Supplementation with small-extracellular vesicles from ovarian follicular fluid during in vitro production modulates bovine embryo development

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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260 EVALUATION OF PORCINE IN VITRO PRODUCTION WITH THE ADDITION OF CHITOSAN TO CULTURE MEDIA

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab260 ◽

2015 ◽

Vol 27 (1) ◽

pp. 219 ◽

Cited By ~ 2

Author(s):

F. García ◽

Y. Ducolomb ◽

S. P. Miranda-Castro ◽

J. F. De la Torre-Sánchez ◽

S. Romo

Keyword(s):

Embryo Development ◽

Oocyte Maturation ◽

Culture Media ◽

Biological Properties ◽

In Vitro Production ◽

Porcine Oocyte ◽

Main Component ◽

Vitro Production ◽

Positive Effect

Chitosan is a partially deacetylated polymer obtained from the alkaline deacetylation of chitin, which is a glucose-based unbranched polysaccharide widely distributed in nature as the main component of exoskeletons of crustaceans and insects. Chitosan has a variety of physicochemical and biological properties resulting in numerous applications. In addition to its lack of toxicity and allergenicity, its biocompatibility, biodegradability, and bioactivity make it a very attractive substance for diverse applications as a biomaterial in pharmaceutical and medical fields. Chitosan stimulates cell growth and it has been used in fibroblast culture, increasing cell proliferation. For these reasons, it is important to evaluate if this polymer has a positive effect on embryo production. The aim of this study was to evaluate porcine oocyte maturation and embryo development, comparing the effect of supplementing different concentrations of chitosan to the maturation (MM) and development media (DM). Cumulus-oocyte complexes (COC) were aspirated from ovarian follicles of slaughtered sows. The COC were matured in supplemented TCM-199 (MM) and incubated for 44 h. All incubations were performed at 38.5°C, with 5% CO2 in air and humidity at saturation. After maturation IVF was performed, frozen-thawed semen from the same boar was used and gametes were co-incubated in MTBM for 7 h. Then, putative zygotes were cultured in NCSU-23 (DM) for 144 h. The following experiments were performed: 1) addition of 0 (control), 35, 50, 100, and 150 ppm chitosan to the MM (n = 1353), 2) addition of 0, 50, 100, and 150 ppm chitosan to the DM (n = 739), 3) addition of 0, 50, 100, and 150 ppm of chitosan to the MM first and then the same concentrations to the DM (n = 702). When chitosan was added to the MM, the highest percentage of matured oocytes (metaphase II) was obtained in the 50 ppm treatment (87%, P < 0.05) when compared with the control, 100, and 150 ppm groups (78, 78, and 82%, respectively). Regarding the percentage of blastocysts, there were no differences when comparing the treatment and the control groups (ranging from 12 to 13%). After addition of chitosan to the putative zygotes in the DM, the percentage of morulae in the 150 ppm treatment was significantly increased with regard to the other groups (54 v. 46%, respectively, P < 0.05). When adding chitosan to both MM and DM, there was no effect on embryo development. It is concluded that the addition of chitosan to the MM at a concentration of 50 ppm significantly improved oocyte maturation and a concentration of 150 ppm in the DM increased the percentage of morulae. Chitosan had a positive effect on oocyte maturation and embryo development. These results justify further investigations to find out if chitosan can be useful as a supplement for chemically defined media.

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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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Bovine Follicular Fluid and Extracellular Vesicles Derived from Follicular Fluid Alter the Bovine Oviductal Epithelial Cells Transcriptome

International Journal of Molecular Sciences ◽

10.3390/ijms21155365 ◽

2020 ◽

Vol 21 (15) ◽

pp. 5365 ◽

Cited By ~ 2

Author(s):

Mohammad Mehedi Hasan ◽

Janeli Viil ◽

Freddy Lättekivi ◽

James Ord ◽

Qurat Ul Ain Reshi ◽

...

Keyword(s):

Gene Expression ◽

Epithelial Cells ◽

Embryo Development ◽

Extracellular Vesicles ◽

Follicular Fluid ◽

Early Embryo ◽

Transcriptional Responses ◽

Early Embryo Development ◽

Sperm Survival

While follicular fluid (FF) is well known to provide an optimal environment for oogenesis, its functional roles following its release into the oviduct during ovulation are currently elusive. We hypothesized that FF and FF-derived extracellular vesicles (EVs) may be conveyors of signals capable of inducing functionally-relevant transcriptional responses in oviductal cells. The aim of this study was, therefore, to evaluate the effect of FF and FF-derived EVs on the transcriptome of primary bovine oviductal epithelial cells (BOECs). We examined the gene expression of BOECs in three conditions: BOECs cultured with FF, FF-derived EVs, and without supplementations. For each condition, cells were cultured for 6 and 24 h. RNA sequencing results revealed that FF had a stronger effect on BOECs gene expression compared to EVs. We detected 488 and 1998 differentially expressed genes (DEGs) with FF treatment in 6 and 24 h, respectively, whereas only 41 DEGs were detected at 6 h following EV treatment. Pathway analysis of the FF-induced DEGs showed that several pathways were highly enriched, notably oxidative phosphorylation, thermogenesis, arachidonic acid metabolism, and steroid hormone biosynthesis. Some of these pathways have a role in sperm survival, fertilization, and early embryo development. In conclusion, the findings of our study demonstrate for the first time that bovine FF and FF-derived EVs can induce changes in the gene expression of the bovine oviductal cells which, although observed in vitro, may be reflective of in vivo responses which may contribute to a favorable periconceptional microenvironment for sperm survival, fertilization, and early embryo development.

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Can extracellular vesicles from bovine ovarian follicular fluid modulate the in-vitro oocyte meiosis progression similarly to the CNP-NPR2 system?

Theriogenology ◽

10.1016/j.theriogenology.2020.06.031 ◽

2020 ◽

Vol 157 ◽

pp. 210-217 ◽

Cited By ~ 2

Author(s):

Elisa M. Pioltine ◽

Mariana F. Machado ◽

Juliano C. da Silveira ◽

Patrícia K. Fontes ◽

Ramon C. Botigelli ◽

...

Keyword(s):

Extracellular Vesicles ◽

Follicular Fluid ◽

Oocyte Meiosis ◽

Ovarian Follicular Fluid

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81 EFFECT OF IN VITRO CULTURE SYSTEM MODIFICATION USING CR1aa MEDIUM ON EMBRYO DEVELOPMENT AND PREGNANCY RATE IN CATTLE

Reproduction Fertility and Development ◽

10.1071/rdv26n1ab81 ◽

2014 ◽

Vol 26 (1) ◽

pp. 154

Author(s):

G. Singina ◽

T. Taradajnic ◽

N. Taradajnic ◽

N. Zinovieva

Keyword(s):

Pregnancy Rate ◽

Embryo Development ◽

Subsequent Pregnancy ◽

Bovine Embryo ◽

In Vitro Production ◽

Developmental Potential ◽

Culture Systems ◽

System 2 ◽

System 1

The culture of in vitro matured and fertilized oocytes is a critical step of in vitro production of bovine embryos. Generally, oocytes are co-incubated with sperm in TALP medium containing different additions and then zygotes are transferred to a medium with another composition. At the same time the effect of the medium alteration on the development of early embryos is unknown. Continual adjustment of fertilized oocytes to the changing culture environment may result in a reduction of their developmental potential. The aim of the present study was to compare effects of two different culture systems on the embryo development and subsequent pregnancy rate in cattle. Slaughterhouse-derived cumulus–oocyte complexes were matured for 24 h in TCM-199 supplemented with 10% FCS, 0.2 mM sodium pyruvate, 10 μg mL–1 porcine FSH, and 5 μg mL–1 ovine LH. Frozen/thawed sperm from different Russian Black Pied bulls were prepared in Sperm-TALP medium by swim-up procedure. In vitro matured oocytes were co-incubated for 18 h with prepared sperm in the modified Fert-TALP medium containing 10 μg mL–1 heparin, PHE (20 μM penicillamine, 10 μM hypotaurine, 1 μM epinephrine), and 0.1% MEM nonessential amino acids. The embryo culture was carried out using 2 systems. A total of 340 presumptive zygotes were incubated in CR1aa medium (Rosenkrans et al. 1994 J. Anim. Sci. 72, 434–437) up to Day 5 post-insemination (System 1) and a total of 442 presumptive zygotes were incubated for 24 h in a fresh Fert-TALP medium without PHE and heparin and then cleaved embryos were transferred to CR1aa medium and incubated until Day 5 post-insemination (System 2). Thereupon, the embryos were transferred to a fresh CR1aa medium supplemented with 5% FCS and cultured for 3 or 5 days. The embryo development was evaluated at Days 2, 8, and 10 for cleavage and blastocyst formation and hatching rates, respectively. A portion of blastocysts (of Grade 1 according to IETS classification) obtained at Day 8 were immediately transferred to recipients or were frozen in 1.5 M ethylene glycol and stored in liquid nitrogen until transplantation. The embryo development data (from 6–8 replicates) were analysed by ANOVA and the embryo transplantation data were analysed using the chi-squared test. The cleavage rates did not differ among Systems 1 and 2 and were 63.6–65.7%. On the other hand, the significant differences between culture Systems 1 and 2 were detected in rates of blastocysts (21.9 ± 1.4 v. 28.8 ± 2.8; P < 0.05) and hatched blastocysts (7.2 ± 1.2 v. 12.3 ± 1.6; P < 0.05). The pregnancy rate for frozen embryos was also higher (but not significantly) in System 2 than in System 1 [26.3% (9/34) v. 16.7% (2/12)], whereas for fresh embryos the similar values of the pregnancy rate were observed [on average 42.9% (6/14)]. Thus the additional 24-h culture of zygotes in Fert-TALP medium favourably affects bovine embryo development in vitro.

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255. In vitro maturation of bovine oocytes in serum-free media

Reproduction Fertility and Development ◽

10.1071/srb05abs255 ◽

2005 ◽

Vol 17 (9) ◽

pp. 102

Author(s):

S. Zhang ◽

A. J. French ◽

R. T. Tecirlioglu

Keyword(s):

Embryo Development ◽

Bovine Serum ◽

Culture Medium ◽

Polar Body ◽

In Vitro Production ◽

Serum Free ◽

Cell Numbers ◽

Defined Culture ◽

Vitro Production

Culture medium supplemented with sera is commonly used for the in vitro production (IVP) of livestock embryos. However, serum induced complications including batch variation, the potential risk of virus and mycoplasma contamination and the implication in the large offspring syndrome in domestic animals impels the development of a serum-free culture system. In this study, we investigated whether replacement of fetal bovine serum (FBS) with bovine serum albumin (BSA) in three maturation media, tissue culture medium-199 (TCM-199), a modified synthetic oviduct fluid (mSOF) routinely used in our laboratories and a commercially available SOF-VC (Vitro Cleave, Cook Australia). Harvested oocytes were matured, parthenogenetically activated and in vitro cultured (Day 7) to measure maturation efficiency, embryo development and quality with the aim of developing a simplified and defined culture medium for the in vitro production of bovine embryos. Abattoir derived cumulus oocyte complexes were matured in TCM-199, mSOF and SOF-VC media supplemented with LH and beta-estradiol in the presence of 15% FBS or 0.08% BSA at 39ºC in 5% CO2 in air. Polar body extrusion was assessed twenty-two hour post maturation and selected MII occytes were activated using calcium ionophore/6-dimethylaminopurine and cultured for seven days in SOF medium supplemented with 0.8% BSA. On day seven, blastocyst development was assessed and randomly selected blastocysts were stained to determine inner cell mass (ICM), trophectoderm (TE) and total cell numbers (TCN). Supplementation with either BSA or FCS did not significantly affect the maturation efficiency, blastocyst rates or differential cell numbers within each maturation media tested. However, maturation efficiency and blastocyst rates were significantly lower (P < 0.01) when oocytes were matured in either mSOF or SOF-VC regardless of FBS or BSA supplementation. From this study, we conclude that BSA effectively replaces FCS and TCM-199 is superior to SOF (mSOF or SOF-VC) in terms of oocyte maturation regardless of protein source. Once matured SOF and TCM-199 parthenogenetically blastocysts were equivalent in terms of embryo development and quality.

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