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

Effects of oocyte age, cumulus cells and injection methods on in vitro development of intracytoplasmic sperm injection rabbit embryos

Zygote ◽  
2004 ◽  
Vol 12 (1) ◽  
pp. 75-80 ◽  
Author(s):  
Yue-Liang Zheng ◽  
Man-Xi Jiang ◽  
Yan-Ling Zhang ◽  
Qing-Yuan Sun ◽  
Da-Yuan Chen
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Polar Body ◽  
Cumulus Cells ◽  
Fertilization Rate ◽  
First Polar Body ◽  
Injection Methods ◽  
Blastocyst Rate ◽  
Repeated Aspiration ◽  
Rabbit Embryos

This study assessed the effects of oocyte age, cumulus cells and injection methods on in vitro development of intracytoplasmic sperm injection (ICSI) rabbit embryos. Oocytes were recovered from female rabbits superovulated with PMSG and hCG, and epididymal sperm were collected from a fertile male rabbit. The oocyte was positioned with the first polar body at 12 o'clock position, and a microinjection needle containing a sperm was inserted into the oocyte at 3 o'clock. Oolemma breakage was achieved by aspirating ooplasm, and the aspirated ooplasm and sperm were re-injected into the oocyte. The injected oocytes were cultured in M199 medium containing 10% fetal calf serum at 38 °C with 5% CO2 in air. The results showed that oocytes injected at 1 h post-collection produced a higher (p<0.05) fertilization rate than those injected at 4 or 7 h post-collection. Blastocyst rate in the 1 h group was higher (p<0.05) than in the 7 h group. Denuded oocytes (group A) and oocytes with cumulus cells (group B) were injected, respectively. Rates of fertilization and development of ICSI embryos were not significantly different (p<0.05) between the two groups. Four ICSI methods were applied in this experiment. In methods 1 and 2, the needle tip was pushed across half the diameter of the oocyte, and oolemma breakage was achieved by either a single aspiration (method 1) or repeated aspiration and expulsion (method 2) of ooplasm. In methods 3 and 4, the needle tip was pushed to the oocyte periphery opposite the puncture site, and oolemma breakage was achieved by either a single aspiration (method 3) or repeated aspiration and expulsion (method 4) of ooplasm. Fertilization rate in method 2 was significantly higher (p<0.05) than in methods 1 and 3. Blastocyst rates were not significantly different (p<0.05) among methods 1, 3 and 4, but method 2 produced a higher (p<0.05) blastocyst rate than method 3.

scholarly journals Developmental competence in vivo and in vitro of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection with fresh or frozen-thawed spermatozoa

Reproduction ◽  
2002 ◽  
pp. 455-465 ◽  
Author(s):  
YH Choi ◽  
CC Love ◽  
LB Love ◽  
DD Varner ◽  
S Brinsko ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Polar Body ◽  
Cumulus Cells ◽  
Fertilization Rate ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
First Polar Body ◽  
Bovine Oocytes

This study was undertaken to evaluate the development of equine oocytes in vitro and in vivo after intracytoplasmic sperm injection (ICSI) with either fresh or frozen-thawed spermatozoa, without the use of additional activation treatments. Oocytes were collected from ovaries obtained from an abattoir and oocytes classified as having expanded cumulus cells were matured in M199 with 10% fetal bovine serum and 5 microU FSH ml(-1). After 24-26 h of in vitro maturation, oocytes with a first polar body were selected for manipulation. Fresh ejaculated stallion spermatozoa were used for the experiment after swim-up for 20 min in sperm-Tyrode's albumen lactate pyruvate. Frozen-thawed spermatozoa from the same stallion were treated in a similar way. Spermatozoa were immobilized and injected into the oocytes using a Piezo drill. Presumptive zygotes were cultured in G1.2 medium for 20 or 96 h after the injection was administered, or were transferred to the oviducts of recipient mares and recovered 96 h later. In addition, bovine oocytes with first polar bodies were injected with the two types of stallion spermatozoa and fixed 20 h after injection to examine pronuclear formation. Fertilization rate (pronucleus formation and cleavage) at 20 h after injection of spermatozoa was not significantly different between fresh and frozen-thawed sperm groups in either equine or bovine oocytes. Pronucleus formation after injection of spermatozoa into bovine oocytes was significantly higher than that for equine oocytes (P < 0.05). There were no significant differences in cleavage rate or average number of nuclei at 96 h between equine oocytes injected with fresh or frozen-thawed spermatozoa. However, embryos developed in vivo for 96 h had a significantly higher number of nuclei in both sperm treatments compared with those cultured in vitro. These results indicate that good activation rates may be obtained after injection of either fresh or frozen-thawed equine spermatozoa without additional activation treatment. Injection of frozen-thawed equine spermatozoa results in similar embryo development to that obtained with fresh equine spermatozoa. In vitro culture of equine zygotes in G1.2 medium results in a similar cleavage rate but reduced number of cells compared with in vivo culture within the oviduct. Bovine oocytes may be useful as models for assessing sperm function in horses.

Different activation treatments for successful development of bovine oocytes following intracytoplasmic sperm injection

Zygote ◽  
2003 ◽  
Vol 11 (1) ◽  
pp. 69-76 ◽  
Author(s):  
S.A. Ock ◽  
J.S. Bhak ◽  
S. Balasubramanian ◽  
H.J. Lee ◽  
S.Y. Choe ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Chromosomal Abnormality ◽  
Polar Body ◽  
Ultrastructural Changes ◽  
Time Interval ◽  
Oocyte Activation ◽  
Group 4 ◽  
Group 5 ◽  
Bovine Oocytes

In this study, the developmental capacity and cytogenetic composition of different oocyte activation protocols was evaluated following intracytoplasmic sperm injection (ICSI) of in vitro matured bovine oocytes. Motile spermatozoa selected by Percoll density gradient were treated with 5 mM dithiothreitol (DTT) and analysed for ultrastructural changes of the head using transmission electron microscopy (TEM). The alterations in sperm morphology after DTT treatment for different times (15, 30 and 60 min) were 10%, 45-55% and 70-85%, respectively. Further, a partial decondensation of sperm heads was observed after DTT treatment for 30 min. Oocytes were injected with sperm treated with DTT for 30 min. In group 1, sperm injection was performed without any activation stimulus to the oocytes. In group 2, sham injection without sperm was performed without activating the oocytes. Oocytes injected with sperm exposed to 5 μM ionomycin for 5 min (group 3), 5 μM ionomycin + 1.9 mM dimethylaminopurine (DMAP) for 3 h (group 4) and 5 µM ionomycin + 3 h culture in M199 + 1.9 mM DMAP (group 5) were also evaluated for cleavage, development and chromosomal abnormality. Cleavage and development rates in groups 1, 2 and 3 were significantly (p < 0.05) lower than those in groups 4 and 5. The incidence of chromosomal abnormality in the embryos treated directly with DMAP after ionomycin (group 4) was higher than in group 5. We conclude that immediate DMAP treatment after ionomycin exposure of oocytes results in arrest of release of the second polar body, and thus leads to changes in chromosomal pattern. Therefore, the time interval between ionomycin and DMAP plays a crucial role in bovine ICSI.

74 IN VIVO SURVIVAL OF DOMESTIC CAT OOCYTES AFTER VITRIFICATION, INTRACYTOPLASMIC SPERM INJECTION, AND TRANSFER TO RECIPIENTS

2008 ◽  
Vol 20 (1) ◽  
pp. 118 ◽  
Author(s):  
M. C. Gómez ◽  
N. Kagawa ◽  
C. E. Pope ◽  
M. Kuwayama ◽  
S. P. Leibo ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Polar Body ◽  
Oocyte Retrieval ◽  
Cumulus Cells ◽  
Domestic Cat ◽  
Minimal Amount ◽  
Vitro Fertilization ◽  
First Polar Body

The ability to cryopreserve female gametes efficiently holds immense economic and genetic implications. The purpose of the present project was to determine if domestic cat oocytes could be cryopreserved successfully by use of the Cryotop method. We evaluated (a) cleavage frequency after in vitro fertilization (IVF) v. intracytoplasmic sperm injection (ICSI) of in vivo- and in vitro-matured oocytes after vitrification, and (b) fetal development after transfer of resultant embryos into recipients. In vivo-matured cumulus–oocyte complexes (COCs) were recovered from gonadotropin-treated donors at 24 h after LH treatment, denuded of cumulus cells, and examined for the presence of the first polar body (PB). In vitro-matured COCs were obtained from ovaries donated by local clinics and placed into maturation medium for 24 h before cumulus cells were removed and PB status was determined. Oocytes were cryopreserved by the Cryotop method (Kuwayama et al. 2005 Reprod. Biomed. Online 11, 608–614) in a vitrification solution consisting of 15% DMSO, 15% ethylene glycol, and 18% sucrose. For IVF, oocytes were co-incubated with 1 � 106 motile spermatozoa mL–1 in droplets of modified Tyrode's medium in 5% CO2/air at 38�C (Pope et al. 2006 Theriogenology 66, 59–71). For ICSI, an immobilized spermatozoon was loaded into the injection pipette, which was then pushed through the zona pellucida into the ooplasm. After a minimal amount of ooplasm was aspirated into the pipette, the spermatozoon was carefully expelled, along with the aspirated ooplasm. After ICSI, or at 5 or 18 h post-insemination, in vivo- and in vitro-matured oocytes, respectively, were rinsed and placed in IVC-1 medium (Pope et al. 2006). As assessed by normal morphological appearance after liquefaction, the survival rate of both in vivo- and in vitro-matured oocytes was >90% (93–97%). For in vitro-matured oocytes, cleavage frequencies after IVF of control and vitrified oocytes were 73% (16/22) and 53% (30/57), respectively, as compared to 68% (19/28) after ICSI of vitrified oocytes (P > 0.05). For in vivo-matured oocytes, cleavage frequencies after IVF of control and vitrified oocytes were 55% (18/33) and 35% (6/17), respectively, compared to 50% (10/20) after ICSI of vitrified oocytes (P > 0.05). At 18–20 h after ICSI, 18 presumptive zygotes and four 2-cell embryos derived from vitrified in vitro-matured oocytes and 19 presumptive zygotes produced from seven in vivo-matured and 12 in vitro-matured vitrified oocytes were transferred by laparoscopy into the oviducts of two recipients at 24–26 h after oocyte retrieval. The two recipients were 9-month-old IVF/ET-derived females produced with X-sperm sorted by flow cytometry. At ultrasonography on Day 22, both recipients were pregnant, with three live fetuses observed in one recipient and one live fetus seen in the second recipient. On Day 63 and Day 66 of gestation, four live kittens were born, without assistance, to the two recipients. The one male and three female kittens weighed an average of 131 g. In summary, in vivo viability of zygotes/embryos produced by ICSI of cat oocytes vitrified by the Cryotop method was demonstrated by the birth of live kittens following transfer to recipients.

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.

44 EFFECT OF CYTOPLAST VOLUME ON FERTILIZATION AND EMBRYO DEVELOPMENT IN PORCINE M-II OOCYTES RECONSTRUCTED WITH KARYOPLASTS AND CYTOPLASTS OBTAINED BY THE 'CENTRI-FUSION' METHOD

2008 ◽  
Vol 20 (1) ◽  
pp. 102
Author(s):  
N. Maedomari ◽  
K. Kikuchi ◽  
M. Fahrudin ◽  
N. Nakai ◽  
M. Ozawa ◽  
...  
Keyword(s):  
Polar Body ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
First Polar Body ◽  
Developmental Capacity ◽  
Sperm Penetration ◽  
Pronuclear Formation ◽  
And Control

Metaphase-II chromosome transfer (M-II transfer) of oocytes is considered to be one of the advanced procedures to improve fertilization and developmental abilities of oocytes with poor cytoplasmic maturation. The aim of this study was to investigate the developmental capacity after IVF and IVC of porcine oocytes reconstructed from karyoplasts and cytoplasts produced by centri-fusion (Fahrudin et al. 2007 Cloning Stem Cells 9, 216–228). In brief, IVM oocytes (Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041) with a visible first polar body were centrifuged at 13 000g for 9 min to stratify the cytoplasm. Then the zonae pellucidae were removed with pronase treatment. Zona-free oocytes were layered on a 300-µL discontinuous gradient of Percoll in TCM-HEPES with 5 µg mL–1 of cytochalasin B. After centrifugation at 6000g for 4 s, fragmented cytoplasms with approximately equal volumes were obtained, stained with Hoechst-33342, and classified into cytoplasm with (K; karyoplast) or without (C; cytoplast) chromosomes. One karyoplast was fused with 0, 1, 2, 3, and 4 cytoplasts (K, K + 1C, K + 2C, K + 3C, and K + 4C, respectively) by an electric stimulation with a single DC pulse (1.5 kV cm–1 for 20 µs) and cultured for 1 h. Zona-free oocytes without any reconstruction served as control oocytes. The diameters of the reconstructed and control oocytes were measured. All specimens were fertilized in vitro with frozen–thawed boar sperm, and cultured using the well of the well (WOW) system (Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–264). Their fertilization status and developmental competence were examined. Data were analyzed by ANOVA followed by Duncan's multiple range tests. The diameter differed significantly among K to K + 4C oocytes (75.0–127.1 µm; P < 0.05), whereas the diameter of K + 2C oocytes was similar to that of the control oocytes (110.5 µm). Regardless of the cytoplast volume, sperm penetration rates (73.1–93.8%) for K to K + 4C oocytes were not significantly different compared to control oocytes (78.0%). Male pronuclear formation rates of K to K + 4C oocytes (92.3–97.1%) were also not different significantly different compared to control oocytes (96.6%). However, monospermy rates of K oocytes was significantly higher (61.6%; P < 0.05) than those of the reconstructed (K + 1C to K + 4C; 18.2–34.9%) and control oocytes (32.9%). The blastocyst formation rates in K, K + 1C, K + 2C, and K + 3C groups (0.0–9.8%; P < 0.05) were significantly lower than those in the control and K + 4C groups (17.8% and 15.3%, respectively; P < 0.05). The total cell numbers per blastocyst in K + 1C and K + 2C groups (7.5 and 8.3 cells, respectively) were significantly lower than in the control, K + 3C, and K + 4C groups (15.3–26.2 cells; P < 0.05). These results suggest that the cytoplast volume of porcine M-II transferred oocytes, produced by reconstruction from a karyoplast and cytoplast(s) and centri-fusion, is important for their ability to develop to the blastocyst stage and influences cell number.

230 ACTIVATION OF IN VITRO-MATURED BOVINE OOCYTES WITH IONOMYCIN AND ROSCOVITINE AFTER INTRACYTOPLASMIC SPERM INJECTION

2008 ◽  
Vol 20 (1) ◽  
pp. 194
Author(s):  
C. B. Fernandes ◽  
L. G. Devito ◽  
L. R. Martins ◽  
T. S. Rascado ◽  
F. C. Landim-Alvarenga
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Kinase Inhibitor ◽  
Polar Body ◽  
Sperm Concentration ◽  
Oocyte Activation ◽  
Cleavage Rate ◽  
Sperm Suspension ◽  
Artificial Activation ◽  
Bovine Oocytes

In all mammalian species studied so far, fertilization induces oocyte activation necessary for pronuclear formation, syngamy, and the beginning of embryonic cleavage. The aim of this experiment was to evaluate the effectiveness of a protocol for artificial activation for bovine oocytes using ionomycin and roscovitine either in combination with intracytoplasmic sperm injection (ICSI) or alone. In this study, ionomycin was used to facilitate the increase of intracellular calcium, due to the release of calcium from intracellular stores. This compound was used in conjuction with roscovitine, a specific cdc2 kinase inhibitor. The success of the treatment was compared with that of oocytes fertilized by IVF. Three replicates were carried out using bovine oocytes harvested from slaughterhouse ovaries. In vitro-matured oocytes were cultured in TCM-199 plus 10% FCS, pyruvate, estradiol, hCG, and gentamicin at 39�C in an atmosphere of 5% of CO2 in air for 20 h. After in vitro maturation, oocytes were divided into 3 groups. For parthenogenetic activation, 100 oocytes were stripped of cumulus cells and placed in H-MEM plus 10% FCS and 5 µm ionomycin for 8 min, maintained in H-MEM plus 10% FCS, 66 mm roscovitine and 7.5 mg mL–1 cytochalasin B for 6 h, and placed into culture. In the ICSI group, oocytes were denuded and transferred to 5-µL H-MEM plus 20% FCS drops. Only MII oocytes were microinjected. The sperm drop was prepared with a mixture of 4 µL polyvinylpyrrolidone (PVP) and 1 µL of the sperm suspension produced by Percoll gradient. For injection, a single normal mobile sperm was aspirated with the tail first. A single oocyte was fixed by holding the pipette to position the polar body at the 6 or 12 o'clock position. The injection pipette was pushed through the zona pellucida and the oolema and the spermatozoan was released into the cytoplasm. After ICSI, the oocytes were subjected to the same activation protocol described earlier and cultured. For IVF, sperm was prepared by swim-up and 100 oocytes were fertilized in Fert-Talp for 18 h (sperm concentration: 1 � 106). All oocytes were cultured in HTF:BME plus 0.6% BSA, 10% FCS, 0.01% myoinositol, and gentamycin at 39�C in an atmosphere of 5% of CO2 in air for 72 h. Cleavage was evaluated visually and the embryos were stained with Hoechst 33342 for estimation of nuclei numbers. The data were analyzed by ANOVA, followed by the Tukey test (P < 0.05). The results showed a cleavage rate of 76% for the IVF group, 57% for the ICSI group, and 51% for the parthenogenic group. The artificial activation proposed was efficient in inducing oocyte activation and cleavage; however, the rates obtained were significantly lower then the ones observed after IVF. Injection of a viable sperm into the oocyte through ICSI did not improve the cleavage rate after activation. This result indicates that the membrane fusion and/or sperm interaction with the oocyte during fertilization is important for the physiological modifications that result in oocyte cleavage in bovine.

6 EQUINE SPERM INDUCES PRONUCLEAR FORMATION BY INTRACYTOPLASMIC SPERM INJECTION IN BOVINE, SWINE, AND FELINE OOCYTES INDEPENDENTLY OF CHEMICAL ACTIVATION ASSISTANCE

2015 ◽  
Vol 27 (1) ◽  
pp. 95
Author(s):  
M. B. Rodríguez ◽  
A. Gambini ◽  
R. J. Bevacqua ◽  
D. F. Salamone
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Electric Pulse ◽  
Polar Body ◽  
Chemical Activation ◽  
In Vitro Production ◽  
Vitro Fertilization ◽  
Chi Squared ◽  
Second Polar Body ◽  
Pronuclear Formation

Interspecific intracytoplasmic sperm injection (ICSI) is a valuable tool to study early events of fertilization in species for which oocyte availability is reduced. Equine in vitro fertilization remains unsuccessful and ICSI is the technique of choice for the in vitro production of high-value embryos. Therefore, the objective of this study was to evaluate the rate of pronuclear (PN) formation after ICSI with stallion sperm in bovine, swine and feline oocytes with or without chemical activation assistance. Ovaries from cows and pigs were collected at abattoirs whereas gonads from female domestic cats were obtained from ovariectomized animals at veterinary sterilization centers. Cumulus-oocyte complexes were matured in TCM-199 supplemented following standard protocols for each species. ICSI was performed in 100-μL drops of TALP-HEPES, using frozen-thawed semen from one stallion. Spermatozoa were held separate in 3-μL droplets of 7% (vol/vol) polyvinylpyrrolidone, where one of them was immobilized by swiping the injection pipette across its tail, and then injected into the matured oocyte. After ICSI, some oocytes were chemically activated with 5 μM ionomycin for 4 min (cow and cat) or with an electric pulse (sow) followed by 3 h in culture medium to allow extrusion of the second polar body and then exposure to 1.9 mM 6-DMAP solution for 3 h. Embryos were cultured in SOF medium. After 17 h of culture, embryos were stained with propidium iodide to identify the percentage of oocytes activated and with PN. Haploid and diploid parthenogenetic controls were included. Cleavage (48 h after activation) and blastocyst formation (7–8 days) of the partenogenetic control groups were assessed. There were no statistical differences (chi-squared analysis) in PN formation between the activated and nonactivated groups within species. When the activated group was compared between the different species, no differences were observed. However, for the nonactivated group, significant differences were observed between species. The feline oocyte showed the higher percentage of PN and activation, whereas the bovine oocyte exhibited the lower rate of PN formation (cat: 22/27, 81.48%; swine: 19/39, 71.64%; cow:18/63, 43.07%). Our results suggest that the feline oocyte can be used as model to study fertilization events associated with the stallion sperm due to the higher efficiency in supporting PN formation. Our results indicate that the equine sperm is capable of inducing PN formation in these 3 species without further chemical activation assistance.

scholarly journals 304INFLUENCE OF ELECTRICAL STIMULUS ON ACTIVATION OF PIG OOCYTES MATURED IN VITRO

2004 ◽  
Vol 16 (2) ◽  
pp. 271
Author(s):  
Chang Sik Park ◽  
Dong Il Jin ◽  
Young June Chang ◽  
Moon Young Kim ◽  
Young Joo Yi
Keyword(s):  
Acetic Acid ◽  
Polar Body ◽  
Stimulus Frequency ◽  
Electrical Stimulus ◽  
Electrical Activation ◽  
Penicillin G ◽  
Oocyte Activation ◽  
Activation Rate ◽  
First Polar Body

Electrically induced activation of pig oocytes deserves particular attention for research on parthenogenesis. The aim of this study was to improve electrical activation of in vitro matured pig oocyte. The medium used for oocyte maturation was TCM-199 supplemented with 26.19mM sodium bicarbonate, 0.9mM sodium pyruvate, 10μgmL−1 insulin, 2μgmL−1 vitamin B12, 25mM HEPES, 10μgmL−1 bovine apotransferrin, 150μM cysteamine, 10IUmL−1 PMSG, 10IUmL−1 hCG, 10ngmL−1 EGF, 0.4% BSA, 75μgmL−1 sodium penicillin G, 50μgmL−1 streptomycin sulfate and 10% pFF. After about 22h of maturation, oocytes were cultured without cysteamine and hormones for 22h at 38.5°C, 5% CO2 in air. Cumulus-free oocytes involving first polar body were selected for activation. For electrical activation, oocytes were rinsed twice in 0.3M mannitol solution supplemented with 0.1mM CaCl2, 0.2mMMgCl2, 0.5mM HEPES and 0.01% BSA, and transferred to a chamber consisting of two electrodes 1mm apart which were overlaid with the same activation solution. Experiment 1 was conducted to investigate the effect of electrical pulse on oocyte activation. Oocytes were activated with DC pulses of 1.0, 1.5, 2.0 and 2.5kVcm−1 for 30, 60 and 90μs, respectively. Experiment 2 was carried out to investigate the effect of electrical stimulus frequency on oocyte activation. Oocytes were activated one, two and three times, with a DC pulse of 1.0kVcm−1 for 60μs. After activation, oocytes were transferred into 500μL NCSU-23 culture medium containing 0.4% BSA and cultured for 20h. Activated oocytes were fixed for 48h in 25% acetic acid (v:v) in ethanol at room temperature, and stained with 1% orcein (w:v) in 45% acetic acid (v:v) to examine pronucleus formation. Data were analyzed by ANOVA and Duncan’s multiple range test using the SAS program. The rate of activation was highest in the DC pulse of 1.0kVcm−1 for 60μs (75.1%) compared with the other durations and strengths (62.5–63.1%). Activation rate by electrical stimulus frequency was highest (76.0%) when oocytes were activated by a one-time pulse. In conclusion, the results suggested that electrical stimulus with a single DC pulse of 1.0kVcm−1 for 60μs might be more efficient than other strengths and durations for activation of pig oocytes.

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