203 EQUINE EMBRYO IN VITRO DEVELOPMENT AFTER INTRACYTOPLASMIC SPERM INJECTION FOLLOWED BY CHEMICAL ACTIVATION

2012 ◽  
Vol 24 (1) ◽  
pp. 214
Author(s):  
J. Jarazo ◽  
A. Gambini ◽  
A. De Stefano ◽  
L. Muredas ◽  
J. G. Oriol ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Chemical Activation ◽  
Chi Square ◽  
Group 4 ◽  
Blastocyst Quality ◽  
The Difference ◽  
Chi Square Analysis ◽  
Vitro Development

Intracytoplasmic sperm injection (ICSI) is an alternative method for producing in vitro-fertilized embryos in horses. Some authors have suggested that using the piezo drill to inject the spermatozoon is required to obtain acceptable blastocyst rates after ICSI. In order to avoid the use of this equipment, the aim of our study was to evaluate 4 different chemical activation protocols and their effect on embryo development. Cumulus–oocyte complexes were recovered from ovaries of slaughtered mares. The maturation medium was DMEM/F12 supplemented with 10% fetal bovine serum (FBS), 1 μL mL–1 of insulin-transferrin-selenium, 1 mM sodium pyruvate, 100 mM cysteamine and 0.1 mg mL–1 of FSH at 39°C in a humidified atmosphere of 6.5% CO2 in air for 24 h. The ICSI was carried out in 20-μL droplets of TALP-HEPES with a 9-μm pipette, using frozen-thawed spermatozoa from 1 stallion. Spermatozoa were held separate in 100-μL droplets of Modified Whittens. Motile spermatozoa were aspirated and transferred to a 5-μL drop of 7% (v/v) polyvinylpyrrolidone, where 1 sperm was immobilized by swiping the injection pipette across its tail; then, the sperm was injected into the oocyte. All injected oocytes were subjected to 8.7 μM ionomycin for 4 min, followed by 1 of 3 further activation treatments: (1) 4-h culture in 1 mM 6-DMAP and 10 mg mL–1 of cycloheximide, starting 3 h after ionomycin; (2) 5-h culture in 10 mg mL–1 of cycloheximide, starting 10 min after ionomycin; (3) An extra incubation with 5 mM ionomycin for 4 min, starting 3 h after ionomycin. Some injected oocytes were left without a further activation protocol (group 4). After activation, injected oocytes were cultured in 100-μL droplets of DMEM/F12 with 5% of FBS at 39°C in a humidified atmosphere of 5% O2, 5% CO2 and 90% N2. Cleavage (48 h after activation) and blastocyst formation (7–8 days) of all experimental groups were assessed. Culture medium was renewed on Day 3 with fresh DMEM/F12 with 5% of FBS. At Day 9, the zona pellucida of some blastocysts was removed and the blastocysts were maintained in culture until Day 15. Blastocyst growth was determined every 24 h. Statistical differences (using chi-square analysis) were observed in cleavage with treatments 1 and 3 when compared to the other groups (1: 30/52, 58%; 2: 8/40, 20%; 3: 9/25, 36%; and 4: 10/38, 26%). There was no difference on blastocyst rates based on injected oocytes (1: 5/52, 9.6%; 2: 2/40, 5%; 3: 1/25, 4%; and 4: 2/38, 5.3%). On Day 7, blastocyst quality did not differ among treatments and on Day 15, blastocysts from groups 3 and 4 reached 1130 μm and 4300 μm, respectively. Despite the difference observed in cleavage, this work suggests that equine blastocysts could be obtained with all of the activation protocols, without the use of the piezo drill. Further studies are required to assess the effect of chemical activation on in vivo development of produced blastocysts to confirm that they are not parthenogenetic. We are grateful to Mr. Willem Melchior, La Vanguardia Polo Club for some financial support and encouragement to undertake this project.

249 AN EFFICIENT AND REPEATABLE IN VITRO FERTILIZATION TECHNIQUE IN THE EQUINE FOR PRESERVATION OF ENDANGERED SPECIES

2015 ◽  
Vol 27 (1) ◽  
pp. 214
Author(s):  
C. Douet ◽  
O. Parodi ◽  
F. Reigner ◽  
P. Barrière ◽  
G. Goudet
Keyword(s):  
Embryo Development ◽  
Intracytoplasmic Sperm Injection ◽  
Embryo Production ◽  
Chi Square ◽  
Vitro Fertilization ◽  
In Vitro Development ◽  
Vitro Development

Most wild equids are currently endangered or threatened, as mentioned in the International Union for the Conservation of Nature Red List, and several domestic horse breeds are at risk of extinction. Genome resource banking requires cryoconservation of semen, oocytes, and/or embryos. Embryo production in equids is limited in vivo because routine induction of multiple ovulation is still ineffective. Embryo production in vitro allows the production of several embryos per cycle that could easily be frozen because of their small size. Intracytoplasmic sperm injection has been widely adopted to generate horse embryos in vitro; however, intracytoplasmic sperm injection is time-consuming and requires expensive equipment and expertise in micromanipulation. Several attempts to establish an efficient IVF technique in the equine were performed, but reported IVF rates remain quite low and no repeatable equine IVF technique was available. Our objective was to develop an efficient and repeatable IVF technique in the equine. Immature cumulus-oocyte complexes (COC) were collected either from slaughtered mares in a local slaughterhouse or from our experimental mares by ovum pick up (OPU). The COC were cultured for 26 h in an in vitro maturation (IVM) medium or in preovulatory follicular fluid (FF) collected by OPU, pre-incubated for 30 min in oviducal fluid collected from slaughtered females, co-incubated for 18 h with fresh spermatozoa treated with procain, and cultured in SOF for 30 h. They were fixed and analysed either after 18 h IVF (experiment 1) or after 30 h in vitro development (experiment 2). In experiment 1, COC were collected from slaughtered mares and analysed after 18 h IVF. Zygotes with 2 pronuclei were observed. The IVF rate was similar for oocytes matured in IVM medium (22/33, 67%) or FF (24/42, 57%; chi-square test, P > 0.05). In experiment 2, COC were collected from slaughtered mares and from experimental mares and analysed after 30 h of in vitro development. We observed zygotes with 2 highly decondensed pronuclei, pronuclei decondensation being the first step of embryo development. For oocytes collected from slaughtered mares, the percentage of zygotes was similar for oocytes matured in IVM medium (8/11, 73%) or FF (10/15, 67%). For oocytes collected by ovum pickup, the percentage was similar for IVM medium (3/5, 60%) or FF (6/8, 75%). We also observed some embryonic structures with several nuclei, but the quality of these embryos was poor. In conclusion, we have established an efficient IVM-IVF technique that allows the first step of embryo development. Because we obtained similar results for 4 years, we consider that this efficient technique is repeatable. Further experiments are in progress to improve the quality of the embryos.

159 THE EFFECT OF DIFFERENT ZWITTERIONIC BUFFERS AND PBS ON IN VITRO DEVELOPMENT AND MORPHOLOGY OF BOVINE EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 187
Author(s):  
J. De la Fuente ◽  
A. Gutiérrez-Adán ◽  
P. Beltrán Breña ◽  
S. S. Pérez-Garnelo ◽  
A. T. Palasz
Keyword(s):  
Embryo Development ◽  
Cell Number ◽  
Apoptotic Cells ◽  
Bovine Embryos ◽  
Chi Square ◽  
Oh Groups ◽  
In Vitro Development ◽  
Chi Square Analysis ◽  
Vitro Development

It is assumed that, contrary to phosphate buffers, zwitterionic buffers are neutral. However, zwitterionic buffers containing hydroxymethyl or hydroxyethyl residues may interact with OH-groups in the media and produce formaldehyde (Shiraishi et al. 1993 Free Radic. Res. Commun. 19, 315-321). Also, it was shown that three zwitterionic buffers tested in this study interact with DNA (Stellwagen et al. 2000 Anal. Biochem. 287, 167-175). Our objective was to evaluate the effect of the following buffers: TES (T), MOPS (M), HEPES (H) (pKa values at 20�C: 7.2-7.5), and PBS on in vitro development and morphology of bovine embryos. Zwitterionic buffers and PBS were prepared at a concentration of 10 mM in TALP medium and the final pH was adjusted to 7.2. Bovine follicular fluid was aspirated from abattoir-derived ovaries and evenly divided into four tubes. Collected oocytes (five replicates) from each tube were processed separately through the entire IVM, IVF, and IVC procedures using washing medium buffered with: PBS (n = 490), Group 1; H (n = 438), Group 2; M (n = 440), Group 3; and T (n = 394), Group 4. All buffers contained 4 mg/mL BSA. Oocytes were matured in TCM-199 + 10% FCS and 10 ng/mL of epidermal growth factor and fertilized in Fert-TALP containing 25 mM bicarbonate, 22 mM sodium lactate, 1 mM sodium pyruvate, 6 mg/mL BSA-FAF, and 10 �g/mL heparin with 1 � 106 spermatozoa/mL. After 24 h, oocytes-sperm co-incubation presumptive zygotes were cultured in SOFaa medium with 8 mg/mL BSA at 39�C under paraffin oil and 5% CO2 in humidified air. Cumulus-oocyte complexes and zygotes were held in designated buffers ?16 min before oocyte maturation, ~7 min after IVM and before IVF, and ~18 min after IVF and before culture. The total time of oocyte/embryo exposure to each buffer was ?41 min. Embryo development was recorded on Days 4, 7, 8, and 9. A total of ten, Day 8 blastocysts were taken randomly from each treatment and fixed in 4% paraformaldehyde for total and apoptotic cells counts, and five blastocysts from each replicate and treatment were frozen for later mRNA analysis. Apoptosis were determined by TUNEL, using commercial In situ Cell Death Detection Kit (Roche Diagnostic, SL, Barcelono, Spain). Embryo development among groups was compared by chi-square analysis. The cleavage rates were not different among the groups: PBS, 70.8%; H, 76.5%; M, 77.5% and T, 73.6%. The number of embryos that developed to d8 cells at Day 4 was higher in M, 36.2%, and PBS, 37.6%, than in H, 30.6%, and T, 29.7%, but was not significantly different. However, more (P < 0.05) blastocysts developed at Days 7, 8, and 9 in H and M than in PBS and T groups (21.9% and 22.9% vs. 16.9% and 14.9%, respectively). No difference was found between groups in total cell number (98.8 � 7, PBS; 111.8 � 11.9, M; 106.8 � 12.9, H; and 104.3 � 9.7, T) and the number of apoptotic cells (9.2 � 1.0, P; 9.2 � 0.8, M; 12.9 � 1.8, H; and 9.7 � 0.9, T). Based on the results of this study, we conclude that within our protocol choice of buffer may affect embryo developmental rates but not morphology.

319 APPROACHES TO IMPROVE INTRACYTOPLASMIC SPERM INJECTION MEDIATED TRANSGENESIS AND MAXIMIZE THE USE OF SEX-SORTED SPERM IN BOVINE

2015 ◽  
Vol 27 (1) ◽  
pp. 248
Author(s):  
N. G. Canel ◽  
R. J. Bevacqua ◽  
M. I. Hiriart ◽  
N. Chavez Rabelo ◽  
L. S. Almeida Camargo ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Transgenic Animals ◽  
Transgenic Animal ◽  
Egfp Expression ◽  
Control Group ◽  
Randomisation Test ◽  
Blastocyst Quality ◽  
Fragmented Nucleus ◽  
Vitro Development

Intracytoplasmic sperm injection (ICSI) mediated transgenesis is an effective tool for transgenic animal production. However, ICSI in cattle remains inefficient. In this work, we assayed approaches to improve egfp expressing blastocysts production by ICSI: the sperm pretreatment with heparin and l-glutathione (Hep-GSH), the use of sex-sorted sperm (SS), the refrozen/thawing of SS sperm, and the combination of these. Quality of ICSI blastocysts was analysed by studying the expression of 4 genes, and the rates of DNA fragmentation. Cumulus-oocyte complexes from slaughtered cow ovaries were in vitro-matured for 21 h. Nonsorted (NS) and sex-sorted (SS) frozen straws were thawed. Some of them were incubated with 80 μM Hep-15 mM GSH for 20 h (Hep-GSH+). The Hep-GSH-control group was not pretreated. Semen samples were co-incubated with 50 ng µL–1 of pCX-EGFP for 5 min before ICSI. Moreover, the SS sperm that are usually discarded after ICSI were cryopreserved and used for ICSI after a second thawing (ICSI SS refrozen). The ICSI NS, sham, and diploid parthenogenetic (Diplo PA) controls were included. Oocytes were activated with 5 µM ionomycin for 4 min, TCM-199 for 3 h (except for diploid PA), and 1.9 mM DMAP for 3 h. Cleavage and blastocyst/egfp expression rates were evaluated on Days 2 and 7 post-ICSI, respectively. Results are shown in Table 1. Relative expression of HMGN1, GLUT5, AQP3, and OCT4 genes from ICSI NS Hep-GSH+ and IVF blastocysts were compared by qPCR. Data were analysed by the pair-wise fixed reallocation randomisation test. None of the 4 genes showed differences between groups. The DNA fragmented nucleus index/blastocyst cell numbers were determined by TUNEL assay, not showing differences between groups (Kruskal–Wallis test, P ≤ 0.05). Means ± s.d. were 29 ± 17/91 ± 27 for ICSI Hep-GSH+; 27 ± 15/63 ± 34 for ICSI Hep-GSH–; 28 ± 17/68 ± 17 for ICSI SS, 28 ± 13/75 ± 24 for ICSI SS refrozen; and 21 ± 13/105 ± 59 for IVF SS control. The Hep-GSH pretreatment can increase blastocyst and transgene expressing blastocysts rates after TM-ICSI, except when SS semen is used. Interestingly, the use of SS sperm for ICSI can be maximized by cryopreservation and reuse of discarded sperm cells. The parameters analysed in this work indicate that the proposed approaches do not affect blastocyst quality. Therefore, Hep-GSH pretreatment of NS sperm and refrozen SS sperm could be applied for TM-ICSI in bovine for the production of transgenic animals. Table 1.In vitro development and egfp expression of ICSI embryos fertilized with nonsorted (NS) and sex-sorted (SS) sperm pretreated with Hep-GSH, refrozen, or both

scholarly journals 320 USE OF SYNTHETIC HYALURONAN OR POLYVINYLPYRROLIDONE FOR INTRACYTOPLASMIC SPERM INJECTION INTO MOUSE OOCYTES

2005 ◽  
Vol 17 (2) ◽  
pp. 310
Author(s):  
P.N. Moreira ◽  
J. De la Fuente ◽  
A.T. Palasz ◽  
A. Gutiérrez-Adán
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Embryo Implantation ◽  
Blastocyst Stage ◽  
Chi Square ◽  
Developmental Rates ◽  
Implantation Rates ◽  
And Migration ◽  
Chi Square Analysis ◽  
Humidified Air

The use of polyvinylpyrrolidane (PVP) in intracytoplasmic sperm injection (ICSI) seems to be exclusively related to its surfactant and colloidal properties. In contrast to PVP, which can be toxic to mouse embryos, hyaluronan (HA) is a biological compound. In addition to its colloidal property, HA plays an important biochemical role in cell proliferation and migration and can be found intracellularly in the cleaving stage of mouse, sheep and primate embryos (Hunter RHF 1994 Mol. Reprod. Dev. 39, 176–181). We expect that the viscoelastic properties of HA in combination with its physiological functions may benefit the ICSI procedure. Oocytes at MII stage were collected from CD-1 mice 14 h after hCG injection (h-pi) and were kept at 37°C in KSOM medium for 30 min before ICSI. Semen used for injection was frozen by direct plunge into liquid nitrogen in M2 medium without cryoprotectants. Samples were thawed at 25°C in the air and mixed (1:5) with M2 medium containing either 10% PVP; 360000 MW (w/v; Sigma, St. Louis, MO, USA) or 60% (v/v) synthetic HA (s-HA; MAP-5; Bioniche Inc, Belleville, ON Canada) with comparable viscosity. Injections were performed at 25°C using a mercury-containing pipette attached to a piezo impact unit (Prime Tech, Ibaraki, Japan). A total of 239 oocytes (115 PVP and 124 s-HA) were injected in groups of ten in four replicates. Individual sperm heads decapitated by the freeze/thaw procedure were injected into oocytes and kept for 15 min at 25°C. Oocytes that survived ICSI were placed in 35 μL drops of KSOM medium (∼15 zygotes per drop) under paraffin oil at 37°C and 5% CO2 in humidified air. Cleavage and developmental rates were recorded at 24, 48, and 96 h after oocyte injection. Embryos which developed to the blastocyst stage were transferred to pseudo-pregnant females mated with vasectomized males. At Day 13, recipient mice were sacrificed and the number of implantations and fetuses were recorded. Data were compared between groups by Chi-square analysis. Significantly (P < 0.05) more embryos survived ICSI in PVP (74%) than in s-HA group (56%), which was primarily related to sperm adhesiveness to the injection pipette. However, there were no differences in developmental rates at any stage of in vitro embryo culture between groups (2 cell, 93 vs. 100%; 4–8 cell, 100 vs. 100%; blastocyst, 44 vs 50%) for PVP and s-HA, respectively. Significant differences (P < 0.05) between groups were observed in embryo implantation rates. When ICSI was performed with s-HA, 29 out of 35 blastocysts (83%) transferred to synchronized recipients were implanted, which was accomplished only by 19 of the 35 from the PVP group (54%). However, there was no difference between groups in the number of fetuses detected (8 (23%) vs. 9 (26%) for PVP and s-HA, respectively). The use of s-HA for mouse ICSI can be a valuable alternative to PVP. Hyaluronan may show further benefit if sperm adhesiveness to the micropipette can be eliminated, and may be superior to PVP if embryo implantation rates in the s-HA group can be sustained. The authors would like to thank Bioniche, Inc., Belleville, ON, Canada for donating MAP-5.

123 AGGREGATION OF CLONED EQUINE EMBRYOS: IMPROVEMENT OF IN VITRO AND IN VIVO DEVELOPMENT

2011 ◽  
Vol 23 (1) ◽  
pp. 166
Author(s):  
A. Gambini ◽  
J. Jarazo ◽  
R. Olivera ◽  
F. Karlanian ◽  
D. F. Salamone
Keyword(s):  
Expression Patterns ◽  
Chi Square ◽  
Serum Progesterone ◽  
Str Analysis ◽  
In Vitro Development ◽  
Group Iii ◽  
Group Ii ◽  
Vitro Development

Development of cloned equine embryo is still inefficient. The aim of our study was to assess the aggregation of zona-free genetically identical cloned embryos as a strategy to improve in vitro and in vivo development. Oocyte collection, maturation, cloning, and activation procedures were performed as described by (Lagutina et al. 2007 Theriogenology 67, 90–98). After activation, reconstructed embryos (RE) were cultured in DMEM/F12 with 5% of FBS in the well of well system in 3 different groups: I, only one RE per well; II, two RE per well; and III, three RE per well. Cleavage and blastocyst formation (7 to 8 days) of all experimental groups was assessed. At day 8, some embryos of each group were either fixed to determine Oct-4 expression by immunocytochemistry or transferred transcervically to a synchronized mare. Pregnancies were assessed by ultrasound from 7 days after embryo transfer until day 45 to 50 of pregnancy every 7 to 10 days, and sizes of vesicles and embryos were measured. In advanced pregnant mares, combined thickness of the uterus and the placenta (CTUP) and serum progesterone levels were also determined. The remaining embryos obtained from each group were maintained in culture from day 7 until day 15. Blastocysts growth was determined every 24 h. In vitro development, on a per-well and RE basis, was compared using the chi-square test. Statistical differences were observed in cleavage among groups I and II (P = 0.0088) and groups I and III (P = 0.0004): (I: 91/111, 82%; II: 74/78, 95%; III: 62/62, 100%). Blastocyst rates differed between groups I and III (I: 10/111, 9%; III: 23/62, 37%); no difference was observed with group II (11/78, 14%). There was no difference on blastocyst rates based on the number of aggregated RE (I: 10/111, 9%; II: 11/156, 7%; III: 23/184, 12.5%). The highest pregnancy rate was obtained in group III (I: 1/3, 33%; II: 2/5, 40%; III: 3/4, 75%). Sizes of vesicles and embryos did not differ statistically in such groups. The CTUP and serum progesterone levels were considered normal (<1.2 cm; >8 ng mL–1, respectively) in ongoing pregnancies. We did not observe any differences in Oct-4 expression patterns among groups. Even though statistical differences were found, surprisingly all embryos grew in vitro until day 15 with good rates and the biggest embryo reached 4.25 mm. Embryo aggregation improved in vitro development of equine cloned embryos until day 7, and pregnancies rates were higher. The in vivo sizes of vesicles and embryos were normal for all groups, and in vitro development beyond day 7 showed the high viability of embryos. To conclude, aggregation of cloned equine embryo does not imply extra oocytes because there is no statistical difference in the number of blastocysts obtained per oocytes used to achieve RE. It is also a good strategy to improve in vitro embryo development without alterations on in vivo progress. This is the first report of pregnancies from aggregated equine cloned embryos, and the first healthy cloned foal from South America, confirmed by STR analysis, was born recently derived from group II. Stumpo, Ignacio, Paola Barboza, and Don Antonio staff.

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.

Births of kittens produced by intracytoplasmic sperm injection of domestic cat oocytes matured in vitro

2000 ◽  
Vol 12 (8) ◽  
pp. 423 ◽  
Author(s):  
M. C. Gómez ◽  
C. E. Pope ◽  
R. Harris ◽  
A. Davis ◽  
S. Mikota ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Polar Body ◽  
Blastocyst Stage ◽  
Domestic Cat ◽  
Oocyte Activation ◽  
Morula Stage ◽  
Cytoplasmic Maturation ◽  
Vitro Development

In Experiment 1, cleavage frequency and in vitrodevelopment of domestic cat embryos produced after in vitro maturation of oocytes obtained from ovaries after ovariohysterectomy (in vivo) with that of oocytes retrieved from follicle-stimulating hormone-treated donors at 24 h after administration of luteinizing hormone (in vivo) and fertilization by intracytoplasmic sperm injection (ICSI) or IVF were compared. In each group presumptive zygotes were assessed for cleavage on IVC Days 1 and 4 and for development to blastocysts on IVC Day 7. In vitro matured oocytes had lower frequencies of meiotic maturation (59.2% v. 66.5%), cleavage at Day 1 (41.4% v. 64.9%) and development to the morula stage at Day 4 (65.8% v. 87.9%) than did in vivo matured oocytes, after ICSI and IVF. Development to the blastocyst stage was lower in in vitro matured oocytes (19.0%) than in vivo matured oocytes (29.5%) after ICSI. In Experiment 2, we evaluated the capacity of sperm injected oocytes without a visible polar body to undergo cleavage and in vitro development. More in vivo matured than in vitro matured oocytes underwent cleavage at Day 1 (46.6% v. 12.6%) and developed to the morula stage by Day 4 (66.7% v. 46.1%), but no blastocysts were obtained at Day 7 in either group. In Experiment 3, we evaluated the in vivo viability of domestic cat embryos derived from ICSI of in vitro matured oocytes. Morula stage embryos were transferred to 18 domestic cat recipients either on Day 4 or 5 after oocyte recovery. A total of 3 domestic cat recipients were pregnant after transfer to recipients on Day 5. Two pregnant cats delivered two normal and healthy live male kittens on Day 68 of gestation and the remaining cat delivered a male kitten on Day 62 that died during the last two days of gestation. These results demonstrate that: (1) inadequate cytoplasmic maturation of in vitro matured domestic cat oocytes is the main cause of deficient oocyte activation; (2) the injection of oocytes without a visible polar body is a useful technique to evaluate oocyte cytoplasmic maturation; and (3) blastocysts obtained after ICSI of in vitro matured oocytes are viable and not a result of parthenogenesis.

377 EMBRYO PRODUCTION BY OVUM PICKUP-INTRACYTOPLASMIC SPERM INJECTION-IVC IN AN EQUINE OVUM PICKUP PROGRAM USING SEMEN FROM FERTILE AND INFERTILE STALLIONS

2010 ◽  
Vol 22 (1) ◽  
pp. 345 ◽  
Author(s):  
S. Colleoni ◽  
R. Duchi ◽  
G. Lazzari ◽  
C. Galli
Keyword(s):  
Embryo Development ◽  
Intracytoplasmic Sperm Injection ◽  
Oocyte Quality ◽  
High Fertility ◽  
Cleavage Rate ◽  
Chi Square ◽  
Female Component ◽  
Chi Square Test

The introduction in equine reproduction of ovum pickup (OPU) combined with intracytoplasmic sperm injection (ICSI), IVC, and embryo transfer, has allowed for the production of offspring from donors and stallions that could not reproduce by conventional techniques. For this reason, we used in our OPU-ICSI-IVC program both fertile stallions and stallions with field records of low or no fertility. Overall, 805 and 584 OPU oocytes were fertilized with sperm from fertile and infertile stallions, respectively. Cleavage rate was statistically lower in the latter group (65.94 v. 59.24%, chi square test; P < 0.05) but embryo development was similar (11.67 v. 8.20% blastocysts/injected oocytes, chi-square test). In order to further investigate the stallion effect on embryo development, we selected 3 stallions with low (A) or no (B, C) fertility in the field and we compared the results of the OPU program with embryo development obtained using oocytes recovered from abattoir ovaries and matured, fertilized, and cultured in vitro as the OPU oocytes. Part of the abattoir oocytes was fertilized with a stallion with known high fertility both in vivo and in vitro (abattoir fertile). Overall, the results (shown in the table) suggest a reduction in the efficiency of stallions A, B, and C compared with to the fertile stallion used as control (10.79, 7.69, and 5.0% v. 17.35%, respectively). For stallions A and B, the efficiency was further reduced in the OPU setting, indicating that the female component can play a role in the overall efficiency of the procedure. In particular, 4 mares out of 8 had a history of no pregnancy and all mares had some rate of inbreeding with the respective stallion used for the ICSI. Instead, the oocytes from the abattoir ovaries were collected in large pools from several mares, representing an average oocyte quality, and the mares were of different breed than the stallions. All data were analyzed by chi-square test and significance was set at P < 0.05. In conclusion, we demonstrated that, for those stallions in which fertility in the field is low or absent, OPU-ICSI-IVP is a suitable choice to obtain embryos, although the efficiency is variable depending not only on the stallion but also on the origin of the oocytes. Table 1.Stallion effect on embryo development of ovum pickup (OPU) and abattoir oocytes This work was supported by Fondazione Cariplo and Regione Lombardia.

Enhancement of ADP-induced Platelet Aggregation by Adrenaline in Vivo and Its Prevention

1973 ◽  
Vol 29 (02) ◽  
pp. 490-498 ◽  
Author(s):  
Hiroh Yamazaki ◽  
Itsuro Kobayashi ◽  
Tadahiro Sano ◽  
Takio Shimamoto
Keyword(s):  
Platelet Count ◽  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
The Other ◽  
Endothelial Surface ◽  
Important Interaction ◽  
Blood Coagulability ◽  
The Difference

SummaryThe authors previously reported a transient decrease in adhesive platelet count and an enhancement of blood coagulability after administration of a small amount of adrenaline (0.1-1 µg per Kg, i. v.) in man and rabbit. In such circumstances, the sensitivity of platelets to aggregation induced by ADP was studied by an optical density method. Five minutes after i. v. injection of 1 µg per Kg of adrenaline in 10 rabbits, intensity of platelet aggregation increased to 115.1 ± 4.9% (mean ± S. E.) by 10∼5 molar, 121.8 ± 7.8% by 3 × 10-6 molar and 129.4 ± 12.8% of the value before the injection by 10”6 molar ADP. The difference was statistically significant (P<0.01-0.05). The above change was not observed in each group of rabbits injected with saline, 1 µg per Kg of 1-noradrenaline or 0.1 and 10 µg per Kg of adrenaline. Also, it was prevented by oral administration of 10 mg per Kg of phenoxybenzamine or propranolol or aspirin or pyridinolcarbamate 3 hours before the challenge. On the other hand, the enhancement of ADP-induced platelet aggregation was not observed in vitro, when 10-5 or 3 × 10-6 molar and 129.4 ± 12.8% of the value before 10∼6 molar ADP was added to citrated platelet rich plasma (CPRP) of rabbit after incubation at 37°C for 30 second with 0.01, 0.1, 1, 10 or 100 µg per ml of adrenaline or noradrenaline. These results suggest an important interaction between endothelial surface and platelets in connection with the enhancement of ADP-induced platelet aggregation by adrenaline in vivo.

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