Caprine blastocyst formation following intracytoplasmic sperm injection and defined culture

Zygote ◽  
1997 ◽  
Vol 5 (3) ◽  
pp. 261-265 ◽  
Author(s):  
L. Keskintepe ◽  
P.C. Morton ◽  
S.E. Smith ◽  
M.J. Tucker ◽  
A.A. Simplicio ◽  
...  
Keyword(s):  
Breeding Season ◽  
Intracytoplasmic Sperm Injection ◽  
Polar Body ◽  
Egg Yolk ◽  
Cumulus Cells ◽  
Essential Amino Acids ◽  
Defined Medium ◽  
First Polar Body ◽  
Defined Culture ◽  
Oviduct Fluid

SummaryExperiments were undertaken to develop intracytoplasmic sperm injection (ICSI) to produce caprine embryos out of the normal breeding season. Oocytes were obtained from 2–6 mm ovarian follicles at slaughter. Selected oocytes with two to four layers of cumulus cells were incubated in 1 ml of H-TCM 199 supplemented with 10 μg each of oFSH and bLH (NHPP, NIDDK, NICHD, USDA) and 20% fetal bovine serum (FBS) in a thermos (38.5°C) for 4.5 h during transportation. Then, oocytes were transferred into 75 μl of freshly prepared maturation medium under paraffin oil and a mixture of 5% O2, 5% CO2 and 90% N2. Approximately 26 h after recovery oocytes were denuded by incubation with hyaluronidase (100 IU/ml) and pipetting and held at 38.5°C for 90 min. Spermatozoa frozen in egg yolk extender were thawed in a 37°C water bath for 15s. Motile fractions were selected by swim-up, then incubated for 90 mm in TALP with 10 μg heparin/ml. Each oocyte was positioned with its first polar body at 6 or 12 o'clock by a holding pipette. Sperm (1 μl) were added to 10 μl medium containing 10% polyvinylpyrrolidone. A sperm cell was aspirated into a pipette, and then injected head-first into the cytoplasm of an oocyte maintained in H-TCM 199 + 20% FBS at 37°C. Injected oocytes were transferred to HM and, after 90 min, cultured in 50 μl of BSA-free synthetic oviduct fluid plus polyvinyl alcohol, citrate and non-essential amino acids. Results demonstrate that caprine blastocysts can be produced outside the breeding season by the use of frozen-thawed semen and injection of sperm cells with broken tails into ova followed by culture in defined medium.

Effects of oocyte age, cumulus cells and injection methods on in vitro development of intracytoplasmic sperm injection rabbit embryos

Zygote ◽  
2004 ◽  
Vol 12 (1) ◽  
pp. 75-80 ◽  
Author(s):  
Yue-Liang Zheng ◽  
Man-Xi Jiang ◽  
Yan-Ling Zhang ◽  
Qing-Yuan Sun ◽  
Da-Yuan Chen
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Polar Body ◽  
Cumulus Cells ◽  
Fertilization Rate ◽  
First Polar Body ◽  
Injection Methods ◽  
Blastocyst Rate ◽  
Repeated Aspiration ◽  
Rabbit Embryos

This study assessed the effects of oocyte age, cumulus cells and injection methods on in vitro development of intracytoplasmic sperm injection (ICSI) rabbit embryos. Oocytes were recovered from female rabbits superovulated with PMSG and hCG, and epididymal sperm were collected from a fertile male rabbit. The oocyte was positioned with the first polar body at 12 o'clock position, and a microinjection needle containing a sperm was inserted into the oocyte at 3 o'clock. Oolemma breakage was achieved by aspirating ooplasm, and the aspirated ooplasm and sperm were re-injected into the oocyte. The injected oocytes were cultured in M199 medium containing 10% fetal calf serum at 38 °C with 5% CO2 in air. The results showed that oocytes injected at 1 h post-collection produced a higher (p<0.05) fertilization rate than those injected at 4 or 7 h post-collection. Blastocyst rate in the 1 h group was higher (p<0.05) than in the 7 h group. Denuded oocytes (group A) and oocytes with cumulus cells (group B) were injected, respectively. Rates of fertilization and development of ICSI embryos were not significantly different (p<0.05) between the two groups. Four ICSI methods were applied in this experiment. In methods 1 and 2, the needle tip was pushed across half the diameter of the oocyte, and oolemma breakage was achieved by either a single aspiration (method 1) or repeated aspiration and expulsion (method 2) of ooplasm. In methods 3 and 4, the needle tip was pushed to the oocyte periphery opposite the puncture site, and oolemma breakage was achieved by either a single aspiration (method 3) or repeated aspiration and expulsion (method 4) of ooplasm. Fertilization rate in method 2 was significantly higher (p<0.05) than in methods 1 and 3. Blastocyst rates were not significantly different (p<0.05) among methods 1, 3 and 4, but method 2 produced a higher (p<0.05) blastocyst rate than method 3.

74 IN VIVO SURVIVAL OF DOMESTIC CAT OOCYTES AFTER VITRIFICATION, INTRACYTOPLASMIC SPERM INJECTION, AND TRANSFER TO RECIPIENTS

2008 ◽  
Vol 20 (1) ◽  
pp. 118 ◽  
Author(s):  
M. C. Gómez ◽  
N. Kagawa ◽  
C. E. Pope ◽  
M. Kuwayama ◽  
S. P. Leibo ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Polar Body ◽  
Oocyte Retrieval ◽  
Cumulus Cells ◽  
Domestic Cat ◽  
Minimal Amount ◽  
Vitro Fertilization ◽  
First Polar Body

The ability to cryopreserve female gametes efficiently holds immense economic and genetic implications. The purpose of the present project was to determine if domestic cat oocytes could be cryopreserved successfully by use of the Cryotop method. We evaluated (a) cleavage frequency after in vitro fertilization (IVF) v. intracytoplasmic sperm injection (ICSI) of in vivo- and in vitro-matured oocytes after vitrification, and (b) fetal development after transfer of resultant embryos into recipients. In vivo-matured cumulus–oocyte complexes (COCs) were recovered from gonadotropin-treated donors at 24 h after LH treatment, denuded of cumulus cells, and examined for the presence of the first polar body (PB). In vitro-matured COCs were obtained from ovaries donated by local clinics and placed into maturation medium for 24 h before cumulus cells were removed and PB status was determined. Oocytes were cryopreserved by the Cryotop method (Kuwayama et al. 2005 Reprod. Biomed. Online 11, 608–614) in a vitrification solution consisting of 15% DMSO, 15% ethylene glycol, and 18% sucrose. For IVF, oocytes were co-incubated with 1 � 106 motile spermatozoa mL–1 in droplets of modified Tyrode's medium in 5% CO2/air at 38�C (Pope et al. 2006 Theriogenology 66, 59–71). For ICSI, an immobilized spermatozoon was loaded into the injection pipette, which was then pushed through the zona pellucida into the ooplasm. After a minimal amount of ooplasm was aspirated into the pipette, the spermatozoon was carefully expelled, along with the aspirated ooplasm. After ICSI, or at 5 or 18 h post-insemination, in vivo- and in vitro-matured oocytes, respectively, were rinsed and placed in IVC-1 medium (Pope et al. 2006). As assessed by normal morphological appearance after liquefaction, the survival rate of both in vivo- and in vitro-matured oocytes was >90% (93–97%). For in vitro-matured oocytes, cleavage frequencies after IVF of control and vitrified oocytes were 73% (16/22) and 53% (30/57), respectively, as compared to 68% (19/28) after ICSI of vitrified oocytes (P > 0.05). For in vivo-matured oocytes, cleavage frequencies after IVF of control and vitrified oocytes were 55% (18/33) and 35% (6/17), respectively, compared to 50% (10/20) after ICSI of vitrified oocytes (P > 0.05). At 18–20 h after ICSI, 18 presumptive zygotes and four 2-cell embryos derived from vitrified in vitro-matured oocytes and 19 presumptive zygotes produced from seven in vivo-matured and 12 in vitro-matured vitrified oocytes were transferred by laparoscopy into the oviducts of two recipients at 24–26 h after oocyte retrieval. The two recipients were 9-month-old IVF/ET-derived females produced with X-sperm sorted by flow cytometry. At ultrasonography on Day 22, both recipients were pregnant, with three live fetuses observed in one recipient and one live fetus seen in the second recipient. On Day 63 and Day 66 of gestation, four live kittens were born, without assistance, to the two recipients. The one male and three female kittens weighed an average of 131 g. In summary, in vivo viability of zygotes/embryos produced by ICSI of cat oocytes vitrified by the Cryotop method was demonstrated by the birth of live kittens following transfer to recipients.

scholarly journals Developmental competence in vivo and in vitro of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection with fresh or frozen-thawed spermatozoa

Reproduction ◽  
2002 ◽  
pp. 455-465 ◽  
Author(s):  
YH Choi ◽  
CC Love ◽  
LB Love ◽  
DD Varner ◽  
S Brinsko ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Polar Body ◽  
Cumulus Cells ◽  
Fertilization Rate ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
First Polar Body ◽  
Bovine Oocytes

This study was undertaken to evaluate the development of equine oocytes in vitro and in vivo after intracytoplasmic sperm injection (ICSI) with either fresh or frozen-thawed spermatozoa, without the use of additional activation treatments. Oocytes were collected from ovaries obtained from an abattoir and oocytes classified as having expanded cumulus cells were matured in M199 with 10% fetal bovine serum and 5 microU FSH ml(-1). After 24-26 h of in vitro maturation, oocytes with a first polar body were selected for manipulation. Fresh ejaculated stallion spermatozoa were used for the experiment after swim-up for 20 min in sperm-Tyrode's albumen lactate pyruvate. Frozen-thawed spermatozoa from the same stallion were treated in a similar way. Spermatozoa were immobilized and injected into the oocytes using a Piezo drill. Presumptive zygotes were cultured in G1.2 medium for 20 or 96 h after the injection was administered, or were transferred to the oviducts of recipient mares and recovered 96 h later. In addition, bovine oocytes with first polar bodies were injected with the two types of stallion spermatozoa and fixed 20 h after injection to examine pronuclear formation. Fertilization rate (pronucleus formation and cleavage) at 20 h after injection of spermatozoa was not significantly different between fresh and frozen-thawed sperm groups in either equine or bovine oocytes. Pronucleus formation after injection of spermatozoa into bovine oocytes was significantly higher than that for equine oocytes (P < 0.05). There were no significant differences in cleavage rate or average number of nuclei at 96 h between equine oocytes injected with fresh or frozen-thawed spermatozoa. However, embryos developed in vivo for 96 h had a significantly higher number of nuclei in both sperm treatments compared with those cultured in vitro. These results indicate that good activation rates may be obtained after injection of either fresh or frozen-thawed equine spermatozoa without additional activation treatment. Injection of frozen-thawed equine spermatozoa results in similar embryo development to that obtained with fresh equine spermatozoa. In vitro culture of equine zygotes in G1.2 medium results in a similar cleavage rate but reduced number of cells compared with in vivo culture within the oviduct. Bovine oocytes may be useful as models for assessing sperm function in horses.

372 SPERM ASTER FORMATION AND BLASTOCYST DEVELOPMENT OF IN VIVO-MATURED BOVINE OOCYTES FERTILIZED BY INTRACYTOPLASMIC SPERM INJECTION

2007 ◽  
Vol 19 (1) ◽  
pp. 301 ◽  
Author(s):  
T. Horiuchi ◽  
M. Takenaka ◽  
C. Kani ◽  
C. Emuta ◽  
Y. Ogata ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Polar Body ◽  
Cumulus Cells ◽  
Blastocyst Development ◽  
Chi Square ◽  
First Polar Body ◽  
Bovine Oocytes ◽  
Sperm Aster

In cattle, activation treatment after intracytoplasmic sperm injection (ICSI) is required to improve cleavage and blastocyst rates (Horiuchi et al. 2002 Theriogenology 57, 1013–1024). The reason why the exogenous activation treatment in bovine ICSI is needed to promote cleavage and blastocyst development is not clear. The objective of this study was to examine the effect of activation treatment on sperm aster formation, cleavage, and blastocyst development of in vivo- and in vitro-matured bovine oocytes following ICSI. In vivo-matured oocytes were collected using transvaginal devices under ultrasound guide at about 29 h after GnRH injection from Japanese Black cows superstimulated with a total 19 mg FSH (Antrin�; Denka Pharmaceutical Co., Kanagawa, Japan) divided into twice daily over 3 days, and treated with 750 �g cloprostenol (Estramate�; Sumitomo Chemical Co., Tokyo, Japan). In a total of 8 aspiration sessions, 131 oocytes were collected; of 116 oocytes with expanded cumulus cells, 84 (72%) had a first polar body and were used for ICSI. On the other hand, in vitro-matured bovine oocytes were prepared by culturing immature follicular oocytes derived from abattoir ovaries. Bull spermatozoa, immobilized by scoring their tails, were injected into in vivo- or in vitro-matured oocytes. At 4 h after ICSI, the oocytes were treated with or without 7% ethanol for 5 min for activation. The injected oocytes were fixed at 8 h after ICSI, and sperm aster formation was examined by using specific antibodies and immunofluorescence microscopy. Data were analyzed by the chi-square test in all experiments. The rate of sperm aster formation in in vivo-matured oocytes was similar regardless of activation treatment (71% vs. 65%), but the rate in in vitro-matured oocytes was significantly (P &lt; 0.05) higher in the group receiving activation treatment than in the non-activation group (57% vs. 19%). Cleavage (88% vs. 88%) and blastocyst rates (59% vs. 47%) of in vivo-matured oocytes after ICSI were also similar, regardless of activation treatment, but cleavage (72% and 20%) and blastocyst rates (19% and 7%) of in vitro-matured oocytes were significantly (P &lt; 0.05) higher in the group receiving activation treatment than in the non-activation group. Moreover, the blastocyst rate of in vivo-matured oocytes was significantly (P &lt; 0.05) higher than the rate in in vitro-matured oocytes. These results show that activation treatment after ICSI of in vivo-matured bovine oocytes is not necessary for cleavage and blastocyst development, and suggest that the necessity of activation treatment in bovine ICSI has relevance to in vitro maturation of bovine oocytes.

98 BLASTOCYST DEVELOPMENT RATE OF CLONED CAT EMBRYOS USING SERIAL CLONING

2007 ◽  
Vol 19 (1) ◽  
pp. 166
Author(s):  
X. J. Yin ◽  
H. S. Lee ◽  
E. G. Choi ◽  
X. F. Yu ◽  
B. H. Choi ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Second Generation ◽  
Assisted Reproductive Technologies ◽  
Polar Body ◽  
Donor Cell ◽  
Fibroblast Cell ◽  
Cumulus Cells ◽  
Reproductive Technologies ◽  
Blastocyst Development ◽  
First Polar Body

Domestic cats are a useful research model to develop assisted reproductive technologies for the conservation of endangered felids. Previously, we produced cloned offspring derived from somatic cell nuclear transfer of ear skin fibroblasts obtained from a deaf, odd-eyed, male Turkish Angora. The aim of this study was to assess the cloning efficiency of the fibroblasts derived from a cloned cat. Fibroblast cell lines were established from 6-mm skin biopsies taken from a deaf, odd-eyed, male Turkish Angora and his clone. The protocol for nuclear transfer was described previously (Yin et al. 2005 Reproduction 129, 245–249). Briefly, cumulus cells were removed from the ova by gently pipetting them into TCM-199 supplemented with 0.1% hyaluronidase. The denuded oocytes were then cultured in TCM-199 supplemented with 0.2 �g mL-1 demecolcine for 1 h and placed into TCM-199 containing 5 �g mL-1 cytochalasin B and 0.2 �g mL-1 demecolcine. The first polar body and protruded chromatin plate were removed with a beveled micropipette. Micromanipulation was used to place a single donor cell nucleus into the perivitelline space of enucleated ova. The ovum-cell couplets were fused and pulse activated. The activated couplets were cultured in 500 �L of CRI medium supplemented with 0.3% BSA for 2 days. The cleaved embryos were cultured in CRII medium supplemented with 10% FBS for 5 days. The cleavage and blastocyst development rates were 38.5% and 3.5% for second generation cloned embryos. A total of 310 second generation cloned embryos were transplanted to 9 surrogates, and 2 pregnancies at 30 days were determined by ultrasonography. One pregnancy was aborted at 40 days of gestation; the second pregnancy continued. These results indicate that the serial cloning of a cat can be generated efficiently up until pregnancy. This work was supported by KOSEF (grant #M10525010001-05N2501-00110).

249 INTRACYTOPLASMIC SPERM INJECTION OF ELAND (TAUROTRAGUS ORYX) AND BONGO (TRAGELAPHUS EURYCERUS)ANTELOPE OOCYTES

2007 ◽  
Vol 19 (1) ◽  
pp. 241 ◽  
Author(s):  
G. Wirtu ◽  
C. E. Pope ◽  
M. C. Gomez ◽  
A. Cole ◽  
D. L. Paccamonti ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Intracytoplasmic Sperm Injection ◽  
Transvaginal Ultrasound ◽  
Polar Body ◽  
Chemical Activation ◽  
Cumulus Cell ◽  
Calcium Ionophore ◽  
Male Gamete ◽  
Assisted Reproductive Technique ◽  
First Polar Body

Intracytoplasmic sperm injection (ICSI) is an assisted reproductive technique applicable in cases of limited male gamete availability. Moreover, it bypasses barriers of the oocyte, thus avoiding poorly understood species-specific capacitation events affecting sperm–egg interaction. In the present study, we evaluated the application of conventional and piezo drill-assisted ICSI and whether subsequent chemical activation is required for initiating embryonic development in eland (Taurotragus oryx) and bongo (Tragelaphus eurycerus) oocytes. Oocytes were collected using transvaginal ultrasound-guided follicular aspiration after gonadotropin-induced ovarian stimulation and incubated in modified TCM-199 medium (Gomez et al. 2000 Reprod. Fertil. Dev. 12, 423) containing 10% FBS. After 3 to 24 h, the cumulus cell layers were removed either by repeated mouth-pipetting and/or by using hyaluronidase. Oocytes with an extruded first polar body were used for ICSI and the other oocytes were returned to culture and evaluated every six hours Piezo drill-assisted (Kimura and Yanagimachi 1995 Biol. Reprod. 52, 709) or conventional (Gomez et al.) ICSI were done as described previously using glass pipettes with internal tip diameters of 9–10 µm. We used frozen–thawed or freshly collected spermatozoa that were kept in HEPES-buffered Tyrode's medium (Gomez et al.) for up to 24 h. Four to 6 h after ICSI, 3 activation treatments were examined: (1) none; (2) 7% ethanol, 5 min; or (3) calcium ionophore (5 µM, 5 min) followed by DMAP (2 mM, 4 h). Then we cultured oocytes in a humidified atmosphere of 5% O2, 5% CO2, and 90% N2 at 38.5°C in one of 3 media: SOF, α-MEM, or CR1aa containing essential and nonessential amino acids and FBS. Fifty-three of 70 (76%) eland oocytes survived after piezo-ICSI, and 13 of 16 (81%) survived after conventional ICSI. For bongo oocytes, 27 of 30 (90%) survived piezo-ICSI and all (n = 8) survived after conventional ICSI. Table 1 outlines cleavage data on Day 2. Generally, embryonic development was arrested at about 10 cells. In summary, eland and bongo oocytes can survive both conventional and piezo drill-assisted ICSI. Activation treatments do not appear to be a prerequisite for initiating cleavage after ICSI in eland and bongo antelope oocytes. Table 1.Cleavage of eland and bongo antelope oocytes after conventional or piezo-ICSI and three activation treatments

228 microRNA REGULATION OF GENES IN BOVINE OOCYTES AND EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 272
Author(s):  
J. P. Barfield ◽  
G. J. Bouma ◽  
G. E. Seidel Jr
Keyword(s):  
Mirna Expression ◽  
Polar Body ◽  
Prostaglandin F2α ◽  
Defined Medium ◽  
Tight Junction Formation ◽  
First Polar Body ◽  
Lysis Buffer ◽  
Bovine Oocytes

Little is known about expression of microRNA (miRNA) in bovine oocytes and pre-implantation embryos. These molecules likely have an important role in regulating development. For example, differences in quality of oocytes matured in vivo v. in vitro might be due, in part, to altered miRNA expression. In Experiment 1, in vivo-matured COC were collected by transvaginal aspiration of 7 superstimulated cows 21 to 23 h after GnRH injection, given 48 h after prostaglandin F2α and the last of 6 FSH injections given b.i.d. Oocytes aspirated from abattoir ovaries were matured in vitro for 23 h in a chemically defined medium. After vortexing, maturation of both groups of oocytes was confirmed by visualization of the first polar body, and oocytes were snap frozen in mirVana lysis buffer (Applied Biosciences, Foster City, CA, USA). In Experiment 2, in vitro-matured oocytes were generated as described. Subsets were fertilized in vitro or activated parthenogenetically by incubation in 5-μM ionomycin for 5 min followed by 10 μg mL-1 cycloheximide plus 5 μg mL-1 cytochalasin B for 5 h. After 18 h and 12 h, respectively, fertilized and activated oocytes were centrifuged at 10 000 × g for 10 min to enable visualization of pronuclei. Zygotes with 2 polar bodies and 2 pronuclei and parthenotes with 2 pronuclei were snap frozen in mirVana lysis buffer. Total RNA was extracted from 30 pooled oocytes for each replicate using the mirVana MiRNA Isolation Kit (Ambion, Inc., Austin, TX, USA). Reverse transcription of RNA was performed using the QuantiMir RT kit (System Biosciences, Mountain View, CA, USA), and miRNA expression was evaluated by real-time PCR using the Mouse miRNome Profiler plate, which contains primers for 384 miRNA (System Biosciences). Three plates were analyzed for each group (30 oocytes per plate). Changes in relative expression levels were analyzed with a t-test of values normalized to miR-181a, which was consistently expressed in all samples. In Experiment 1, compared with in vitro-matured oocytes, in vivo-matured oocytes had 11-fold higher (P = 0.02) expression of miR-375, which targets numerous genes involved in electron transport chain and oxidative phosphorylation pathways according to the bioinformatic database mirGator. MiR-291a-5p, miR-494, miR-539, and miR-547 were expressed in in vivo-matured oocytes only; the converse was found for miR-575-5p. Results from Experiment 2 are in the table. Major pathways associated with potential targets of the detected miRNA include TGF-beta signaling, Wnt signaling, tight junction formation, DNA replication reactome, steroid biosynthesis, mRNA processing binding reactome, and glutamate metabolism. Several of these candidate miRNA might be important for regulation of bovine oocyte maturation and embryo development. Table 1.Experiment 2: Fold change expression of miRNA

287 RESCUE AND IN VITRO MATURATION OF FOLLICULAR OOCYTES IN CHINCHILLA LANIGER

2007 ◽  
Vol 19 (1) ◽  
pp. 259 ◽  
Author(s):  
G. Aiudi ◽  
M. Cinone ◽  
F. Maritato ◽  
A. De Sandro Salvati ◽  
M. E. Dell'Aquila
Keyword(s):  
Breeding Season ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Metaphase Plate ◽  
Meiotic Maturation ◽  
Current Therapy ◽  
Embryo Cryopreservation ◽  
Vaginal Smear ◽  
First Polar Body

The chinchilla is a hystricomorph rodent with a natural habitat in the Andes mountains of Chile (see review by Boussarie 2002 Proc. 27th WSAVA Congr.). For most of the chinchilla subspecies, the decline in the natural population can be attributed to human destruction of the native ecosystems and hunting for fur. Chinchillas are listed as a protected endangered species, at immediate risk of extinction. In Europe, chinchillas are reared for pets and fur production. The female has a seasonal polyestrous reproductive activity with a breeding season from November to May. The estrous cycle length is variable (28–41 days), with an estrous duration of 2 days. After a gestation of about 112 days, a litter of 1 to 6 young is born (see reviews by Morrow 1986 in Current Therapy in Theriogenology 2, W.B. Saunders; and Collot 1998 in Proc. I EVSSAR Congr.). Reproductive biotechniques in this species could play an important role in managing both captive and natural populations as well as in sustaining and improving genetic and global biodiversity. The specific aim of this preliminary work was to standardize an efficient in vitro maturation (IVM) procedure for Chinchilla laniger oocytes so that it will be possible, in the future, to perform IVF and embryo cryopreservation and transfer. Oocytes from 4 cyclic breeding females were recovered by slicing ovaries, obtained by ovariohysterectomy, and were matured in vitro according to the procedure described for bovine oocytes by Dell'Aquila et al. (2002 Mol. Reprod. Dev. 63, 210–222). Two trials of 2 estrous subjects each were performed, on the basis of behavioral signs of estrous and vaginal cytology (Harris-Schorr staining), in the early and late breeding seasons. During estrus, the vaginal smear consisted of superficial cells, further neutrophils, and small and large intermediates, whereas parabasal cells were not found. At the end of the culture time, oocytes were stained with Hoechst 33258 and evaluated for the stage of meiotic maturation. Three out of 4 oocytes recovered in November (75%) reached full meiotic maturation, showing the second metaphase plate and the first polar body (PB) extruded. The fourth oocyte, showing the first PB together with multiple pronuclear structures, was classified as activated. On the contrary, none out of 12 oocytes recovered in May reached full maturation. Of them, 7 (58%) remained at the germinal vesicle stage, 2 (17%) reached metaphase I, and 3 (25%) showed abnormally dispersed chromatin configuration. To our knowledge, this is the first study reporting that chinchilla oocytes can be matured in vitro by bovine IVM procedures. Even though the number of oocytes was poor, we can hypothesize that oocytes from C. laniger are best collected in the breeding season when subjected to an IVM technique.

Intracytoplasmic sperm injection in dysmorphic human oocytes

Zygote ◽  
1995 ◽  
Vol 3 (4) ◽  
pp. 283-288 ◽  
Author(s):  
Mina Alikani ◽  
Gianpiero Palermo ◽  
Alexis Adler ◽  
Massimo Bertoil ◽  
Marlena Blake ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Smooth Endoplasmic Reticulum ◽  
Polar Body ◽  
Light Microscopic Level ◽  
Human Oocytes ◽  
Fertilisation Rate ◽  
Polar Bodies ◽  
First Polar Body ◽  
Multiple Abnormalities ◽  
Further Development

SummaryFertilisation and development of dysmorphic human oocytes recovered from hyperstimulated ovaries have been evaluated following intracytoplasmic sperm injection (ICSI) for treatment of male infertility. A total of 2968 oocytes at metaphase II of meiosis were injected, of which 806 (27.2%) were dysmorphic at the light microscopic level. Cytoplasmic abnormalities included granularity, areas of necrosis, organelle clustering, vacuoles, and accumulating saccules of smooth endoplasmic reticulum. Anomalies of the first polar body and zona pellucida, as well as non-spherical shapes of oocytes, were also noted. Contrary to previous findings linking some dysmorphisms to non-assisted fertilisation failure, in this study no single abnormality led to a reduction in the fertilisation rate, nor was fertilisation compromised in oocytes with multiple abnormalities. The incidence of normal fertilisation (two pronuclei and two polar bodies) was 69% in both the dysmorphic and non-dysmorphic oocytes. While overall pregnancy and implantation results were not altered in the group of patients (n = 242) in whom at least one dysmorphic oocyte was injected, exclusive replacement of embryos which originated from dysmorphic oocytes led to a higher incidence of early pregnancy loss. It is concluded that aberrations in the morphology of human oocytes – most probably a product of controlled ovarian stimulation – are of little or no consequence to fertilisation or early cleavage after ICSI. It is possible, however, that these embryos have a reduced potential for implantation and further development.

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