Prognostic value of first polar body morphology on fertilization rate and embryo quality in intracytoplasmic sperm injection

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.

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First polar body morphology before ICSI is not related to embryo quality or pregnancy rate

Human Reproduction ◽

10.1093/humrep/deh433 ◽

2004 ◽

Vol 19 (10) ◽

pp. 2334-2339 ◽

Cited By ~ 62

Author(s):

P.M. Ciotti ◽

L. Notarangelo ◽

A.M. Morselli-Labate ◽

V. Felletti ◽

E. Porcu ◽

...

Keyword(s):

Pregnancy Rate ◽

Embryo Quality ◽

Polar Body ◽

Body Morphology ◽

First Polar Body

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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 ◽

10.1530/rep.0.1230455 ◽

2002 ◽

pp. 455-465 ◽

Cited By ~ 74

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.

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

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab272 ◽

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 frozenâthawed 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, frozenâthawed 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

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Intracytoplasmic sperm injection: correlation of oocyte grade based on polar body, perivitelline space and cytoplasmic inclusions with fertilization rate and embryo quality

Human Reproduction ◽

10.1093/humrep/12.8.1750 ◽

1997 ◽

Vol 12 (8) ◽

pp. 1750-1755 ◽

Cited By ~ 159

Author(s):

P. Xia

Keyword(s):

Intracytoplasmic Sperm Injection ◽

Embryo Quality ◽

Polar Body ◽

Fertilization Rate ◽

Perivitelline Space ◽

Cytoplasmic Inclusions

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315 EFFICIENCY OF CHEMICAL OR ELECTRICAL ACTIVATION OF BOVINE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab315 ◽

2006 ◽

Vol 18 (2) ◽

pp. 265

Author(s):

M. P. Milazzotto ◽

W. B. Feitosa ◽

R. Simões ◽

C. M. Mendes ◽

M. E. O. A. Assumpção ◽

...

Keyword(s):

Nuclear Transfer ◽

Embryo Quality ◽

Calcium Influx ◽

Polar Body ◽

Chemical Activation ◽

Electrical Activation ◽

Activation Process ◽

Oocyte Activation ◽

Blastocyst Formation ◽

First Polar Body

Activation of in vitro matured oocytes is essential for the success of nuclear transfer embryo production. Oocyte activation is promoted by the release of intracellular calcium and influx of extracellular ions, and can be chemically induced by calcium ionophores such as A23187 (CA) or ionomycin (IO). Electrical stimulation (EL) is an essential stage in nuclear transfer protocols for the fusion of enucleated oocytes with the donor's cell nucleus. Moreover, EL can be used as an alternative method to induce calcium influx through the formation of pores in the plasma membrane. This work aimed to evaluate the effect of electrical pulse vs the use of different calcium ionophores (A23187 or ionomycin) as primary agents of bovine oocyte activation, with or without the addition of BSA, on the rate of blastocyst formation and blastocyst quality. BSA was used to quench the activation process after a 5-min exposure to CA or IO. Cumulus-oocyte complexes were matured in TCM-199 medium with FCS and hormones for 18 h at 38.5�C and 5% CO2 in air. After removal of cumulus cells, oocytes presenting the first polar body were selected and maintained in SOFaa medium to complete 24 h of maturation. They were then divided into five treatments groups 1-CA (CA 5 mM, 5 min); 2-CAB (CA 5 mM, 5 min; BSA, 5 min); 3-IO (IO 5 mM, 5 min); 4-IOB (IO 5 mM, 5 min; BSA, 5 min); and 5-EL (EL 1.5 kV/cm, 20 �s, 2 pulses). After treatments, oocytes were kept in 6-dimethylaminopurine for 3 h and cultured in SOFaa medium for 7 days at 38.5�C and 5% CO2 in air. Rates of cleavage and blastocyst were evaluated respectively on Days 2 and 7 of culture. To evaluate embryo quality, Hoechst 33342/propidium iodide staining was used. Data were evaluated by ANOVA and submitted to LSD test for embryo rates and t-test for embryo quality. Four replicates were carried out with a total of 89 oocytes per treatment. There was a difference (P < 0.05) in rate of development to blastocyst between treatments 1-CA (54.4%a), 3-IO (51.4%a), and 5-EL (54.5%a) compared with 4-IOB (18.3%b). Treatment 2-CAB (39.8%ab) did not show any difference from the others. There was no difference (P > 0.05) among treatments in total number of cells: 1-CA (63.1a), 2-CAB (57.2a), 3-IO (60.9a), 4-IOB (72.4a), and 5-EL (58.4a). However, there was a difference (P < 0.01) in the percentage of viable cells between treatments 1-CA (49.9%a), 2-CAB (45.8%a), 3-IO (64.9%a), and 4-IOB (50.9%a) in comparison to 5-EL (82.7%b). In conclusion, BSA, when associated with IO, had a negative effect on embryonic developmental rates. The different calcium ionophores used and the BSA did not improve embryo quality. Although there were no significant differences between electrical and chemical activation on the rate of blastocyst formation, it is important to point out that higher quality embryos were achieved by using electrical activation. This work was supported by FAPESP 03/00156-9.

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249 INTRACYTOPLASMIC SPERM INJECTION OF ELAND (TAUROTRAGUS ORYX) AND BONGO (TRAGELAPHUS EURYCERUS)ANTELOPE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab249 ◽

2007 ◽

Vol 19 (1) ◽

pp. 241 ◽

Cited By ~ 1

Author(s):

G. Wirtu ◽

C. E. Pope ◽

M. C. Gomez ◽

A. Cole ◽

D. L. Paccamonti ◽

...

Keyword(s):

Embryonic Development ◽

Intracytoplasmic Sperm Injection ◽

Transvaginal Ultrasound ◽

Polar Body ◽

Chemical Activation ◽

Cumulus Cell ◽

Calcium Ionophore ◽

Male Gamete ◽

Assisted Reproductive Technique ◽

First Polar Body

Intracytoplasmic sperm injection (ICSI) is an assisted reproductive technique applicable in cases of limited male gamete availability. Moreover, it bypasses barriers of the oocyte, thus avoiding poorly understood species-specific capacitation events affecting sperm–egg interaction. In the present study, we evaluated the application of conventional and piezo drill-assisted ICSI and whether subsequent chemical activation is required for initiating embryonic development in eland (Taurotragus oryx) and bongo (Tragelaphus eurycerus) oocytes. Oocytes were collected using transvaginal ultrasound-guided follicular aspiration after gonadotropin-induced ovarian stimulation and incubated in modified TCM-199 medium (Gomez et al. 2000 Reprod. Fertil. Dev. 12, 423) containing 10% FBS. After 3 to 24 h, the cumulus cell layers were removed either by repeated mouth-pipetting and/or by using hyaluronidase. Oocytes with an extruded first polar body were used for ICSI and the other oocytes were returned to culture and evaluated every six hours Piezo drill-assisted (Kimura and Yanagimachi 1995 Biol. Reprod. 52, 709) or conventional (Gomez et al.) ICSI were done as described previously using glass pipettes with internal tip diameters of 9–10 µm. We used frozen–thawed or freshly collected spermatozoa that were kept in HEPES-buffered Tyrode's medium (Gomez et al.) for up to 24 h. Four to 6 h after ICSI, 3 activation treatments were examined: (1) none; (2) 7% ethanol, 5 min; or (3) calcium ionophore (5 µM, 5 min) followed by DMAP (2 mM, 4 h). Then we cultured oocytes in a humidified atmosphere of 5% O2, 5% CO2, and 90% N2 at 38.5°C in one of 3 media: SOF, α-MEM, or CR1aa containing essential and nonessential amino acids and FBS. Fifty-three of 70 (76%) eland oocytes survived after piezo-ICSI, and 13 of 16 (81%) survived after conventional ICSI. For bongo oocytes, 27 of 30 (90%) survived piezo-ICSI and all (n = 8) survived after conventional ICSI. Table 1 outlines cleavage data on Day 2. Generally, embryonic development was arrested at about 10 cells. In summary, eland and bongo oocytes can survive both conventional and piezo drill-assisted ICSI. Activation treatments do not appear to be a prerequisite for initiating cleavage after ICSI in eland and bongo antelope oocytes. Table 1.Cleavage of eland and bongo antelope oocytes after conventional or piezo-ICSI and three activation treatments

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