211 IN VIVO MATURATION AND IN VITRO FERTILIZATION OF ALPACA OOCYTES

2011 ◽  
Vol 23 (1) ◽  
pp. 204 ◽  
Author(s):  
W. Huanca ◽  
R. L. Condori ◽  
M. A. Chileno ◽  
J. Cainzos ◽  
J. J. Becerra ◽  
...  
Keyword(s):  
Cumulus Cells ◽  
Hypodermic Needle ◽  
High Humidity ◽  
Cleavage Rate ◽  
Sodium Pyruvate ◽  
Vitro Fertilization ◽  
Collection Rate ◽  
Follicular Response

The objectives of the study were to evaluate the ovarian follicular response, cumulus–oocyte complex (COC) collection rate, fertilization, and culture of COC collected from alpacas after treatment with 2 different gonadotropins. Female alpacas were assigned to Group 1 (n = 8), 200 mg of FSH (Folltropin, Bioniche, Belleville, Ontario, Canada) divided b.i.d. for 3 days, plus a single IM dose of 1000 IU of hCG (Chorulon, Intervet, Salamanca, Spain) 24 h after the last FSH treatment; or Group 2 (n = 10), 750 IU of eCG (Folligon, Intervet) as a single dose, plus a single IM dose of 1000 IU of hCG on Day 3 after eCG treatment (Day 0 = start of the superstimulatory treatment). At 20 to 22 h post-hCG treatment, the ovaries were surgically exposed and COC were aspirated from follicles ≥6 mm and evaluated. The COC with a homogeneous cytoplasm and 2 or more layers of cumulus cells were transferred to plates with a 40-μL drop of TCM-199 maturation medium supplemented with 10% FCS (vol/vol) plus 0.5 μg mL–1 of FSH, 10 μg mL–1 of hCG, 0.2 mM sodium pyruvate, 50 μg mL–1 of gentamicin, and 1 μg mL–1 of oestradiol under mineral oil with 10 to 12 oocytes/drop and maturated 24 h at 39°C in an atmosphere of 5% CO2 and high humidity. After maturation, COC were removed and fertilized in vitro using epididymal sperm. Testes were collected from mature males from a slaughterhouse and transported to the laboratory. The caudal epididymide was isolated. A prick was made on the convoluted tubules with a sterile hypodermic needle and the fluid, rich in spermatozoa, was aspirated in syringes containing 2 mL of Tris-fructose egg yolk extender. Motile spermatozoa were obtained by centrifugation at 600 × g on a Percoll discontinuous gradient (45.0:22.5%) for 10 min. The supernatant was then removed by aspiration and the pellet was resuspended in TL-HEPES and centrifuged again at 300 × g for 5 min. The pellet was resuspended in TL-stock. Gametes were co-incubated for 18 h at 39°C with 5% CO2 and high humidity. Presumptive zygotes were cultured in KSOM medium supplemented with 1 mM glutamine, 0.3 mM sodium pyruvate, 50 μg mL–1 of gentamicin, EDTA, essential and nonessential amino acids, and BSA for 3 days and cultured in SOF medium for 7 days. Embryo development was evaluated at 72 h and 7 days. Data were subjected to ANOVA. The number of follicles ≥6 mm did not differ at the time of COC collection (19.3 ± 5.7 and 21.5 ± 7.3), and the number of COC collected was 16.7 ± 5.3 and 17.3 ± 6.6 for the FSH group and the eCG group, respectively. The cleavage rate was 45.2 and 42.1% for the FSH group and the eCG group, respectively, at 72 h of culture, and the blastocyst stage at Day 7 (22.2 v. 19.3) did not differ between treatments. In conclusion, the FSH and eCG treatments did not differ in the ovarian follicular response, COC collection rate, fertilization, and culture of COC. Both gonadotropins can be used in the IVF protocol for alpacas. Grant 064 FINCyT-PIBAP 2008 and Grant 032-2009 PROCYT–CONCYTEC.

341 IN VITRO MATURATION AND IN VITRO FERTILIZATION OF ALPACA (VICUGNA PACOS) OOCYTES: EFFECT OF TIME OF INCUBATION ON NUCLEAR MATURATION AND CLEAVAGE

2010 ◽  
Vol 22 (1) ◽  
pp. 327 ◽  
Author(s):  
W. Huanca ◽  
R. Condori ◽  
J. Cainzos ◽  
M. Chileno ◽  
L. Quintela ◽  
...  
Keyword(s):  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Hypodermic Needle ◽  
Cleavage Rate ◽  
Sodium Pyruvate ◽  
Maturation Time ◽  
Vitro Fertilization ◽  
Nuclear Maturation ◽  
Maturation Medium

Experiments were carried out to evaluate the effect of incubation time on nuclear maturation (Experiment 1) and determine the cleavage rate of alpaca oocytes after of IVF time (Experiment 2) In Experiment 1, CCOs were collected from slaughterhouse ovaries and transported to the laboratory in a thermos flask containing a saline solution 0.9% with antibiotic antimycotic at 35°C. CCOs were aspirated from follicles >2 mm and pooled in a conical tube to sedimentation previous to evaluation under stereomicroscope and CCOs with a cytoplasm homogeneous and 2 or more layers of cumulus cells were transferred to plates with a 40-μL drop of maturation medium TCM-199 supplemented with 10% FCS (v : v) plus 0.5 μg mL-1 FSH, 10 μg mL-1 hCG, 0.2 mM sodium pyruvate, 50 μg mL-1 gentamicine, and 1 μg mL-1 Estradiol under mineral oil with 10-12 oocytes/drop. Oocytes were incubated under the following maturation times: 30, 34, and 38 h at 39°C in an atmosphere of 5% CO2 and high humidity. After each maturation time, CCOs were removed from maturation medium and washed with PBS supplemented with 10% FCS and 1 mgmL-1 of hyaluronidase and fixed in ethanol: acetic acid (3 : 1). Oocytes were placed on the slide with minimum medium and stained with 1% orcein for 5 min The slides were examined under a phase contrast microscope at × 400 to evaluate status of nuclear maturation and classified as germinal vesicle (GV); metaphase I (M-I), anaphase-telophase; metaphase II (M-II) and degenerated. Experiment 2: The same maturation method as Experiment 1 was used. Testes were collected of mature males from slaughterhouse and transported to the laboratory. Caudal epididymide was isolated. A prick was made on the convoluted tubules with a sterile hypodermic needle and the fluid, rich in spermatozoa, was aspirated in syringes containing 2 mL of Tris-fructose egg yolk extender. Motile spermatozoa were obtained by centrifugation: 700 g on a Percoll discontinuous gradient (22.5 :45.0%) for 25 min. The supernatant was removed by aspiration and pellet (containing viable spermatozoa) was resuspended in TL stock. Spermatozoa and oocytes were co-incubated for 18-20 h at 39°C with 5% CO2 and then cultivated in TCM-199 supplemented with 10% FCS (v: v), 0.2 mM sodium pyruvate, and 50 μg mL-1 gentamicine and evaluated at 48 h. Data were subjected to ANOVA. For Experiment 1, the proportions of oocytes reaching M-II stage was 18.9 ± 15.7, 42.9 ± 16.2, and 65.8 ± 8.1% for the 30, 34, and 38 h of culture, respectively, with difference to maturation time (P < 0.05). For Experiment 2, the cleavage rate was 9.5, 7.7, and 15.4% to 30, 34, and 38 h after of fertilization time 48 h culture. These results indicate that 38 or more h is required for the maturation and fertilization of alpaca oocytes. Grant 064 FINCyT-PIBAP 2008.

208 IN VITRO FERTILIZATION UNDER SIMULATED STRESS AND SUBSEQUENT IN VITRO EMBRYO PRODUCTION IN THE PIG

2011 ◽  
Vol 23 (1) ◽  
pp. 203
Author(s):  
R. González ◽  
Y. Brandt
Keyword(s):  
Cumulus Cells ◽  
Early Embryo ◽  
Reproductive Hormones ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Vitro Fertilization ◽  
Endocrine Profile ◽  
Serum Gonadotropin

Fertilization is a crucial step for successful reproduction and can be negatively influenced by stressful situations. It is generally accepted that stress affects reproduction, altering the endocrine profile of the female. An altered hormonal environment where the oocyte is developing could affect critical processes such as fertilization. Using a mixed in vivo–in vitro system, we assessed the ability of the oocyte to undergo fertilization and early development after exposure to blood plasma from sows that had experienced simulated stress through repeated injections of adrenocorticotropic hormone (ACTH) before ovulation (known concentrations of cortisol and reproductive hormones as well as exact ovulation time assessed by ultrasonography). Oocytes (n = 926, 7 replicates) collected from abattoir ovaries were matured in TCM-199 with BSA supplemented with hormones (10 IE mL–1 of pregnant mare serum gonadotropin and 5 IE mL–1 of hCG) and insulin-transferrin-selenium (5 μL mL–1) for 24 h, followed by 22 h without supplements. During IVF, gametes were exposed to 10% of pooled plasma (n = 3 per treatment) collected approximately 1 h before ovulation from ACTH-treated sows (A group), nontreated control sows (C group), or media with BSA (B group) for 24 h. Fresh semen was added at 5 × 105 cells mL–1. Afterward, the remaining cumulus cells and sperm were removed from oocytes by vortexing (1 min), and presumptive zygotes were placed in culture medium (porcine zygote medium). Cleavage rate was assessed at 48 h post-insemination (hpi) and the embryos (n = 433, 7 replicates) were cultured up to Day 7 and stained with Hoechst 33342 (10 μg mL–1) to count the total number of nuclei. In addition, non-cleaved oocytes were stained at 48 hpi with Hoechst to assess sperm-zona binding. Binding to the zona was assessed only in oocytes found to be matured. Statistical analysis was done using Kruskal-Wallis ANOVA and the Mann-Whitney U test. The number of spermatozoa bound to the zona pellucida was higher in the B group, and binding was notably negatively affected in the ACTH group (0.43 ± 0.18, 35.93 ± 2.50, and 3.44 ± 1.04 for the A, B, and C group, respectively; P < 0.001). Cleavage rate (over total number of presumptive zygotes) in the A group (30.71 ± 3.76%) was significantly lower than in the control groups (59.93 ± 4.0 and 52.2 ± 5.31% for the B and C group, respectively; P < 0.01). Blastocyst rate expressed over the total number of embryos was reduced in the A group (9.40 ± 5.20%) compared with the controls (27.10 ± 5.79 and 25.66 ± 5.28% in the B and C group, respectively; P < 0.05). However, no differences were found in the total number of nuclei in the blastocysts. The results suggest that fertilization is a sensitive event that could be negatively influenced by stress, subsequently affecting early embryo development. A reduced number of spermatozoa attached to the zona and a lower number of embryos and lower blastocyst development were observed in the simulated-stress group. Further studies would help to elucidate which (in the oocyte, spermatozoon, or both) mechanisms are being affected by ACTH-simulated stress around fertilization. Data are expressed as mean ± SEM. Funded by Formas.

scholarly journals Features of the preparation of biological material for genome editing in cattle

2019 ◽  
Vol 191 (12) ◽  
pp. 40-44
Author(s):  
A. Barkova ◽  
M. Modorov ◽  
G. Isaeva ◽  
A. Krivonogova
Keyword(s):  
Genome Editing ◽  
Biological Material ◽  
Genetic Material ◽  
Cumulus Cells ◽  
Vitro Fertilization ◽  
Expected Time ◽  
Donor Material ◽  
Material Transportation

Abstract. To carry out genome editing in cattle, an effective and well-functioning system for obtaining gametes, fertilizing eggs and their cryopreservation is necessary. Aim of the work: review and research of present-day existing methods of obtaining, insemination and cryopreservation of donor material, in order to provide genome editing in cows. Methods and materials. The work is completed according to the theme No. 0532-2019-0001 “Development of complex technology of marker-based genome selection of agricultural animals” within State Order of Ministry of Education and Science of the Russian Federation. The analysis of open scientific literature on the issues of in vitro fertilization in animals, cryopreservation of oocytes and embryons, sperm preparation and methods of insemination of cows’ oocytes, and cryopreservation of oocytes and embryons of animals is done. Features of the preparation of biological material of cattle for genome editing by microinjection into ooplasm are described. Results of research and duscussion. At present time there are two ways to obtain donor material from cattle: from live animals and taking ovaries after slaughtering cows. Material transportation is carried out at a temperature of 30–37 °C depending on the distance to the laboratory and expected time period of transportation. Oocyte-cumulus complexes can be removed by ovarian dissection and aspiration of visible follicles. In both cases, immature eggs are predominantly obtained. Subsequent ripening is carried out in vitro using special media in a CO2 incubator. The culture medium for oocyte maturation should contain hormones that mimic the peak of LH (luteinizing hormone), which occurs in vivo during the maturation of oocytes before ovulation. To accumulate a certain number of eggs at the stage of MII, it is recommended to carry out their cryopreservation by the method of vitrification, having previously released the oocyte from the cumulus cells. After thawing, oocytes need to be incubated for 2–3 hours 38.5 °C in 5–6.5% CO2 to restore the spindle. In order to make editing more effective, the introduction of genetic material is recommended to be carried out in parallel with the fertilization method “icsi”. In humans, mice and rabbits, an injection of sperm into the cytoplasm is sufficient to activate the oocyte, however, in cattle, just micro-injection of the sperm is not enough and often the male pronucleus does not form. To solve the problem, various methods are used, including freezing-thawing of sperm, resulting in damage of a membrane, or addition of heparin-glutathione into the medium that increases decondensation of the sperm DNA.

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.

256 EFFECT OF SPERM SELECTION ON THE RATE OF IN VITRO FERTILIZATION IN ALPACA (VICUGNA PACOS)

2015 ◽  
Vol 27 (1) ◽  
pp. 217
Author(s):  
E. Mellisho ◽  
V. Rivas ◽  
J. Ruiz ◽  
G. Mamani
Keyword(s):  
Extended Period ◽  
Cumulus Cells ◽  
Sperm Selection ◽  
Cleavage Rate ◽  
Slow Cooling ◽  
Vitro Fertilization ◽  
Progressive Motility ◽  
Frozen Semen ◽  
Washing Method

In alpacas, improvement of reproductive efficiency of male camelids is limited by the small size of the testes, extended period of ejaculation, and low quality of semen. This study was designed to determine the effect of 2 sperm preparation treatments before IVF on the cleavage rate. The sperm was obtained by slicing the head of the epididymis of slaughtered male alpacas (n = 8), diluting in Tris-yolk-glycerol, and freezing with the slow-cooling method. Frozen semen straws per each male were thawed in a water bath at 37°C for 15 s and evaluated for percentage of progressive motility (32 ± 8.6%) and concentration (66.5 ± 24 × 106 sperm mL–1) post-thawing. Sperm selection by the swim-up method was performed by centrifugation at 1077 × g for 5 min with washing sperm medium eliminating the supernatant; sperm were settled in inclined tube with fertilization medium (without capacitating agent) for 60 min, after which 100 μL from the surface was recovered for use in IVF. The washing method consisted in repeated washing (twice) of sperm in washing sperm medium and fertilization medium by centrifugation at 1077 × g for 5 and 3 min, respectively, and recovery of 50 μL from the bottom of the tube for use in IVF. Sperm selected by swim-up or washing methods had similar characteristics of progressive motility (18 and 23%); however, the concentration was higher for the washing v. swim-up method (52 v. 14 × 106 sperm mL–1, respectively). Cumulus-oocyte complexes (COC) were recovered from 278 ovaries of alpacas killed at abattoirs and classified (Grade 1 and 2) for in vitro maturation (38.5°C at 5% CO2 in air for 27 h in 50 μL of 10 COC per drop). A total of 839 oocytes cultured for 27 h in maturation medium were partially stripped out of cumulus cells by gentle aspiration with a pipette. Sperm suspensions in Fert TALP medium (5 μL) from each treatment group were added to each fertilization drop with 10 oocytes per drop of 45 μL obtaining a final concentration of 10 × 106 sperm mL–1 and cultivated for 72 h until their evaluation. The data for the 13 repetitions of the rate of cleavage (2 to 8 cells) were converted to angular values (angle = arcsin √%) with the object of normalizing the distribution of the data; the analysis of variance was performed (complete randomised design with sub-sampling, P < 0.05) using SAS® version 8.0 for Windows. The rate of cleavage (cell division) did not show statistical differences (P = 0.67) for the swim-up method (37%; 155/421) v. washing method (35%; 147/418). The methods of sperm selection (swim-up and washing) did not affect the rate of IVF.

264 TESTOSTERONE IN THE OOCYTE CULTURE DOES NOT ALTER SEX-RATIO OF IN VITRO PRODUCED BOVINE EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 229
Author(s):  
C. Díez ◽  
P. Bermejo-Alvarez ◽  
A. Gutiérrez-Adan ◽  
J. N. Caamaño ◽  
M. Muñoz ◽  
...  
Keyword(s):  
Sex Ratio ◽  
Cumulus Cells ◽  
Basic Medium ◽  
Bovine Embryos ◽  
Experimental Conditions ◽  
Vitro Fertilization ◽  
Grant Support ◽  
High Concentrations

The production of sex-known offspring is a main objective in reproductive biotechnology. It has been reported that bovine ova developed in follicles with high concentrations of testosterone in vivo yielded significantly more male embryos in vitro (Grant V et al. 2008 Biol. Reprod. 78, 812–815). In this work we aimed to test the effects of testosterone on sex ratio of bovine embryos produced in fully in vitro conditions. Immature bovine cumulus–oocyte complexes (COCs; n = 750) from slaughterhouse ovaries were cultured in 199 HNaCO3 with polyvinyl alcohol (PVA) 0.1 mg mL–1 as a basic medium. Culture was made in two steps, a 24 h meiotic arrest (roscovitine 25 μm), and a subsequent in vitro maturation period with FSH-LH for 24 h. Testosterone (T-86500, Sigma-Aldrich, St. Louis, MO, USA) was added throughout the entire oocyte culture at 0, 30, 300, and 1500 nm. After in vitro fertilization (Day 0), zygotes were freed of cumulus cells by pipetting, and subsequently cultured in SOF + 6 g L–1 BSA up to Day 3. At this time, embryo development was recorded, and all embryos having 3 or more cells were treated with pronase to remove the zona pellucida. Zona-free embryos were washed in PBS containing PVA 0.1 mg mL–1 and individually frozen at –80°C until sex analysis by PCR (Bermejo-Alvarez P et al. 2008 Biol. Reprod. doi:10.1095/biolreprod.108.070169). A total of 252 embryos from 5 replicates were sexed. Data for development and sex-ratio are presented as % LSM ± SD. There were no interactions between testosterone treatment, embryonic sex, and embryonic stage analyzed. Testosterone did not affect development rates (P > 0.05) at any stage: cleavage (47.8 ± 6.8, 56.5 ± 6.8; 50.9 ± 6.8; 62.2 ± 6.8), 3 to 4 cells (40.6 ± 5.2, 45.8 ± 5.2; 37.8 ± 5.2; 47.7 ± 5.2) and >5 cells rates (24.5 ± 4; 27.3 ± 4; 21.3 ± 4; 25.3 ± 4) for 0, 30, 300, and 1500 nm testosterone, respectively. Cumulative percentages of male embryos were as follows: 53 ± 8 (n = 56), 42.6 ± 8 (n = 52), 53.6 ± 6 (n = 81) and 57.6 ± 8 (n = 63) for 0, 30, 300, and 1500 nm groups respectively (P > 0.05). These results show that the testosterone effects on oocyte ability to select Y-chromosome bearing spermatozoa are not reproducible in vitro under the present experimental conditions. Grant support: MEC, project AGL2008-01530; RTA2008-0082; M. Muoz is supported by FICYT.

Presence of cumulus cells during in vitro fertilization protects the bovine oocyte against oxidative stress and improves first cleavage but does not affect further development

Zygote ◽  
2005 ◽  
Vol 13 (2) ◽  
pp. 177-185 ◽  
Author(s):  
A. Nader Fatehi ◽  
Bernard A.J. Roelen ◽  
Ben Colenbrander ◽  
Eric J. Schoevers ◽  
Bart M. Gadella ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Hydrogen Peroxide ◽  
Formation Rate ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Physical Contact ◽  
Blastocyst Formation ◽  
Cleavage Rate ◽  
Vitro Fertilization

The present study was conducted to evaluate the function of cumulus cells during bovine IVF. Oocytes within cumulus–oocyte complexes (COCs) or denuded oocytes (DOs) were inseminated in control medium, or DOs were inseminated in cumulus cell conditioned medium (CCCM). DOs exhibited reduced cleavage and blastocyst formation rates when compared with intact COCs. The reduced blastocyst formation rate of DOs resulted from reduced first cleavage but subsequent embryo development was not changed. Live-dead staining and staining for apoptotic cells revealed no differences in blastocysts from oocytes fertilized as COC or DO. Fertilization of DOs in CCCM partially restored the cleavage rate, suggesting that factors secreted by cumulus cells are important for fertilization but that physical contact between oocytes and cumulus cells is required for optimal fertilization and first cleavage. Exposure of COCs to hydrogen peroxide shortly before fertilization reduced the cleavage rate, but did not lead to enhanced death of cumulus cells or oocyte death. Exposure of DOs to hydrogen peroxide, however, resulted in oocyte death and a complete block of first cleavage, suggesting that cumulus cells protect the oocyte against oxidative stress during fertilization.

Technical Report: Effect of commercially available PMSG on maturation, fertilization and embryo development of buffalo oocytes in vitro

2001 ◽  
Vol 13 (6) ◽  
pp. 355 ◽  
Author(s):  
P. S. P. Gupta ◽  
S. Nandi ◽  
B. M. Ravindranatha ◽  
P. V. Sarma
Keyword(s):  
Embryonic Development ◽  
Genetic Manipulation ◽  
Culture Media ◽  
Cell Monolayer ◽  
Basic Research ◽  
Cost Effective ◽  
Cumulus Cells ◽  
Cleavage Rate ◽  
Vitro Fertilization

In vitro fertilization (IVF) technology provides an opportunity to produce embryos for genetic manipulation, embryo transfer and basic research in developmental physiology, and can be exploited for emerging biotechnologies such as transgenesis and cloning. In the present study, the effects of different concentrations of commercially available pregnant mare serum gonadotrophin (PMSG) (Folligon; Intervet, International B.V., Boxmeer, Holland) in oocyte culture media, on maturation, fertilization and embryonic development of buffalo oocytes in vitro were investigated. Oocytes aspirated from abattoir-derived ovaries were cultured in media containing TCM-199 + PMSG at 0, 2.5, 20, 30, 40 and 50 IU mL–1 in presence or absence of steer serum (10%) for 24 h in a CO2 incubator. The maturation rate was assessed on the basis of degree of expansion of cumulus cells. The matured oocytes were inseminated with 9–10 x 106 spermatozoa mL–1 in Brackett and Oliphant medium and the cleavage rate was recorded 40–42 h after insemination. Uncleaved oocytes were stained with aceto-orcein for evaluation of fertilization rates. The cleaved embryos were further cultured in TCM-199 + 10% steer serum on buffalo oviducal cell monolayer for 7 days. Maturation, fertilization, cleavage and embryonic development were significantly higher (P<0.05) in oocytes cultured in TCM-199 + 10% steer serum supplemented with 40 and 50 IU PMSG mL–1. It is concluded that commercially available PMSG can effectively be used in place of pure follicle-stimulating hormone for in vitro maturation of buffalo oocytes, making it cost effective for IVF studies.

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.

68 EFFECT OF CO-CULTURE DURING FERTILIZATION OF BUFFALO (BUBALUS BUBALIS) IN VITRO-MATURED DENUDED OOCYTES VITRIFIED BY CRYOTOP

2008 ◽  
Vol 20 (1) ◽  
pp. 115
Author(s):  
L. Attanasio ◽  
A. De Rosa ◽  
L. Boccia ◽  
R. Di Palo ◽  
G. Campanile ◽  
...  
Keyword(s):  
Survival Rate ◽  
In Vitro Fertilization ◽  
Survival Rates ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Control Group ◽  
Cleavage Rate ◽  
Vitro Fertilization ◽  
Group B

Although removal of cumulus cells improves the efficiency of vitrification of buffalo (Bubalus bubalus) in vitro-matured (IVM) oocytes (Gasparrini et al. 2007 Anim. Reprod. Sci. 98, 335–342), the lack of cells impairs the fertilization process. Therefore, the aim of the present work was to evaluate the influence of a somatic support during in vitro fertilization (IVF) of buffalo vitrified denuded matured oocytes. Since IVF on a cumulus cells monolayer was inefficient, we verified the effects of co-culture with cumulus-enclosed oocytes (COCs). IVM buffalo oocytes (n = 316) were vitrified by the Cryotop� method (Kuwayama and Kato 2000, J. Assist. Reprod. Genet. 17, 477 abst) that was recently proven suitable for buffalo oocyte cryopreservation (Attanasio et al. 2006 Reprod. Domest. Anim. 41, 302–310). Denuded buffalo oocytes were equilibrated in 10% ethylene glycol (EG) and 10% dimethyl sulfoxide (DMSO) for 3 min, transferred into 20% EG and 20% of DMSO in TCM199 with 20% fetal calf serum (FCS) + 0.5 m sucrose, loaded on Cryotops, and plunged into liquid nitrogen within 25 s. For warming, oocytes were exposed for 1 min to 1.2 m sucrose and then to decreasing concentrations of the sugar (0.6, 0.4, 0.3 m for 30 s) in TCM199 + 20% FCS. Oocytes were rinsed and allocated to IVM drops for 1.5 h. Survival rate was evaluated at this point and the oocytes that had survived (292/316 = 92.4%) were split into 2 fertilization groups: (A) approximately 5 buffalo oocytes per 50-µL drop of IVF medium, and (B) approximately 3 buffalo oocytes + 3 bovine fresh COCs per 50-µL drop of IVF medium. Since buffalo COCs easily lose their cells following IVF, for better identification we used bovine COCs that have a brighter and more compact cumulus mass. In vitro fertilization and culture were carried out as previously described (Gasparrini et al. 2007). As control, buffalo oocytes (n = 104) were in vitro-matured, fertilized, and cultured up to the blastocyst stage. On Day 1, survival rate was evaluated in the two vitrification groups; cleavage and blastocyst rates were recorded on Days 5 and 7, respectively, in all groups. The experiment was repeated 4 times. Differences in the percentages of survival, cleavage, and blastocyst formation among treatments were analyzed by chi-square test. Within vitrification groups, despite similar survival rates on Day 1 (90.6% v. 93.3%, respectively, in Groups A and B), cleavage rate was significantly improved in Group B compared to Group A (59.2% v. 45.4%, respectively; P < 0.01). Interestingly, the cleavage rate in Group B was not significantly different from that recorded in the control group (71.0%). Although blastocysts were produced in both vitrification groups (3.6% v. 4.1%, respectively, in Groups A and B), the yield was significantly lower than that of the control group (29.0%, P < 0.01). In conclusion, co-culture with bovine COC during fertilization improves the capability of buffalo denuded vitrified oocytes to cleave.

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