221 EFFECT OF ROCKING ON IN VITRO PRODUCTION OF BOVINE EMBRYOS

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.

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Effects of in vitro maturation media on in vitro fertility of porcine oocytes and early development of embryos

Vietnam Journal of Biotechnology ◽

10.15625/1811-4989/18/2/15020 ◽

2020 ◽

Vol 18 (2) ◽

pp. 249-255

Author(s):

Nguyen Viet Linh ◽

Nguyen Thi Hiep

Keyword(s):

Follicular Fluid ◽

In Vitro Maturation ◽

Subsequent Development ◽

Developmental Competence ◽

In Vitro Production ◽

Vitro Fertilization ◽

Fetal Bovine ◽

Vitro Production ◽

Normal Fertilization

In pigs, embryo productivity is still lower than that in other livestocks. One of the reasons is incomplete maturation of porcine oocytes in in vitro conditions. Therefore in vitro maturation (IVM) plays a crucial role in in vitro production of porcine embryos. It provides prerequisite condition to in fertilization and subsequent development of porcine embryos. In a previous study, effects of NCSU-37-based medium and TCM-199-based media supplemented with porcine follicular fluid (pFF) or Fetal Bovine Serum (FBS) on in vitro maturation of Landrace oocytes collected in Vietnam have been compared, suggesting that NCSU-37 medium supplemented with 10% of porcine follicular fluid (pFF) had the highest rate of oocytes reach to metaphase II stage in comparison to those of the other two TCM-199-based media. In the present study, further experiments were carried out to evaluate the contribution of IVM media on fertilization capability and developmental competence. Porcine oocytes matured in vitro in 3 media: NCSU-37 supplemented with 10% pFF, TCM-199 supplemented with either 10% pFF or 10% FBS were subjected to in vitro fertilization and subsequent in vitro culture to monitor fertility and embryo development. The results showed that penetration and normal fertilization rates in both TCM-199 groups are both higher than that of NCSU-37 group. Moreover, the cleavage and blastocyst rates, and cell numbers of blastocysts which is a criterion for embryo quality were all higher in TCM-199 groups, especially in the group supplemented with pFF. It might be concluded that TCM-199 media supplemented with either pFF or FBS are suitable for effective in vitro maturation of Landrace porcine oocytes collected in Vietnam.

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

Scientific Messenger of LNU of Veterinary Medicine and Biotechnologies ◽

10.32718/nvlvet-a9311 ◽

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.

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EFFECT OF THE USE OF TWO SPERM SELECTION TECHNIQUES FOR IN VITRO PRODUCTION OF ALPACA EMBRYOS

SPERMOVA ◽

10.18548/aspe/0009.10 ◽

2021 ◽

Vol 11 (1) ◽

pp. 67-72

Author(s):

Mijail Contreras Huamani ◽

◽

Mary Naveros ◽

Cesar Olaguivel

Keyword(s):

In Vitro Fertilization ◽

Cumulus Cells ◽

Sperm Selection ◽

Percoll Gradient ◽

In Vitro Production ◽

Vitro Fertilization ◽

Maturation Medium ◽

Blastocyst Rate ◽

Vitro Production

The objective of this research was to evaluate the effect of the use of two sperm selection techniques for in vitro production of alpaca embryos. The ovaries and testis were collected from the local slaughterhouse and transport to 37 ° C in saline solution (0.9%) supplemented with gentamicin. Quality I, II and II oocytes were incubated in a maturation medium for 32 h at 38.5 ° C and 5% O2 and 5% CO2. For in vitro fertilization, sperm from the epididymis were selected using the Percoll gradient and Swim up technique. 18h after the oocytes were incubated with the sperm, these were denuded from the cumulus cells and cultured in SOFaa culture medium for 7 days. Morula and blastocyst rate and their morphological quality are evaluated at day 7 of culture. From a total of 370 ovaries, 1,137 oocytes were recovered, making an average of 3.6 oocytes / ovary. After the maturation and fertilization process and in vitro culture, the blastocyst rate was 8.43 ± 6.04% and 3.89 ± 1.75%, for oocytes fertilized with sperm selected with Percoll gradient and Swim up, respectively, not finding significant statistical differences (p> 0.05), between the groups. In conclusion, the in vitro fertilization of alpaca oocytes with spermatozoa selected with two selection techniques (percoll and swim up) did not significantly influence the quantity and quality of morulae and blastocysts at day 7 of embryo culture.

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264 EFFECT OF FROZEN SEMEN BATCH ON THE IN VITRO PRODUCTION OF BOVINE EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab264 ◽

2010 ◽

Vol 22 (1) ◽

pp. 289

Author(s):

M. B. Fernandes ◽

T. L. G. Torregrossa ◽

R. B. Prado ◽

R. A. Vila ◽

F. P. Elias ◽

...

Keyword(s):

Embryo Development ◽

Bos Indicus ◽

Cumulus Cells ◽

Development Rate ◽

In Vitro Production ◽

Vitro Fertilization ◽

Significance Level ◽

Frozen Semen ◽

Vitro Production

Within an in vitro production controlled system, bulls differ with respect to their semen potential in generating embryos when the variables of maternal effect are minimized (Marquant-le-Guienne and Humblot 1992 Ann. Zootech. 41, 361-370). We have tested the hypothesis that even with this variation among bulls, there is also an intra-bull variation, according to the frozen semen batch used in the in vitro fertilization, identified with the date of ejaculate and its freezing. In an embryo commercial production system, over 12 months, 10 619 viable oocytes were obtained by ultrasound-guided follicular aspiration from 642 Nelore cows (Bos indicus). The oocytes were matured in vitro for 24 h in TCM-199 supplemented with 0.5 μg mL-1 FSH, 50 μg mL-1 LH, and 10% fetal bovine serum. They were then inseminated for 18 hours in IVF-TALP medium, using the semen from 4 bulls (A to D) subdivided into 4 frozen batches (I to IV) and selected by 45/90% Percoll gradient. Putative zygotes surrounded in cumulus cells were transferred in CR2aa medium drops (Rosenkrans and First 1994 J. Anim. Sci. 72, 434-437) for 163 h at 39°C in a humidified atmosphere of 5% CO2 in air. The oocyte distribution, the total number of blastocysts, and the embryo development rate by each bull and respective batch are described in Table 1. The chi-square test was measured with a significance level of P < 0.05 and showed that there is a difference between the used batches of each bull regarding the development rate of blastocysts 163 h after IVF Therefore, there is intra-bull variation in the ability to develop in vitro embryos according to the batch of frozen semen. Table 1.Viable oocytes (VO), total blastocysts (TB), and embryo development rate (%E) by bull and batch used in IVF

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177 EFFECT OF DIFFERENT FERTILIZATION MEDIA ON IN VITRO BOVINE EMBRYO DEVELOPMENT USING FLOW-CYTOMETRICALLY-SORTED FEMALE SPERM

Reproduction Fertility and Development ◽

10.1071/rdv26n1ab177 ◽

2014 ◽

Vol 26 (1) ◽

pp. 203

Author(s):

L. B. Ferré ◽

Y. S. Bogliotti ◽

J. L. Chitwood ◽

P. J. Ross

Keyword(s):

In Vitro Fertilization ◽

Embryo Development ◽

Bovine Serum ◽

Fetal Bovine Serum ◽

Cumulus Cell ◽

Bovine Embryo ◽

Vitro Fertilization ◽

Female Sex ◽

Fetal Bovine

High demand exists for in vitro-derived bovine embryos fertilized with female sex-sorted sperm by seedstock and commercial cattle producers. The aim of this study was to evaluate different fertilization media on in vitro fertilization performance using female sex-sorted semen. Ovaries were collected from a slaughterhouse and oocytes aspirated from 2- to 6-mm follicles. Cumulus-oocyte complexes containing compact and complete cumulus cell layers were selected and matured in groups of 50 cumulus-oocyte complexes in 400 μL of M199 medium supplemented with ALA-glutamine (0.1 mM), Na pyruvate (0.2 mM), gentamicin (5 μg mL–1), epidermal growth factor (50 ng mL–1), oFSH (50 ng mL–1), bLH (3 μg mL–1), cysteamine (0.1 mM), and 10% fetal bovine serum for 22 to 24 h. Fertilization (Day 0) was carried out using female sex-sorted semen selected with a discontinuous density gradient and diluted to a final concentration of 1 × 106 sperm mL–1. Three different fertilization media, M199 (Gibco 11043–023, Grand Island, NY, USA), SOF (Tervit et al. 1972 J. Reprod. Fertil. 30, 493–497), and TALP (Parrish et al. 1988 Biol. Reprod. 38, 1171–1180), were assayed along with 3 female sex-sorted bulls. All fertilization media were supplemented with fructose (90 μg mL–1), penicillamine (3 μg mL–1), hypotaurine (11 μg mL–1), and heparin (20 μg mL–1). After 18 h, presumptive zygotes were denuded and cultured in groups of 15 to 20 in 50-μL drops of KSOM-BSA for 9 days. On Day 3, 3% fetal bovine serum was added. Low oxygen tension (5% O2) was used for the entire culture period. On Days 7 and 9 blastocysts and hatched embryos, respectively, were morphologically evaluated according to IETS standards and recorded. Results are shown in Table 1. Data was compared by chi-squared analysis. Fertilization media affected cleavage rate and subsequent embryo development, quality, and hatching ability. The SOF and TALP fertilization media produced significantly more and higher quality embryos than M199. Table 1.In vitro fertilization performance after oocyte fertilization using sex-sorted sperm

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Polyspermic penetration in porcine IVM - IVF systems

Reproduction Fertility and Development ◽

10.1071/rd02076 ◽

2003 ◽

Vol 15 (3) ◽

pp. 167 ◽

Cited By ~ 56

Author(s):

Hiroaki Funahashi

Keyword(s):

Zona Pellucida ◽

Blastocyst Stage ◽

Developmental Competence ◽

In Vitro Production ◽

Vitro Fertilization ◽

Final Maturation ◽

Morphological Differences ◽

Vitro Production ◽

Boar Spermatozoa

Although techniques for in vitro production of porcine embryos have proceeded very rapidly during the past decade, polyspermic penetration still remains a persistent obstacle to porcine in vitro fertilization (IVF) systems. Considerable research on in vitro polyspermic penetration in porcine in vitro-matured (IVM) oocytes has been undertaken to try to solve this problem. In the current paper, recent advancements in overcoming the problems of polyspermy in porcine IVF systems are reviewed. Partial induction of the acrosome reaction of boar spermatozoa in IVF media that contain caffeine is likely to be one of the major causes of polyspermy. A reduction in the number of incompletely acrosome-reacted spermatozoa, which can bind tightly to the zona pellucida and mask free sperm receptors of the zona pellucida, could reduce the incidence of polyspermic penetration; however, morphological differences in the reaction of the zona pellucida have been observed between IVM and ovulated oocytes, which suggests that altered zona morphology may be another cause of polyspermic penetration. It has been shown that the developmental ability of polyspermic porcine embryos to the blastocyst stage is similar to that of normal embryos but that developmental competence to term is much lower. To overcome the current problems of polyspermy, it is suggested that future efforts should be focused on controlling boar sperm function and/or sperm–zona binding to achieve the final maturation associated with normal zona modifications of porcine oocytes at fertilization.

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Health of Human and Livestock Conceived by Assisted Reproduction

Twin Research ◽

10.1375/twin.4.5.412 ◽

2001 ◽

Vol 4 (5) ◽

pp. 412-416 ◽

Cited By ~ 4

Author(s):

Manon Ceelen ◽

Jan P.W. Vermeiden

Keyword(s):

In Vitro Fertilization ◽

Assisted Reproduction ◽

Perinatal Outcome ◽

Current Knowledge ◽

Gestation Length ◽

In Vitro Production ◽

Breeding Programs ◽

Vitro Fertilization ◽

Vitro Production

AbstractAssisted reproduction is used to resolve infertility problems in human and in breeding programs to generate livestock. Except for gestation length and birth weight, perinatal outcome of children conceived by In Vitro Fertilization is similar to that of spontaneously conceived children. However, large offspring syndrome observed after In Vitro Production in livestock is quite alarming. The distinct parts of assisted reproduction (oocyte maturation, fertilization and culture) have been found to contribute to abnormal fetal growth and development. Genomic imprinting is suggested to be involved in the induction of the aberrant phenotypes observed after assisted reproduction. Furthermore, current knowledge on postnatal health of offspring conceived by assisted reproduction and speculations on potential longterm effects of In Vitro Fertilization will be described.

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