scholarly journals 289EFFECT OF FERTILIZATION TIME OF PIG OOCYTES MATURED IN-VITRO BY BOAR SPERM STORED AT 4°C

2004 ◽  
Vol 16 (2) ◽  
pp. 264
Author(s):  
Y.J. Yi ◽  
M.Y. Kim ◽  
Y.J. Chang ◽  
D.I. Jin ◽  
C.S. Park
Keyword(s):  
In Vitro Fertilization ◽  
Room Temperature ◽  
Sperm Concentration ◽  
Egg Yolk ◽  
Penicillin G ◽  
In Vitro Production ◽  
Vitro Fertilization ◽  
Boar Sperm ◽  
Sperm Penetration

The use of boar sperm stored at 4°C may be a useful tool for in vitro production of pig embryos. Therefore, this study was undertaken to investigate the effect of fertilization time of pig oocytes matured in-vitro by boar sperm. The sperm-rich fraction (30–60mL) was slowly cooled to room temperature (20–23°C) by 2h after collection. Semen was transferred into 15-mL tubes, centrifuged at room temperature for 10min at 800g, and the supernatant solution was poured off. The concentrated sperm was resuspended with 5mL of the LEN (11.0g lactose hydrate, 20mL egg yolk, 0.05g N-acetyl-D-glucosamine and 100mL distilled water) diluent to provide 1.0×109 spermmL−1 at room temperature. The resuspended semen was cooled in a refrigerator to 4°C. The medium used for oocyte maturation was TCM-199 supplemented with 26.19mM sodium bicarbonate, 0.9mM sodium pyruvate, 10μgmL−1 insulin, 2μgmL−1 vitamin B12, 25mM HEPES, 10μgmL−1 bovine apotransferrin, 150μM cysteamine, 10IUmL−1 PMSG, 10IUmL−1 hCG, 10ngmL−1 EGF, 0.4% BSA, 75μgmL−1 sodium penicillin G, 50μgmL−1 streptomycin sulfate and 10% pFF. After about 22h of maturation, oocytes were cultured without cysteamine and hormones for 22h at 38.5°C, 5% CO2 in air. Oocytes were inseminated with boar sperm stored at 4°C for 2 days after collection. Oocytes were coincubated for 1, 3, 6 and 9h in 500μL TBM fertilization media with 1×106mL−1 sperm concentration. Thereafter, oocytes were transferred into 500μL NCSU-23 culture medium containing 0.4% BSA for further culture of 6, 48 and 144h, fixed and stained for the evaluation of fertilization parameters and developmental ability. Data were analysed by ANOVA and Duncan’s multiple range test using the SAS program. The rates of sperm penetration and male pronuclear formation were higher in the fertilization times of 6 and 9h than in those of 1 and 3h. The percentage of polyspermic oocytes was highest in fertilization time of 9h compared with other incubation times. The rates of cleaved oocytes were higher in the fertilization times of 6 and 9h (85.0 and 84.6%) compared with those of 1 and 3h (61.1 and 76.8%). The percentage of blastocyst formation from the cleaved oocytes was highest in the fertilization time of 6h (33.6%) than in that of 1, 3 and 9h (11.4, 23.0 and 29.6%). Mean cell numbers per blastocyst were 32.9±3.3, 27.6±2.7, 26.3±2.2 and 24.4±1.8 in the fertilization times of 6, 9, 3 and 1h, respectively. In conclusion, we found out that boar sperm stored at 4°C could be used for in vitro fertilization of pig oocytes matured in vitro. Also, we recommend the coincubation time of 6h in 500μL TBM fertilization medium with 1×106mL−1 sperm concentration for in vitro fertilization of pig oocytes matured in vitro.

Protamine dissociation before decondensation of sperm nuclei during in vitro fertilization of pig oocytes

Reproduction ◽  
2000 ◽  
pp. 247-256 ◽  
Author(s):  
A Shimada ◽  
K Kikuchi ◽  
J Noguchi ◽  
K Akama ◽  
M Nakano ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Morphological Changes ◽  
Affinity Purification ◽  
Immunohistochemical Analysis ◽  
Sperm Chromatin ◽  
Vitro Fertilization ◽  
Boar Sperm ◽  
Sperm Penetration ◽  
Sperm Nuclei

The correlation between morphological changes and the dynamics of protamine in boar sperm chromatin during in vitro fertilization of pig oocytes matured in vitro was assessed. For this purpose, protamine was purified from boar sperm nuclei and an antiserum against protamine was developed. After affinity purification, the antiserum reacted exclusively with boar protamine during western blotting, showing no crossreactivity with core histones. Immunohistochemical evaluation revealed that only fully developed spermatid nuclei in boar testes stained strongly with the antiserum. When pig oocytes matured in vitro were fertilized in vitro, sperm penetration was observed in 37% of oocytes at 2 h after insemination and the penetration rate increased to 99% by 5 h after insemination, accompanied by an increase in polyspermic penetration. Paraffin wax sections of the inseminated oocytes were examined by immunohistochemical analysis with the antiserum. The proportion of condensed sperm nuclei that reacted with the antiserum was 87% of the sperm nuclei that penetrated by 2 h after insemination, and this decreased to 20 and 13% at 3 and 5 h after insemination, respectively. However, none of the decondensing sperm nuclei or male pronuclei reacted with the antiserum during the entire insemination period. These results indicate that a specific antiserum against boar protamine can be raised and, using this serum, it has been demonstrated that protamine is dissociated from boar sperm nuclei before decondensation during in vitro fertilization.

Calibration of sperm concentration for in vitro fertilization in a mouse reprotoxicity model

2019 ◽  
Vol 55 ◽  
pp. 58-61 ◽  
Author(s):  
Hanne Skovsgaard Pedersen ◽  
Ying Liu ◽  
Leslie Foldager ◽  
Henrik Callesen ◽  
Knud Larsen ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Sperm Concentration ◽  
Vitro Fertilization

scholarly journals Modern achievements of the development of scientific research at obtaining in vitro sheep embryos

2020 ◽  
Vol 22 (93) ◽  
pp. 60-68
Author(s):  
O. M. Sharan ◽  
V. Yu. Stefanyk ◽  
S. G. Shalovylo
Keyword(s):  
In Vitro Fertilization ◽  
Embryo Culture ◽  
Biologically Active ◽  
In Vitro Production ◽  
Embryo Production ◽  
Vitro Fertilization ◽  
Maturation In Vitro ◽  
Key Factor ◽  
Vitro Production

New literature data on research aimed at improving the in vitro production of sheep embryos presents in the article. An analysis of the achievements of scientists from different countries to increase the efficiency of the main stages of embryo production in vitro: maturation of oocytes in vitro, their in vitro fertilization and in vitro embryo culture. In the literature experience has shown that the efficiency of oocyte maturation in vitro is significantly influenced by the experience and qualifications of scientists, the age of the egg donor, the improvement of the environment by adding roscovitin to inhibit meiosis, α-linolenic acid, cerium dioxide nanoparticles (CeO2 NPs) and sericin to accelerate nuclear maturation and increase the number of oocytes of the second meiotic metaphase (MII). The main factors influencing the effectiveness of in vitro fertilization have been identified, and the parameters of the limited time of fertilization ability of sperm and the ability of oocytes to fertilize, which is called the “fertile span”, have been determined. The main effective medium that increases the effectiveness of in vitro fertilization – synthetic oviduct fluid (SOF) with the addition of heparin and serum of cattle or sheep. The main parameters of sheep embryo culture in vitro are presented with the definition of the most commonly used media and their influence on embryonic development. Potential ways to improve the production of sheep embryos in vitro with the determination of morphological evaluation of categories of oocytes, methods of synchronization of their maturation in vitro are also highlighted. At the same time, literature data on the synchronization of oocyte-cumulus complexes with the use of a large number of inhibitors of meiotic division are presented, which according to many scientists may be a key factor in improving the efficiency of sheep embryo production in vitro. In addition, the results of studies of many scientists on the expansion of the fertile gap of oocytes of sheep cultured in vitro using certain biologically active substances were analyzed. In conclusion, the prospect of using the technology of in vitro production of sheep embryos in biomedical research is highlighted.

Effects of Isatis root polysaccharide on boar sperm quality during liquid storage and in vitro fertilization

2019 ◽  
Vol 210 ◽  
pp. 106178 ◽  
Author(s):  
Zhiqiang Ren ◽  
Weike Shaoyong ◽  
Qian Li ◽  
Lu Ma ◽  
Junying Xiao ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Sperm Quality ◽  
Vitro Fertilization ◽  
Liquid Storage ◽  
Boar Sperm

Effect of Astragalus polysaccharide addition to thawed boar sperm on in vitro fertilization and embryo development

2018 ◽  
Vol 121 ◽  
pp. 21-26 ◽  
Author(s):  
Xiao-gang Weng ◽  
Ming-ming Cai ◽  
Yu-ting Zhang ◽  
Yan Liu ◽  
Zheng-ling Gao ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Embryo Development ◽  
Vitro Fertilization ◽  
Astragalus Polysaccharide ◽  
Boar Sperm

221 EFFECT OF ROCKING ON IN VITRO PRODUCTION OF BOVINE EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 209
Author(s):  
Y. Serita ◽  
C. Kubota ◽  
T. Kojima
Keyword(s):  
In Vitro Fertilization ◽  
Embryo Development ◽  
Developmental Competence ◽  
In Vitro Production ◽  
Humid Atmosphere ◽  
Vitro Fertilization ◽  
Rate Of Development ◽  
Fetal Bovine ◽  
Vitro Production

This study tested whether embryo development yield using in vitro fertilization (IVF) could be improved by rocking cultures. Bovine ovaries were obtained at a slaughterhouse and transported to the laboratory within 6 h. Cumulus–oocyte complexes were collected and 20–25 were transferred in 100-μL drops of TCM-199 containing 10% fetal bovine serum and antibiotics under paraffin oil. Maturation was for 20–24 h at 38.5°C under 5% CO2 and 95% air in a humid atmosphere (IVM). In vitro fertilization was carried out for 6 h using frozen–thawed sperm from a single bull in modified Brackett and Oliphant (BO) medium. Presumptive zygotes were cultured in CR1aa supplemented with 10 mg mL–1 of BSA or 5% FBS for 9 d at 38.5°C under 5% CO2, 5% O2, and 90% N2 in a humid atmosphere (IVC). Rocking was performed to a height of 6 cm every 7 s using a Profile Rocker (New Brunswick Scientific Co., Edison, NJ, USA) in an incubator. Dishes were placed at a 15-cm distance from the fulcrum of the rocker. The conventional method (no rocking) served as a control, and every experiment was replicated 3 times. For Experiment 1, the effect of the period of rocking on developmental competence was examined when COC or zygotes were subjected to rocking for IVM, IVF, or IVC (IVM-move, IVF-move, and IVC-move). There were no significant differences in rates of oocyte maturation, cleavage, and development for IVM-move v. the control, or for rate of development between IVC-move and the control. However, the rate of fertilization for IVF-move was higher than that of the control (88.9 v. 67.5%; P < 0.01), and the rate of development was higher for IVF-move than for the control (39.0 v. 25.7%; P < 0.05). For Experiment 2, the effect of rocking frequency during IVF on development was determined. The IVF cultures were rocked every 7, 3.5, and 1.5 s (IVF-1move, IVF-2move, IVF-3move). The rates of cleavage on IVF-1move, IVF-2move, IVF-3move, and the control were 74.3, 69.8, 68.8, and 60.4%, and the rates of development were 39.0, 48.3, 26.2, and 25.7%, respectively. The rates of development on IVF-1move and IVF-2move were significantly different from the control and IVF-3move (P < 0.01). These results showed that rocking during IVF improved fertilization and embryo yield, and that frequency of rocking affected embryo development.

scholarly journals 266IN VITRO-DERIVED EMBRYO PRODUCTION WITH SEXED AND UNSEXED SEMEN FROM DIFFERENT BULLS

2004 ◽  
Vol 16 (2) ◽  
pp. 253 ◽  
Author(s):  
L. Ferré ◽  
C. Ohlrichs ◽  
D. Faber
Keyword(s):  
In Vitro Fertilization ◽  
Embryo Development ◽  
Calf Serum ◽  
Sperm Concentration ◽  
Reproductive Technologies ◽  
Chi Square ◽  
Vitro Fertilization ◽  
Sexed Semen ◽  
Chi Square Analysis

The production of pre-sex-selected calves by in vitro fertilization (IVF), using sexed semen, does show some benefits due to the small quantity of sperms needed for the process as compared to other reproductive technologies. The objective of this study was to determine differences among bulls and sperm concentrations in embryo development with sexed and unsexed semen. Follicles ranging from 2 to 6mm in diameter were aspirated from slaughterhouse ovaries. COC were selected and matured in groups of maximum of 30 in 1.8mL of TCM-199, supplemented with 10% fetal calf serum, 0.01UmL−1 bFSH, 0.01UmL−1 bLH and 10μLmL−1 penicillin-streptomycin for 24h at 38.5°C. Fertilization (Day 0) was carried out in micro-drops (50μL) with TALP-FERT medium containing PHE (3μgmL−1 penicillamine, 11μgmL−1 hypotaurine and 0.18μgmL−1 epinephrine), 10μLmL−1 non-essential amino acid and 2μgmL−1 heparin. Frozen/thawed sexed (female) and non-sexed sperms from five bulls were selected in a discontinuous percoll gradient. Sperm concentration was 1×106 for non-sexed semen and 1×106 or 2×106 for sexed semen. After 18–20h, presumptive zygotes were denuded and cultured in groups of 10 in 50-μL micro-drops of SOF citrate with 5% FCS (Holm P et al., 1999 Theriogenology 52, 683–700) under paraffin oil in a 5% O2, 5% CO2, 90% N2 atmosphere with high humidity. On Day 7, blastocysts (BL) were morphologically evaluated and recorded. Results are shown in Table 1. Data was compared by chi-square analysis. Sexed frozen bovine sperm can be used successfully in IVF systems. More research needs to be done to optimize and standardize bovine in vitro fertilization with sexed semen. Table 1 Results of comparisons between bulls, sperm concentrations, cleavage and embryo development

Sign in / Sign up

Export Citation Format

Share Document