152 Effect of biphasic in vitro maturation with C-type natriuretic peptide on meiosis arrest and in vitro embryo production of oocytes from prepubertal and adult sheep

2022 ◽  
Vol 34 (2) ◽  
pp. 314
Author(s):  
A. Gil ◽  
M. Ferrer ◽  
M.-T. Paramio ◽  
D. Izquierdo
Keyword(s):  
Natriuretic Peptide ◽  
In Vitro Maturation ◽  
Embryo Production ◽  
Adult Sheep ◽  
In Vitro Embryo Production

186 SUPPLEMENTATION WITH LOW DOSES OF DIMETHYL SULFOXIDE DURING IN VITRO MATURATION RESULTS IN IMPROVED IN VITRO EMBRYO PRODUCTION IN CATTLE

2017 ◽  
Vol 29 (1) ◽  
pp. 201
Author(s):  
A. E. Ynsaurralde ◽  
M. Suvá ◽  
R. Bevacqua ◽  
S. Munilla ◽  
C. Luchetti ◽  
...  
Keyword(s):  
Dimethyl Sulfoxide ◽  
Embryo Development ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Predictive Equation ◽  
Embryo Production ◽  
Vitro Fertilization ◽  
First Polar Body ◽  
In Vitro Embryo Production

Oocyte in vitro maturation (IVM) is crucial for subsequent in vitro embryo production. It involves acquisition of competence for fertilization and embryo development. Therefore, its optimization could have a direct impact on in vitro embryo development. Dimethyl sulfoxide (DMSO) is commonly used as solvent or vehicle, but also increases the membrane permeability and behaves as a scavenger of cytotoxic free radicals. The aim of this study was to evaluate the effect of DMSO supplementation during bovine oocyte maturation on subsequent in vitro embryo development and to determine the optimal usage dose with no toxic effect. To this aim, cumulus-oocyte complexes were collected from slaughterhouse ovaries and IVM in TCM 199 containing 10% fetal bovine serum, 10 µg mL−1 of FSH, 0.3 mM sodium pyruvate, 100 mM cysteamine, and 2% antibiotic-antimycotic. The oocytes were incubated for 24 h at 6.5% CO2 in humidified air at 38.5°C. For Experiment 1, IVM medium was supplemented with DMSO at concentrations of 0, 0.1, 0.5, 1, or 10% (vol/vol) DMSO (n = 241, 195, 42, 192, 172 oocytes) and IVM rate was determined by presence of the first polar body. For Experiment 2, 0, 0.1, 0.25, 0.5, 0.75, 1, or 10% (vol/vol) DMSO (n = 446, 322, 65, 194, 77, 250, 39 oocytes) was supplemented to IVM medium and cleavage and blastocyst rates were determined to establish the optimal usage dose. In vitro fertilization was performed according to Brackett and Oliphant (1975), with 16 × 106 spermatozoa/mL for 5 h. Afterwards, presumptive zygotes were cultured in SOF for 7 days at 38.5°C and 5% O2. Cleavage and blastocyst rates were determined on Days 2 and 7, respectively. Results were statistically analysed using Fisher’s exact test by GraphPad Prism software (GraphPad Software Inc., La Jolla, CA, USA). Also, the percentage of blastocyst was adjusted to DMSO concentration using the R software quadratic regression model. The optimum usage dose was determined by calculating the maximum of the estimated predictive equation. In vitro maturation in 10% DMSO resulted in significantly lower first polar body extrusion rates (0% = 74%a, 0.1% = 73%a, 0.5% = 83%a, 1% = 66%a, and 10% = 8%b; different letters indicate statistical differences) and lower cleavage rates (0% = 75%a, 0.1% = 77%a, 0.25% = 80%a, 0.5% = 79%a, 0.75% = 78%a, 1% = 77%a, and 10% = 3%b) than the other treatments. Furthermore, blastocyst production was higher for the 0.25 and 0.5% (vol/vol) supplemented DMSO groups (0% = 26%b, 0.1% = 37%ab, 0.25% = 40%a, 0.5% = 41%a, 0.75% = 34%ab, 1% = 23%b, and 10% = 0%c). The predictive equation results indicate that the maximum percentage of blastocysts is obtained with a concentration of 0.458% (vol/vol) of DMSO. In conclusion, DMSO supplementation during IVM of bovine oocytes had a positive effect on in vitro development. Further studies will be carried out to elucidate its mechanism of action.

228 IN VITRO EMBRYO PRODUCTION AFTER EXPOSURE OF BOVINE OOCYTES TO DIFFERENT TRANSPORTATION MEDIA AND PERIODS: PRELIMINARY RESULTS

2013 ◽  
Vol 25 (1) ◽  
pp. 262
Author(s):  
T. C. Rossi ◽  
J. Rossi ◽  
T. M. Miyauchi ◽  
C. A. C. Fernandes ◽  
L. S. A. Camargo ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Penicillin G ◽  
Controlled Atmosphere ◽  
Time Data ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Fetal Bovine ◽  
In Vitro Embryo Production ◽  
Main Effects

This study was designed to evaluate the effect of different oocyte transportation media and time. Immature oocytes were recovered from slaughterhouse bovine ovaries. Oocytes (n = 492) of quality grades I to III were randomly allotted to one of the following transportation media based on TCM-199, either buffered with HEPES (control) or buffered with NaHCO3 and added with FSH/LH (maturing). Both media were supplemented with pyruvate, penicillin G (10 000 IU), streptomycin (0.05 mg mL–1), and 10% fetal bovine serum (FBS). In control medium, the oocytes were kept for 1 or 8 h at 37°C, and thereafter were transferred to maturing medium until the maturation period (24 h) was completed, under controlled atmosphere (5% CO2) and temperature, in an incubator. The maturing oocytes were distributed in two types of equipment developed for oocyte and embryo transportation, with and without 5% CO2, and kept for 1, 8, or 24 h before maturation or fecundation (IVF) procedures. The oocytes kept for 24 h in the transportation device were placed directly for IVF. All procedures used for in vitro maturation, IVF, and in vitro culture were the same as those adopted for commercial in vitro embryo production at Biotran LTDA (Alfenas, Minas Gerais, Brazil). The cleavage rate was evaluated on Day 3 post-insemination, and the blastocyst production was evaluated on Day 7. The statistical model included the main effects of treatment (control and maturing with or without 5% CO2), time, replicate, and the interaction of media × time. Data (3 replicates) were analysed by ANOVA and differences were identified by Tukey’s test. The time before in vitro maturation at the incubator negatively (P < 0.007) affected cleavage rates (76.4 ± 16.9 v. 58.8 ± 13.2 and 52.2 ± 18.5%, respectively, for 1, 8, and 24 h). However, treatment had no effects (P = 0.3) on cleavage (66.9 ± 15.0, 57.8 ± 19.7, and 67.7 ± 19.8% for the control and maturation with and without 5% CO2, respectively). Similarly, blastocyst production rates differed (P < 0.04) between 1 h (33.4 ± 14.7) and 24 h (19.3 ± 17.0%), whereas blastocyst production at 8 h did not show significant effects (19.3 ± 17.0). Although it was not significant (P > 0.05), only 14.1% of the oocytes kept in medium 2 with a controlled atmosphere for 24 h became embryos. This difference was probably related to the trend in treatment effect (P = 0.06) and the lower rate of embryo production (22.2 ± 13.6%) in this treatment compared with the control (33.8 ± 18.1%) and maturation without 5% CO2 (29.8 ± 17.3%). These results showed that 24 h of transportation is detrimental for the oocyte development potential and that the buffered medium used in this study with 5% CO2 did not efficiently maintain embryo production. Supported by CNPq, CAPES, FAPEMIG, and Biotran LTDA.

183 IN VITRO EMBRYO PRODUCTION: A TOOL TO PRESERVE THE THREATENED WOOD BISON (BISON BISON ATHABASCAE)

2016 ◽  
Vol 28 (2) ◽  
pp. 222
Author(s):  
M. P. Cervantes ◽  
J. M. Palomino ◽  
M. Anzar ◽  
R. J. Mapletoft ◽  
G. Mastromonaco ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Animal Health ◽  
Developmental Competence ◽  
Bison Bison ◽  
High Humidity ◽  
Embryo Production ◽  
Wood Bison ◽  
In Vitro Embryo Production

In vitro embryo production is being developed as a tool to restore genetic diversity and eliminate endemic disease in wood bison. In a recent study in wood bison, we found that more oocytes reached maturity after 30 h v. 24 h of in vivo maturation following hCG treatment (Cervantes et al. 2014 Reprod. Fertil. Dev. 26, 199). An additional 4 h of in vitro maturation after an in vivo maturation period of 30 h also had a positive effect on developmental competence. The present study was designed to test the hypothesis that extending the in vivo maturation time (i.e. extending the interval between hCG treatment and cumulus-oocyte complex (COC) collection) from 30 to 34 h will improve in vitro embryo production in wood bison. Follicular wave development was synchronised among female wood bison (n = 28, 6 to 10 years old) by transvaginal follicular ablation. The study was done in 4 replicates (n = 7 bison per replicate). Bison were given FSH 1 day (300 mg) and 3 days (100 mg) after ablation for ovarian superstimulation, and hCG (2500 IU) 5 days after ablation to induce COC maturation in vivo. Bison were divided randomly into 2 groups (n = 14/group) in which COC were collected transvaginally at either 30 h or 34 h after hCG treatment. Expanded COC from the 30 h group were fertilised after 4 h of in vitro maturation, while expanded COC from the 34 h group were fertilised immediately. Oocytes and sperm were co-incubated (Day 0 = day of fertilization) for 18 h at 38.5°C in 5% CO2 in air and high humidity. Presumptive zygotes were cultured in 4-well dishes containing 500 μL well–1 of CR1aa medium at 38.5°C, 5% CO2, 5% O2, 90% N2 and high humidity, and assessed on Days 3, 7, and 8 (Day 0 = day of fertilization). Data were compared between groups by Chi-squared analysis. No effect of replicate was found. Compared to the 30 h group, the 34 h group had a greater cleavage rate [55/74 (74%) v. 49/86 (57%); (P < 0.05)], and a greater blastocyst rate on Day 7 [25/74 (34%) v. 9/86 (10%); (P < 0.05)] and Day 8 [(40/74 (54.1%) v. 32/86 (37.2%); (P < 0.05)]. We concluded that an extended period of in vivo maturation is beneficial for embryo production after in vitro fertilization in wood bison. We thank Vetoquinol Canada for providing FSH (Folltropin-V) and hyaluronan (MAP-5) and thank Merck Animal Health for hCG (Chorulon).

248 IN VITRO EMBRYO PRODUCTION USING IN VIVO-MATURED OOCYTES COLLECTED TRANSVAGINALLY FROM WOOD BISON (BISON BISON ATHABASCAE)

2015 ◽  
Vol 27 (1) ◽  
pp. 213
Author(s):  
M. P. Cervantes ◽  
J. M. Palomino ◽  
M. Anzar ◽  
R. J. Mapletoft ◽  
G. Mastromonaco ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Animal Health ◽  
Calf Serum ◽  
Developmental Competence ◽  
High Humidity ◽  
Embryo Production ◽  
Wood Bison ◽  
In Vitro Embryo Production

Reproductive technologies are being developed to help conserve the genetic diversity of wood bison, a threatened species. To date, the efficiency of in vitro embryo production in bison is very low and appears to be related to inadequate in vitro conditions for oocyte maturation. Recently, we have attempted to circumvent the problem by inducing oocyte maturation in vivo and found that more than one-third of superstimulated oocytes collected 30 h after administration of hCG were at metaphase II (Cervantes et al. 2013 Reprod. Fertil. Dev. 25, 283; Cervantes et al. 2014 Reprod. Fertil. Dev. 26, 199). We hypothesise that additional maturation time in vitro, after in vivo maturation, will allow the remaining oocytes to reach the MII stage, and thus improve in vitro embryo production in wood bison. The objective of this study was to determine the effect of an additional 4 h of in vitro maturation on the developmental competence of oocytes collected 30 h after hCG treatment. Wood bison cows (n = 24) were superstimulated by the administration of 300 mg of FSH (Folltropin-V) diluted in 0.05% hyaluronan on the day of follicular wave emergence and 100 mg of FSH in hyaluronan 2 days later. Bison were administered 2500 IU of hCG (Chorulon) IM 2 days after the last dose of FSH. Transvaginal ultrasound-guided follicle aspiration was performed 30 h after hCG treatment to collect cumulus-oocyte complexes (COC). Expanded COC (with no evidence of degeneration) were selected and assigned randomly to 2 groups (n = 38 COC/group) in which IVF was done immediately, or after 4 h of in vitro maturation in TCM 199 with 5% calf serum, 5 μg mL–1 pLH, 0.5 μg mL–1 pFSH, and 0.05 μg mL–1 gentamicin, at 38.5°C, 5% CO2 and high humidity. In vitro fertilization (Day 0) was done with frozen-thawed wood bison semen (dose 5 × 106 sperm mL–1) in Brackett-Oliphant medium at 38.5°C, 5% CO2, and high humidity. Presumptive zygotes were cultured in CR1aa plus 5% calf serum, at 38.5°C and in 5% CO2, 5% O2, and 90% N2 and high humidity. Cleavage was recorded on Day 3, and blastocyst formation was recorded on Days 7 and 8. Cleavage and blastocyst rates (calculated from the total number of oocytes submitted to IVF) were compared between groups by chi-square analysis. No difference was detected between groups (immediate fertilization v. after an additional 4 h in vitro) in cleavage rate on Day 3 (55.3 v. 60.5%, respectively, P = 0.82), or blastocyst rate on Day 7 (13.2 v. 23.7%, respectively, P = 0.37). However, the blastocyst rate on Day 8 was higher in the COC group exposed to an additional 4 h of in vitro maturation (18.4 v. 44.7%, respectively, P = 0.03). Results support the hypothesis that an additional short period of in vitro maturation improves the developmental competence of oocytes collected after 30 h of in vivo maturation.We thank Bioniche Animal Health for providing FSH (Folltropin-V) and hyaluronan (MAP-5), and Merck Animal Health for hCG (Chorulon).

scholarly journals In vitro embryo production in small ruminants

2010 ◽  
Vol 39 (suppl spe) ◽  
pp. 409-413 ◽  
Author(s):  
Vicente José de Figueirêdo Freitas ◽  
Luciana Magalhães Melo
Keyword(s):  
Molecular Biology ◽  
Oocyte Maturation ◽  
Genetic Improvement ◽  
In Vitro Maturation ◽  
Small Ruminants ◽  
Northeastern Brazil ◽  
Improvement Program ◽  
Embryo Production ◽  
In Vitro Embryo Production

This paper reviews the technical bases of in vitro embryo production in small ruminants with special attention to the results obtained by our group in Northeastern Brazil. The laparoscopic oocyte recovery in hormonally treated live animals indicates a promising future for the application of this technique to genetic improvement program. New molecular biology tools should provide information to improve the efficiency of in vitro maturation. Furthers efforts have to be made to improve the oocyte maturation and to standardize the semen-capacitating process.

In vitro embryo production using ovaries removed from culled cows

2001 ◽  
Vol 81 (4) ◽  
pp. 589-591 ◽  
Author(s):  
G. Giritharan ◽  
R. Rajamahendran
Keyword(s):  
Dairy Cows ◽  
Key Words ◽  
Estrous Cycle ◽  
Recovery Rate ◽  
In Vitro Maturation ◽  
Follicle Stimulating Hormone ◽  
Embryo Production ◽  
Oocyte Recovery ◽  
In Vitro Embryo Production

The objective was to utilize in vitro maturation and fertilization to maximize embryo production from culled dairy cows. Ovaries were removed (via a colopotomy technique) from culled Holstein cows (n = 21) 2 d after standing estrus (T1), 2 d after treatment with 40 mg follicle stimulating hormone (FSH) given at standing estrus (T2), or at an unknown stage of the estrous cycle (T3).Treatment with FSH increased (P < 0.05) oocyte recovery rate (mean ± SEM, 14.6 ± 2.2, 25.4 ± 3.4, and 11.1 ± 2.6 oocytes ovary–1), for groups T1, T2 and T3, respectively, and the number of blastocysts obtained (1.6 ± 0.5, 4.4 ± 0.4 and 1.6 ± 0.6 blastocysts ovary–1). Culled dairy cows of high genetic merit are potential embryo donors, with an average of six to eight transferable embryos obtained from each FSH-treated cow. Key words: FSH treatment, oocyte recovery, IVM, IVF, culled cows

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