Cleavage, development and competence of sheep embryos fertilized by intracytoplasmic sperm injection and in vitro fertilization

ELSEVIER

CLEAVAGE, DEVELOPMENT AND COMPETENCE OF SHEEP EMBRYOS FERTILIZED BY INTRACYTOPLASMIC SPERM INJECTION AND IN VITRO FERTILIZATION M.C. G6mez, la J.W. Cart, 2 G. Evans 1 and W.M.C. Maxwell 1 ~Department of Animal Science, University of Sydney, NSW 2006 Australia 2Sydney IVF, 40'Connell St, Sydney, NSW 2000 Australia Received for publication: August 21, 1997 Accepted: January 6, 1998 ABSTRACT More abnormal fertilization has been found in sheep oocytes after intracytoplasmic sperm injection (ICSI) than after in vitro fertilization (IVF). Although the birth of a normal lamb has been reported, the efficiency of blastocyst production is low. We therefore evaluated the cleavage, development and viability of sheep embryos obtained from ICSI, IVF and sham injection. In vitro matured oocytes either injected or inseminated with spermatozoa were ~ s s e d for cleavage 1 and 4 d after injection or insemination, and for development to blastocyst after 7 d of culture. A total of 699 oocytes was injected (ICSI); 198 (30.6%) were activated and 55 (8.5%) developed to the blastocyst stage. Of the 17 recipient ewes with 1, 2, 3 or 4 embryos, 15 (88.2%) were pregnant on Day 18; of these 17 recipients, 7 (41.1%) and 6 (35.2%) ewes remained pregnant on Days 45 and 110, respectively. Two normal lambs were born, one ewe died on Day 110 with 2 normal male fetuses, another ewe aborted on Day 90 and 4 pregnancies were maintained. A total of 517 oocytes was inseminated (IVF); 296 (62%) were activated and 90 (18.8%) reached the blastocyst stage. A total of 19 ewes received 1, 2, 3 or 4 embryos; of these, 13 (68.4%) were pregnant on Day 18, and 8 (42.1%) ewes remained pregnant on each of Days 45 and I10. Three ewes delivered 5 lambs. Five pregnancies were maintained. A total of 156 oocytes was sham injected, 38 (24.3%) were activated and no blatocysts were obtained after culture. The results of this study showed that blastocysts obtained after ICSI are potentially viable and are not a result of parthenogenesis. © 1998 by ElsevierScience Inc.

Key words: sheep, ICSI, IVF, loss pregnancy

Acknowledgments The r ~ c h was funded by grants from The University of Sydney and Colombian Institute of Development in Science and Technology-Colciencias. The authors thank Ms. K. Hea,vJnan, Mr. A. Souter, Ms. A. Harrington and Mr. P. Gooden for their technical support, aCorrespondence and reprint requests. Theriogenology 49:1143-1154, 1998 © 1998 by Elsevier Science Inc.

0093-691X/98/$19.00 PII S0093-691X(98)00062-4

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INTRODUCTION Different reproducn_'ve techniques have been used to obtain embryos and produce sheep offspring with in vitro fertilization (1VF) (4,11,15,16,19,21 ), intracytoplasmic sperm injection (ICSI; 5) and nuclear transfer (26,27), but a low number of blastocysts have been formed using the 2 latter procedures. A low rate of blastocyst formation has been reported in cattle (6, 10) and mice (1) after ICSI of in vitro matured oocytes. In the mouse, researchers have improved the rate of blastocyst formation by modifying the sperm treatment (13) or sperm injection technique (12). However, a lmmher of factors such as oocyte maturation, the culture system 7 oocyte activatian aud abnol:mal fertilization could be involved in the low number of blastocysts formed during culUtre after ICSI. In vitro intervention or manipulation of embryos call interfere with normal embryonic development in some mnmmalian ~ ] e s (28). Abnormal fertiliTntion has been found in sheep oocyt~ after the use of ]CS] unlike that after conveufiOllal IVF (9). Abnormal embryos have also been obtained after ICSI in human~ (2,3,7,20), and a relaticm~hip between chgomosomal abnormsliti~ and embryo preimplanmtion has been demostrated (3). Low embryo viabili~ has been reported after pronuclear gene injecion (24) and nuclear transfex (27) in shoep oocytes. However, such abnormalities could also be expressed as a failure of implantation or later loss, which has been r_%ported after ICSI in hnmanx (3,29) or after IVF in cattle (14,18). The aim of the pro,z,ent study was to compare the cleavage, development and viabifity of ~h~*~. embryos obtain o~rJ from ICSI and IVF, and to determine the relationship between blastocyst quality and embryonic survival in vivo. b MATERIALS AND METHODS Oocyte Collection and Preparation c Ovaries from adult sheep were obtained from an abattoir and transported (25°C) to the laboratory within 2 h of slaughter. The ovaries were rinsed with phosphate buffered saline (PBS) supplemented with 0.3 mg/mL penicillin G-potassium salt, 0.25 mg/mL streptomycin sulfate and 0.25 mg/mL neomycin, and washed a further 3 times in PBS medium at 30°C. Oocytes were aspirated from follicles (2 to 8 mm in diameter) using a 1-mL syringe and21-g needle, and collected into tissue o~_~,_re medium 199 (M-199 with Earle's salts, Gibco-BRL. N.Y. USA), supplemented with Hepes (free acid) 15mM, sodium Hepes 15mM, 0.33 mg/mL b

Since this ~ was accepted. 3 and 6 normal lambs have been born from the ICSI and IVF treatments, respectively. CAll chemicals were obtained from Sigma Chemical Company (st. Louis, MO, USA) unless

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sodium bicarbonate, 0.01 mg/mL heparin sodium salt (Grade l-A: from porcine inter'hal mucous), 0.075 mg/mL penicillin G-potassium salt, 0.05 mg/mL streptomycin sulfate, 0.08 mg/mL kanamycin monosulfate and 10% fetal bovine sexum {FBS). After rinsing 3 times in this medium, oocytes with a homogenous ooplasm and surrounded by several layers of cumulus cells were matured in multiweU dishes (Nunclon; Inter Med, Roskilde, Denmark). Each well contained 50 cumulus-oocyte complexes (COC) and 500 #L of maturation medimn-199 (TCM-199 Gibco-BRL NY, USA) containing 10% v/v FBS, 10 ug/mL FSH (Folla'opin-V:Vetrepharm, Essedon, Victoria, AnsWalla) and 10 ug/mL LH (Lutrophin-V: Vetrepharm). The wells were covered by mineral oil, and the oocytes were cultured for 20 to 24 h in 5% CO2 in humidified air at 39°C. After maturation, the oocytes allocated for ICSI, IVF or sham injection were denuded of their c~,mul,s and corona cells by aspiration through a narrow hand-drawn pipette. The cumulus-free oocytes were washed and transferred to multiwell dishes. Each well contained 500/zL of bicarbonate-buffered synthetic oviduct fluid medium (BSOF; 22) supplemented with 2% sheep serum (v/v; collected and processed from a ewe on Day 3 of the estrons cycle), 0.1 mg/mL pyruvic acid, 0.15 mg/mL L-glutamine, 0.08 mg/mL kanamycin monosulfate~ 0.075 mg/mL penicillin G-potassium salt, and 0.05 mg/mL streptomycin sulfate. The oocytes were cultured under mineral oil at 39°C in 5% CO2 in air prior to microinjection or insemination. Sperm Preparation Pooled fresh semen from 3 different rams was diluted (1:20 semen:medium) in Hepes buffered modified SOF (HSOF; 25) supplemented with 3mg/mL (0.3%) BSA-fraction V, 2.1 mg/mL sodium bicarbonate, 0.72 mg/mL D-glucose (Analar, BDH Chemicals, Victoria, Australia), 0.06 mg/mL pyruvic acid, 0.25 mg/mL L-glutamine and 0.12 mg/mL kanamycin monosulfate, 0.075 mg/mL penicillin G-potassium salt, and 0.05 mg/mL slreptomycin sulfate, and held for 1 h at room temperature. Two-layer Percoll gradients (90 to 45%) were prepared in 10-mL centrifuge tubes: 2 mL of 90% Percoll were placed in the bottom of the tube, and 2 mL of 45 % Percoll were carefully layered on the top, avoiding mixing of the 2 layers; 200 #L of the semen ~mples were then placed at the top of the Percoll gradient. The samples were centrifuged at 3000 g for 15 min. The top layer of Percoil was discarded, and the sperm pellet (containing spermatozoa) was resuspended in 3mL of HSOF and centrifuged again at 600 G for 6 rain. The supernatant was discarded, and the spermatozoa were purified by the "swimup" technique. Spermatozoa for IVF were incubated for 1 h and the spermatozoa for ICSI were incubated between 1 to 4 h, depending on the time required to complete the injec~on of oocytes. For ICSI, 10 #L of medium with spermatozoa were aspirated from the sperm treatment and diluted (1:1) with 10% (v/v) polyvinyl pyrrolldone (PVP, MW 360.000). This reduced velocity and facilitated the capture of a spermatozoon. A small drop (about 2 #L) of this sperm suspension was kept under mineral oil in a plastic PelTi dish before injection into oocytes.

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Oocyte Fertilization and Culture After ICSI, the oocytes were cultured in groups of 35 to 50 in 500 #L of BSOF medium supplemented with 2% sheep serum and covered by mineral oil for 19 to 21 h in 5% CO2 in air at 39°C. The embryos of each group were then washed 3 times and cultured in 500/~L of synthetic oviduct fluid medium (SOF) supplemented with 3 mg/mL falIy acid-free BSA (Pent_ex 81-001-2, Lot # 98, Miles INC. ]~ankank~e, ~ USA), 20 p.L/mL essential aminoacids (MEM Aminoacids Solutions without L-glutamine, BILL. Gibco, NY, USA), 10 i ~ / m L nonessential aminoacids (MEM nonessential aminoacids solution, BRL), 0.03 mg/mL pyruvic acid, 0.04 mg/mL L-glutamine, 0.08 mg/mL kanamycin monosulfate, 0.075 mg/mL penicillin G-potassium salt and 0.05 mg/mL s~eptomycin s]df~te, nnder ~ oil at 39°C in 5% CO2, 5% 02, 90% N2 in a desecator for up to 10 d. On Day 4 after injection, 0.27 mg/mL glucose was added to the culture medium. For sham injection, as an activation control, oocytes were injected and cultured by the same procedure as described for ICSI except that no spermatozoon was injected. For IVF, the oocytes were co-incubated with 0.6 -1.0 xl06sperm/mL in groups of 50 in 500 ~L of BSOF medium supplemented with 2% sheep serum, covered with mineral oil in 5% CO2 in air at 39°C for 19 to 21 h. The oocytes were then washed in SOF medium to removed the spermatozoa and cultured in SOF medium as for ICSI for a total of 10 d. Embryos at the blastocyst stage following culUne were Iransferred to surrogate ewes or were fixed to determine cell numbers. Microinjection Procedure The holding pipette was made from borosilicate glass capillary tubing (lmm; SDR, Clinical Technology, Sydney, NSW, Australia). This was hand-pulled into a microt~e and broken to an outside diameter of 150 to 200 #m. The tip was then heat-polished using a microforge (Model MF-9; Narishige Instrtunent, Tokyo, Japan) until the inside ¢l_i~mS,~.was 30 to 80 pm. The injection pipette was made from thin-walled capillary tubing using a microelectrode puller (Sutter 1)87, Su~er, Novota, CA, USA) to give an internal and external diameter of the microinjection pipette of 8 to 10 #m and 10 to 15 #m, respectively. A bevel of ~ 40 ° was ground using a pipette grinder (EG-4, Nacishige). To facilitate the injection procedure, both holding and microinjection pipettes were bent to an angle of -30 ° to allow for horizontal manipu~on. One droplet of sperm-PV]' suspension (2 v.L) and 1 droplet of H199 medium (10 ~,L) containing 5 oocytes were placed in the bottom of a 9-cm Petri. dish modified with 4 holes that were covered with a ~la~ coverslip to ~ con'mafibility with Nomarski optics. These droplets were covered by mineral oil. Both ICSI and the sham injection were carried out on the heated stage (39°C) of an inverted microscope (Diaphot; Nikon, Tokyo, Jat3an) at x 100 mat,nification n.~ing Nomarski optics

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(Nikon). The holding and injection pipettes were placed in a tool holder and were connected to a micrometer-type mL_~mmjector0M-5A/B; Narashige). The movements of the pipettes were controlled by 2 coarse positioning manipulators (3D Manipulator MNZ; Narishige) and with 2 three-dimensional hydraulic remote-control micro-manipulators (MO-302 and MO 202, Narishige). For ICSI, the injection mieropipette was moved to the bottom of the drop containing the sperm-PVP. A single spermatozoon was immobilized by striking the tail with the pipette and aspirated tail first into the tip of the injection pipette. Both pipettes were then moved to the drop containing the oocytes. Metaphase II oocytes were held under negative pressure on the holding pipette, with the polar body at the 6 or 12 o'clock position. The microi__%i~tion pipette was pushed through the zuna pellucida and into the cytoplasm of the oocyte at the 3 o'clock position. After aspirating the cytoplasm into the injection pipette (to ensure plasmalemma breakage), the spermatozoon was deposited with a minimum amount of PVP medium ( < 5 pL). The injection pipette was withdrawn, and the oocyte released from the holding pipette. Injected oocytes were washed twice in BSOF medium and incubated in 5 % CO2 in air at 39°C. Assessment of Embryos The number of oocytes cleaving for each Ueatment (ICSI, sham injection and IVF) were assessed visually (light microscopy, x 100 magnification) on Day 1 (day of insemination= Day 0) and Day 4; while the number of oocytes developing to the blastocyst stage was determined on Days 6, 7 and 8. Some blastocysts from each Ireatment were removed on Days 6 to I0 in order to determine the number of cells. Four development stages were assigned: blastocyst (blastocoele cavity form); expanded blastocyst (enlarge zona pellucida); hatching blastocyst; and hatched blastocyst. Hoechst staining was used to determine the cell numbers: 5 #L of Bisbenzimide 100x Hoeschst stain (Bisbenzimide H-33342, Calbiochem, La Jolla, CA, USA) were added to the SOF medium containing the blastocysts, and the embryos were incubated for 30 min at 39°C. The embryos were then washed in HSOF medium and mounted on clean slides and covered with coverslips supported on Vaseline. The number of cells were counted under an Olympus Fluorescent Microscope under UV illnminatJon. After fluorescence assessment, the stone embryos were fixed in ~hancq:acetic acid (3:1) for 24 h, stained with 0.5% orcein, and examined under phase contrast microscopy (x 400 magnification). The number of cells was counted and compared with the results obtained by fluorescence Hoeschst staining. Estrus Synchronization, Embryo Transfer, Pregnancy Detection and Parturition Induction Six Mature Merino and 41 Corriedale ewes were synchronized to control eslrus and ovulation using intrava~nal sponges impregnated with Flugestone acetate (30 mg Chronogest; Intervet, Castle Hill, NSW, Australia) which were inserted for 12 d. At the time that sponges were removed, the ewes were given a single im injection of 400 IU (Merino) or 600 IU

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(Corriedale) PMSG (Folligon; Intervet) to induce ovulation. On Day 5 after ovulation the ewes were starved overnight. Embryo transfers were carried out by mini-laparotomy 6 d following estrus. Recipient ewes were treated with a local anesthetic (5 mL Lignomav (20 mg/mL); Illium, Troy Laboratories, Smithfield, NSW, Australia) and with a sedative anesthetic (0.4 mL Rompun (20 mg/mL; Bayer AG, Leverkusen, Germany) plus 3 mL Ketamil (100 mg/mL; Ilium Troy Lab), both administered intramuscularly. The uterus of each recipient was located by laparoscopy (8), and the uterine horn ipsilateral to a corpus luteum was exteriorized through a 1.5-cm mid-venWal incision.The horn was punctured with a sterilepaper clip, and I tO 4 blastocysts from the same group (ICSI or IVF) on the same cleavage day (Day 7 or 8) and at any development stage (blastocyst, expanded blastocys, hatching blastocyst or hatched blastocyst) were transfered in 150 #L SOF medium using a Tom Cat catheter (Open end 51/2 inches, Sherwood Medical, St. Louis, MO, USA) attached to a 1-mL syringe. The horn was reUlrned into the abdon~inal cavity, the incision sutured, and the ewe injected intramuscularly with antibiotics (5 mL Penstrep; Ilium, Troy Lab). Seventen and 19 ewes recieved blastocysts resulting from ICSI and IVF, respectively. To assess pre~,nancy status, blood samples were collected from recipient ewes on Day 18+1, and plasma progesterone levels were determined by RIA. Ewes with a plasma progesterone concentration ~ 1.0 ng/mL were considered to be pregnant. These ewes were scanned by ultrasound on Days 45 and ll0 for the presence of 1 or more fetuses. Parturition was induced in pregnant ewes on Day 148 by administration of 15 rag, im estradiol monobenzoate (Intervet, Australia). The ewes were penned for birth, and obstetrical assistance was given when necessary. Statistical Analysis The data on embryo cleavage, blastocyst formation and pretmancies were analyzed by Chisquare with Yates' correction. Analysis of variance (ANOVA) was also used to test the difference between ICSI, IVF and sham injection for embryo cleavage and total activation rates. Comparison of the number of blastocyst cells between ICSI and IVF was analyzed by the general linear model (GLM) procedure. The linear standar deviation (LSD) te~ was used to d_et__,~-mineany differeaces between individual me~_n~. All analyses were performed by the Minitab (version 8.2) computer program. RESULTS Data on the development of oocytes after ICSI, IVF and sham injection are presented in Table 1. Of 699 microinjected oocytes, 52 (7.4%) lysed after 1 d of culture after ICSI, which was lower than for sham-injected oocytes (37/157, 23.5%; P<000.1). The proportion of oocytes that had cleavage 96 h (Day 4) after insemination was 62%, which was higher than for ICSI (42.6%) or for sham injection (31.6%; P<0.05). The number of oocytes that reached

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the blastocyst stage after IVF (18.8%) was also higher than for ICSI (8.5%) or for sham injection (0%; P<0.001). Table 1. Development of sheep oocytes after ICSI, IVF and sham injection Group No. of No. of oocytes No. of normal embryos at developmant Treatment oocytes lysed

Day1 ICSI IVF

699 517

52 (7.4%) a 40 (7.7%) a

SHAM

157

37 (23.5%) b

Day4

~y7

198(30.6%) a 275 (42.5%) a 55 (8.5%) a 176(36.8%) b 296(62.0%) b 90~1&8%) b 32 (26.6%) a

38 (31.6%) a

0 (0%)c

a,b,c. Different superscripts within the same column indicate statistical significance (P < 0.05).

The development of blastocysts occurred predominantly on Days 6 and 7 of culture, with only 1 hatched blastocyst obtained after ICSI on Day 10 of culture (Figure 1).

40

- - o - ICSI --4~ IVF

,, 30 o 20 10 0 Day 6

Day 7

Day 8 Day of culture

Day 9

Day 10

Figure I. Blastocyst development on Days 6 to I0 after culture.

Of the 55 and 90 blastocyst obtained after ICSI and WF, respectively, 8 and 20 embryos were stained with Hoechst 33342 and fixed with ethanol: acetic acid at Days 6 to 10 after microinjection or insemination, to determine cell n_-__m_bers.Significant differences were found in cell numbers at each stage of development between ICSI and IVF (P<0.001; Table 2) procedures.

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Table 2. Cell numbers in blastocysts, expanded blastocysts, hatching blastocysts and hatched blastocysts after ICSI and IVF

Group

Development stage

ICSI

43 +18 a

67.5 +--3a

96.0 +t9 a

75 +7 a

IVF

68 +-27b

96.8 -'~32b

185.5 :i:78b

196 -+-22b

a,b. Different superscripts within the same column indicate statistical significance (P < 0.001).

A mean of 2.2 and 2.3 embryos was transferred to a total of 17 and 19 ewes for ICSI and IVF treatments, respectively. There were no statistical differences in the pregnancy rates determined by RIA on Day 18 (15/17, 88.2% and 13/19, 68.4%) and by ultrasound on Day 45 (7/17, 41.1% and 8/19, 42.1%) or Day 110 (6/17, 35.2% and 8/19, 42.1%) between ICSI and IVF, respectiveAy. A high rate of embryonic loss was obtained after both ICSI (9/15; 60%) and IVF (5/13, 38.4%) procedures. All loss of embryos following IVF occurred before Day 45 (5/13, 38.4%), whereas that following ICSI (8/15, 53.3%) occurred before Day 45, and 6.7% (1/15) occurred before Day 90. There was no difference in the implantation rate of embryos formed after ICSI (9/38, 23.6%) and IVF (13/44, 29.5%). Two normal lambs were born from the ICSI treatment, the ewes received eswadiol benzoate on Day 148 of pretmsncy, and normal parturition occurred 28 h later; one ewe carrying twin fetuses from ICSI died on Day 110 of unknown causes; one twin and three singleton pretmancies were maintained. After the transfer of IVF-derived embryos, 3 ewes delivered 5 lambs, 1 ewe had triplets, of which 2 lambs died at birth and the relmining 2 lambs were normal; 5 ewes maintained their pregnancies to term. DISCUSSION The results of this study show that sheep oocytes injected with a single spermatozoon are capable of development to the blastocyst stage. In contrast, sham-injected oocytes cleaved parthenogenetically up to 16 cells, and none of them developed to the blastocyst stage, suggesting that blastocysts obtained from ICSI are pontenlially viable and not a result of parthenogenesis. The rate of blastocyst development obtained in this study was lower after ICSI than after IVF. Similar rates of blastocyst development have been reported after ICSI in cattle (6, 10). One reason for the low blastocyst development rate after ICSI could be that oocyte activation was inadequate. Tesarik et al. (23) in humans and Goto et al. (10) in cattle reported that the main cause of fertiliT~tioll failure after ICSI was the failure of oocyte activation. This was improved when the oocytes were exposed to an artifu:ial oocyte activation with 10 #L of calcium ionophore (23). In the present study, the total activation rates after ICSI

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(42.5%) and sham injection (31.6%) were similar, but were lower than the total activation rate after IVF (62%). This suggests that it is possible to induce mechanical activation by the injection process, but that manipulation alone is inadequate to cause proper oocyte activation in comp~_,ison with IVF. Therefore, new methods to activate oocytes after ICSI are needed to improve the rates of fertilization and blastocyst formation. Another consideration is the role of in vi~o oocyte maturation in the activation of oocytes in culture. It has been reported that the rates of in vitro maturation, fertilization and cleavage are similar for prepuberhal and adult oocytes in sheep, but prepubertal oocytes compared with adult oocytes have a different capacity for in vitro development (16). This suggests that chromosomal maturation in vitro is adequat_e to allow normal fertilization rates, but cytoplasmic maturation is not sufficient to allow higher rates of blastocyst formation. In 1VF, activation is ip~uced by the fertilizing spermatozoon which facilitates a more normal physiological response in the oocyte than when activation is induced by mechanical injection. Ina_~eq~ate in vitro cytoplasmic rna~,ration combined with inadeq~,ate activation could be reflected in lower sperm decondensation and lower embryo development to reach the blastocyst stage after ICSI. Thus, further improvement of techniq~_les for in vitro raamration of sheep oocytes may also result in better fertilization and blastocyst formation rates after ICSI. A high proportion of embryos was arrested on Day 4 after cleavage (ICSI; 80% and IVF; 43%). Goto et al. (10) and Chen (6) reported 79 and 76%, respectively, of embryos arrested, as a proportion of oocytes cleaved. There are many possible causes of arrest of embryooic development at the 16-cell or morula stage, such as inadequate in vitro oocyte maturation, the injection procedure or the manipulations associated with injection, embryo abnormalitie~ or the presence of parthenogenetic embryos. Abnormal fertilization has been found after sperm injection in humans (7,2,3,20) and sheep (9). In our study, we found a lower cell number in blastocyst stage embryos derived from ICSI than from IVF that may be related to abnormal fertilization or chromosomal abnormalities. It was not possible to correlate the number of blastocyst cells with the proportion of lost embryos and pre~ancies, and embryonic loss occurred after both ICSI and IVF. There are a number of reports of embryonic loss after Wansfer of in vitro produced embryos. In cattle, Renard et al. (18) reported a direct negative effect of culture on development of the inner cell mass, resulting in the loss of embryos at around Days 21 to 35 (50%) and at the time of implantation (46.3%). MacMillan et aL (14) reported a 62% embryo loss before Day 60. Almeida et al. (3) reported chromosomal abnormalities in 49% of human embryos cultured in vitro and a high correlation of abnormalities with embryo loss during preimplantation development. Similarly, in our present study, embryo loss mainly occurred after the implantation stage (Day 21) but before Day 45 (ICSI, 53.3%; IVF, 38.4%). These results suggest that embryo culture may directly affect the viability of the embryos compared with that of the level expected of late embryo loss after natural mating (6%, 17). It would be necessary to conduct detailed chromosome analysid of embryos produced by ICSI and to subject control IVF-produced embryos to the same culture

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conditions as for ICSI, omitting the injection procedure itself, to detect abnormalities and to properly assess the effects of ICSI on the cell numbers in blastocyst stage embryos. The present study has demonstrated that embryos obtained after ICSI are potentially viable, but have a lower capacity for in vitro development than IVF embryos. Further investigation on cytopla_~_,:-_icmaturation and oocyte activation is necessary to improve the rates of fertilization and development to the blastocyst stage after ICSI. REFERENCES 1. Ahmadi A, Ng SC, Liow SL, Ali J, Bongso A, Ratnam SS. Intracytoplasmic sperm injection of mouse oocytes with 5mM Ca2+ at different intervals. Hum Reprod 1995; 10:431-435. 2. AI-Hasani S, Ludwig M, Diedrich K, Bauer O, Kupker W, Diedrich C, Slamm R, yilma7 A. Preliminary results on the incidence of pobjploidy in cryopreseved human oocytes after ICSI. Proc 12th Ann Mtg EHSRE. 1996; 50 abstr. 3. Almeida PA, Bolton VN. The relationship between chromosomal abnormality in the human preimplantation embryo and development in vitro. Regmxl Fertil Dev 1996; 8:235-241. 4. Baldassarre H, Fumes CC, de MaWs DG, Pessi H. In vitro production of sheep embryos using laparoscopic folliculc~_entesis: alternative gonadotrophin treatments for stimulation of oocyte donors. Theriogenology 1996; 45:707-717. 5. Catt SL, Catt JW, G6mez MC, Maxwell WMC, Evans G. The birth of a male lamb derived from an in vitro matured oocyte fe~iliTx~d by intra-cytoplasmic i~eetion of a single presumptive male sperm. Vet Record 1996; 139:494-495. 6. Chen SH, Seidel GE, Jr. Bovine oocyte activation after intracytoplasmic sperm injection. Theriogenology 1997; 47:248 abstr. 7. Cohen J, Levron J, Palermo G, Munn6 S, Adler A, Alikani M, SchaUman Ca, Sultan K, Willadsen SM. Atypical activation and fertiliTation patterns in human. Theriogenology 1995; 43:129-140. 8. Evans G, Maxwell WMC. Intrauterine insemination. In: Salamon's artificial insemination of Sheep and Goats. Sydney, Butterworths 1987; 154-166. 9. G6mez MC, Cart SL, Gillan L, Catt JW, Maxwell WMC, Evans G. Time course of p~_-_uc!eax formation after insemination in vitro and intracytopl&~.n sperm injection of in vitro matured sheep oocytes. Proc 13th ICAR, Conf 1996; 2:10-9 abstr. 10. Goto K, Kinoshita A, Nakanishi Y, Ogawa K. Blastocyst formation following intracytoplasmic sperm injection of in-vitro derived spermatids into bovine oocytes. Hum Reprod 1996; 11:824-829. 11. Kelk DA, Gartley CJ, King WA. Birth of lambs from embryos produced by in vitro maturation, fertilization and culture of oocytes. Theriogenology 1992; 37:237 abs~.

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12. Kimura Y, Yana~machi R. Intracytoplasmic sperm injection in the mouse. Biol Reprod 1995; 52:709-720. 13. Lacham-Kaplan O, Trounson A. Intracytoplasmic sperm injection in mice: increased fertilization and development to term after induction of the acrosome reaction. Hum Reprod 1995; 10:2642-2649. 14. McMillan WH, Peterson AJ, Hall DRH, Donnison MI. Embryo and recipient contributions to embryo loss to Day 60 in heife~ receiving either one or two in vitroproduced embryos. Tberiogenology 1997; 47:370 abstr. 15. Moor RM, Trounson AO. Hormonal and follicular factors affecting maturation of sheep oocytes in vitro and their subsequent developmental capacity. J Reprod Ferfil 1977; 49:101-109. 16. O'Brien JK, CaR SL, Ireland KA, Maxwell WMC, Evans G. In vitro and in vivo developmental capacity of oocytes from prepubertal and adult sheep. Theriogenology 1997; 47:1433-1443. 17. Quinh'van TD, Martin CA, Taylor WB, Cairney IM. Estimates of pre- and perinatal mortality in the New Zealand Romney Marsh ewe. II. Pre-and perinatal loss in those ewes that conceived to second service and those that returned to second service and were mated a third time. J Reprod Fertil 1966; 11:391-398. 18. Renard JP, Heyman Y, Ozil J. Importance of gestation losses after non-surgical transfer of cultured and non-cultured bovine blastocyst. Vet Rec 1980; 107:152-153. 19. Slavik T, Fulka J, Goll I. Pregnancy rate after the transfer of sheep embryos originated from randomly chosen oocytes matured and fertilized in vitro. Theriogenology 1992; 38:749-756. 20. Staessen C, Van Steirteghem AC. The chromosomal constitution of embryos developing from abnormally fertilized oocytes after intracytoplasmic sperm injection and conventional in-vitro fertilization. Hum Reprod 1997; 12:321-327. 21. Texvit HR, Rowson LEA. Birth of lambs after culture of sheep ova in vitro for up to 6 days. J Reprod Fertil 1974; 38:177-179. 22. Tervit HR, Whittin~ham DG, Rowson LEA. Successful culture in vitro of sheep and cattle ova. J RgprodFertil 1972; 30:493-497. 23. Tesafik J, Sousa M. More than 90% fertilization rates after intracytoplasmic sperm injection and artificial induction of oocyte activation with calcium ionophore. Fertil Steril 1995; 63:343-349. 24. Walker SK, Heard TM, Seamark RF. In vitro culture of sheep embryos without co-culture: successes and persectives. Theriogenology 1992; 37:111-126. 25. Walker SK, Lami~ RJ, Seamark RF. Culture of sheep zygotes in sythetic oviduct fluid medium with different concentrations of sodium bicarbonate and hepes. Theriogenology 1989; 32:797-804. 26. Willadsen SM. Nuclear transplantation in sheep embryos. Nature 1986; 320:63-65. 27. Wilmat I, Schnieke AE, McWhir J, Kind AJ, Campbell KHS. Viable offspring derived from fetal and adult mammalian cells. Nature 1997; 385:810-813.

1154

Theriogenology

28. Wright RW Jr, Ellington JE. Morphological and physiological differences between in vi~o and in vivo produced preimplantation embryos from livestock species. Theriogenology 1995; 44:1167-1189. 29. Zinaman MI, Clegg ED, Brown CC, O'connor J, Selevan SG. Estimates of human fertifity and pret~nancy loss. Fcrtil Steril 1996; 65:503-509.