengan LP 45.6% (rerata FR 70.97%). Tidak ditemukan hubungan yang bermakna antara kadar AMH dengan FR, dinyatakan dengan nilai p=0.977. Kadar AMH terhadap FR bila dipisahkan menurut protokol terapi yang diberikan tidak menunjukkan perbedaan yang bermakna pada masing-masing protokol (LP p=0,763; SP p=0,843). Mengenai hubungan antara protokol IVF dengan FR juga tidak diperoleh perbedaan yang signifikan secara statistik dengan nilai p=0,27 (RR 1.17 (0.62-2.15); CI 95%). Penggobatan menggunakan LP (p=0,770) maupun SP (p=0.845) tidak memberikan pengaruh yang bermakna terhadap FR pada setiap kategori AMH. Kesimpulan: Tidak ada pengaruh kadar AMH dan protokol terapi terhadap FR. Kata kunci : In Vitro Fertilization, Fertilization Rate, Anti-Mullerian Hormone, Protokol Stimulasi Ovarium

43 SHORT- AND LONG-LASTING EFFECTS OF TRICHOSTATIN A TREATMENT OF SCNT EMBRYOS IN CATTLE

2011 ◽  
Vol 23 (1) ◽  
pp. 127 ◽  
Author(s):  
I. Lagutina ◽  
R. Duchi ◽  
S. Colleoni ◽  
G. Lazzari ◽  
C. Galli
Keyword(s):  
Trichostatin A ◽  
Blastocyst Stage ◽  
Development Project ◽  
Full Term ◽  
Control Group ◽  
Cloning Efficiency ◽  
Chi Square ◽  
Developmental Potential ◽  
Student’S T

Both preimplantation and full-term development of mouse somatic cell nuclear transfer (SCNT) embryos are significantly enhanced by treatment with trichostatin A (TSA), an inhibitor of histone deacetylase (Kishigami et al. 2006 Biochem. Biophys. Res. Commun. 340, 183–189; Rybouchkin et al. 2006 Biol. Reprod. 74, 1083–1089). The present study was designed to examine the effect of TSA treatment on preimplantation and full-term development of bovine cloned embryos. To investigate the effect of TSA on bovine NT embryos development, we treated them with 50 nM TSA during the first 10 h after activation. Bovine NT-embryos were reconstructed using adult fibroblasts of 2 female donors (A and B) with significantly different in vitro cloning efficiency (respectively, 84/245; 34.3% v. 155/298; 52.1% blastocyst D7, P ≤ 0.05, chi-square test). TSA treatment significantly improved blastocyst rate in A, however did not affect development in B (56.3% and 50.5%, respectively). The level of acetylated histone H3K9 10 h after activation detected by anti-acH3K9 antibody was significantly increased after TSA-treatment in A (P ≤ 0.05, Student’s t-test) but did not change in B, thus demonstrating that the levels of histone acetylation in cloned embryos correlate with their in vitro developmental potential. To evaluate the long-lasting effect of TSA-treatment on the full-term development of cloned embryos, SCNT embryos derived from 4 female donor animals were reconstructed. 196 TSA-treated embryos at the blastocyst stage were transferred into 98 recipients and 2 calves (2%) were born. In the control group, 167 embryos were transferred into 141 recipients and 3 calves (2.1%) were born. Our data show that cell lines demonstrate different susceptibility to TSA that may affect reprogramming of the somatic genome with low level of acetylation resulting in higher in vitro embryo development. However, TSA does not improve overall cloning efficiency in cattle, measured as full-term development. Project partly supported by EU grants Plurisys (n 22348), Xenome (LSHB-CT-2006-037377) and Regione Lombardia.

272 BIRTH OF A CALF AFTER INTRACYTOPLASMIC SPERM INJECTION WITH FROZEN - THAWED EPIDIDYMAL SPERM FROM A POSTMORTEM BULL

2008 ◽  
Vol 20 (1) ◽  
pp. 216
Author(s):  
C. A. Guerrero ◽  
J. Smith ◽  
J. W. Lynn ◽  
K. R. Bondioli ◽  
R. A. Godke
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Polar Body ◽  
Fertilization Rate ◽  
Post Injection ◽  
Oocyte Activation ◽  
Epididymal Sperm ◽  
Bull Calf ◽  
First Polar Body ◽  
Pronuclear Formation

The use of postmortem epididymal sperm for intracytoplasmic sperm injection (ICSI) will allow a more effective use of valuable gametes if a breeding male dies unexpectedly. The objective of this study was to determine pronuclear formation and embryo development rates of frozen–thawed bovine epididymal sperm-injected oocytes. Epididymal sperm were harvested by multiple incisions in the cauda epididymides of an abattoir-derived mature, mixed breed beef bull within 5 h postmortem and frozen in 7% glycerol. Oocytes were matured in vitro for 21 h, selected for extrusion of the first polar body, and centrifuged at 6000g to assist in visualizing the microinjection procedure. Oocytes were injected with either frozen–thawed epididymal sperm, frozen–thawed ejaculated sperm (laboratory control), or were sham-injected (control). Piezo-injected oocytes were chemically activated 4 h post-injection in 7% ethanol for 5 min (Treatment A) or exposure to 5 μm ionomycin for 5 min followed by incubation in 10 μg mL–1 of cycloheximide for 5 h (Treatment B). The sperm-injected oocytes were cultured in CR1aa medium from day 0 to day 3 post-injection and then in CR1aa medium supplemented with 5% fetal bovine serum from day 3 to day 8 of in vitro culture. Pronuclear formation was assessed 18 to 20 h after sperm injection. A summary of oocyte activation by treatments indicated that ethanol was more successful than the ionomycin + cycloheximide treatment (Table 1). Cleavage and blastocyst rates were assessed on day 3 and day 8 of culture, respectively. A significantly higher (P ≤ 0.05) fertilization rate was achieved when ejaculated (43%) rather than epididymal (31%) sperm was used in the ICSI procedure. However, this difference in fertilization rate was only noted when ethanol was used for the exogenous activation. Furthermore, the blastocyst rate for epididymal sperm-injected oocytes was significantly greater when using ethanol (14%) compared with ionomycin followed by cycloheximide (4%). The birth of a live bull calf (42.2 kg; 292-day gestation) resulted from the nonsurgical transfer of 2 ethanol-activated Grade 1 day ICSI blastocysts into each of 2 beef recipient females (50%). To our knowledge, this is the first calf produced by piezo ICSI using cryopreserved bovine caudal epididymal sperm. We can conclude that postmortem epididymal sperm can be collected from genetically valuable males and used for the production of offspring using piezo ICSI. Table 1. Summary of bovine oocyte activation using ethanol and ionomycin + cycloheximide (Iono + Cyclo) treatments

132 TIMING OF THE FIRST ZYGOTIC CLEAVAGE WAS NOT RELATED TO THE SHIFT IN SEX RATIO OF BOVINE BLASTOCYST IN VITRO

2009 ◽  
Vol 21 (1) ◽  
pp. 165
Author(s):  
K. Zaorska ◽  
E. Pers-Kamczyc ◽  
D. Lechniak
Keyword(s):  
Sex Ratio ◽  
Positive Influence ◽  
Control Group ◽  
Fish Analysis ◽  
Chi Square ◽  
Experimental Conditions ◽  
Female Ratio ◽  
Developmental Potential ◽  
Frozen Semen

It has been shown that embryos which cleaved earlier (<30 h postinsemination, hpi) have greater chance to develop to blastocyst. It has been shown that in in vitro conditions male embryos develop faster than females. Further, spermatozoa with the Y chromosome more frequently penetrate oocytes during the first 6 hpi. The aim of this experiment was to investigate the sex-related embryo growth rate in relation to the timing of the first zygotic cleavage and GH presence during IVM. Oocytes were matured in TCM-199 supplemented with fatty acid free BSA and hormones (FSH and GH) and then inseminated (Parrish et al. 1998 Biol. Reprod. 38, 1171–1180), whereas embryos were cultured in sequential media (Lane et al. 2005 Theriogenology 60, 407–419). All embryos that cleaved by 30 hpi (early cleavers, EC) were selected and cultured separately. The remaining embryos cleaved by 48 hpi (non early cleavers, NEC) were also incubated in separate drops. Blastocysts of proper morphology were collected at 176 hpi and subjected to sex determination by PCR (AMGL gene). The significance of developmental stage, timing of the first zygotic cleavage and GH supplementation in relation to the sex ratio was evaluated by the chi-square test. All straws with frozen semen of 2 bulls used in this experiment were derived from single ejaculates. The sex ratio of sperm samples used for IVF was evaluated by FISH with locus-specific probes. The experiment was done on 266 embryos obtained from 1249 oocytes. A significant predominance of male blastocysts regardless of the experimental conditions was observed [the male to female ratio (M:F) 2:1, P < 0.001]. FISH analysis revealed that there was no deviation from the expected 1:1 ratio of X and Y spermatozoa in sperm samples used for IVF. When the timing of the first zygotic cleavage was considered, shift in M:F ratio in favor of males (P < 0.01) in blastocysts derived from EC zygotes was noticed. Due to a very low number of NEC embryos, this category was not included in analysis. Although the M:F ratio was shifted towards males, the rate of female embryos was greater in the control group (25F/38M) v. GH group (16F/39M) of EC expanded blastocysts. This phenomenon was not observed among hatched blastocysts; however, GH presence caused an increase (P < 0.01) in the number of females at this stage. Therefore GH may stimulate embryonic growth, especially embryos of reduced quality, through its positive influence on cytoplasmic oocyte maturation. In conclusion, the predominance of male blastocysts observed in this study may be attributed to the applied IVF procedure because the X:Y ratio in spermatozoa was not different from the expected 1:1 ratio. Moreover, significantly fewer females among analyzed blastocysts may suggest that the developmental potential of female embryos in applied in vitro conditions was somehow reduced when compared with males. This became evident when the transition from expanded to hatched blastocysts was observed. This work was supported by the project No. N302 046 31/3780 of the Ministry of Science and Higher Education, Poland.

Evaluation of the developmental potential of metaphase I oocytes from stimulated intracytoplasmic sperm injection cycles

2011 ◽  
Vol 23 (3) ◽  
pp. 433 ◽  
Author(s):  
Mei Li ◽  
Yuan Li ◽  
Shui-Ying Ma ◽  
Huai-Liang Feng ◽  
Hui-Jun Yang ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Control Group ◽  
High Quality ◽  
Developmental Potential ◽  
Short Period ◽  
Group A ◽  
Group B ◽  
Group D ◽  
Metaphase I

The objective of the present study was to evaluate the developmental potential and clinical application value of metaphase I (MI) oocytes obtained from stimulated intracytoplasmic sperm injection (ICSI) cycles. ICSI was performed on MI oocytes immediately after denudation (Group A), or on in vitro-matured (IVM) oocytes following culture; oocytes in culture were further divided into two groups, being cultured for either 3–5 h (Group B) or 24–28 h (Group C). Metaphase II oocytes from the same cycle(s) isolated for ICSI served as the control group (Group D). The rates of normal fertilisation, cleavage and high-quality embryos were compared among the four groups. High-quality embryos were transferred whenever possible, and pregnancy rates were evaluated. Results showed that normal fertilisation rates for Groups B, C and D were significantly higher than that of Group A (68.6%, 57.8%, 74.5% and 30.1%, respectively; P < 0.01). The rate of high-quality embryos in Group B was comparable with Group D; the rate for Group C was significantly lower than that of the other groups (P < 0.05). Two clinical pregnancies were achieved after transfer of embryos from IVM oocytes. In vitro maturation of MI oocytes for a short period of time may increase the number of available embryos; however, overnight in vitro culture of MI oocytes did not improve results.

scholarly journals 46 Effect of the addition of α-tocopherol to in vitro maturation media of bovine oocytes

2020 ◽  
Vol 98 (Supplement_2) ◽  
pp. 2-3
Author(s):  
Theisy P Acosta Pérez
Keyword(s):  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Control Group ◽  
Chi Square ◽  
Cumulus Expansion ◽  
Minimum Concentration ◽  
Maturation Rate ◽  
Chi Square Test ◽  
System Data

Abstract α-tocopherol is known to be a powerful antioxidant, in this regard, it was added to bovine oocyte in vitro maturation media to evaluate its effect on oocyte maturation. Oocytes (n = 624) aspirated from ovaries of slaughtered cows were classified by quality and divided in four categories according to cytoplasm appearance and cumulus cells layers. Oocytes were washed in TCM-199 supplemented with fetal bovine serum (FBS) and FSH, then distributed in maturation media (TCM-199 supplemented with FBS, FSH and gentamicin). Three experimental groups of α-tocopherol (50, 100 and 200 mM) and a control group without α-tocopherol were used. Maturation was carried 22 h at 38.5°C in a 5% CO2 atmosphere. Oocytes were examined to determine cumulus expansion as categorical data (expansion or no expansion), as well as cumulus expansion Index (CEI). For CEI determination oocytes were graded 0 to 4, being 0 those with null expansion and 4 those with a noticeable cell expansion, then the number of oocytes were multiplied by the grade given and a sum of the totals was obtained, the new total was divided by the total of oocytes in the group and the result obtained corresponded to the CEI of the group. Results were analyzed with Chi Square test (for maturation rates) and an ANOVA (for the CEI) using the SAS system, data are presented as mean ± standard error. There was no statistical difference between control and α-tocopherol groups (P &gt;0.05). Numerically, the control group showed a higher maturation rate (100%) and obtained a higher CEI (2.44±0.20), followed by the 50 mM group (98.16%; 2.39±0.13), the groups 200 mM (97.40%; 2.00±0.14) and 100 mM (96.25%; 2.06±0.24) were the lowest. The addition of the minimum concentration (50 mM) of α-tocopherol to the maturation media could improve maturation rates without exposing oocytes to toxic effects.

Effect of vitrification technique and assisted hatching on rabbit embryo developmental rate

Zygote ◽  
2009 ◽  
Vol 17 (1) ◽  
pp. 57-61 ◽  
Author(s):  
M. Popelková ◽  
Z. Turanová ◽  
L. Koprdová ◽  
A. Ostró ◽  
S. Toporcerová ◽  
...  
Keyword(s):  
Cell Number ◽  
Total Cell ◽  
Control Group ◽  
Assisted Hatching ◽  
Hatching Rate ◽  
Total Cell Number ◽  
Developmental Potential ◽  
Significant Difference ◽  
Rabbit Embryos

SummaryThe aim of the study was to determine the efficiency of two vitrification techniques followed by two assisted hatching (AH) techniques based on post-thaw developmental capacity of precompacted rabbit embryos and their ability to leave the zona pellucida (hatching) during in vitro culture. The total cell number and embryo diameter as additional markers of embryo quality after warming were evaluated. In vivo fertilized, in vitro cultured 8–12-cell rabbit embryos obtained from superovulated rabbit does were cryopreserved by two-step vitrification method using ethylene glycol (EG) as cryoprotectant or by one-step vitrification method with EG and Ficoll (EG+Ficoll). Thawed embryos were subjected to enzymatic or mechanical AH. Vitrified EG group showed significantly lower (P < 0.05) blastocyst rate (22.5%) and hatching rate (15%) than those vitrified with EG + Ficoll (63 and 63% resp.) and that of control (97 and 97% respectively). Significantly lower values of total cell number (P < 0.05) as well as embryo diameter (P < 0.01) in EG group compared with EG + Ficoll and control group were recorded. No significant difference was found in developmental potential of warmed embryos treated by either mechanical or enzymatic AH. The present study demonstrates that the EG + Ficoll vitrification protocol provides superior embryo survival rates over the EG vitrification protocol for 8–12-cell stage precompacted rabbit embryos. No positive effect of either mechanical or enzymatic AH on the post-thaw viability and quality of rabbit embryos in vitro was observed.

Sign in / Sign up

Export Citation Format

Share Document