Beneficial effects of melatonin on in vitro embryo production from juvenile goat oocytes

2018 ◽  
Vol 30 (2) ◽  
pp. 253 ◽  
Author(s):  
Sandra Soto-Heras ◽  
Montserrat Roura ◽  
Maria G. Catalá ◽  
Irene Menéndez-Blanco ◽  
Dolors Izquierdo ◽  
...  
Keyword(s):  
Embryo Quality ◽  
Cell Number ◽  
Blastocyst Development ◽  
Embryo Production ◽  
Melatonin Concentration ◽  
Oocyte Competence ◽  
Beneficial Effects ◽  
In Vitro Embryo Production ◽  
Ovarian Follicular Fluid

Melatonin is a universal antioxidant that improves in vitro embryo production in several species. The aims of this study were to determine the melatonin concentration in the ovarian follicular fluid (FF) of juvenile goats and the effect of melatonin during in vitro maturation (IVM) on embryo development. The FF melatonin concentration was 0.57­–1.07 × 10−9 M, increasing with follicular diameter. Oocytes were matured, fertilised and cultured under conventional conditions. Blastocyst development, embryo quality and levels of reactive oxygen species (ROS) and reduced glutathione were assessed. In Experiment 1 different melatonin concentrations (10−3, 10−7, 10−9, 10−11 M) were added to the IVM medium, which contained cysteamine as antioxidant, and no differences were observed. In Experiment 2, melatonin (10−7 M) was tested in the presence or absence of cysteamine (experimental groups: melatonin, cysteamine, melatonin + cysteamine, non-antioxidant). The melatonin group presented a higher blastocyst rate than the non-antioxidant group (28.9 vs 11.7%; P < 0.01) and a higher total cell number than the cysteamine group (225.1 vs 129.0; P < 0.05). Oocytes from the melatonin and cysteamine groups had lower ROS levels than those from the non-antioxidant group. This study shows that melatonin is an interesting tool for improving oocyte competence in juvenile goats as it increases embryo production and quality.

135 Use and dose of porcine follicle-stimulating hormone for ovarian superstimulation prior to ovum pickup and in vitro embryo production in pregnant Holstein heifers

2019 ◽  
Vol 31 (1) ◽  
pp. 192
Author(s):  
R. V. Sala ◽  
L. C. Carrenho-Sala ◽  
M. Fosado ◽  
E. Peralta ◽  
D. C. Pereira ◽  
...  
Keyword(s):  
Limited Information ◽  
Dominant Follicle ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Oocyte Competence ◽  
Treatment Groups ◽  
Oocyte Recovery ◽  
In Vitro Embryo Production

The benefit of superstimulation with exogenous FSH before ovum pickup for in vitro embryo production has been the subject of significant controversy. In addition, there is limited information on different dose regimens. Thus, the objective of the present study was to evaluate the effect of dose of porcine (p)-FSH during superstimulation before ovum pickup (OPU) on in vitro embryo production in pregnant heifers. Pregnant Holstein heifers (n=36) were assigned to a complete 3×3 crossover design. Three treatment groups were evaluated as follows: p-FSH 0mg (FSH0), p-FSH 160mg (FSH160) and p-FSH 300mg (FSH300). Three sessions of OPU were performed on each animal at 48, 62 and 76 days of gestation, with a washout interval between sessions of 14 days. Follicular wave emergence was synchronized by dominant follicle removal. Heifers in the FSH0 group received no further treatment, whereas the remaining groups received a total of 4 injections 12h apart as follows: FSH160 (48.0, 42.7, 37.3 and 32.0mg) or FSH300 (90.0, 80.0, 70.0 and 60.0mg), beginning 36h after dominant follicle removal. Ovum pickup was performed in all heifers 40h after the last p-FSH injection. Heifers were subjected to OPU for oocyte recovery, and number of follicles was determined. Recovered oocytes were processed and in vitro embryo production performed. Differences between treatment groups were evaluated by generalized linear mixed models. Data are presented (Table 1) as mean±standard error of the mean. There was no effect of days in gestation for any of the outcomes evaluated (P&gt;0.05). Follicle numbers at the time of oocyte recovery were different (P&lt;0.01) between groups. Heifers in the FSH300 group had a greater (P&lt;0.05) number of medium, large and total follicles than heifers in the FSH0 group, whereas heifers in the FSH160 were intermediate. Total number of recovered, viable and cleaved oocytes were greater (P&lt;0.01) in FSH300- than in FSH160- and FSH0-treated heifers. Cleavage rate and blastocyst development rate were not different (P&gt;0.10) between groups. The number of grade 1 and 2 blastocysts was greater in FSH300- than in FSH160- and FSH0-treated heifers (P&lt;0.03). In summary, the use of 300mg of p-FSH before OPU in pregnant heifers increases the number of follicles, oocytes and blastocysts produced per heifer with no detrimental effect on oocyte competence. Table 1.Ovum pickup and in vitro embryo production in pregnant heifers treated with different doses of porcine FSH

PSX-A-10 Late-Breaking: Effects of paternal high energy diets on blastocyst development during in vitro embryo production in the bovine

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 363-363
Author(s):  
Dylan B Davis ◽  
Zachary Seekford ◽  
Mackenzie Dickson ◽  
Lucas Gonçalves ◽  
Samir Burato ◽  
...  
Keyword(s):  
High Gain ◽  
High Energy ◽  
Carcass Composition ◽  
Adaptation Period ◽  
Blastocyst Development ◽  
Embryo Production ◽  
Ad Libitum ◽  
In Vitro Embryo Production ◽  
To Receive

Abstract The objective of this study was to evaluate the effect of paternal high energy diets on blastocyst development during in vitro embryo production (IVP). Eight sires were stratified by body weight (initial BW = 946 ± 85 kg) and randomly assigned to the same diet (NEm = 2.10, NEg = 1.44, CP = 14.1%, NDF = 16.6%, DM basis) fed at two different inclusion rates while having ad libitum access to bermudagrass hay (NEm = 1.02, NEg = 0.45, CP = 10.2%, NDF = 71.6). After a 10-d adaptation period, sires were individually fed to receive 0.5% (MAINT) or 1.25% [High gain (HG)] of their BW daily for 67 days. At the end of the feeding period, semen was collected through electroejaculation and frozen. Antral follicles were aspirated from ovaries obtained from a slaughterhouse and utilized for IVP in 4 independent replicates (n = 2,227 total oocytes). Cleavage rates were evaluated 48 h after fertilization and blastocyst development rates were evaluated after 7 days of embryo culture. The proposed treatments successfully induced differences in BW gain (P &lt; 0.01; 2.28 vs -0.04 kg/d) and carcass composition (Rump fat: 1.63 vs. 0.41 cm, P = 0.08; Rib fat: 1.06 vs. 0.41 cm, P = 0.02; intramuscular fat: 3.5 vs. 3.0%, P = 0.36; for HG vs. MAINT sires, respectively). There was a significant decrease in cleavage rates (69.9 ± 2.5 vs. 65.0 ± 2.7; P &lt; 0.04), blastocyst rate as a percentage of oocytes (16.7 ± 2.9 vs. 11.5 ± 2.1; P &lt; 0.01), and blastocyst rates as a percentage of cleaved structures (24.1 ± 3.8 vs. 11.5 ± 2.1; P &lt; 0.01) for HG compared with MAINT sires. In conclusion, sires fed diets that induce highly anabolic conditions had impaired blastocyst development compared to sires fed a maintenance diet.

158 IMPACT OF MATURED CATTLE OOCYTES AT HIGHER INCUBATION TEMPERATURE ON IN VITRO EMBRYO PRODUCTION

2016 ◽  
Vol 28 (2) ◽  
pp. 209
Author(s):  
M. Nkadimeng ◽  
E. van Marle-Koster ◽  
K. P. M. Lekola ◽  
M. L. Mphaphathi ◽  
M. M. Seshoka ◽  
...  
Keyword(s):  
Incubation Temperature ◽  
Cell Types ◽  
Cell Number ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Developmental Potential ◽  
Cell Numbers ◽  
Significant Difference ◽  
In Vitro Embryo Production

Heat stress during IVF is associated with reduced fertility in cattle oocytes. It may, however, enhance thermo-tolerance or cause detrimental effects on a variety of cell types or organisms, depending on the duration and intensity of the thermal challenge. The aim of this study was to evaluate the developmental potential of cumulus-oocyte complexes (COC) matured for 18 or 24 h and incubated at 39°C or 41°C. A total of 1000 immature oocytes were collected at slaughter from indigenous South African cow ovaries. The COC were randomly allocated (100/treatment) into 2 maturation times (18 or 24 h) and cultured in M199 + FSH-LH-estradiol medium under oil at 100% humidity and 5% CO2 at 39°C or 41°C. Post maturation, oocytes were subjected to normal subsequent embryo conditions. The Bracket and Oliphant medium was used for IVF. All matured oocytes were fertilised for 6 h with frozen-thawed Nguni bull semen at a concentration of 265 × 106. The presumptive zygotes from each treatment were cultured into SOF-BSA medium under oil and incubated at 39°C for assessment of cleavage rate 48 h post IVF. After Day 7 of culture, blastocyst were stained (Hoechst 33323) for nuclei cell count. Statistical analyses was performed using Genstat® software of SAS (SAS Institute, Cary, NC, USA; P < 0.05). Oocytes that were matured for 18 h in 41°C resulted in more 8-cell embryos (41%) compared with those incubated at 39°C (21.6%). However, no difference was observed for cleavage rate at both maturation times and incubation temperatures (41 or 39°C). There was more morula formation from oocytes matured for 18 h (19.6%) and 24 h (19.0%) at 41°C compared to 39°C (8.4%) group. The results further showed more blastocyst formation during 18 h at 41°C (15.2%) than at 39°C (7.4%) and during 24 h at 41°C (11.2%), 39°C (11.4%). However there was no difference in the nuclei cell number during 18 h at 41°C (45.2), 24 h (45.8), and 18 h at 39°C (43.4) of maturation. Thus, there was a significant difference in the nuclei cell numbers at 24 h on 39°C (n = 133.2) and 41°C (n = 45.8). In conclusion, oocytes that were matured for 18 and 24 h at 41°C or for 18 h at 39°C developed further to blastocyst stage on in vitro embryo production, however, with low nuclei cell numbers due to accelerated maturation temperature or shortened maturation period.

scholarly journals The effect of pre-maturation culture using phosphodiesterase type 3 inhibitor and insulin, transferrin and selenium on nuclear and cytoplasmic maturation of bovine oocytes

Zygote ◽  
2015 ◽  
Vol 24 (2) ◽  
pp. 219-229 ◽  
Author(s):  
A.L.S. Guimarães ◽  
S.A. Pereira ◽  
N.R. Kussano ◽  
M.A.N. Dode
Keyword(s):  
Embryo Development ◽  
Cumulus Cell ◽  
Cell Number ◽  
Blue Staining ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Oocyte Competence ◽  
Phosphodiesterase Type ◽  
Type 3

SummaryThis study aims to evaluate if a pre-maturation culture (PMC) using cilostamide as a meiotic inhibitor in combination with insulin, transferrin and selenium (ITS) for 8 or 24 h increases in vitro embryo production. To evaluate the effects of PMC on embryo development, cleavage rate, blastocyst rate, embryo size and total cell number were determined. When cilostamide (20 μM) was used in PMC for 8 or 24 h, 98% of oocytes were maintained in germinal vesicles. Although the majority of oocytes resumed meiosis after meiotic arrest, the cleavage and blastocyst rates were lower than the control (P < 0.05). When the cilostamide concentration was lowered (10 μM) and oocytes were arrested for 8 h, embryo development was improved (P < 0.05) and was similar (P > 0.05) to the control. The deleterious effect of 20 μM cilostamide treatment for 24 h on a PMC was confirmed by lower cumulus cell viability, determined by trypan blue staining, in that group compared with the other groups. A lower concentration (10 μM) and shorter exposure time (8 h) minimized that effect but did not improve embryo production. More studies should be performed to determine the best concentration and the arresting period to increase oocyte competence and embryo development.

157 EFFECT OF MELATONIN ON EMBRYO QUALITY OF BOVINE OOCYTES SUBJECTED TO HEAT SHOCK

2016 ◽  
Vol 28 (2) ◽  
pp. 208
Author(s):  
N. G. Alves ◽  
I. J. Ascari ◽  
L. S. A. Camargo ◽  
J. Jasmin ◽  
C. C. R. Quintão ◽  
...  
Keyword(s):  
Heat Shock ◽  
Embryo Quality ◽  
Linear Models ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Quality Data ◽  
Trophoblast Cells ◽  
Melatonin Concentration

The aim of this study was to evaluate the effect of different concentrations of melatonin added to in vitro maturation (IVM) medium of oocytes subjected to heat shock on embryo quality. Immature oocytes aspirated from ovaries obtained from a slaughterhouse were selected and randomly allocated in factorial experiment design (3 × 2). Three concentrations of melatonin (0, 10–6, and 10–4 M; M5250, Sigma, St. Louis, MO, USA) were added to the IVM medium and 2 incubation conditions (conventional: 24 h at 38.5°C and 5% CO2; heat shock: 12 h at 41°C followed by 12 h at 38.5°C and 5% CO2) were tested, resulting in treatments: M1 (0 M; 38.5°C; n = 15), M2 (10–6 M; 38.5°C; n = 15), M3 (10–4 M; 38.5°C; n = 15), M4 (0 M; 41°C; n = 15), M5 (10–6 M; 41°C; n = 15), and M6 (10–4 M; 41°C; n = 15). The IVM was performed in a Nunc plate (144444 – Thermo, Fisher Scientific Inc., Pittsburgh, PA, USA) containing 400 µL of TCM-199 (Invitrogen, Carlsbad, CA, USA) supplemented with 20 µg mL–1 of FSH (Pluset®, Calier Laboratories, Barcelona, Spain) and 10% oestrus cow serum. Oocytes were IVF in FERT-TALP medium for 20–22 h and incubated at 38.5°C and 5% CO2. After IVF, the presumptive zygotes were denuded and cultured in CR2aa medium supplemented with 2.5% FCS (Nutricell, Campinas, Brazil) in an incubator at 38.5°C under 5% CO2, 5% O2, and 90% N2, and saturated humidity for 8 days. Blastocysts with 8 days post-fertilization from different treatments were fixed in 4% paraformaldehyde in PBS for 1 h and analysed by TUNEL assay (Deadend™ Fluorometric TUNEL System, Promega, Madison, WI, USA) to evaluate embryonic quality. Data were analysed by generalised linear models considering the Poisson distribution and using the Proc Genmod of SAS software (version 9.1; SAS Institute Inc., Cary, NC, USA) considering effects of melatonin concentration, incubation conditions, and interaction between the factors. Values shown are the mean ± s.e.m. The interaction between melatonin concentration and incubation conditions was no significant (P > 0.05). The total number of cells was not affected (P > 0.05) by melatonin, but it was decreased (P < 0.05) by heat shock (M1 = 117 ± 6.7a; M2 = 118 ± 4.2a; M3 = 120 ± 6.3a; M4 = 102 ± 6.2b; M5 = 106 ± 5.7b; M6 = 108 ± 8.9b). Melatonin and heat shock did not affect (P > 0.05) the index of embryo apoptotic cells (M1 = 15.3% ± 2.0; M2 = 15.5% ± 1.3; M3 = 13.6% ± 2.0; M4 = 14.9% ± 1.5; M5 = 13.3% ± 1.3; M6 = 13.5% ± 1.2) and the index of trophoblast cells (M1 = 74.6% ± 2.3; M2 = 75.0% ± 1.7; M3 = 75.2% ± 1.9; M4 = 78.4% ± 2.3; M5 = 76.4% ± 3.0; M6 = 75.2% ± 2.6). The melatonin and heat shock affected the index of the inner cell mass (ICM; P < 0.05), and the heat shock reduced the index of the ICM of oocytes not treated with melatonin (M1 = 25.4% ± 2.3a; M2 = 25.0% ± 1.7a; M3 = 24.8% ± 1.8a; M4 = 21.6% ± 2.3b; M5 = 23.6% ± 3.0a; M6 = 24.8% ± 2.6a). In conclusion, melatonin supplementation to the medium IVM of oocytes subjected to heat shock had no effect on blastocyst total cell number, general apoptotic index, or index of the trophoblast cells, but increased index of the ICM. Research was supported by Fapemig, CNPq, Embrapa, and CAPES.

scholarly journals Oocyte Source and Hormonal Stimulation forIn VitroFertilization Using Sexed Spermatozoa in Cattle

2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
Giorgio A. Presicce ◽  
Jie Xu ◽  
Guochun Gong ◽  
Juan F. Moreno ◽  
Sanjeev Chaubal ◽  
...  
Keyword(s):  
Hormone Treatment ◽  
Intramuscular Administration ◽  
Blastocyst Development ◽  
Embryo Production ◽  
Hormonal Stimulation ◽  
In Vitro Embryo Production ◽  
Donor Oocytes

The aim of this study was to investigate the efficiency of in vitro embryo production in cattle utilizing sexed sperm from two bulls and oocytes recovered by OPU. Twenty donor animals were employed in eight OPU replicates: the first four OPU trials were conducted on animals without hormone treatment, and the last four were run on the same animals, following FSH subcutaneous and intramuscular administration. A higher rate of blastocyst development was recorded in stimulated, as compared to nonstimulated animals, (25.2% versus 12.8%, ). Ocytes derived from slaughterhouse (SH) ovaries were also fertilized with sperm from the same bulls. Overall, non-sexed sperm used with oocytes derived from SH ovaries was significantly more efficient for blastocyst development than was sexed sperm with these same SH derived oocytes and sexed sperm with stimulated donor oocytes (39.8% versus 25.0% and 25.2%, ). In conclusion, the use of sexed sperm with OPU-derived oocytes resulted in a significantly higher blastocyst development when donors were hormonally stimulated; furthermore, the level of efficiency achieved was comparable to that attained when the same sexed sperm was tested on oocytes derived from SH ovaries.

365 QUALITY ASSESSMENT OF BOVINE CRYOPRESERVED SPERM AFTER SEXING BY FLOW CYTOMETRY AND ITS USE FOR IN VITRO EMBRYO PRODUCTION

2010 ◽  
Vol 22 (1) ◽  
pp. 339
Author(s):  
J. O. Carvalho ◽  
R. Sartori ◽  
G. M. Machado ◽  
G. B. Mourão ◽  
M. A. N. Dode
Keyword(s):  
Flow Cytometry ◽  
Sperm Motility ◽  
Membrane Integrity ◽  
Fertilization Rate ◽  
Computer Assisted ◽  
Blastocyst Development ◽  
Embryo Production ◽  
Acridine Orange Staining ◽  
In Vitro Embryo Production

Several studies using sex-sorted sperm by flow cytometry have shown that its fertility is reduced. Therefore, this study evaluated structural and functional characteristics of sperm sexed by flow cytometry. In addition, in vitro embryo production (IVP) and development was assessed when frozen-thawed unsorted and sex-sorted sperm from 4 Nellore bulls. Each ejaculate was separated into three fractions: non-sexed (NS), sexed for X-sperm (SX), and sexed for Y-sperm (SY). After thawing, each sample was analyzed for sperm motility by computer-assisted semen analysis (CASA, Berkeley, CA), sperm head agglutination, sperm morphology, membrane integrity by propidium iodide (PI) and 6-carboxy-fluorescein diacetate (CFDA) staining, acrosome integrity by peanut agglutinin (PNA), capacitation by chlortetracycline (CTC), and chromatin integrity by acridine orange staining. Then, the samples were placed in 45 : 90% (NS90) or 45 : 60% (NS60, SX, and SY) Percoll™ gradients. After Percoll™ centrifugation, sperm pellets were analyzed or used for IVP. All analyses were replicated independently three times. For IVP, 2,271 in vitro matured oocytes were used. To assess fertilization rate, presumptive zygotes were fixed and stained with lacmoid at 18 h post-insemination (hpi). Cleavage was evaluated at Day 2 (48 hpi) and blastocyst development at Days 6, 7, 8, and 9 of culture. Data were analyzed using generalized linear models. No differences (P > 0.05) were observed between SX and SY groups for e sperm variables evaluated either before or after Percoll™. However, non-sexed sperm had higher sperm motility, greater percentage of sperm with intact membranes, and greater percentage of live sperm with intact acrosomes than sexed sperm (P < 0.05). An effect of Percoll™ was observed in the non-sexed samples, with those submitted to 45 : 90% gradient having higher motility, greater percentage of cells with intact membrane, and lower recovery rate than those submitted to a 45 : 60% gradient. No differences among groups were observed for fertilization rate, being 74.0 ± 5.7, 63.2 ± 5.1, 67.2 ± 5.7, and 55.4 ± 5.9% for NS90, NS60, SX, and SY, respectively. Group NS90 showed a greater cleavage rate than did the SY group, while groups NS60 and SX had similar rates to the others. Blastocyst development rates on Day 6 to Day 9 were greater for group NS90. For example, on Day 8 the blastocyst rate was 34.9 ± 3.6, 22.2 ± 3.2, 18.1 ± 3.3, and 14.8 ± 2.9% forNS90, NS60, SX, and SY groups, respectively. All groups showed similar embryonic developmental stages on Day 6 to Day 9. Although sex-sorting affected sperm characteristics, it did not cause a decrease with in vitro fertility. However, differences in blastocyst rates between groups NS60 and NS90 indicated that the sperm selection protocol affected embryo production. Financial support: Embrapa Genetic Resources and Biotechnology.

30 Vitrification of Equine Embryos: Application in a Commercial Cloning Program

2018 ◽  
Vol 30 (1) ◽  
pp. 154
Author(s):  
G. Vichera ◽  
R. Jordan ◽  
V. Arnold ◽  
D. Dobler ◽  
R. Olivera
Keyword(s):  
Breeding Season ◽  
Production Efficiency ◽  
Embryo Quality ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Pregnancy Rates ◽  
Embryo Production ◽  
In Vitro Embryo Production ◽  
The One

During a commercial horse cloning program, a critical point is the availability and maintenance of suitable recipient mares for a large quantity of embryo transfers. Usually, pregnancy rates and viable births off the breeding season decrease significantly, whereas the rate of in vitro embryo production remains constant. For this reason, an efficient vitrification system allows continuous embryo production throughout the year with the advantage of doing the transfers only during the breeding season. The vitrification technique evaluated in this study was the one described by Kuwayama et al. (2007 Theriogenology 67, 73-80). By using this method, we compared post-warming recovery efficiency, pregnancy rates, and viable foaling rates in 2 experimental groups: cloned blastocysts vitrified off-season and transferred in breeding season (VC, n = 337), and non-vitrified cloned blastocysts also transferred in breading season (no-VC, n = 516). To achieve this, equine oocytes were collected from slaughterhouse ovaries, matured, enucleated, and fused to a donor cell according to Olivera et al. (2016 PLoS One 11, e0164049, 10.1371/journal.pone.0164049). The reconstructed embryos (RE) were cultured in a well-of-the-well system by adding 3 RE per well for 7 to 8 days to reach the blastocyst stage, at which they were vitrified as mentioned above. During the breeding season, blastocysts were warmed and transferred in couples in a single cycling receptive mare. Pregnancies were confirmed by transrectal ultrasonography 15 days post-transfer. All variables were analysed by Fisher test (P < 0.05). The warming recovery rate was 91% (308/337) for cloned blastocysts. In addition, pregnancy and viable birth rates were similar for the VC and no-VC groups: 15.6% (24/154) v. 16.7% (43/258) for pregnancy rates, respectively, and 37.5% (9/24) v. 37.2% (16/43) for foaling rates, respectively. In summary, 9 viable cloned foals were obtained with off-season embryos warmed and transferred during the breeding season, showing that vitrification did not affect embryo quality. Hence, the proposed strategy provides the ability to maximize production efficiency of equine clones by generating a large number of pregnancies without stopping in vitro embryo production at any time of the year.

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