177 Equine Sperm Decondensation and Blastocyst Formation After Conventional versus Piezo-Driven Intracytoplasmic Sperm Injection

2018 ◽  
Vol 30 (1) ◽  
pp. 228
Author(s):  
J. G. Brom-de-Luna ◽  
R. M. Salgado ◽  
H. L. Resende ◽  
H. S. Canesin ◽  
K. Hinrichs
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Sperm Head ◽  
Injection Technique ◽  
Blastocyst Formation ◽  
Dapi Staining ◽  
Vitro Fertilization ◽  
Significant Difference ◽  
Head Area ◽  
Sperm Decondensation

Intracytoplasmic sperm injection (ICSI) is currently the most effective method for in vitro fertilization in the horse. There are 2 main techniques, conventional (Conv) ICSI and piezo-driven (Piezo) ICSI. Many laboratories reporting good equine blastocyst rates (>20% per injected oocyte) use Piezo ICSI, but it is not known whether the Piezo confers an advantage. We compared sperm decondensation and blastocyst formation between the 2 techniques. A blunt, 6-µm inner diameter needle, loaded with 10 µL of Fluorinert was used for Piezo ICSI; 1 pulse was used to penetrate the oolemma. Frozen-thawed semen from one stallion was used. In experiment 1, in vitro-matured equine oocytes were randomly assigned to Conv or Piezo ICSI, performed concurrently by separate operators. Blastocyst formation was evaluated on Days 7 to 10 and confirmed by DAPI staining. Data were analysed by Fisher’s exact test. There was no significant difference in blastocyst rates (32/82, 39% for Conv and 35/87, 40% for Piezo; P > 0.1). In experiment 2, equine sperm head decondensation after ICSI was evaluated using porcine oocytes, due to scarcity of equine oocytes. Porcine oocytes were recovered from slaughterhouse-derived ovaries and matured in vitro using a biphasic maturation culture system. Mature oocytes were subjected to Conv or Piezo ICSI, using equine sperm treated with a mitochondrial stain to allow identification of the sperm tail. The oocytes were fixed in 4% paraformaldehyde immediately after injection (0 h) or were cultured for 3, 6, 9, or 18 h after injection, and then fixed. Fixed oocytes (15-22 per treatment per period) were stained with DAPI and the area of the sperm head, in arbitrary units, determined using ImageJ software (National Institutes of Health, Bethesda, MD, USA). The medians were compared using the Mann-Whitney U-test. Sperm heads in the Piezo treatment increased in area over time, from a median of 1242 at 0 h to 54,991 at 18 h. In contrast, sperm heads in the Conv treatment largely failed to decondense, having median areas of 1275 at 0 h and 1510 at 18 h. Sperm head area was significantly greater for sperm in the Piezo than in the Conv treatment for all time periods except 0 h (P < 0.05). Because this conflicted with the blastocyst results obtained with horse oocytes, we conducted experiment 3 to examine sperm head decondensation after ICSI in horse oocytes. Oocytes (8 to 12 per treatment per period) were fixed 0, 6, or 18 h after ICSI. There was no difference between techniques in sperm head area at any time (median values for 0, 6 and 18 h of 1280, 4323, and 57,185 respectively for Piezo and 1326, 1604, and 62,558 for Conv; P > 0.2). These results indicate that there is a species-specific difference in processing of sperm after ICSI, dependent on injection technique. Further work evaluating sperm from additional stallions, as well as porcine sperm, is necessary to determine whether sperm source affects these results. Research supported by the Clinical Equine ICSI Program, Texas A&M University.

160 Exposure to Cadmium Affects Oocyte and Embryo Competence in Cattle

2018 ◽  
Vol 30 (1) ◽  
pp. 219
Author(s):  
C. De Canditiis ◽  
N. Pagano ◽  
V. Franco ◽  
I. Paradiso ◽  
É. C. Dos Santos ◽  
...  
Keyword(s):  
Embryo Development ◽  
Cumulus Cells ◽  
Early Embryo ◽  
Dapi Staining ◽  
Vitro Fertilization ◽  
Nuclear Maturation ◽  
Significant Difference ◽  
Vitro Model ◽  
Hoechst Staining

There is a growing worldwide concern regarding the increased release of the heavy metal cadmium (Cd) in the environment, due to several industrial processes, as it is known to affect health. Among other heavy metals, Cd is widely recognised to influence the reproductive system at different levels, interfering with both gametes and embryo functions in several species (Thompson and Bannigan, 2008 Reprod. Toxicol. 25, 304-315). The in vitro model can be used to mimic environmental conditions allowing us to evaluate their effect on oocyte maturation and early embryo development. Therefore, the aim of this study was to evaluate the influence of different Cd concentrations on nuclear maturation, apoptosis in cumulus cells, and cleavage and blastocyst yields in cattle. For this purpose, abattoir-derived bovine oocytes were in vitro matured, fertilized, and cultured according to standard procedures (Rubessa et al. 2011 Theriogenology 76, 1347-1355). In particular, oocytes were matured with 0 (control; n = 126), 0.1 μM (n = 139), 1 μM (n = 134), and 10 μM of Cd (n = 135), at 39°C under humidified air with 5% CO2, 7% O2, and 88% N2. For each replicate, after 22 h of maturation, a representative sample of oocytes (n = 10 per each group) was used to evaluate nuclear maturation by 4′,6-diamidino-2-phenylindole (DAPI) staining and another sample (n = 10 per each group) to assess cumulus-cells complex apoptosis by TUNEL/Hoechst staining (Pocar et al. 2005 Reproduction 130, 857-868). The remaining oocytes were in vitro fertilized and cultured with 0 (n = 106), 0.1 μM (n = 119), 1 μM (n = 114), and 10 μM (n = 115) Cd. The experiment was repeated 3 times. On Day 8 post-IVF, the blastocyst yields were recorded. Differences among groups were analysed by ANOVA, with the least significant difference method used as a post hoc test. Data are presented as means ± SE. Unexpectedly, the exposure of oocytes to Cd during IVM did not affect the percentage of oocytes undergoing nuclear maturation (on average 96.3 ± 2.3). In contrast, concentrations of 1 and 10 μM Cd increased the percentage of apoptotic cumulus-cells in cumulus–oocyte complexes (COC) compared with the control (3.4 ± 0.4, 10.6 ± 1.8, 15.0 ± 0.9, 16.7 ± 4.0, respectively, with 0, 0.1, 1, and 10 μM; P < 0.05). It is worth pointing out that with the highest concentration, cumulus expansion did not occur and cumulus cells appeared detached from the oocyte. Likewise, 1 and 10 μM Cd decreased cleavage rates compared with the control (68.7 ± 1.8, 54.3 ± 5.0, 58.5 ± 4.2 and 2.8 ± 2.6, respectively, with 0, 0.1, 1, and 10 μM Cd; P < 0.01). Finally, blastocyst yields decreased when oocytes were treated with 0.1 μM Cd and no development to blastocyst was observed at the 2 higher concentrations (35.1 ± 1.7, 26.2 ± 3.1, 0, 0, respectively, with 0, 0.1, 1, and 10 μM; P < 0.01). In conclusion, exposure to Cd during maturation negatively affects bovine COC, as indicated by the increased apoptotic index in cumulus cells, without influencing the nuclear maturation process. Furthermore, the presence of Cd during in vitro fertilization and culture severely impairs both the fertilization and post-fertilization embryo development.

scholarly journals 146COMPARISON OF EMBRYO CULTURE MEDIA FOR BLASTOCYST DEVELOPMENT AFTER INTRACYTOPLASMIC SPERM INJECTION OF IN VITRO-MATURED EQUINE OOCYTES

2004 ◽  
Vol 16 (2) ◽  
pp. 195
Author(s):  
Y.H. Choi ◽  
D.D. Varner ◽  
K. Hinrichs
Keyword(s):  
In Vitro Culture ◽  
Embryo Culture ◽  
Intracytoplasmic Sperm Injection ◽  
Culture Media ◽  
Polar Body ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Vitro Fertilization ◽  
Significant Difference

Research on in vitro culture of equine embryos has been scant, due to failure of equine in vitro fertilization to be repeatably successful. We have recently obtained high fertilization rates of equine oocytes via intracytoplasmic sperm injection (ICSI) using a piezo drill (Choi et al., 2002 Reproduction 123, 455–465). Culture of presumptive zygotes in G1.2/2.2 medium resulted in 63% cleavage and an average of 15 cells at 4d, but only 2 to 9% blastocyst development at 7 days (Choi et al., 2003 Theriogenology 59, 1219–1229). In the present study, we evaluated the effect of two different culture media, G1.3/G2.3 v. DMEM/F-12, with or without FBS, on blastocyst development after ICSI. Oocytes were collected from slaughterhouse-derived ovaries by follicular scraping and were matured in vitro for 24h in M199 with 10% FBS and 5μUmL−1 FSH. After culture, oocytes having a polar body (198/305; 65%) were fertilized by ICSI with frozen-thawed equine sperm using a piezo drill. Presumptive zygotes were cultured in 1 of 4 media: G1.3/G2.3 (which includes 0.8% BSA) with or without 10% FBS, or in DMEM/F-12 with 0.5% BSA, with or without 10% FBS. Culture was performed in microdroplets at 5μL/zygote under oil at 38.2°C in an atmosphere of 5% CO2, 5% O2 and 90% N2 for 7.5 days. In G1.3/2.3 treatments, G1.3 media were completely refreshed at 48h, zygotes were transferred to G2.3 (with or without FBS as per the first stage) at 96h, and were completely refreshed with the same media at 144h. In DMEM/F-12 treatments, media were completely refreshed every other day. Three to 5 replicates were performed in each treatment, and data were analyzed by chi-square test. There were no significant differences in cleavage rates (59–64%) among treatments. The rate of development to blastocyst, per oocyte injected, in G1.3/G2.3/BSA (1/49, 2%) was significantly lower (P&lt;0.05) than that for the other three treatments: G1.3/2.3/BSA/FBS (9/49, 18%), DMEM/F-12/BSA (9/50, 18%), or DMEM/F-12/BSA/FBS (10/50, 20%). There was no significant difference in blastocyst development among the latter three treatments. These findings indicate that G1.3/2.3 media with BSA only do not adequately support growth of equine embryos. Development of up to 20% of injected oocytes to the blastocyst stage in G media supplemented with FBS, in DMEM/F-12/BSA or in DMEM/F-12/BSA/FBS represents the highest in vitro equine blastocyst rate in medium alone (i.e. without co-culture) yet reported. The success of DMEM/F-12 as an embryo culture medium may provide a relatively simple basis for equine in vitro culture programs. To determine whether this medium was able to support further developmental competence, we cultured equine embryos resulting from nuclear transfer of in vitro-matured oocytes in DMEM/F-12+10% FBS (without BSA). We transferred 4 resulting blastocysts to recipient mares by transcervical transfer; one pregnancy is ongoing at 230d gestation at the time of this writing. This work was supported by the Link Equine Research Endowment Fund, Texas A&amp;M University.

scholarly journals In Vitro Fertilization Outcome After Intracytoplasmic Sperm Injection with Fresh and with Frozen-Thawed Epididymal Spermatozoa

KnE Medicine ◽  
2016 ◽  
Vol 1 (1) ◽  
Author(s):  
Hilma Putri Lubis
Keyword(s):  
In Vitro Fertilization ◽  
Intracytoplasmic Sperm Injection ◽  
Oocyte Retrieval ◽  
Epididymal Sperm ◽  
Vitro Fertilization ◽  
Fertilization Rates ◽  
Sperm Aspiration ◽  
Significant Difference ◽  
Epididymal Spermatozoa

<p><strong>Introduction</strong></p><p>Testicular epididymal sperm aspiration (TESA) is one of the method  to retrieve sperm from the testes in men with azoospermia. The aim of the study is to compare the In vitro fertilization (IVF) outcome of intracytoplasmic sperm injection (ICSI)-ET cycles with fresh testicular epididymal spermatozoa obtained on the same day with  oocyte retrieval and with frozen-thawed testicular epididymal spermatozoa.</p><p><strong>Material &amp; Methods</strong></p><p>A retrospective comparative analysis of  patients who underwent fresh TESA and frozen-thawed TESA in ICSI-ET cycles from January 2012 to December 2014 in Halim Fertility Center was done. Fresh testicular epididymal sperm aspiration (fresh TESA) was performed on the same day with oocyte retrieval in 28 cycles and the frozen-thawed testicular epididymal sperm aspiration (frozen-thawed TESA) was used in 30 cycles.  </p><p><strong>Results</strong></p><p>The two groups were comparable in terms of the ages of male and female patients, etiology of infertility and duration of infertility. Fertilization rates in fresh TESA group were 53,5% and in frozen-thawed TESA group, fertilization rates were 50%. There was no statistically significant difference between the groups. Clinical pregnancy rates in fresh TESA group were 35,7%  and in frozen-thawed TESA group, clinical pregnancy rates were 26,7% and statistically there was no significant difference between the groups.</p><p><strong>Conclusion</strong></p>There is no significant difference in the in vitro fertilization outcome of intracytoplasmic sperm injection (ICSI)-ET cycles between fresh TESA and frozen-thawed TESA .

scholarly journals comparison between Cleavage Stage versus Blastocyst Stage Embryo Transfer in an Egyptian Cohort Undergoing in vitro Fertilization: A possible Role for Laser Assisted Hatching

2011 ◽  
Vol 5 ◽  
pp. CMRH.S7735 ◽  
Author(s):  
Sherif F. Hendawy ◽  
TA Raafat
Keyword(s):  
Embryo Transfer ◽  
Intracytoplasmic Sperm Injection ◽  
Blastocyst Transfer ◽  
Assisted Hatching ◽  
Vitro Fertilization ◽  
Significant Difference ◽  
Implantation Rates ◽  
Group Ii ◽  
In Pregnancy

Background Extended in vitro embryo culture and blastocyst transfer have emerged as essential components of the advanced reproductive technology armamentarium, permitting selection of more advanced embryos considered best suited for transfer. Aim of study The aim of this study was to compare between cleavage stage and blastocyst stage embryo transfer in patients undergoing intracytoplasmic sperm injection, and to assess the role of assisted hatching technique in patients undergoing blastocyst transfer. Patients and methods This study was carried out on two groups. Group I: 110 patients who underwent 120 cycles of intracytoplasmic sperm injection with day 2-3 embryo transfer—for unexplained infertility or male factor within the previous 3 years. Their data obtained retrospectively from medical records. Group II: 46 age matched infertile female patients undergoing 51 intracytoplasmic sperm injection cycles for similar causes. Patients in Group II were further subdivided into 2 equal subgroups; Group Ila (23 patients), which had laser assisted hatching and Group IIb (23 patients), which did not have assisted hatching. All patients had an infertility workup including basal hormonal profile, pelvic ultrasound, hysterosalpingogram and/or laparoscope and semen analysis of the patient's partner. All patients underwent controlled ovarian hyperstimulation: Using long protocol of ovulation induction. Laser assisted hatching was done for blastocysts of 23 patients. Results Comparison between both groups as regards the reproductive outcome showed a significant difference in pregnancy and implantation rates, both being higher in group II ( P < 0.05) Comparison between both subgroups as regards the reproductive outcome showed a highly significant difference in pregnancy and implantation rates, both being higher in Group IIa ( P < 0.01). There was also a significantly higher rate of multiple pregnancies among Group IIa ( P < 0.05). Conclusion Blastocyst transfer is a successful and improved alternative for patients with multiple failed in vitro fertilization attempts, associated with a significant increase in pregnancy and implantation rates. Furthermore, laser assisted hatching increases implantation and clinical pregnancy rates.

scholarly journals 321 DEVELOPMENT IN VIVO AND IN VITRO OF PORCINE OOCYTES FERTILIZED BY INTRACYTOPLASMIC INJECTION OF A FREEZE - DRIED SPERM HEAD

2005 ◽  
Vol 17 (2) ◽  
pp. 311
Author(s):  
M. Nakai ◽  
K. Kikuchi ◽  
A. Takizawa ◽  
M. Ozawa ◽  
J. Noguchi ◽  
...  
Keyword(s):  
Sperm Head ◽  
Developmental Competence ◽  
Blastocyst Formation ◽  
Vitro Fertilization ◽  
In Vitro Development ◽  
Sperm Suspension ◽  
Freeze Dried ◽  
Vitro Development

The present study investigated the development in vivo and in vitro of in vitro matured porcine oocytes injected with a freeze-dried (FD) boar sperm head. In mice, DNA damage was induced during the holding period after rehydration and before sperm injection (Wakayama, T. and Yanagimachi, R. 1998, Nat. Biotechnol., 16, 639–641). Here, we examined the relationship between duration of rehydration of FD sperm and in vitro development of FD sperm-injected porcine oocytes. We also assessed the in vivo developmental competence of the injected oocytes after embryo transfer. Ejaculated boar spermatozoa were suspended in Pig-FM (Suzuki, K. et al. 2002, Int. J. Androl. 25, 84–93) and sonicated for 1 min to separate sperm heads from the tails. An aliquot (100 μL) of the sperm suspension was put into a glass tube and then pre-cooled at −40°C for 6 h. Each tube was attached to a freeze-dry system (DuraDry μP, FTS Systems, Stone Ridge, NY, USA) for 12 h. The ampules were closed and stored at 4°C for more than 7 days before use. For rehydration, 100 μL of distilled water was added into the ampules. In Experiment I, we injected FD sperm heads which were kept for 0–60, 60–120, or 120–180 min after rehydration. At 1 h after the injection, the injected oocytes were stimulated with a DC pulse and cultured for 6 days. The rate of blastocyst formation and the number of cells in the blastocysts were examined. Embryos after in vitro fertilization (IVF) were evaluated as a control. As shown in Table 1, the rates of blastocyst formation were not different (by χ2 test) for duration of rehydration and the control. However, the cell numbers of FD groups were lower (P < 0.05; by Student's t-test) than that in the control. In Experiment II, oocytes injected with a single FD sperm head and stimulated were transferred to both oviducts of a total of ten recipient gilts. Two recipients were diagnosed as pregnant at Day 30 of gestation. At Day 39, one of the pregnant recipients had an abortion, and two fetuses were recovered. The other pregnancy was not maintained. The results suggest that oocytes fertilized with a single FD sperm head have competence to be implanted and to develop to the early fetal stage, and also that the duration for rehydration does not influence in vitro developmental ability in pigs. Table 1. Effects of the duration from rehydration of freeze-dried sperm heads to the injection of the heads into in vitro matured oocytes on in vitro development of the oocytes in pigs

scholarly journals The role of Anti-Mullerian hormone in predicting fertilization and pregnancy rates following in vitro fertilization-embryo transfer (IVF-ET) and intracytoplasmic sperm injection (ICSI) cycles at a public fertility centre in Nigeria

2020 ◽  
Author(s):  
Safiyya Faruk Usman ◽  
Olubunmi Peter Ladipo ◽  
J.A.F Momoh ◽  
Chris Ovoroyeguono Agboghoroma ◽  
Nabila Datti Abubakar
Keyword(s):  
In Vitro Fertilization ◽  
Embryo Transfer ◽  
Intracytoplasmic Sperm Injection ◽  
Pregnancy Rates ◽  
Vitro Fertilization ◽  
Confounding Variables ◽  
Significant Difference ◽  
Ivf Et

AbstractObjectiveTo determine the role of Anti-Mullerian Hormone (AMH) in predicting fertilization and pregnancy rates following in vitro fertilization-embryo transfer (IVF-ET) and intracytoplasmic sperm injection (ICSI) treatment cycles.MethodsThis was a prospective cohort study of one hundred and fifty consecutive women undergoing IVF-ET/ICSI that were recruited from February 1, 2017 to October 31, 2018 at the Fertility centre of the National Hospital, Abuja, Nigeria. Participants’ plasma AMH were assayed and were followed up till achieving fertilization and pregnancy. Association between AMH levels, fertilization and pregnancy rates was assessed using univariable and multivariable logistic regression modelling to adjust for confounding variables.ResultsThe mean age and mean AMH level of the participants were 36 ± 4.2 years and 1.74 ± 2.35ng/ml respectively. There was a statistically significant association between AMH level and age (P <0.001), duration of infertility (P =0.026), cause of infertility (P =0.035), number of oocytes retrieved (P =<0.001), number of embryos generated (P =<0.001) and type of treatment (P =<0.001). However, there was no significant difference in the fertilization rates (adjusted odds ratio [AdjOR] 0.36, 95% confidence interval [CI] 0.23–4.30; P =0.533) and pregnancy rates (AdjOR 0.26, 95% CI 0.04–2.00; P =0.210) at different plasma levels of AMH.ConclusionPlasma AMH level was not a predictor of fertilization and pregnancy rates among our cohort of patients who had IVF/ICSI treatment cycles.

131 EFFECT OF BREED AND FROZEN - THAWED RAM SEMEN ON IN VITRO FERTILIZATION AND OVINE EMBRYONIC DEVELOPMENT

2011 ◽  
Vol 23 (1) ◽  
pp. 170 ◽  
Author(s):  
K. C. Lehloenya ◽  
N. Mahoete ◽  
J. P. C. Greyling ◽  
T. L. Nedambale
Keyword(s):  
In Vitro Fertilization ◽  
Embryonic Development ◽  
Developmental Stages ◽  
Germplasm Conservation ◽  
Egg Yolk ◽  
Blastocyst Formation ◽  
Cleavage Rate ◽  
Vitro Fertilization ◽  
Significant Difference

Ovine embryonic development was evaluated 8 days following in vitro fertilization, after using fresh or frozen–thawed Merino and indigenous (Pedi and Zulu) sheep semen. Semen used was collected twice weekly over a 3-month period with the aid of an electro-ejaculator. Following collection, semen samples were evaluated and semen with acceptable sperm motility and a percentage live sperm of 60% diluted with an egg yolk-based extender (Egg-Yolk Citrate). Semen samples were cryopreserved in straws with a programmable freezer to –130°C and then plunged into liquid nitrogen (–196°C) until used for IVF. Fresh and frozen–thawed semen was used to fertilize the matured oocytes in vitro. A total of 791 oocytes were fertilized using fresh semen and 802 oocytes fertilized using frozen–thawed semen. No significant differences were recorded between the fresh and frozen–thawed semen regarding the embryonic developmental stages. The performance of fresh and frozen–thawed semen followed the same trend, with the cleavage rate gradually declining with the progression in time and the embryonic developmental stage. The lowest developmental rate recorded was the occurrence of blastocyst formation, ranging between 0.4 ± 0.4% and 2.6 ± 1.0%. Regarding breed, no significant difference was observed from cleavage to the 2- to 4-cell stages. The use of fresh and frozen–thawed Zulu semen resulted in a significantly (P < 0.05) higher percentage of 8-cell development compared with the Pedi semen. However, the 8-cell embryonic stage recorded with the use of the Zulu ram semen (fresh and frozen–thawed), did not differ significantly from that of the Merino breed. No significant difference between the breeds regarding blastocyst formation was recorded. The overall cleavage rate, 2- to 4-cell, and blastocyst embryonic developmental stages following the use of fresh and frozen–thawed semen from the different rams were generally lower than those recorded by other researchers. The low blastocyst rates obtained warrant more research regarding the in vitro embryo production technique in order to improve the ovine blastocyst formation rate. The study was funded by the University of the Free State and conducted at the Germplasm Conservation and Reproduction Biotechnologies ARC.

183 CANINE EMBRYOS OBTAINED BY INTRACYTOPLASMIC SPERM INJECTION

2014 ◽  
Vol 26 (1) ◽  
pp. 206 ◽  
Author(s):  
S. Chastant-Maillard ◽  
K. Reynaud ◽  
S. Thoumire ◽  
M. Chebrout
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Fetal Calf Serum ◽  
Selection Process ◽  
Calf Serum ◽  
Germinal Vesicle ◽  
Sperm Head ◽  
Cell Stage ◽  
Vitro Fertilization

In vitro fertilization encounters 2 specific difficulties in the canine species, with no puppies born to date: low penetration rates (10–50%) and high polyspermia (around 50% of fertilized oocytes; Saint-Dizier et al. 2001 J. Reprod. Fert. Suppl. 57, 147–150). The objectives of the study were to test whether intracytoplasmic sperm injection (ICSI), which overcomes these 2 obstacles, could allow production of canine embryos, using in vivo- or in vitro-matured oocytes. The time of ovulation was determined on 8 Beagle bitches from our experimental kennel by blood progesterone assay and transabdominal ultrasound examination. After ovariohysterectomy 82 to 100 h after ovulation, 58 metaphase II (MII) oocytes were collected by tubal flushing. In parallel, 88 oocytes from 6 anoestrus bitches were matured in vitro (M199 + 20% fetal calf serum for 72 h in 5% CO2 at 38°C). Sperm was collected from 1 Beagle dog with excellent fertility record at natural mating. The sperm was diluted 1 : 100 in PBS/BSA without any selection process. Intracytoplasmic sperm injection was performed at 38°C in M199 HEPES + 20% BSA (4-μm injection pipette; 120-μm holding pipette). One motile spermatozoon of normal morphology was injected per oocyte. Injected oocytes were cultured in vitro for 48 h after injection (M199 + 20% fetal calf serum in 5% CO2 at 38°C) in 4-well open dishes. Oocytes were then fixed and DNA and tubulin were stained for observation by confocal microscopy (Chebrout et al. 2012 Microsc. Microanal. 18, 483–492). Among the 58 MII oocytes recovered in vivo, 7.4% lysed at injection and 20% degenerated during the 48 h after injection. Among the 40 injected oocytes still alive, 6 fragmented (15%) and 4 developed as embryos [10%; 2-pronuclei (n = 2), 2-cell and 6-cell). None of the other oocytes showed decondensed female chromatin. Among the 88 oocytes incubated for in vitro maturation, 13 (14.8%) reached MII. These were successfully injected; 48 h after injection, 3 were embryos at the 2-cell stage and 10 were at the MII stage with a condensed sperm head. Fifty-one non-mature oocytes were injected; 31 were at the germinal vesicle (GV) stage and the stage of others was not determined. Of the GV oocytes, 71% degenerated during culture after injection. The 9 surviving oocytes were still at the GV stage with condensed sperm head 48 h after injection. In conclusion, canine embryos can be obtained through ICSI. Nevertheless, this procedure induced low activation rates. Development at later stages, especially after transfer into a recipient female, is to be evaluated, in particular for in vitro-produced MII oocytes, of lower cytoplasmic competence (Viaris et al. 2008 Reprod. Fert. Dev. 20, 626–639).

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