scholarly journals Porcine embryos produced after intracytoplasmic sperm injection using xenogeneic pig sperm from neonatal testis tissue grafted in mice

2008 ◽  
Vol 20 (7) ◽  
pp. 802 ◽  
Author(s):  
Ali Honaramooz ◽  
Xiang-Shun Cui ◽  
Nam-Hyung Kim ◽  
Ina Dobrinski
Keyword(s):  
Embryo Development ◽  
Intracytoplasmic Sperm Injection ◽  
Animal Species ◽  
Blastocyst Stage ◽  
Immunodeficient Mice ◽  
Neonatal Pig ◽  
Neonatal Testis ◽  
Porcine Spermatozoa ◽  
Testis Tissue

Embryo development after homologous intracytoplasmic sperm injection (ICSI) with sperm from testis tissue xenografts from pigs or any other farm animal species has not been evaluated critically. Here, we report development of porcine embryos in vitro following ICSI with sperm retrieved from xenografted neonatal pig testis. Small pieces of testis tissue from newborn piglets were grafted under the back skin of castrated immunodeficient mice (n = 4) and the xenografts were collected 8 months after grafting. Spermatozoa were recovered by mincing of the grafted tissue. For comparison, testicular, epididymal and ejaculated spermatozoa were also collected from mature boars. Oocytes injected with xenogeneic spermatozoa were either fixed to determine fertilisation processes (n = 89 in five replicates) or allowed to develop in vitro (n = 143 in four replicates). Xenogeneic porcine spermatozoa were fertilisation competent (24% v. 58%, 68%, 62% or 0% for xenogeneic v. control testicular, epididymal and ejaculated spermatozoa or no spermatozoa, respectively) and embryos developed to the blastocyst stage (8% v. 22%, 27%, 25% or 0%, respectively). These results demonstrate that porcine spermatozoa derived from immature testis tissue xenografted into mice are fertilisation competent, albeit at a lower rate than testicular, epididymal or ejaculated spermatozoa from control boars, and support embryo development after ICSI.

Use of time-lapse imaging to evaluate morphokinetics of in vitro equine blastocyst development after oocyte holding for two days at 15°C versus room temperature before intracytoplasmic sperm injection

2019 ◽  
Vol 31 (12) ◽  
pp. 1862 ◽  
Author(s):  
N. A. Martino ◽  
G. Marzano ◽  
A. Mastrorocco ◽  
G. M. Lacalandra ◽  
L. Vincenti ◽  
...  
Keyword(s):  
Embryo Development ◽  
Intracytoplasmic Sperm Injection ◽  
Room Temperature ◽  
Time Lapse ◽  
Blastocyst Stage ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Group 13 ◽  
Time Lapse Imaging

Time-lapse imaging was used to establish the morphokinetics of equine embryo development to the blastocyst stage after invitro oocyte maturation (IVM), intracytoplasmic sperm injection (ICSI) and embryo culture, in oocytes held overnight at room temperature (22–27°C; standard conditions) before IVM. Embryos that developed to the blastocyst stage underwent precleavage cytoplasmic extrusion and cleavage to the 2-, 3- and 4-cell stages significantly earlier than did embryos that arrested in development. We then determined the rate of blastocyst formation after ICSI in oocytes held for 2 days at either 15°C or room temperature before IVM (15-2d and RT-2d treatment groups respectively). The blastocyst development rate was significantly higher in the 15-2d than in the RT-2d group (13% vs 0% respectively). The failure of blastocyst development in the RT-2d group precluded comparison of morphokinetics of blastocyst development between treatments. In any condition examined, development to the blastocyst stage was characterised by earlier cytoplasmic extrusion before cleavage, earlier cleavage to 2- and 4-cell stages and reduced duration at the 2-cell stage compared with non-competent embryos. In conclusion, this study presents morphokinetic parameters predictive of embryo development invitro to the blastocyst stage after ICSI in the horse. We conclude that time-lapse imaging allows increased precision for evaluating effects of different treatments on equine embryo development.

Speed of in vitro embryo development affects the likelihood of foaling and the foal sex ratio

2020 ◽  
Vol 32 (5) ◽  
pp. 468 ◽  
Author(s):  
A. Claes ◽  
J. Cuervo-Arango ◽  
S. Colleoni ◽  
G. Lazzari ◽  
C. Galli ◽  
...  
Keyword(s):  
Sex Ratio ◽  
Embryo Development ◽  
Intracytoplasmic Sperm Injection ◽  
Odds Ratio ◽  
Blastocyst Stage ◽  
Embryo Production ◽  
Time Required

The success of invitro embryo production (IVEP) in horses has increased considerably during recent years, but little is known about the effect of the speed of invitro embryo development. Blastocysts (n=390) were produced by intracytoplasmic sperm injection of IVM oocytes from warmblood mares, cryopreserved, thawed and transferred into recipient mares on Days 3, 4, 5 or 6 after ovulation. The time required for invitro-produced (IVP) embryos to reach the blastocyst stage was recorded (Day 7 vs Day 8). The likelihood of foaling was affected by the speed of invitro embryo development and recipient day after ovulation at transfer. The odds ratio for foaling was ~0.63 for transfer of Day 8 (46%) compared with Day 7 (56%) IVP blastocysts. The highest likelihood of pregnancy (72%) and foaling (60%) was observed when IVP blastocysts were transferred to recipient mares on Day 4 after ovulation. Finally, the sex (colt:filly) ratio was higher after transfer of Day 7 (71%:29%) than Day 8 (54%:46%) IVP blastocysts, suggesting that the speed of embryo development is sex dependent. In conclusion, the speed of invitro embryo development in our IVEP system affects the likelihood of foaling and the sex of the foal.

182 EFFECTS OF MITOCHONDRIAL ACTIVITY OF INJECTED SPERM ON EARLY DEVELOPMENT IN BOVINE INTRACYTOPLASMIC SPERM INJECTION-DERIVED EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 205
Author(s):  
Y. Nagao ◽  
H. Yamamoto ◽  
B. Sarentonglaga ◽  
K. Ogata ◽  
M. Yamaguchi ◽  
...  
Keyword(s):  
Embryo Development ◽  
Intracytoplasmic Sperm Injection ◽  
Laser Scanning ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Mitochondrial Activity ◽  
Early Embryo Development

Intracytoplasmic sperm injection (ICSI) has become the method of choice for bovine ovum pick-up and IVF. However, there are many difficulties with the ICSI technique to obtain viable fetuses. One of the major problems associated with this technique is our lack of knowledge concerning the status of the sperm mitochondria when injected into the oocyte and its effect on embryo development. First, we examined the mitochondrial activity of sperm that had been activated by culturing with methyl-β cyclodextrin (MBCD), in ICSI and in IVF. In vitro-matured oocytes and JC1-labelled sperm were used for the ICSI and IVF. The fluorescence intensity of injected/penetrated sperm mitochondria was measured using confocal laser scanning microscopy. Then, the relative membrane potential of the mitochondria was analysed by a ratiometric method. Second, the reactive oxygen species (ROS) production and capacitation status of the sperm exhibiting normal motility and of the sperm that had been activated by culturing with MBCD were analysed. The ROS levels produced by the sperm were estimated using the luminol assay. The chlortetracycline stain was used to evaluate capacitation status of the sperm. Third, the effect of ROS produced by these sperm types upon embryogenesis following ICSI and IVF was studied. Early developing embryos were examined with a stereomicroscope for cleavage and development to the blastocyst stage after 7 days of culture. Chromosome samples stained with Giemsa solution from the blastocysts were used to analyse the chromosomal integrity. Data were analysed by t-test for Experiments 1 and 2, and ANOVA with Fisher's PLSD test for Experiment 3. The mitochondrial activity immediately after ICSI was higher than at 3 h after insemination (immediately after sperm penetration) in IVF (P < 0.05). The sperm exhibiting activation were capacitated and produced more ROS than the sperm exhibiting normal motility (P < 0.05). The rates of cleaved embryo and blastocyst after ICSI with activated sperm were the same as that in ICSI with normal motility sperm and in IVF (cleaved rate: 66.7, 71.8, and 85.0%, respectively; blastocyst rate: 24.4, 23.3, and 32.0%, respectively). However, chromosomal integrity of blastocysts derived from ICSI with activated sperm was lower than that for ICSI with normal motility sperm or for IVF (23.1, 75.0, and 63.6%, respectively; P < 0.01). In conclusion, capacitated, activated sperm induced chromosomal aberrations during early embryo development following ICSI. Conceivably, the selection of sperm exhibiting progressive motility, which is expected to be activated and to fertilize, would not always be better for early embryo development and fetal growth following ICSI due to the ROS derived from the sperm mitochondria. Injection of sperm exhibiting normal motility, or of mitochondria reduced activated sperm, could improve the quality of ICSI-derived embryos.

scholarly journals Analysis of Morphokinetic Parameters of Feline Embryos Using a Time-Lapse System

Animals ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 748
Author(s):  
Joanna Kochan ◽  
Agnieszka Nowak ◽  
Barbara Kij ◽  
Sylwia Prochowska ◽  
Wojciech Niżański
Keyword(s):  
Embryo Development ◽  
Embryo Quality ◽  
Time Lapse ◽  
Blastocyst Stage ◽  
Cavity Formation ◽  
Non Invasive ◽  
Morphokinetic Parameters ◽  
Morphological Defects ◽  
Development In Vitro

The aim of this study was to analyze the morphokinetic parameters of feline embryos using a time lapse system. Oocytes matured in vitro were fertilized (IVF) and in vitro cultured in a time lapse-system (Primo Vision®, Gothenburg, Sweden). The first cell division of embryos occurred between 17 h post insemination (hpi) and 38 hpi, with the highest proportion of embryos (46%) cleaving between 21 and 24 hpi. The timing of the first cleavage significantly affected further embryo development, with the highest development occurring in embryos that cleaved at 21–22 hpi. Embryos that cleaved very early (17–18 hpi) developed poorly to the blastocyst stage (2%) and none of the embryos that cleaved later than 27 hpi were able to reach the blastocyst stage. Morphological defects were observed in 48% of the embryos. There were no statistically significant differences between the timing intervals of the first cleavage division and the frequency of morphological defects in embryos. Multiple (MUL) morphological defects were detected in more than half (56%) of the abnormal embryos. The most frequent single morphological defects were cytoplasmic fragmentation (FR) (8%) and blastomere asymmetry (AS) (6%). Direct cleavage (DC) from 1–3 or 3–5 blastomeres, reverse cleavage (RC) and vacuoles were rarely observed (2–3%). The timing of blastocyst cavity formation is a very good indicator of embryo quality. In our study, blastocyst cavity formation occurred between 127–167 hpi, with the highest frequency of hatching observed in blastocysts that cavitated between 142–150 hpi. Blastocysts in which cavitation began after 161 h did not hatch. In conclusion, the timing of the first and second cleavage divisions, the timing of blastocyst cavity formation and morphological anomalies can all be used as early and non-invasive indicators of cat embryo development in vitro.

scholarly journals 136 EVIDENCE OF A DIRECT EFFECT OF P4 ON IVF-DERIVED BOVINE 8-CELL EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 219 ◽  
Author(s):  
C.E. Ferguson ◽  
T.R. Davidson ◽  
M.R.B. Mello ◽  
A.S. Lima ◽  
D.J. Kesler ◽  
...  
Keyword(s):  
Embryo Development ◽  
Direct Effect ◽  
Blastocyst Stage ◽  
Control Group ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Direct Role ◽  
Treatment Groups ◽  
And Control

There has been much debate over a direct role for progesterone (P4) in early bovine embryo development. While previous attempts to supplement bovine embryos in vitro with P4 produced results that vary and are often contradictory, this may be a response of administering P4 at inappropriate times. Therefore, the objective of these experiments was to determine if P4 could exert a direct effect on developing IVF-derived bovine embryos when administered at an appropriate time of embryo development. In Exp. I, IVF-derived bovine 8-cell embryos were randomly allotted to treatments: (1) control, CR1aa medium (n = 168); (2) vehicle, CR1aa + ETOH (0.01%) (n = 170); and (3) P4, CR1aa + ETOH + P4 (20 ng/mL in 50-μL droplet) (n = 173). In Exp. II, IVF-derived bovine 8-cell embryos were randomly allotted to treatments: (1) control, CR1aa medium (n = 160); (2) vehicle, CR1aa + DMSO (0.01%) (n = 180); and (3) P4, CR1aa + DMSO (0.01%) + P4 (20 ng/mL in 50-μL droplet) (n = 170). All embryos were evaluated on Days 6 to 9 post-insemination and rates calculated from 8-cell embryos. In Exp. I, ETOH tended to have a detrimental effect with significantly fewer (P < 0.05) embryos (53%) developing to the blastocyst stage on Day 7 compared with the control (62%) and P4 (71%) groups. At Day 7, significantly more embryos cultured in P4 (71%) developed to the blastocyst stage compared with the control group (62%). P4 treatment significantly increased the number of Grade 1 blastocysts (25%) on Day 7 compared with vehicle (15%) and control (17%) groups. At the end of culture, there were also significantly more Day 9 hatched blastocysts in the P4 group (33%) compared with vehicle (22%) and control (21%) groups. Supplementing P4 in the culture medium increased the rate of development, resulting in significantly more blastocysts (8%) on Day 6 and hatched blastocysts (21%) on Day 8 compared with vehicle (3% and 12%) and control (0% and 8%) groups, respectively. In Exp. II, there were no significant differences between treatment groups for Day 7 blastocysts (control 54%, DMSO 61%, P4 57%) and Day 9 hatched blastocysts (control 46%, DMSO 51%, P4 46%). However, there were significantly more Grade 1 blastocysts in the P4 group (22% and 36%) on Days 6 and 8 compared with vehicle (11% and 23%) and control (13% and 23%) groups, respectively. The lack of improvement in Day 7 blastocysts and Day 9 hatched blastocysts rates leads to further uncertainty in understanding the P4 vehicle interactions. In conclusion, the results of these two experiments indicate that P4 can exert a direct effect on the developing IVF-derived bovine embryo; however, due to P4 vehicle interactions; other inert vehicles need to be explored to further evaluate the direct effects of P4 on the developing bovine embryo.

Mutations in ZP4 are associated with abnormal zona pellucida and female infertility

2021 ◽  
pp. jclinpath-2020-207170
Author(s):  
Xiaoli Wei ◽  
Youzhu Li ◽  
Qicai Liu ◽  
Wensheng Liu ◽  
Xiaohong Yan ◽  
...  
Keyword(s):  
Embryo Development ◽  
Zona Pellucida ◽  
Intracytoplasmic Sperm Injection ◽  
Treatment Strategies ◽  
Suction Pressure ◽  
Human Female ◽  
Female Reproduction ◽  
Efficient Treatment ◽  
Whole Exome

BackgroundThe zona pellucida (ZP) of human oocytes plays essential protective roles in sperm–egg interactions during fertilisation and embryo development. ZP4-null female rabbits exhibit a thin and irregular ZP, which severely impairs embryo development and fertility. However, the effects of ZP4 defect on human female reproduction remain unknown.Methods and resultsWe performed whole-exome sequencing in 26 female patients with abnormal (thin and irregular) ZP and identified heterozygous variants in ZP4 (OMIM: 613514) from 3 patients (approximately 11%). No ZP4 variant was found in the 30 control women with proven fertility. We constructed ZP4-mutated plasmids and found that the variants reduced the secretion of ZP4 in vitro. Lower suction pressure facilitated egg retrieval, and intracytoplasmic sperm injection (ICSI) was a desirable treatment for ZP4-mutated patients with abnormal ZP.ConclusionsWe identified ZP4 as a novel gene for human abnormal ZP and found that lower suction pressure and ICSI are efficient treatment strategies.

175 BOVINE EMBRYO DEVELOPMENT AND MRNA EXPRESSION IN BLASTOCYSTS CULTURED IN ISOLATED MOUSE OVIDUCTS MAINTAINED IN SOF OR KSOM

2006 ◽  
Vol 18 (2) ◽  
pp. 195
Author(s):  
D. Rizos ◽  
B. Pintado ◽  
J. de la Fuente ◽  
P. Lonergan ◽  
A. Gutierrez-Adan
Keyword(s):  
Embryo Development ◽  
Relative Abundance ◽  
Culture Medium ◽  
Embryo Quality ◽  
Blastocyst Stage ◽  
Glut 1 ◽  
Gene Transcripts ◽  
Culture Environment ◽  
Chi Square Analysis

It is well known that modification of the post-fertilization culture environment of mammalian pre-attachment embryos can affect blastocyst quality, manifested in terms of morphology, cryotolerance, and relative abundance of certain gene transcripts. Culture of in vitro-produced bovine zygotes in the ewe oviduct leads to the development of blastocysts of a quality similar to those derived totally in vitro (Rizos et al. 2002 Biol. Reprod. 66, 589-595). However, such a system has disadvantages from a practical and animal welfare point of view. The isolated mouse oviduct (IMO) culture system is a potential alternative and has been successfully used in the in vitro culture of mouse, rat, hamster, and pig embryos from the one-cell stage to the morula/blastocyst stage. The aim of this study was to examine (1) the development of bovine zygotes in the IMO maintained in two different media (SOF and KSOM) in organ culture, and (2) the quality of the resultant blastocysts assessed in terms of the relative abundance of transcripts for several genes that have been previously implicated in embryo quality. Mouse oviducts were isolated from adult Swiss females (CD1, Harlan) the day after mating with an intact male. Approximately 10-15 presumptive bovine zygotes, produced by in vitro oocyte maturation and fertilization, were transferred to the ampullae of the isolated oviducts and were cultured in Transwell plates (Costar, Corning, NY, USA) over 1.1 mL of culture medium (SOF, n = 241 or KSOM, n = 320) at 39�C in an atmosphere of 5% CO2 in air at maximum humidity. A control group of embryos was cultured in droplets (25 �L) of the same culture medium and conditions in parallel (SOF, n = 278, KSOM, n = 225). Five replicates (=days of bovine ovary collection) were carried out. Following 6 days of culture, embryos were recovered from the oviducts/culture drops and blastocysts were snap-frozen in liquid nitrogen. Quantification of all gene transcripts was carried out by real time quantitative RT-PCR. Data on embryo development were analyzed by chi-square analysis and differences in transcript abundance by ANOVA. Culture in the IMO did not affect the proportion of zygotes developing to the blastocyst stage compared to the respective control droplets (SOF: 21.0 vs. 21.9%; KSOM: 22.0 vs. 22.2%). Culture in the IMO in SOF resulted in an increase (P d 0.05) in the abundance of transcripts for Oct-4 and SOX and reduced abundance of Glut-1, Na/K transporter, Cx43, and survivin, compared to control embryos. In contrast, culture in the IMO in KSOM resulted in increased abundance of transcripts for Glut-1, Cx43, Oct-4, and survivin and a reduced expression of Na/K transporter and SOX. Transcripts for G6PDH, IFN, and E-Cad were unaffected by culture environment. In conclusion, culture in the IMO leads to alterations in the relative abundance of transcripts that have been previously associated with embryo quality following culture in the ewe oviduct. However, the effect is dependent on the basal medium used.

35 IN VITRO BOVINE EMBRYO DEVELOPMENT AFTER NUCLEAR TRANSFER BY HANDMADE CLONING USING A MODIFIED WOW CULTURE SYSTEM

2006 ◽  
Vol 18 (2) ◽  
pp. 126 ◽  
Author(s):  
C. Feltrin ◽  
F. Forell ◽  
L. dos Santos ◽  
J. L. Rodrigues
Keyword(s):  
Embryo Development ◽  
Culture System ◽  
Nuclear Transfer ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Inner Diameter ◽  
Group 2 ◽  
Group 1 ◽  
Handmade Cloning

The effect of the microenvironment on embryo development during in vitro culture of zona-free embryos after nuclear transfer is still unclear. The aim of this experiment was to determine the effect of the dimensions of the well (WOW; Vajta et al. 2000 Mol. Reprod. Dev. 55, 256-264) culture system on the in vitro development of handmade cloned bovine embryos to the blastocyst stage. Appropriately ground steel needles were pressed slightly by hand to the bottom of the well of a polystyrene four-well dish (176740, Nunc, Life Technologies AS, Roskilde, Denmark). Embryos were produced by the handmade cloning (HMC) technique (Vajta et al. 2003 Biol. Reprod. 68, 571-578) with modifications, using primary cultures of skin fibroblast cells from an adult cow as nuclear donors. Cumulus-oocyte complexes were in vitro-matured in M-199 supplemented with 10% estrous cow serum (ECS), FSH, hCG, and estradiol (E2) for 17 h. After maturation, cumulus cells were removed by pipetting. Following zona pellucida removal in 0.5% protease (Sigma, Brazil), zona-free oocytes were incubated for 15 min in 5 mg/mL cytochalasin B (Sigma) and subsequently hand-bisected and screened for nuclear material under UV light after incubation in 10 mg/mL bisbenzimide (Hoechst 33342). Next, two enucleated halves and one donor cell were aggregated after a quick exposure to phytohemagglutinin (PHA) and subsequently fused by two electrical DC pulses of 1 kV/cm for 20 �s, in a BTX 453 chamber coupled to an ECM 2001 Electro Cell Manipulator System (BTX, Inc., San Diego, CA, USA), with additional exposure to brief pre- and post-fusion AC pulses of 15 V. Reconstructed embryos were chemically activated in 5 mM ionomycin (Sigma) for 5 min, followed by 2 mM 6-DMAP (Sigma) for 2.5 h. Finally, activated reconstructed cloned embryos were in vitro-cultured in one of two WOW culture systems (larger vs. smaller micro-wells) in 4-well plates containing 400 mL modified SOF medium supplemented with 10% ECS, under mineral oil, at 5% CO2, 5% O2 and 90% N2, and 39�C for 7 days. In Group 1 (large-size micro-well), embryos were cultured in individual cylindrical micro-wells with an inner diameter and depth of approximately 280 and 250 mm, respectively, whereas in Group 2 (small size micro-well), embryos were cultured in individual conical micro-wells with approximately 130 mm inner diameter and 150 mm depth. Data analysis was performed by the chi-square test. After four replicates, cleavage rates were significantly higher (P < 0.05) in Group 2 (51/63, 80.9%) than in Group 1 (43/67, 64.1%). Embryo development to the blastocyst stage was also greater (P < 0.05) in the small micro-wells (16/63, 25.3%) than in the large ones (8/67, 11.9%). In summary, these results show a significant increase in cleavage and blastocyst developmental rates in handmade cloned embryos cultured in a modified WOW system using individual small size micro-wells, suggesting that a small, tighter micro-well provides favorable in vitro conditions for embryo development.

374 THE EFFECTS OF POLYVINYLPYRROLIDONE ON BOVINE SPERM FUNCTION AND EMBRYO DEVELOPMENT

2007 ◽  
Vol 19 (1) ◽  
pp. 302 ◽  
Author(s):  
Y. Kato ◽  
M. Fukushima ◽  
A. Kenmotsu ◽  
K. Chikazawa ◽  
Y. Nagao
Keyword(s):  
Embryonic Development ◽  
Embryo Development ◽  
Staining Method ◽  
Blastocyst Stage ◽  
Sperm Function ◽  
Bovine Sperm ◽  
Developmental Capacity ◽  
Chromatin Integrity ◽  
Triple Staining

In assisted reproduction by ICSI, PVP has been successfully used to replicate the viscosity of sperm solution, thus facilitating the handling and immobilization of spermatozoa. Sperm is suspended in medium containing polyvinylpyrrolidone (PVP), then injected into the oocytes together with a small amount of the medium in ICSI. However the effects of PVP on sperm function and embryo development have not been investigated in detail. In the present study, we investigated the effects of PVP solution on sperm function and embryonic development. Frozen–thawed spermatozoa from a Japanese Black bull and immature COCs from slaughterhouse bovine ovaries were used for all experiments. In experiment 1, bovine sperm was cultured in SOF or SOF containing 10% PVP. For detection of sperm acrosomal and chromatin integrity, sperm cultured in each medium were stained by the triple staining method and acridine orange after 0, 15, 30, and 60 min of culture. In experiment 2, zygotes were injected with PVP solution and cultured in vitro; subsequent cleavage and development to blastocysts were examined. In experiment 3, zygote injected with PVP solution was fixed by 4% paraformaldehyde after 1–3 h of PVP injection. The location of PVP solution in zygote was observed. In experiment 4, two-cell embryos were microinjected with a solution of dextran conjugated with fluorescein (FITC-dextran) and cultured in vitro. The location of FITC-dextran in the embryo was examined. In experiment 1, acrosome reactions of the sperm were enhanced after 15 min of incubation in PVP solution (P &lt; 0.05), but chromatin integrity of the sperm was not influenced (P &gt; 0.05). In experiment 2, PVP suppressed the development of the zygote to 2-cell, morula and blastocyst (75.0%, 35.1%, and 26.3% vs. 61.3%, 20.2%, and 12.9% for control and PVP group, respectively, P &lt; 0.05). In experiment 3, the locations of PVP solution in the zygote were observed 1–3 h after injection. In experiment 4, FITC-dextran was observed in ICM at the blastocyst stage. These findings suggest that PVP affects the acrosome but not the chromatin of sperm in ICSI. PVP solution exists locally in embryos injected and affects the developmental capacity of the embryos.

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