86 EFFECT OF CRYOPROTECTANT EXPOSURE, VITRIFICATION, AND WARMING TIME OF BOVINE CUMULUS OOCYTE COMPLEXES ON IN VITRO FERTILIZATION AND EMBRYONIC DEVELOPMENT

2011 ◽  
Vol 23 (1) ◽  
pp. 148
Author(s):  
J. R. Prentice ◽  
J. Singh ◽  
R. J. Mapletoft ◽  
M. Anzar
Keyword(s):  
Calf Serum ◽  
Cumulus Cells ◽  
Control Group ◽  
Time Interval ◽  
Crystal Formation ◽  
Ice Crystal ◽  
Vitro Fertilization ◽  
Vitrification Solution ◽  
High Concentrations

Despite the importance of cryoprotectants for avoiding ice crystal formation, the high concentrations required for vitrification may be toxic to bovine oocytes. During warming (thawing), the removal of permeating cryoprotectants from cells can lead to osmotic injury, and the most appropriate time interval for warming and cryoprotectant removal from vitrified oocytes is currently uncertain. The present study aimed to evaluate the effect of cryoprotectant exposure, vitrification, and warming time of bovine cumulus oocyte complexes (COC) on fertilization and ability to develop as embryos in vitro. Follicles <8 mm in diameter were aspirated from slaughterhouse-derived bovine ovaries. Cumulus oocyte complexes with ≥3 layers of cumulus cells and a uniform cytoplasm were selected, washed 3 times in Dulbecco’s PBS + 5% newborn calf serum (CS), and randomly divided into 4 groups: 1) control group: no treatment; 2) VS1 group: COC were exposed to vitrification solution 1 [VS1: 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) in TCM-199 + 20% CS] for 5 min; 3) VS1+VS2 group: COC were exposed to VS1 for 5 min followed by vitrification solution 2 (VS2: 15% EG, 15% DMSO, and 0.5 M sucrose in TCM-199 + 20% CS) for 30 s; and 4) vitrified group: COC were exposed to VS1 and VS2, and then vitrified in liquid nitrogen using cryotops. The COC in VS1, VS1+VS2, and vitrified groups were exposed to a warming solution (0.5 M sucrose in TCM-199) for 1 or 5 min. The COC from all groups were in vitro matured (IVM) for 22 h in TCM-199 containing 5% CS, 5 μg mL–1 LH, 0.5 μg mL–1 FSH, and 0.05 μg mL–1 gentamicin at 38.5°C, 5% CO2, and high humidity, incubated with frozen–thawed sperm in Brackett-Oliphant capacitating medium for 18 h, and the presumptive zygotes were cultured in Charles Rosenkrans 1 amino acids (CR1aa) + 5% CS for 9 days. Data were analysed using Proc Glimmix in SAS® 9.2 (SAS Institute Inc., Cary, NC, USA). Cleavage and blastocyst rates in the vitrified group (25 and 2%, respectively) were significantly lower (P < 0.0001) than in control (75 and 27%), VS1 (68 and 19%), or VS1+VS2 (63 and 22%) groups. Cleavage and blastocyst rates did not differ among non-vitrified groups (P > 0.05). In VS1, VS1+VS2, and vitrified groups, warming time had no effect on cleavage or blastocyst rates (P > 0.05). In conclusion, although cryoprotectant exposure and warming times had no apparent adverse effect, vitrification of bovine COC drastically reduced cleavage and blastocyst rates. Further studies are required to understand how vitrification of bovine COC affects subsequent fertilization and embryo development. This study was supported by the Canadian Animal Genetic Resources Program, Agriculture and Agri-Food Canada.

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.

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.

165 FETAL CALF SERUM INFLUENCES CYCLIC GMP PATHWAY, WHICH IN TURN AFFECTS THE LIPID CONTENT IN IN VITRO-MATURED BOVINE OOCYTES

2014 ◽  
Vol 26 (1) ◽  
pp. 196
Author(s):  
K. R. L. Schwarz ◽  
R. C. Botigelli ◽  
F. C. Castro ◽  
M. R. Chiaratti ◽  
C. L. V. Leal
Keyword(s):  
Lipid Content ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Control Group ◽  
Maturation Medium ◽  
Cgmp Pathway ◽  
Bovine Oocytes ◽  
Synthesis And Degradation

The sensitivity of IVP embryos to cryopreservation is often associated with lipid accumulation in the cytoplasm induced by the presence of fetal calf serum (FCS) during culture. Intracellular levels of cyclic (c)AMP and cGMP are involved in the regulation of lipolysis in adipocytes; high levels stimulate lipolysis whereas low levels lead to lipogenesis. Both nucleotides are present in bovine oocytes, together with the enzymes for their synthesis and degradation. The aim of this study was to analysis the effect of FCS on the cGMP pathway and the influence of cGMP on cytoplasmic lipids in bovine oocytes. In experiments 1 and 2, cumulus–oocyte complexes (COC) were cultured for 24 h in maturation medium with different proportions of FCS (2 and 10%) and a control group was matured with 0.4% BSA. After this period, transcripts for cGMP pathway were assessed by real-time PCR (GUCY1B3 and PDE5, cGMP synthesis and degradation enzymes, respectively; experiment 1) in oocytes and cumulus cells, and cGMP levels were measured in COC using commercial enzyme immunoassay kits (EIA; experiment 2). In experiments 3 and 4, COC were matured for 24 h with 0.4% BSA and different concentrations of the phosphodiesterase (PDE)5 inhibitor (0, 10–7, and 10–5 M sildenafil) to inhibit cGMP degradation and a control group was matured with 0.4% BSA. The nucleotide levels were measured in COC (experiment 3) and the oocytes were stained with Nile Red (1 μg mL–1) for evaluation of lipid content (experiment 4). Statistical analyses were performed by ANOVA followed by Tukey post hoc test using SAS software (SAS Institute Inc., Cary, NC, USA). Data for gene expression from 5 replicates and for cGMP measurements and lipid content from 3 replicates were log10-transformed into before analyses. The level of significance was 5%. The presence of FCS reduced GUCY1B3 expression in both cells and increased PDE5A in cumulus cells (P < 0.05). In experiment 2, the groups treated with 2 (0.64 fmol/COC) and 10% FCS (1.04 fmol/COC) showed decreased cGMP levels compared with control (9.46 fmol/COC; P < 0.05). In experiment 3, inhibition of PDE5A increased cGMP levels in the treated groups (36 and 56 fmol/COC for 10–7 and 10–5 M sildenafil, respectively) compared with control (9.5 fmol/COC; P < 0.05). Therefore, sildenafil showed inverse effects compared with FCS (experiment 2). In experiment 4, oocytes treated with 10–7 and 10–5 M sildenafil showed a reduced lipid content compared with controls (11.6 ± 9.4 v. 13.9 μm2 fluorescence intensity, respectively; P < 0.05). The results suggest that FCS in maturation medium affects the cGMP pathway, interfering with the transcription of genes that control its levels, which in turn results in nucleotide reduction. Inhibition of PDE5 increases cGMP levels and reduces the lipid content of oocytes, indicating that changes in this pathway caused by FCS may affect lipid metabolism of oocytes. More studies are underway to better understand this mechanism. The authors acknowledge FAPESP 2012/00170-0 for financial support.

205 EFFECT OF OSTEOPONTIN ON CLEAVAGE AND BLASTOCYST RATES IN BUFFALO SPECIES (BUBALUS BUBALIS)

2009 ◽  
Vol 21 (1) ◽  
pp. 200
Author(s):  
S. Di Francesco ◽  
E. Mariotti ◽  
M. Rubessa ◽  
G. Campanile ◽  
R. Di Palo ◽  
...  
Keyword(s):  
Embryo Development ◽  
Cumulus Cells ◽  
Bubalus Bubalis ◽  
The Other ◽  
Control Group ◽  
Single Chain ◽  
Cleavage Rate ◽  
Chi Square ◽  
Vitro Fertilization

It was previously reported that osteopontin (OPN), an acidic single-chain phosphorylated glycoprotein found in the oviductal fluid in cattle (Gabler C et al. 2003 Reproduction 126, 721–729), is able to facilitate fertilization in this species (Gasparrini B et al. 2008 Reprod. Fertil. Dev. 20(Suppl. I), 180 abst). The present study aimed to investigate whether the addition of OPN to the fertilization medium would affect both cleavage and postfertilization embryo development in the buffalo. To assess the influence of OPN on cleavage and blastocyst rates, in vitro-matured oocytes were fertilized in modified Tyrode’s albumin lactate pyruvate medium (Lu KH et al. 1987 Vet. Rec. 121, 259–260) supplemented with penicillamine, hypotaurine, and heparin, in the presence of 0.0 (n = 258), 0.1 (n = 263), 1 (n = 261), and 10 μg mL–1 (n = 264) of OPN. In vitro fertilization was carried out with frozen–thawed spermatozoa from a bull already tested for IVF. After 20 to 22 h of co-incubation at 38.5°C and 5% CO2 in air, putative zygotes were gently pipetted to remove cumulus cells, washed, and transferred, 10 per droplet, into 20 μL of SOF medium including essential and nonessential amino acids and BSA (Tervit HR et al. 1972 J. Reprod. Fertil. 30(3), 493–497), in a controlled gas atmosphere consisting of 5% CO2, 7% O2, and 88% N2, in humidified air, at 38.5°C. The culture medium was changed on Day 5 (Day 0 = day of insemination), when cleavage rate was assessed and embryos were moved into fresh medium for an additional 2 days. On Day 7, development rates into blastocysts of superior quality were recorded. Differences in the percentages of both cleavage and blastocyst rates among groups were analyzed by chi-square test. Significantly higher cleavage rates (59.3, 70.3, 71.6, and 42.4%, respectively, in the control group and in the groups with 0.1, 1, and 10 μg mL–1 of OPN; P < 0.01) were observed in the groups with 0.1 and 1 μg mL–1 of OPN compared with the other groups. Likewise, higher blastocyst rate percentages (17.4, 27.4, 29.9, and 9.5%, respectively, in the control group and in the groups with 0.1, 1, and 10 μg mL–1 of OPN; P < 0.01) were observed in the groups with 0.1 and 1 μg mL–1 of OPN compared with the other groups. In conclusion, these results showed that addition of low concentrations of OPN in the fertilization medium improved both cleavage and postfertilization embryo development in the buffalo, whereas the higher concentration resulted in impaired late-stage embryo development.

scholarly journals 213EFFECTS OF TIME OF MATURATION AND SHEEP SERUM ON IN VITRO FERTILIZATION IN THE ENDANGERED ELD'S DEER (CERVUS ELDI THAMIN) OOCYTES

2004 ◽  
Vol 16 (2) ◽  
pp. 228
Author(s):  
B. Siriaroonrat ◽  
P. Comizzoli ◽  
N. Songsasen ◽  
R.E. Spindler ◽  
S.L. Monfort ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Calf Serum ◽  
Genetic Material ◽  
Cumulus Cells ◽  
Fertilization Success ◽  
Estrous Cycles ◽  
Vitro Fertilization ◽  
Offspring Production ◽  
Oviduct Fluid

The Eld’s deer, native to Southeast Asia, is threatened with extinction. Although artificial insemination is effective for offspring production, in vitro fertilization (IVF) would be more useful for rapidly disseminating genetic material from valuable founders. The objectives of this study were to: 1) determine if oocytes recovered from exogenous gonadotropin-treated hinds require additional in vitro maturation;; and 2) assess if fertilization is enhanced by supplementing Deer Synthetic Oviduct Fluid (DSOF;; Berg DK et al., 2003 Theriogenology 59, 189–205) with 1-day postestrus sheep serum (SS). Estrous cycles in Eld’s deer hinds (n=10) were synchronized with PGF2α analog (Lutalyse™, 500mg), followed by a 14-day intravaginal CIDR-G insertion;; ovine FSH (Ovagen™; 0.05 unit×8 injections) was administered at 12-h intervals beginning 84h before CIDR-removal. COCs (n=160) were retrieved laparoscopically 40–46h post-CIDR-removal and either fixed or matured in vitro (for 12h v. 24h) in TCM-199 (Earle’s salt) supplemented with 0.33mM pyruvate, 2mM glutamine, 100IUmL−1 penicillin, 100μgmL−1 streptomycin, 10% fetal calf serum, 5μgmL−1 FSH and LH and 1μgmL−1 E2 (5% CO2, 38.5°C). After 12- or 24-h IVM, cumulus cells were partially removed and oocytes (n=110) fertilized in DSOF with pooled frozen-thawed sperm (3 males;; 2×106 motile sperm mL−1), in the absence or presence of SS (20%, v/v). Additional oocytes (n=18) were used for parthenogenetic control. At 20-h postinsemination, presumptive zygotes were fixed and stained (Hoechst 33342) to assess fertilization success (presence of two pronuclei). Data were analyzed by ANOVA. Overall, 16.0±2.6 (mean±SEM) COCs were recovered/female. The majority of COCs were of excellent quality (grade I; 67.7±3.8%). At time of aspiration, 85% of the oocytes (n=11/13) were in metaphase I stage, 7.5% in telophase and 7.5% degenerate. No parthenogenic activation was observed. Likewise, no polyspermy was observed in any treatment. Fertilization was higher (P&lt;0.05) in oocytes matured for 24h and fertilized in the absence (64.4±3.1%) compared to presence (26.9±11.2%) of SS. In the absence of SS, a higher (P&lt;0.05) proportion of oocytes were fertilized after 24h (64.4±3.1%) compared to 12h (27.1±9.0%) IVM. There was no effect (P&gt;0.05) of SS on fertilization among oocytes subjected to 12-h IVM (27.1±9.0% v. 12.5±9.5%). When SS was present during fertilization, no difference (P&gt;0.05) was observed among oocytes matured for 12 or 24h. Results demonstrate that: 1) Eld’s deer oocytes require an additional 24-h IVM to complete maturation;; 2) DSOF supports sperm-oocyte interaction;; and 3) SS is not essential for successful fertilization. (Supported by Morris Animal Foundation.)

361 EFFECT OF HORMONES, EGF AND β-MERCAPTOETHANOL ON IN VITRO MATURATION OF CAPRINE OOCYTES

2010 ◽  
Vol 22 (1) ◽  
pp. 337 ◽  
Author(s):  
P. Yadav ◽  
S. D. Kharche ◽  
A. K. Goel ◽  
S. K. Jindal ◽  
M. C. Sharma
Keyword(s):  
In Vitro Maturation ◽  
Calf Serum ◽  
Basic Medium ◽  
Control Group ◽  
Vitro Fertilization ◽  
Base Medium ◽  
Maturation Medium ◽  
Maturation Rate ◽  
Group 5

In vitro maturation of oocytes is an integral part of in vitro culture system. In almost all studies of mammalian in vitro maturation, the basic medium is supplemented with serum and hormones. The maturation medium and selection of protein supplements, growth factors, antioxidants and hormones for IVM play an important role in subsequent in vitro fertilization and in vitro embryo development. The objective of the present experiment was to study the effect of exogenous addition of hormones, epidermal growth factor, insulin and β-mercaptoethanol to the maturation medium for in vitro maturation of caprine oocytes. A total of 1540 oocytes were collected from slaughtered goat of 1.5 to 2.5 year of age and randomly divided in to treatment groups. Group 1; COCs were matured in TCM-199 medium containing 10% calf serum (NCS) and 3 mg mL-1 BSA (used as a base medium) for control, Group 2; COCs were matured in a base medium supplemented with hormones (5 μg mL-1 FSH, 5 μg mL-1 LH and 1 μg mL-1 estradiol-17β), Group 3β COCs were matured in a base medium supplemented with 50 ng mL-1 insulin, Group 4β COCs were matured in a base medium supplemented with hormones and 10 ng mL-1 EGF, Group 5; COCs were matured in a base medium supplemented with hormones and 50 mM β-mercaptoethanol and Group 6; COCs were matured in a base medium supplemented with hormones and 50 ng mL-1 insulin. These COCs were matured at 38.5°C in 5% CO2 in air for 27 h. After the maturation, oocytes were separated from cumulus and corona cells by treatment with 0.1% hyaluronidase and by passing through a fine pipette. They are them fixed in 2.5% glutaraldehyde, stained with DAPI and observed under fluorescent microscope for evidence of nuclear maturation. The maturation rates in groups 1 to 6 were 33.6%, 38.0%, 39.7%, 60.0%, 37.4%, and 44%, respectively. Statistical analysis (ANOVA) after arcsin transformation revealed that the maturation rate in group 4 was statistically significant (P < 0.05) as compared to those in groups 1, 2, 3, 5, and 6. The results suggest that the supplementation of EGF in maturation medium significantly enhances the in vitro maturation rate of caprine oocytes.

124 CO-CULTURE WITH BOVINE INTACT CUMULUS - OOCYTE COMPLEXES DURING IN VITRO FERTILIZATION OF BUFFALO DENUDED OOCYTES COMPLETELY RESTORES THEIR FERTILIZING AND DEVELOPMENTAL COMPETENCE

2011 ◽  
Vol 23 (1) ◽  
pp. 167
Author(s):  
M. De Blasi ◽  
M. Rubessa ◽  
L. Boccia ◽  
S. Di Francesco ◽  
M. V. Suárez Novoa ◽  
...  
Keyword(s):  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Negative Control ◽  
Developmental Competence ◽  
Control Group ◽  
Chi Square ◽  
Oocyte Vitrification ◽  
Vitro Fertilization ◽  
Chi Square Test

Removal of cumulus cells is necessary for several technologies such as vitrification, intracytoplasmic sperm injection, and nuclear transfer. However, it is known that the presence of cumulus cells during IVF of buffalo oocytes is fundamental for fertilization and embryo development (Gasparrini et al. 2007 Anim. Reprod. Sci. 98, 335–342; Nandi et al. 1998 Theriogenology 50, 1251–1262). The aim of this work was to evaluate whether co-culture with intact bovine cumulus–oocyte complexes (COC) during IVF would restore the developmental competence of denuded buffalo oocytes. Due to the scarce availability of buffalo ovaries, the somatic support was provided by bovine cumulus cells. Abattoir-derived COC were matured in vitro according to our standard procedures (Gasparrini et al. 2006, Theriogenology, 65, 275–287) and randomly distributed in 3 fertilization groups: 1) a control group of COC (n = 122), 2) a negative control of denuded oocytes (DO; n = 119), and 3) DO co-cultured with in vitro matured bovine COC (DO+COC; n = 103) in a 1:1 ratio (3 bovine COC + 3 denuded buffalo oocytes/50 μL drop). Fertilization was carried out with frozen–thawed spermatozoa from a tested bull in TALP medium supplemented by 0.2 mM penicillamine, 0.1 mM hypotaurine, and 0.01 mM heparin at 38.5°C under a controlled gas atmosphere of 5% CO2 in humidified air. After fertilization the zygotes were cultured in SOF medium including essential and nonessential amino acids and 8 mg mL–1 BSA, at 38.5°C under humidified 5% CO2, 7% O2, and 88% N2, up to the blastocyst stage. On Day 5 and on Day 7 (Day 0 = IVF) cleavage and blastocyst rates were respectively recorded. Data were analysed by chi-square test. As expected, cleavage and blastocyst rates were lower (P < 0.01) in DO (36.1 and 9.2%, respectively) compared with the control (67.2 and 27.1%, respectively). However, co-culture during IVF (DO+COC) significantly increased (P < 0.01) both parameters compared with DO, giving cleavage (70.9%) and blastocyst (27.2%) rates similar to the control. The results of this study demonstrated that co-culture with bovine intact COC during IVF of buffalo denuded oocytes completely restores their fertilizing capability and blastocyst developmental competence. We conclude that this may be a suitable strategy for preserving the developmental competence of oocytes devolved to technologies, such as oocyte vitrification, that require cumulus removal.

202 EFFECT OF OSTEOPONTIN ON IN VITRO EMBRYO DEVELOPMENT IN CATTLE

2008 ◽  
Vol 20 (1) ◽  
pp. 180 ◽  
Author(s):  
B. Gasparrini ◽  
E. Monaco ◽  
L. Boccia ◽  
A. De Rosa ◽  
L. Attanasio ◽  
...  
Keyword(s):  
Embryo Development ◽  
Bovine Milk ◽  
Cumulus Cells ◽  
Control Group ◽  
Single Chain ◽  
Chi Square ◽  
Vitro Fertilization ◽  
Chi Square Test ◽  
Oviduct Fluid

Osteopontin (OPN) is an acidic single-chain phosphorylated glycoprotein found both in the oviduct fluid (ODF) and oviductal epithelium in cattle, which is believed to facilitate fertilization. It was recently reported that addition of a rabbit polyclonal immunoglobulin G antibody against purified bovine milk OPN with sperm oocytes, bovine oocytes, or both decreased (P < 0.05) fertilization compared with the in vitro-fertilized control (Goncalves et al. 2007 Theriogenology 67, 468–74). The aim of the present work was to determine the effect of in vitro addition of OPN to the fertilization medium on both cleavage and postfertilization embryo development in cattle. In the first experiment, in vitro-matured oocytes were fertilized in modified TALP medium in the presence of 0.0, 0.1, 1.0, or 10 µg mL–1 of OPN. In a second experiment, matured oocytes were in vitro-fertilized in modified TALP medium in the presence of 0.0, 10, 20, or 40 µg mL–1 of OPN. In vitro fertilization was carried out with frozen–thawed spermatozoa from a bull previously tested for IVF. After 20 to 22 h of coincubation at 39�C, 5% in CO2 in air, presumptive zygotes were vortexed to remove cumulus cells, washed, and transferred, 30 to 50 per well, into 400 µL of SOF modified medium. Zygotes were incubated in a humidified mixture of 5% CO2, 7% O2, and 88% N2 in air at a temperature of 39�C. On Day 7 of development (Day 0 = day of insemination), cleavage and development rates into transferable embryos (TE)–tight morulae (TM) and blastocysts (Bl) of superior quality were recorded. Differences in the percentages of both cleavage and blastocyst rates among groups were analyzed by a chi-square test. In experiment 1, numerically higher percentages of TM–Bl (29.5, 29.5, 30.5, and 37.5%, respectively, in the control group and in the groups with 0.1, 1, and 10 µg mL–1 of OPN; P = 0.25) and Bl (28.6, 27.5, 29.1, and 36.7, respectively, in the control group and in the groups with 0.1, 1, and 10 µg mL–1 of OPN; P = 0.24) were observed with 10 µg mL–1 of OPN. In experiment 2, significantly more cleavage (80.0 v. 71.3%; P < 0.05) and higher percentages of TM–Bl (44.6 v. 34.5%; P < 0.05) and Bl (39.2 v. 30.6%; P = 0.06) were observed with 10 µg mL–1 of OPN v. the control. Combined analysis from both experiments showed an overall effect of 10 µg mL–1 of OPN v. the control in the percentages of TM–Bl and Bl (respectively, 41.1 v. 33.3% and 37.7 v. 30.5%; P < 0.05). These results indicate that it is possible to improve the efficiency of bovine in vitro embryo production by adding the oviductal protein OPN.

Effect of cyanocobalamin on oocyte maturation, in vitro fertilization, and embryo development in mice

Zygote ◽  
2020 ◽  
pp. 1-8
Author(s):  
Tamana Rostami ◽  
Fardin Fathi ◽  
Vahideh Assadollahi ◽  
Javad Hosseini ◽  
Mohamad Bagher Khadem Erfan ◽  
...  
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
Cumulus Cells ◽  
Treated Group ◽  
Blastocyst Stage ◽  
Control Group ◽  
Vitro Fertilization ◽  
Maturation In Vitro

Summary The aim of this study was to investigate the effect of cyanocobalamin supplementation on in vitro maturation (IVM), in vitro fertilization (IVF), and subsequent embryonic development competence to the blastocyst stage, and in vitro development of mouse 2-cell embryos. Cumulus cells were prepared from mouse cumulus–oocyte complexes (COCs) and incubated for 24 h in an in vitro culture (IVC) medium that contained different concentrations of cyanocobalamin (100, 200, 300 or 500 pM). We collected 2-cell embryos from superovulated NMRI mice and cultured them in the same concentrations of cyanocobalamin (100, 200, 300 or 500 pM). After 42 h of IVM, we observed significantly increased oocyte maturation in the 200 pM cyanocobalamin-treated group compared with the control group (P < 0.0001). Mature oocytes cultured in 200 pM cyanocobalamin were fertilized and cultured in IVC medium with cyanocobalamin (100, 200, 300 or 500 pM) during early embryogenesis. The matured oocytes that were cultured in 200 pM cyanocobalamin had significantly higher 2-cell development rates compared with the control oocytes (P < 0.01). Embryos obtained from in vitro mature oocytes and in vivo fertilized oocytes that were cultured in 200 pM cyanocobalamin had significantly greater frequencies of development to the blastocyst stage and a significant reduction in 2-cell blocked and degenerated embryos compared with the control embryos (P < 0.0001). Embryos derived from oocytes fertilized in vivo with 200 pM cyanocobalamin had a higher percentage of blastocyst embryos compared with those derived from matured oocytes cultured in vitro (P < 0.0001). These finding demonstrated that the effects of cyanocobalamin on oocyte maturation, fertilization, and embryo development in mice depend on the concentration used in IVC medium.

73 VITRIFICATION OF BOVINE OOCYTES: EFFECT OF PACKAGING AND EQUILIBRATION TIME ON NUCLEAR MATURATION

2009 ◽  
Vol 21 (1) ◽  
pp. 136
Author(s):  
J. R. Prentice ◽  
J. Singh ◽  
O. Dochi ◽  
M. Anzar
Keyword(s):  
Complex Structure ◽  
Calf Serum ◽  
Control Group ◽  
Equilibration Time ◽  
Treatment Groups ◽  
Body Tissues ◽  
Vitrification Solution ◽  
Chi Square Analysis ◽  
Bovine Oocytes

The conservation of female animal genetics is challenging because of the scarcity of oocytes and their sensitivity to cryopreservation techniques. During slow, controlled freezing procedures, intracellular ice crystallization often leads to cell damage. Vitrification as an alternate method of cryopreservation exposes cells to a higher concentration of cryoprotectants with an ultra-rapid cooling rate, leading them to an ice-crystal-free, solid glasslike structure. The vitrification procedure has been used successfully for the cryopreservation of embryos and other body tissues, but very few reports of successful oocyte cryopreservation exist because of their complex structure. The present study was designed to compare two packaging methods (Cryotop v. 0.25-mL straw) and two equilibration times (10 v. 0 min) for vitrification of bovine oocytes. COC were aspirated from follicles <8 mm in diameter on bovine ovaries collected from a slaughterhouse. COC with ≥3 layers of cumulus cells and a uniform cytoplasm were selected, washed in Dulbecco’s phosphate-buffered saline (DPBS) + 5% calf serum (CS), and divided into five equal groups. In the control group, the COC were washed in TCM-199 + 5% CS and matured in vitro in TCM-199 containing 5% CS, 5 μg mL–1 of LH, 0.5 μg mL–1 of FSH, and 0.05 μg mL–1 of gentamicin at 38.5°C, 5% CO2, and high humidity for 22 h. In the treatment groups, half the COC were equilibrated with vitrification solution 1 [VS1: TCM-199, 7.5% ethylene glycol (EG), 7.5% DMSO, and 20% CS] for 10 min. After equilibration, COC were exposed to vitrification solution 2 (VS2: TCM-199, 15% EG, 15% DMSO, 20% CS, and 17.1% sucrose) for 30 s. The remaining half of the COC were directly exposed to VS2 without equilibration in VS1. Groups of five equilibrated or nonequilibrated COC were either loaded in a 0.25-mL straw or placed on Cryotop and plunged in liquid nitrogen. The COC were thawed by immersing straws and Cryotops into 37°C thawing solution (TCM-199, 20% CS, and 17.1% sucrose) for 1 min, and were washed and matured in vitro, as described above. After maturation, the COC were denuded using 0.3% hyaluronidase in Ca-Mg free DPBS and mounted on slides. The oocytes were fixed in ethanol:acetic acid (3:1) for 24 h, stained with aceto-orcein for 20 min, and evaluated for stage of maturation. The data (maturation rates) were analyzed using chi-square analysis. In the control group, 61% (n = 54) of the oocytes reached the metaphase-II (M-II) stage. In the treatment groups, more (P < 0.001) oocytes vitrified on Cryotops reached the M-II stage than those vitrified in straws (23.4%; n = 107 v. 9.4%; n = 116). The effect of equilibration time was not significant (P > 0.05) in either packaging method. In conclusion, vitrification of bovine oocytes using the Cryotop method provides an alternative for the cryopreservation of bovine oocytes. Moreover, bovine oocytes can be successfully vitrified without equilibration. This study was supported by the Canadian Animal Genetic Resources Program, Agriculture and Agri-Food Canada.

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