Effect of vitrification of in vitro matured prepubertal goat oocytes on embryo development after parthenogenic activation and intracytoplasmic sperm injection

This work studies the effect of vitrification of in vitro matured (IVM) prepubertal goat oocytes on: 1) oocyte damage assessed by reactive oxygen species (ROS) level and apoptosis and 2) embryo development after Intracytoplasmic sperm injection (ICSI) and Parthenogenic Activation (PA). Oocytes were IVM in supplemented TCM-199 for 22–24 h. Control group oocytes matured during 24 h were directly used for the analysis after IVM. Vitrified/warmed IVM-oocytes were vitrified after 22 h of IVM in 15% ethylene glycol (EG), 15% dimethyl sulfoxide (Me₂SO) and 0.5 M sucrose and after subjected to warming procedure. Oocyte ROS level was measured by staining denuded IVM-oocytes with 10 μM 2′7′ dichlorodihydrofluorescein diacetate. Apoptosis was analyzed by Annexin V (AV) Apoptosis Detection kit and Propidium iodide (PI) signal and oocytes were classified as: Live (AV⁻ PI⁻), early apoptotic (AV⁺ PI⁻), dead non-apoptotic (AV⁻ PI⁺) and necrotic (AV⁺ PI⁺). Developmental competence of vitrified/warmed oocytes was assessed by PA (5 min in 5 μM Ionomycin plus 4 h in 2 mM 6-Dimethylaminopurine), and by ICSI fertilization. Presumptive zygotes were in vitro cultured for 8 days in commercial media BO-IVC. Vitrified/warmed oocytes showed higher ROS levels (P < 0.0001), lower live oocytes (44 vs. 66%; P: 0.0025) and higher dead non-apoptotic oocytes (33 vs. 13% P: 0.023) compared to control. No differences were found on normal zygote formation (2 PN) (32 vs. 25%) or blastocyst development (0 vs. 4%) after ICSI fertilization. However, after PA, significant differences were found in cleavage rate (59 vs.78%; P < 0.0343) and blastocyst formation (1 vs. 25%; P < 0.0001). In conclusion, vitrification reduced oocyte competence by increasing dead oocytes and ROS levels.     

Viable piglets generated from porcine oocytes matured in vitro and fertilized by intracytoplasmic sperm head injection

Intracytoplasmic sperm injection (ICSI) of a nonmotile cell into the ooplasm for assisted fertilization is a highly specialized procedure for producing the next generation. The production of piglets by ICSI has succeeded when in vivo-matured oocytes have been used as recipients. Our objective was to generate viable piglets by using porcine oocytes matured in vitro and fertilized by ICSI after evaluating the efficacy of using donor spermatozoa in which the acrosome had been artificially removed by treatment with calcium ionophore A23187 (Ca-I). The rate of acrosomal loss in spermatozoa was increased significantly as the duration of treatment with 10 micro M Ca-I was prolonged for 30-120 min (Ca-I treated; 55.6-78.6%), whereas the rate was not different as the duration of incubation without Ca-I was prolonged for 30-120 min (control; 45.3-58.4%). On the sixth day of in vitro culture after injection of the sperm head and subsequent stimulation with an electrical pulse, the rates of blastocyst formation were not significantly different between the two groups: the rates for oocytes injected with Ca-I-treated sperm heads (incubated for 120 min) and for those injected with control sperm heads were 8.6% and 4.0%, respectively. The mean cell numbers of the blastocysts were not significantly different between the two groups (25.6 and 22.7, respectively). Within 2 h after the stimulation, the injected oocytes were transferred to estrous-synchronized recipients. The three recipients that received oocytes injected with Ca-I-treated sperm heads (77-150 oocytes per recipient) were not pregnant, whereas two of the four recipients given oocytes injected with control sperm heads (55-100 oocytes per recipient) were pregnant. One of these farrowed three (a male and two female) healthy piglets. The results demonstrate clearly that in vitro-matured oocytes injected with sperm heads are developmentally competent and can produce viable piglets. They also suggest that removal of the acrosome from the spermatozoon before injection does not affect the development of the blastocyst in vitro. This might not also improve the production of piglets in vivo.     

Offspring derived from intracytoplasmic injection of transgenic rat sperm

The objective of the present study was to produce rat offspring by intracytoplasmic sperm injection (ICSI) using a Piezo-driven micromanipulator. Transgenic male rats carrying a green fluorescent protein gene (GFP: homozygous) were used as sperm donors. The epididymal spermatozoa were suspended and sonicated in m-KRB medium and were frozen in the same medium at –20°C until use. When the sperm heads were aspirated into injection pipettes 7–10m in diameter and introduced into oocytes from the Wistar strain, no offspring resulted from the transfer of 59 eggs. In contrast, the sperm heads were hung on the tip of injection pipettes 2–4m in diameter and introduced into the oocytes, use of Piezo resulting in the production of 18 transgenic offspring carrying the GFP gene from 181 eggs transferred. The oocytes from the Sprague–Dawley strain also supported full-term development following ICSI with three offspring resulting from 163 transferred eggs. In an additional ICSI trial, spermatozoa from infertile transgenic rats carrying human lactalbumin with the thymidine kinase gene (LAC3: heterozygous) were used. The spermatozoa of the LAC3 transgenic rats appeared to be defective and immotile because of the expression of thymidine kinase in the testes, and no ICSI offspring resulted from 218 transferred eggs. These results suggest that ICSI is applicable in rats when Piezo-driven smaller pipettes are used to inject sperm heads together with a limited amount of the surrounding medium and that the ability of isolated sperm heads to participate in normal embryo development is maintained under the cryopreservation conditions employed.     

Effects of oocyte activation and sperm preparation on the development of porcine embryos derived from in vitro-matured oocytes and intracytoplasmic sperm injection

The objective was to determine the effects of various methods of oocyte activation and sperm pretreatment on development of porcine embryos derived from in vitro-matured oocytes and intracytoplasmic sperm injection (ICSI). The second polar body was extruded in the majority (>78.4%) of in vitro-matured (IVM) oocytes 4h after electrical pulse activation. In embryos generated by ICSI and sham-ICSI, a combination of an electrical pulse, with various chemical activators 4 h later, improved (P < 0.05) blastocyst formation rate compared to activation only with a pulse. Treatment with 6-dimethylaminopurine (DMAP) after electrical activation significantly increased the oocyte activation rate. The effects of exposure of sperm to repeated freeze-thaw cycles (without cryoprotectant) on oocyte activation and the effects of sperm pre-incubated with dithiothreitol (DTT) or Triton X-100 on early embryo development were also examined. Blastocyst formation rates after ICSI did not differ between motile sperm and those rendered immotile by one-time freezing and thawing without cryoprotectant. However, sperm rendered immotile by three cycles of freezing/thawing without cryoprotectant had a significantly lower blastocyst formation rate. Although oocytes injected with sperm pre-incubated with Triton X-100 had a higher normal fertilization rate than those pre-incubated with DTT or one-time frozen/thawed sperm, rates of blastocyst formation and cell numbers were similar among the three groups. In conclusion, various methods of oocyte activation and sperm preparation significantly affected the developmental capacity of early porcine embryos derived from IVM and ICSI.     

Viable rabbits derived from reconstructed oocytes by germinal vesicle transfer after intracytoplasmic sperm injection (ICSI)

Abnormal oocyte spindle due to the improper function of ooplasm is associated with female infertility of advanced maternal age. A possible way to overcome this problem is to transfer an oocyte germinal vesicle (GV) which contains genetic materials of a patient with a history of poor embryo development to the cytoplast from a donor oocyte. Here we demonstrate that GV transfer is feasible using a rabbit model. When the GVs were transferred to auto- or hetero-cytoplasts of GV stage oocytes, around 80% of the reconstructed oocytes could mature in vitro and 7.1-9.4% of the oocytes developed to blastocyst stage after intracytoplasmic sperm injection (ICSI). Transfer of 93 fertilized eggs reconstructed via GV transfer into six recipients resulted in two live offspring. Results of this experiment indicate that GV transfer can potentially become a new approach in treatment of infertility because of advanced maternal age.     

In vitro development of vitrified buffalo oocytes following parthenogenetic activation and intracytoplasmic sperm injection

The objective of this study was to investigate the potential of swamp buffalo oocytes vitrified-warmed at the metaphase of the second meiotic cell division (M-II) stage to develop to the blastocyst stage after parthenogenetic activation (PA) or intracytoplasmic sperm injection (ICSI). In Experiment 1, we examined the effects of exposure time of oocytes to cryoprotectants (CPA) on their in vitro development after PA. In vitro matured (IVM) oocytes were placed in 10% dimethylsulfoxide (DMSO) + 10% ethylene glycol (EG) for 1 min and then exposed to 20% DMSO + 20% EG + 0.5 M sucrose for 30 s, 45 s or 60 s (1 min + 30 s, 1 min + 45 s and 1 min + 60 s groups, respectively). The oocytes were then exposed to warming solution (TCM199 HEPES + 20% FBS and 0.5M sucrose) for 5 min and then washed in TCM199 HEPES + 20% FBS for 5 min. IVM oocytes without CPA treatments served as a control group. The viability assessed by fluorescein diacetate (FDA) staining was 100% in all groups. The developmental rates after PA to the blastocyst stage between 1min+30s (16%) and control (26%) groups did not differ significantly, but they were significantly higher than those in 1 min + 45 s (10%) and 1 min + 60 s (2%) groups. In Experiment 2, we examined the effect of two CPA exposure times, 1 min + 30 s and 1 min + 45 s on the in vitro development after PA of oocytes vitrified by the microdrop method. The viabilities in vitrified 1 min + 30 s, 1 min + 45 s and the control (without CPA treatments) groups were not different (97%, 95% and 100%, respectively). The development of surviving oocytes to the blastocyst stage in the vitrified 1 min + 30 s group (8%) was significantly higher than that in the vitrified 1 min + 45 s group (4%) and significantly lower than those in control group (26%). In Experiment 3, we examined the effect of two CPA exposure times, 1 min + 30 s and 1 min + 45 s on in vitro development after ICSI of vitrified oocytes. Viabilities in vitrified oocytes among 1 min + 30 s, 1 min + 45 s and control groups were not different (96%, 91% and 100%, respectively). After ICSI, vitrified-warmed oocytes were activated and oocytes with the second polar body were cultured for 7 days. The development of ICSI oocytes to the blastocyst stage in the vitrified 1 min + 30 s group (11%) was significantly higher than that in the vitrified 1 min + 45 s (7%) group and significantly lower than those in control group (23%). In conclusion, our study demonstrated that the 1 min + 30 s CPA treatment regimen could yield the highest blastocyst formation rates after PA and ICSI for oocytes vitrified by the microdrop method.     

Fertilization and development in vitro of bovine oocytes following intracytoplasmic injection of heat-dried sperm heads

This study investigated the development of bovine oocytes following intracytoplasmic injection of sperm heads from spermatozoa dried by heating. When sperm suspension was heated in a dry oven at 50, 56, 90, and 120 degrees C, the mean amounts of residual water were about 0.3 g water/g dry weight within 8 h, 6 h, 1.5 h, and 20 min of heating, respectively. Oocyte activation, cleavage of oocytes, and development of cleaved embryos to the morula stage were better in oocytes injected with spermatozoa stored at 25 degrees C for 7-10 days following drying at 50 and 56 degrees C than at 90 and 120 degrees C; however, only a small proportion of oocytes developed to the blastocyst stage. When spermatozoa were dried at 50 degrees C for 16 h, activation, male pronucleus (MPN) formation, cleavage, and development to the morula stage were less good than when spermatozoa were dried for 8 and 10 h and no blastocysts were obtained. The development of oocytes was significantly better when spermatozoa were stored for 7-10 days at 4 degrees C than 25 degrees C after drying at 50 degrees C for 8 h. Longer storage (7 days-12 mo) of heat-dried spermatozoa at 4 degrees C did not affect MPN formation in activated oocytes, but blastocyst development was significantly lower when spermatozoa were stored for 3 mo or more. These results demonstrate that bovine oocytes can be fertilized with heat-dried spermatozoa and that the fertilized oocytes can develop at least to the blastocyst stage.     

Fertilization and development of quail oocytes after intracytoplasmic sperm injection

The present study was conducted to establish the intracytoplasmic sperm injection (ICSI) method for in vitro fertilization and development in quail. The efficiency of fertilization of oocytes was compared 1) between spontaneous and premature ovulation and 2) among testicular round spermatids, elongated spermatids, and immature and mature spermatozoa. The oocytes were injected with a single spermatozoon or spermatid and cultured for 24 h. Cell division was histologically observed with hematoxylin-eosin (HE) and a nucleus-specific fluorescent dye (DAPI). Five of 30 (16.6%) and 4 of 30 (13.3%) oocytes injected with mature sperm were fertilized in the spontaneous and induced ovulation group, respectively. Those embryos showed development at stages II-VII. Half the number (three of six) of the oocytes injected with testicular spermatozoa were fertilized and developed to stages IV-VII, and two of five oocytes injected with elongated spermatids were fertilized and developed to stage VI. All ooocytes injected with round spermatids were unfertilized. The results demonstrate that intracytoplasmic injection of a single sperm into quail oocyte can activate the oocyte and lead to fertilization. Oocytes prematurely ovulated are capable of fertilizing with mature sperm as are those spontaneously ovulated. In addition, the results suggest that the testicular round spermatids may not possess sufficient oocyte-activating potency but that the elongated spermatids and immature spermatozoa are competent to participate in fertilization and early embryonic development in quail.     

Developmental capacity of in vitro matured and fertilized oocytes from prepubertal and adult goats

The developmental competence of oocytes from prepubertal and adult goats was studied through in vitro maturation, fertilization and embryo culture up to the blastocyst stage. Oocytes were recovered from antral follicles of prepubertal and adult goat ovaries, with or without ovarian stimulation with exogenous FSH. The effect of different sources of granulosa cells during IVM on the developmental competence of prepubertal goat oocytes was also noted. Oocytes were matured for 27 h at 38.5 degrees C in 5% CO(2) in air in 50-microl microdrops in TCM199 supplemented with 20% estrus goat serum, FSH, LH and estradiol-17beta or in 2 ml of the same medium supplemented with granulosa cells. Matured oocytes were inseminated with freshly ejaculated spermatozoa following capacitation At 24 h post-insemination, the oocytes were transferred to a granulosa cell monolayer, and early embryo development was evaluated until Day 10. Results show that the developmental ability of embryos from prepubertal goats after IVM and IVF is similar to those from adult goats. Treatment of the prepubertal and adult goats with FSH did not improve the developmental capacity of the resulting embryos. On studying the addition of different sources of granulosa cells to a maturation system of 2 ml of medium, a significantly positive effect of the cells from primed females was observed on the percentage of maturation, on embryo cleavage and on the percentage of embryos that overcame the in vitro developmental block from 8 to 16 cells.     

Development of in vivo-matured porcine oocytes following intracytoplasmic sperm injection

The objective of this study was to assess the development of porcine ova fertilized by intracytoplasmic sperm injection (ICSI). Allyl trenbolone (Regumate) was used to synchronize estrus in 13 postpuberal gilts. Gilts were superovulated with pregnant mare serum gonadotropin and hCG. Ova were aspirated from 5- to 8-mm follicles at 36 h after hCG. Cumulus cells were removed by blunt dissection and pipetting in Beltsville embryo culture medium (BECM) supplemented with 0.1% hyaluronidase. Sperm were washed and resuspended in BECM + 8% polyvinylpyrrolidone. Ova (n = 237) that exhibited a polar body were centrifuged at 15 000 x g for 6 min and injected with a single spermatozoon. One hundred fifty-four ova were cultured in NCSU-23 medium in a 5% CO(2) in air environment for 168 h. Ova were fixed in acetic acid/ethanol and stained with 1% orcein. Sixty-nine ICSI ova were cultured for 24 h and transferred (mean = 23) to three recipients. Eighty-one ova (69%) that survived ICSI cleaved within 48 h. Thirty-eight percent (31/81) of these ova became blastocysts (mean +/- SEM = 24.7 +/- 1.1 cells). One recipient gave birth to three pigs. These results demonstrate that porcine embryos derived from ICSI can develop into live pigs.     

In vitro fertilization by intracytoplasmic sperm injection

Intracytoplasmic sperm injection (ICSI) is the latest, and by far the most efficient, variant of micromanipulation-assisted fertilization, whereby a single spermatozoon is selected, aspirated into a microinjection needle and injected to the oocyte cytoplasm. The development of this technique is mainly linked to application in human assisted reproduction for which it enables fertilization with defective spermatozoa that would not otherwise be able to penetrate an oocyte by their proper means. Because ICSI by-passes many steps of the natural fertilization process, it offers an extremely interesting model for the study of basic mechanisms underlying fertilization. This is particularly true for oocyte activation, whose mechanism needs to be revisited in light of the current ICSI research. The massive application of ICSI in human infertility treatment also represents a huge laboratory in which the impact of different genetic and epigenetic anomalies of the male gamete on fertilization and embryonic development can be studied. BioEssays 21:791–801, 1999. © 1999 John Wiley & Sons, Inc.     

Fertilization of mouse oocytes from in vitro-matured preantral follicles using classical in vitro fertilization or intracytoplasmic sperm injection

Early preantral mouse follicles with a diameter of 110-160 microm were cultured in vitro for 10 or 12 days. Mature oocytes were retrieved following hCG, and fertilization was attempted either by in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Two-cell and blastocyst formation rates and blastocyst cell numbers were compared between 10-day and 12-day in vitro-matured oocytes versus in vivo-matured oocytes. Uncleaved IVF oocytes were subjected to chromosome analysis. The 2-cell formation rate was significantly improved by ICSI compared with IVF both in 10-day (72.1% versus 56.1%; P = 0.03) and 12-day cultures (74.1% versus 54.5%; P = 0.028). Cytogenetic analysis of uncleaved MII oocytes following IVF showed that about 30% of MII oocytes showed no sign of sperm penetration. The blastocyst formation rate was significantly lower in 12-day versus 10-day cultures, whether fertilization was by IVF (40.7% versus 62.4%, P = 0.016) or by ICSI (32.5% versus 57.1%, P = 0.035). Blastocyst cell numbers from IVF and ICSI 10-day groups were similar and both significantly higher (P < 0.001) than from IVF 12-day cultures. All above expressed values were significantly higher for in vivo-matured oocytes. In conclusion, fertilization of oocytes from in vitro-matured mouse preantral follicles can be optimized with ICSI, giving significantly higher 2-cell formation rates than IVF. Blastocyst formation rate was not influenced by the technique of fertilization but rather by the extent of the in vitro culture period. Best results on preimplantation development of oocytes for in vitro-matured preantral follicles were obtained with ICSI on oocytes from 10-day in vitro cultures.     

Embryo development of prepubertal goat oocytes fertilised by intracytoplasmic sperm injection (ICSI) according to oocyte diameter

The aim of this study was to evaluate embryo development of prepubertal goat oocytes fertilised by ICSI according to their diameter. Three experiments were carried out to achieve this objective. In all experiments, oocytes were matured in TCM199 supplemented with hormones, cysteamine and serum for 27 h at 38.5 degrees C. In Experiment 1, we studied the nuclear stage of goat zygotes produced by conventional ICSI and IVF using 20 nM ionomycin plus 10 microM heparin as sperm treatment. A group of Sham-injected oocytes was used as control. Results showed differences in the percentage of 2 PN (zygotes with male and female pronuclei) between ICSI, IVF and Sham (40.9, 26.6 and 3.0%, respectively; P<0.05). In Experiment 2, we evaluated the embryo development of prepubertal goat oocytes produced by ICSI and IVF after 192 h of culture in SOF medium. The percentage of morulae plus blastocysts obtained was higher in the ICSI than in the IVF group (13.4 and 5.1%, respectively; P<0.05). In Experiment 3, IVM-oocytes were classified in four groups depending on their diameter (Group A: <110 microm; Group B: 110-125 microm; Group C: 125-135 microm; Group D: >135 microm), fertilised by ICSI and cultured for 192 h. Results showed a positive correlation between oocyte diameter and embryo development (morulae+blastocysts: Group A: 0%; Group B: 6.2%; Group C: 46.4% and Group D: 33.3%). In conclusion, sperm treatment with ionomycin plus heparin using the conventional ICSI protocol improved fertilisation rates in comparison to IVF. Oocytes smaller than 125 microm were unable to develop up to blastocyst stage.     

In vitro maturation and intracytoplasmic sperm injection of oocytes collected from hormonally stimulated common wombats, Vombatus ursinus

Porcine FSH/LH stimulation successfully induced development of multiple large (>or=4mm) antral follicles in 10 of 11 common wombats. A mean of 5.5 metaphase II (MII) oocytes were aspirated from wombats that were stimulated during the follicular phase of the oestrous cycle (n=3) or after pouch young removal (n=3). Three subadults (n=3) and two anoestrus adults did not produce MII oocytes despite pFSH/pLH administration. In vitro maturation of immature oocytes at the time of aspiration doubled the number of MII oocytes that could be collected from pFSH/pLH stimulated wombats. Immature oocytes with cumulus attached, matured more readily to the MII stage than immature oocytes without cumulus. Following intracytoplasmic sperm injection (ICSI), approximately 5% of the oocytes that were MII at the time of collection cleaved. Approximately 5% of those that were matured by in vitro maturation (IVM) formed two polar bodies following ICSI, although they not cleave. Parthenogenesis cannot be excluded. This demonstrates that assisted reproductive technologies may be applicable to the common wombat.     

Activation regimens to prepare bovine oocytes for intracytoplasmic sperm injection

Activation of bovine oocytes to produce a single haploid pronucleus in preparation for intracytoplasmic sperm injection (ICSI) has been investigated with various combinations of ionomycin and 6-dimethylaminopurine (DMAP). Effects were evaluated by immunocytochemical staining, chromosomal analysis and assessment of development in vitro. Oocytes matured in vitro were exposed to: ionomycin alone (single or repeated treatments, Groups 1 and 2 respectively), ionomycin followed by DMAP (immediately or after a 3-h delay, Groups 3 and 4), or no treatment (control, Group 5). They were then co-cultured in M199 with bovine oviductal epithelial cells. Activation rates were not significantly different among groups but significantly fewer oocytes in Group 3 extruded a second polar body than in Groups 1, 2, and 4. Most parthenotes (60% to 80%) in Groups 1, 2, and 4 were haploid, whereas 82% in Group 3 were mixoploid or polyploid. Most of the parthenotes (88%) in Group 4 formed a single pronucleus besides extruding the second polar body and were therefore more suitable for ICSI than those of Groups 1 and 2 in which condensed chromosomes predominated. The respective rates of oocyte cleavage in Groups 1 to 4 were 24%, 36%, 70%, and 75%; corresponding blastocyst rates were 1%, 5%, 17%, and 8%. There were significantly fewer cells in the parthenotes of Groups 1, 2, and 4 than of Group 3, or of embryos produced by in vitro fertilization. Thus, delaying the addition of DMAP after ionomycin decreases chromosomal abnormalities and produces a high proportion of activated oocytes suitable for ICSI. Mol. Reprod. Dev. 50:485–492, 1998. © 1998 Wiley-Liss, Inc.     

Development of in vitro-matured oocytes from porcine preantral follicles following intracytoplasmic sperm injection.

The objective of this study was to assess fertilization and embryonic development following intracytoplasmic sperm injection (ICSI) of oocytes from porcine preantral follicles matured in vitro. Also, another aim was to describe actin filament distribution during fertilization and embryonic development of those oocytes after ICSI as one of the factors assessed. Preantral follicles isolated from prepubertal porcine ovaries were cultured in a system that supports follicular development. After in vitro maturation, the oocytes were fertilized by ICSI or conventional fertilization in vitro (IVF). Actin filaments of the fertilized oocytes and embryos produced by ICSI or IVF were stained by rhodamine-phalloidin and visualized by fluorescence microscopy. ICSI resulted in 64% fertilization of porcine preantral follicle oocytes matured in vitro. Of those, 51% of the fertilized oocytes cleaved and 21% developed to the blastocyst stage. No significant differences in percentages of oocyte fertilization, cleavage, and blastocyst formation were observed between ICSI and IVF (53%, 45% and 16%, respectively). Actin filament distribution during fertilization and embryonic development of ICSI- or IVF-fertilized oocytes from porcine preantral follicles was similar to that of oocytes derived from antral follicles and fertilized by standard IVF. These results indicate that oocytes from porcine preantral follicles matured in vitro following ICSI can undergo fertilization and subsequent embryonic development.     

Blastocyst formation rates in vivo and in vitro of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection

This study was conducted to evaluate in vivo and in vitro development of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection. Oocytes were collected from slaughterhouse-derived ovaries, matured in vitro, and injected with frozen-thawed stallion sperm. In vivo development was assessed after transfer of injected oocytes to the oviducts of recipient mares. Mares were killed 7.5-8.5 days after transfer and the uterus and oviducts flushed for embryo recovery. Of 132 injected oocytes transferred, 69 (52%) were recovered; of these, 25 (36%) were blastocysts with a blastocoele and capsule. In vitro development was assessed in three culture systems. Culture of zygotes in modified Chatot, Ziomek, Bavister medium with BSA containing either 5.5 mM glucose for 7.5 days or 0.55 mM glucose for 3 days, followed by 3 mM glucose for 2 days, then 4.3 mM glucose for 2.5 days, did not result in blastocyst formation. Culture of zygotes in Dulbecco modified Eagle medium (DMEM)/F-12 with 10% fetal bovine serum with and without coculture with equine oviductal epithelial explants yielded 16% and 15% blastocyst development, respectively. Development to blastocyst was significantly lower in G1.3/2.3/BSA than in DMEM/F-12/BSA or in either medium with 10% added serum (2% vs. 18%, 18% or 20%; P < 0.05), suggesting that requirements for equine embryo development differ from those for other species. These results indicate that in vitro-matured equine oocytes are sufficiently competent to form 36% blastocysts in an optimal environment (in vivo). While we identified an in vitro culture system that provided repeatable blastocyst development without coculture, this yielded only half the rate of development achieved in vivo.     

Factors affecting fertilization of porcine oocytes following intracytoplasmic injection of sperm

The objective of this study was to optimize intracytoplasmic sperm injection (ICSI), and to assess the effects of membrane-damaged sperm on development of porcine oocytes following ICSI. For optimization of development following the ICSI process, sperm injected oocytes were activated 0.5-1.0 hr after ICSI with 1 x 30 micros pulse of 1.2, 1.7, 2.2, and 2.7 kV/cm DC in experiment 1. After 7-days of culture ICSI oocytes activated with [x1]2.2 kV/cm produced more blastocyts ([x2]34.4%, P < 0.05) than other treatment groups. In experiment 2, oocytes were activated with 1 x 30 micros pulse of 2.2 kV/cm at either 0, 1.5, 3, or 6 hr after ICSI. Oocytes activated 1.5 hr after [x3]ICSI yielded more blastocysts (27.6%)[M4] than in other treatments. In experiment 3, sperm were briefly exposed to 0.1% Triton X-100 to induce membrane damage. Live-dead staining of Percoll-sorted untreated spermatozoa (frozen-thawed) used in this study showed that over 96% were "alive" whereas none were "alive" after Triton X-100 treatment. The rate of development to blastocyst of oocytes injected with Triton X-100 treated sperm combined with electrical activation (EA) at 1 x 30 micros pulse of 2.2 kV/cm (EA, 40.0%) was the best, when compared with those injected with untreated sperm plus EA (P < 0.05). In experiment 4, the development rate of oocytes to the blastocyst stage ([x5]32.1%) following injection of a sperm head only was not significantly different from that of oocytes injected with whole sperm (31.0%). In conclusion, we found that an intact membrane and tail structures of pig spermatozoa are not essential for embryo development by ICSI, and furthermore, dead porcine spermatozoa, at an early stage of necrosis caused by plasma membrane damage, support better embryo development than do live non-damaged sperm.     

Activation of bovine oocytes following intracytoplasmic sperm injection (ICSI)

In the human and the mouse, intracytoplasmic sperm injection (ICSI) apparently triggers normal fertilization and may result in offspring. In the bovine, injection of spermatozoa must be accompanied by artificial methods of oocyte activation in order to achieve normal fertilization events (e.g., pronuclear formation). In this study, different methods of oocyte activation were tested following ICSI of in vitro-matured bovine oocytes. Bovine oocytes were centrifuged to facilitate sperm injection, and spermatozoa were pretreated with 5 mM dithiothreitol (DTT) to promote decondensation. Sperm-injected or sham-injected oocytes were activated with 5 microM ionomycin (A23187). Three hours after activation, oocytes with second polar bodies were selected and treated with 1.9 mM 6-dimethylaminopurine (DMAP). The cleavage rate of sperm-injected oocytes treated with ionomycin and DMAP was higher than with ionomycin alone (62 vs 27%, P < or = 0.05). Blastocysts (2 of 41 cleaved) were obtained only from the sperm-injected, ionomycin + DMAP-treated oocytes. Upon examination 16 h after ICSI, pronuclear formation was observed in 33 of 47 (70%) DMAP-treated oocytes. Two pronuclei were present in 18 of 33 (55%), while 1 and 3 pronuclei were seen in 8 of 33 (24%) and 7 of 33 (21%) oocytes, respectively. In sham-injected oocytes, pronuclear formation was observed in 15 of 38 (39%) with 9 (60%) having 2 pronuclei. Asa single calcium stimulation was insufficient and DMAP treatment could result in triploidy, activation by multiple calcium stimulations was tested. Three calcium stimulations (5 microM ionomycin) were given at 30-min intervals following ICSI. Two pronuclei were found in 12 of 41 (29%) injected oocytes. Increasing the concentration of ionomycin from 5 to 50 microM resulted in a higher rate of activation (41 vs 26%). The rate of metaphase III arrest was lower while the rate of pronuclear formation and cleavage development was higher in sperm-injected than sham-injected oocytes, suggesting that spermatozoa contribute to the activation process. Further improvements in oocyte activation following ICSI in the bovine are necessary.