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.     

Piezo-actuated mouse intracytoplasmic sperm injection (ICSI)

The mouse is a genetically tractable model organism widely used to study mammalian development and disease. However, mouse metaphase II (mII) oocytes are exquisitely sensitive and intracytoplasmic sperm injection (ICSI) with conventional pipettes generally kills them. This problem can be solved with piezo-actuated micromanipulation, in which the piezo-electric effect (crystal deformation in response to an externally applied voltage) propels a microinjection needle tip forward in a precise and rapid movement. Piezo-actuated micromanipulation enhances the penetration of membranes and matrices, and mouse ICSI is a major application. Here we describe a comprehensive, step-by-step mouse piezo ICSI protocol for non-specialists that can be completed in 2-4 h. The protocol is a basic prelude to multiple applications, including nuclear transfer cloning, spermatid injection, blastocyst injection, mII transgenesis, and streamlining micromanipulation in primates and livestock. Moreover, piezo ICSI can be used to obtain offspring from 'dead' (non-motile) sperm, enabling trivial sperm freezing protocols for mouse strain storage and shipment.     

Understanding fertilization through intracytoplasmic sperm injection (ICSI)

Since the establishment of in vitro fertilization, it became evident that almost half of the couples failed to achieve fertilization and this phenomenon was attributed to a male gamete dysfunction. The adoption of assisted fertilization techniques particularly ICSI has been able to alleviate male factor infertility by granting the consistent ability of a viable spermatozoon to activate an oocyte. Single sperm injection, by pinpointing the beginning of fertilization, has been an invaluable tool in clarifying the different aspects of early fertilization and syngamy. However, even with ICSI some couples fail to fertilize due to ooplasmic dysmaturity in relation to the achieved nuclear maturation marked by the extrusion of the first polar body. More uncommon are cases where the spermatozoa partially or completely lack the specific oocyte activating factor. In this work, we review the most relevant aspects of fertilization and its failure through assisted reproductive technologies. Attempts at diagnosing and treating clinical fertilization failure are described.     

Oocyte activation and parthenogenetic development of bovine oocytes following intracytoplasmic sperm injection.

Development of bovine oocytes after intracytoplasmic sperm injection (ICSI) was investigated. Oocytes were matured for 24-26 h in vitro and injected with isolated sperm heads. When treated with 7% ethanol (v/v) for 5 min, 71.7% of ICSI oocytes were activated as shown by the resumption of meiosis and the formation of female pronuclei. However, 41.5% of injected sperm heads remained condensed at 18-20 h after injection into the ooplasm. The incidence of decondensing sperm and that of male pronuclei at this stage were 15.1% and 26.4%, respectively. A total of 55.5% of oocytes reached the 2-cell stage following sperm head injection and 54.7% after sham-ICSI; these percentages were not significantly different from those following in vitro fertilisation (IVF) (73.1%). The percentage of 2-cell embryos reaching the 8-cell stage following ICSI was 37.5%, and 27.6% after sham-ICSI, which were significantly lower (p < 0.01) than the equivalent percentage following IVF (62.4%). The percentages of parthenogenetic embryos reaching the 2-cell, 4-cell and 8-cell stages following ICSI were 56.4%, 48.9% and 30.0%, respectively. These results indicate that the low rate of normal embryonic development of bovine oocytes following ICSI is largely due to the parthenogenetic activation of the oocytes.     

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

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 microM ionomycin for 5 min (group 3), 5 microM ionomycin + 1.9 mM dimethylaminopurine (DMAP) for 3 h (group 4) and 5 microM 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.     

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.     

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.     

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.     

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.     

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.     

ICSI精子遗传和表观遗传学缺陷

卵胞浆内精子注射(Intracytoplasmic sperm injection, ICSI)技术可用于男性少精、弱精、精子畸形、无精子和常规体外受精周期失败等, 克服了精子数量不足甚至直接从附睾、睾丸获取精子来治疗不育。该技术直接将单个精子注射入卵子, 因违背自然受精的生物学法则而具有很大的遗传风险。文章对ICSI精子遗传缺陷和表观遗传缺陷及其相关疾病进行综述, 可进一步认识ICSI精子遗传与表观遗传缺陷导致后代遗传风险增加的分子的机理, 文章阐述了ICSI精子有待于通过DNA甲基化、组蛋白乙酰化等表观遗传因子进行严格质量控制, 切实降低ICSI遗传及表观遗传缺陷风险的必要性。     

Full-term development of golden hamster oocytes following intracytoplasmic sperm head injection

The golden hamster is the mammalian species in which intracytoplasmic sperm injection (ICSI) was first tried to produce fertilized oocytes. Thus far, however, there are no reports of full-term development of hamster oocytes fertilized by ICSI. Here we report the birth of hamster offspring following ICSI. Keys to success were 1) performing ICSI in a dark room with a small incandescent lamp and manipulating both oocytes and fertilized eggs under a microscope with a red light source and 2) injecting sperm heads without acrosomes. All oocytes injected with acrosome-intact sperm heads died within 3 h after injection, while those oocytes injected with acrosomeless sperm heads survived injection. Under illumination with red light in a dark room, the majority of the oocytes injected with acrosomeless sperm heads were fertilized normally (77%), cleaved (91%), and developed into morulae (49%). Of the 47 morulae transferred to five recipient females, nine (19%) developed to live offspring.     

Production of piglets using intracytoplasmic sperm injection (ICSI) with flowcytometrically sorted boar semen and artificially activated oocytes

The purpose of the present study was to develop a protocol for the successful production of piglets employing intracytoplasmic sperm injection (ICSI) with flowcytometrically sexed spermatozoa and artificially activated porcine oocytes. In vitro matured oocytes were fertilized by ICSI using non-sorted frozen/thawed epididymal semen. Oocytes were either activated by CaCl(2), Ca(2+)-ionophore or electrical pulse. Activation and fertilization rates of sperm injected oocytes stimulated by CaCl(2)-injection were significantly higher than those without activation (70.4% versus 45.9%; 49.9% versus 33.2%, respectively; P<0.001). Activation rate of sham injected oocytes increased in parallel (11.2% versus 26.3%, P<0.05), parthenogenetic development remained low (2.8% versus 8%). Co-incubation in Ca(2+)-ionophore did not improve activation rates as compared to non-activated oocytes (44.8% versus 42.5%). Fertilization rate decreased as compared to non-treated sperm injected oocytes (36.8% versus 24.5%, P<0.05). Activation of oocytes with a single electrical pulse resulted in significantly higher activation rates in all groups of oocytes as compared to non-stimulated ones (sperm injected oocytes: 65.6% versus 43.1%, P<0.001; sham injected oocytes: 48.5% versus 5.6%, P<0.001; control oocytes: 50.7% versus 0.0%, P<0.001). Fertilization rates (32.3% versus 48.2%) and parthenogenetic development (0.7% versus 38.9%, 0.0% versus 30.9%, P<0.001) increased significantly in parallel. In addition, in four replicates of flowcytometrically sorted Y-chromosome bearing spermatozoa were injected into in vivo matured oocytes, activated with 1.2 pl of a 30 mM CaCl(2) solution. On average 85.3 fertilized oocytes were transferred surgically into four recipients. Pregnancies delivered a total of 13 male piglets. These are the first piglets born from ICSI with sorted spermatozoa.     

Cytochalasin B treatment of mouse oocytes during intracytoplasmic sperm injection (ICSI) increases embryo survival without impairment of development

Summary Intracytoplasmic sperm injection (ICSI) is a technique commonly used in clinical and research settings. In mouse oocytes, conventional ICSI has a poor survival rate caused by a high level of lysis. Cytochalasin B (CB) is a toxic microfilament-inhibiting agent that is known to relax the cytoskeleton and enhance the flexibility of oocytes. CB has been used widely in nuclear transfer experiments to improve the success rate of the micromanipulation, however information describing the use of CB in ICSI is limited. Here, we demonstrated that the addition of 5 μg/ml CB to the manipulation medium of ICSI procedure significantly improved the survival rate of the ICSI embryos (80.74% vs. 89.50%, p < 0.05), and that there was no harm for the in vitro or in vivo development. The birth rates and birth weights were not significantly different between the CB-treated and -untreated groups. Interestingly, the microfilaments of the ICSI embryos were almost undetectable immediately after CB treatment; however, they gradually re-appeared and had fully recovered to the normal level 2 h later. Moreover, CB did not disturb spindle rotation, second polar body formation or pronuclei migration, and had no effect on the microtubules. We thus conclude that ICSI manipulation in CB-containing medium results in significantly improved survival rate of mouse ICSI embryos, and that short-term treatment with CB during ICSI manipulation does not have adverse effects on the development of ICSI embryos.     

Fertilization of porcine oocytes following intracytoplasmic spermatozoon or isolated sperm head injection

We demonstrated normal fertilization processes (as determined by pronuclear formation, pronuclear apposition and syngamy) in porcine oocytes either following intracytoplasmic spermatozoon (ICSI) or isolated sperm head injection. Microtubule organization and chromatin configuration were investigated in these oocytes during the first cell cycle. Following ICSI, the microtubular aster was organized from the neck of the spermatozoon and filled the whole cytoplasm. These male-derived microtubules appear to move both pronuclei to the center of oocytes. These cytoskeletal changes are analogous to those seen following conventional fertilization. In contrast, following isolated sperm head injection, the sperm aster was not seen. Instead, the microtubule matrix was organized from the cortex and then filled the whole cytoplasm in all cases in normally fertilized oocytes following injection (n = 35). This organization is similar to what has been shown in the parthenogenetically activated oocytes. Chromosome analysis revealed that the oocytes injected with isolated sperm heads were fertilized normally. At 7 days following injection, the incidence of blastocoele formation following ICSI (38%) and isolated sperm head injection (22%) was higher than that following sham injection (2%). These results suggested that successful fertilization and preimplantation development occurred in porcine oocytes following either ICSI or isolated sperm head injection. Our results also indicated that fertilization processes can occur by self-assembled microtubules within cytoplasm in the absence of a sperm centrosome. Mol. Reprod. Dev. 51:436–444, 1998. © 1998 Wiley-Liss, Inc.     

Meiotic competence of equine oocytes and pronucleus formation after intracytoplasmic sperm injection (ICSI) as related to granulosa cell apoptosis

Follicle atresia and granulosa cell apoptosis may be related to oocyte meiotic and developmental competence. We analyzed the relationships among granulosa cell apoptosis, initial cumulus morphology, oocyte nuclear maturation in vitro, and pronucleus formation after intracytoplasmic sperm injection (ICSI) in the horse. For each follicle, the size was measured and granulosa cells were used for DNA laddering analysis. Oocytes were evaluated for cumulus morphology, cultured for in vitro maturation, and submitted to ICSI. Apoptosis was categorized as absent, intermediate, or advanced according to the relative concentrations of two DNA fragments at 900 and 360 base pairs (bp). In 98 oocyte-follicle pairs, 52 oocytes were classified as expanded (Exp), 39 as compact (Cp), and 7 as having a partial (P) cumulus. Advanced apoptosis was detected in 55% (54/98) of follicles; 37% (36/98) of follicles showed an intermediate level of apoptosis; and 8 follicles (8%) were nonapoptotic. Follicle size was not significantly correlated with granulosa cell apoptosis (P > 0.05). Significantly more Exp than Cp oocytes originated from follicles with advanced apoptosis (P < 0.001). The proportion of oocytes maturing in vitro was significantly higher in oocytes issuing from apoptotic follicles than in oocytes issuing from healthy follicles (P < 0.05). The proportion of normally (two pronuclei) or abnormally fertilized oocytes (one or greater than two pronuclei, or partially decondensed sperm) did not differ in relation to granulosa cell apoptosis. We conclude that, in the mare, granulosa cell apoptosis is related to cumulus expansion and an increase in oocyte meiotic competence but has no effect on the proportion of meiotically competent oocytes that activate after ICSI. These results provide selection criteria for horse oocytes used in assisted reproductive techniques so that embryo production may be maximized.     

精子和卵母细胞质量控制对山羊卵胞质内精子注射（ICSI）受精的影响

ABSTRACT Effects of sperm and oocyte quality control on the efficiency of ICSI of in vitro matured goat oocytes were studied in this paper. The results showed that when injected intracytoplasmically, spermatozoa from caput, corpus and cauda epididymidis resulted in similar rates of fertilization, cleavage and morulae/blastocysts, but when injected subzonally, spermatozoa from caput and corpus gave rise to significantly lower rates of fertilization and embryo development than spermatozoa from the cauda epididymidis and ejaculates. When dead spermatozoa collected from semen that had been preserved in different ways were used for ICSI, those dead from liquid storage at 20 degrees C for 24 h gave rise to the best, but those dead from liquid storage at 5 degrees C for 15 days produced the poorest fertilization and embryo development. When spermatozoa were treated with different concentrations of Triton X-100 before ICSI, significantly higher rates of fertilization, cleavage and morulae/blastocysts were obtained with 0.0005% Triton X-100 than with other concentrations and manual immobilization. Oocytes were classified as of good and poor qualities by treatment in hypertonic sucrose solution, and rates of fertilization and embryo development were significantly higher in the good than in the poor oocytes after ICSI. Post-injection activation of oocytes with either A23187 or ionomycin/6-DMAP significantly increased the rates of fertilization, cleavage and morulae/blastocysts after ICSI. It is therefore concluded that (i) epididymal maturation mainly endowed spermatozoa with the capacity to fuse with the egg plasma membrane; (ii) different methods of semen storage caused different impairment of sperm fertilizing capacity; (iii) pre-injection treatment of spermatozoa with proper concentrations of Triton X-100 might be used to replace manual immobilization for ICSI; (iv) oocyte quality was a major factor influencing the efficiency of ICSI; (v) post-injection activation treatment of oocytes improved fertilization and embryo development after ICSI.     

Analysis of different factors influencing the intracytoplasmic sperm injection (ICSI) yield in pigs

Intracytoplasmic sperm injection (ICSI) in pigs is a technique with potential application in diverse fields of animal production and biomedicine. Even though there are some cases of live offspring resulting from this technique, its yield is still quite low compared to other species. The aim of this study was to evaluate different factors affecting the ICSI performance. This was done by studying (1) the sequence of culture media for the oocytes after injection; (2) modifications in the in vitro maturation system (IVM) through meiotic inhibitors such as roscovitine, and changes in the IVM time; (3) oocyte activation through injection of inositol triphosphate (InsP(3)) together with the sperm. In vitro matured oocytes were employed. All the ICSI experiments were performed with fresh ejaculated semen. Results showed that porcine ICSI zygotes give an improved proportion of two-cell embryos using the sequence IVF medium-embryo culture medium (NCSU-23) rather than transferring directly to NCSU-23. Pronuclear formation ability was not affected by prematuration, but a faster embryo development was observed in roscovitine treated oocytes. In relation to IVM times, oocytes matured for 36 h can achieve better fertilization percentages than oocytes matured for 44 h. These results were independent of the roscovitine treatment. Finally, no influence on embryo development was observed until the blastocyst stage with the use of the InsP(3) as an exogenous activating factor.     

Incubation of sperm heads impairs fertilization and early embryo development following intracytoplasmic sperm injection (ICSI) by decreasing oocyte activation in mice

When intracytoplasmic sperm injection (ICSI) is performed in mice, isolation of sperm heads is usually performed prior to injections in order to increase the efficiency of the procedure. Consequently, the isolated sperm heads undergo an inevitable incubation in vitro. However, little is known about the effects of this incubation step on fertilization and embryo development following ICSI. When we incubated sperm heads at 37 °C, there was a significant time-dependent decrease in fertilization and blastocyst formation. Moreover, the DNA integrity of the sperm heads was maintained over 12 h incubation. Using assisted oocyte activation, these defects in fertilization and embryo development were rescued. Taken together, incubation of sperm heads following isolation can affect the oocyte-activating capacity of sperm thereby compromising fertilization and embryo development associated with ICSI.     

Birth of normal calves after intracytoplasmic sperm injection of bovine oocytes: a methodological approach

Intracytoplasmic sperm injection (ICSI) is advantageous when only very few spermatozoa are available for insemination. Bovine spermatozoa were injected individually into matured oocytes using a piezo electric actuator. Spermatozoa were "immobilized", by scoring their tails immediately before injection, or "killed", by repeated freezing and thawing. About 4 h after ICSI, the oocytes with two polar bodies (activated by sperm injection) were selected and treated 5 min with 7% ethanol before further culture. When examined 19-21 h after ICSI, nearly 90% of the oocytes were fertilized normally (two pronuclei and two polar bodies) irrespective of the sperm treatment (immobilization or killing) prior to ICSI, but subsequent preimplantation embryo development was much superior (cleavage 72%: blastocysts 20%) after ICSI with immobilized spermatozoa than by using killed spermatozoa (cleavage 28%; blastocysts 1%). Ethanol activation of bovine oocytes with two polar bodies 4 h after ICSI improved the cleavage (33% versus 72%) and blastocyst (12% versus 20%) rates markedly (P < 0.05). Five normal calves were born after transplantation of ten blastocysts to ten surrogate cows. These results show that piezo-ICSI using immobilized spermatozoa, combined with ethanol treatment of sperm-injected oocytes, is an effective method to produce bovine offspring.