199 EFFECT OF HEPARIN CONCENTRATION ON BOVINE PREIMPLANTATIONAL DEVELOPMENT IN VITRO USING SEX-SORTED SPERM

The objective of this study was to examine the effect of heparin on bovine IVF and to improve the efficiency of IVF production by using sex-sorted sperm. The fertility performance of sex-sorted and unsorted semen from 4 bulls was compared to determine the optimal heparin concentration during preimplantational embryo development. A total of 7615 matured bovine oocytes were randomly allocated among different heparin concentrations (0, 2.5, 5, 10, 20, 40, 60, 80, and 100 μg mL–1) in Brackett-Oliphant medium and coincubated with either sex-sorted or unsorted sperm for 6 h. Presumptive zygotes were cultured in CR1aa+ 6 mg mL–1 of BSA in 5% O2 , 5% CO2 and 90% N2 at 39°C until Day 8 (Day 0, culture post-IVF). Cleavage rates at Day 2 and embryo development to blastocyst (BL) at Day 8 were recorded. Data (4 replicates) were analyzed by a general linear model (SPSS 11.0, SPSS Inc., Chicago, IL). The optimal heparin concentration for each treatment was determined as the lowest value from those groups that resulted in the highest BL rates. The results (Table 1) demonstrated that a differential requirement of heparin concentration was important for the highest preimplantational BL development between sexed sperm and unsorted control within each bull. By optimizing heparin concentration, in 3 out of 4 (75%) bulls, the in vitro BL development with sex-sorted sperm could be increased to a level that was comparable to the highest BL rate from unsorted sperm (bulls A, B, and C, P > 0.05). A higher heparin concentration was required for optimal BL development in bulls A and C; however, a lower concentration was desirable for bulls B and D, indicating that a partial capacitation to the sperm may have taken place in bulls B and D during the sorting process, as reported by Lu and Seidel (2004 Theriogenology 62, 819–830). The fertility of sorted sperm from bull D (1 out of 4, 25%) was adversely affected, even after heparin optimization for BL development (P < 0.05). This result suggests that sperm sorting could affect the IVF fertility of sorted sperm in a bull-specific manner, but it was not significant for all bulls. Table 1. Blastocyst (BL) development in bovine IVF after heparin optimization using sorted and unsorted sperm This project was supported by the SBIR program under a USDA Cooperative State Research, Education, and Extension Service (CSREES) grant to F. Du (USDA #2006-03069).

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87 COAGULANSIN-A VIA HEAT SHOCK PROTEIN 70 INDUCTION SHOWS BENEFICIAL EFFECTS ON THE DEVELOPMENT OF BOVINE EMBRYOS IN VITRO

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab87 ◽

2016 ◽

Vol 28 (2) ◽

pp. 173

Author(s):

I. Khan ◽

K.-L. Lee ◽

A.-N. Ha ◽

P.-R. Park ◽

S.-H. Song ◽

...

Keyword(s):

Heat Shock ◽

Heat Shock Protein ◽

Embryo Development ◽

Heat Shock Protein 70 ◽

Terminal Deoxynucleotidyl Transferase ◽

Bovine Embryo ◽

Hsp 70 ◽

Development In Vitro ◽

Bovine Oocytes

Coagulansin-A (withanolide) is the steroidal lactone obtained from Withania coagulans, which belong to Solanaceae family. The coagulansin-A induces heat shock protein 70 (HSP-70), which acts as a cellular antioxidant. This study was conducted to investigate the effect of coagulansin-A on bovine oocytes maturation and embryo development in vitro. All these oocytes were aspirated from the ovaries obtained from Korean Hanwoo cows at a local abattoir. To analyse the possible beneficial effect of coagulansin-A on bovine oocytes maturation in vitro, 355 oocytes per group (control and treatment) in seven replicates were subjected with three concentrations i.e. (1, 5, and 10 µM) of coagulansin-A. The coagulansin-A was added in the in vitro-matured (IVM) media for 20 to 22 h followed by IVF for 18 to 22 h, and after fertilization the fertilized oocytes were transferred to IVC1 media for 3 days. After 3 days, the cleavage rate was checked and the 8-cell stage embryos were transferred to IVC2 media and embryo development was checked at Day 8. The culture was carried out at 5% CO2 and 38.5°C. The results indicated that among the three concentrations of Coagulansin-A, only 5 µM remarkably (P < 0.05) improved embryo development (Day 8 blastocyst), being 27.30% and 40.01% for control and treated groups, respectively. This concentration also significantly (P < 0.05) encouraged the activation of HSP-70, having 16.44 arbitrary units (AU) and 35.41 AU integral optical density (IOD) for control and treated groups, respectively. The immunofluorescence analysis revealed that 5 µM coagulansin-A supplementation significantly (P < 0.05) inhibited oxidative stress and inflammation during bovine embryo development in vitro by decreasing IOD of 8-Oxoguanosine (8-OxoG) from 28.12 AU in control to 18.06 AU for the treated group and nuclear factor kappa B (NF-kB) IOD (P < 0.05) from 42.25 AU to 21.80 for control and treated groups, respectively. Additionally, the results obtained from terminal deoxynucleotidyl transferase TUNEL assay confirmed that coagulansin-A treatment reduced the bovine embryo DNA damage significantly (P < 0.05) from 7.4 ± 0.375 to 5.7 ± 0.287 and improved the embryo quality (P < 0.05) with mean cell numbers of 127.7 ± 4.161 and 150.1 ± 3.624 per embryo for control and coagulansin-A treated groups, respectively. This study provides new information regarding the mechanisms by which coagulansin-A promotes bovine oocyte maturation and embryo development in vitro.

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Maintenance of meiotic arrest in bovine oocytes using the S-enantiomer of roscovitine: effects on maturation, fertilization and subsequent embryo development in vitro

Reproduction ◽

10.1530/rep.1.00299 ◽

2005 ◽

Vol 129 (1) ◽

pp. 19-26 ◽

Cited By ~ 32

Author(s):

Pilar Coy ◽

Raquel Romar ◽

Rebecca R Payton ◽

Lisa McCann ◽

Arnold M Saxton ◽

...

Keyword(s):

Embryo Development ◽

Lens Culinaris ◽

Germinal Vesicle ◽

Fluorescein Isothiocyanate ◽

Cortical Granule ◽

Nuclear Maturation ◽

Development In Vitro ◽

Hoechst Staining ◽

Bovine Oocytes

The overall objective was to evaluate the effectiveness of the S-enantiomer of roscovitine (inhibitor of p34cdc2/cyclin B kinase) to maintain bovine cumulus–oocyte complexes at the germinal vesicle (GV) stage for extended times after removal from antral follicles without compromising subsequent maturation, fertilization and embryo development. Oocytes were cultured in 0, 12.5, 25 or 50 μmol/l S-roscovitine for 24 h. Hoechst staining showed that 50 μmol/l S-roscovitine maintained >90% of oocytes at the GV stage and inhibited gonadotropin-induced cumulus expansion. Fewer oocytes underwent nuclear maturation after in vitro maturation (Hoechst staining) when cultured in 50 μmol/l S-roscovitine for 66 versus 21 or 42 h. Zona pellucida (ZP) hardening (pronase resistance), cortical granule types (lens culinaris agglutinin–fluorescein isothiocyanate), nuclear maturation and fertilization with frozen-thawed spermatozoa (Hoechst staining) were assessed after culture of oocytes in 50 μmol/l S-roscovitine for 0, 24 or 48 h. Neither ZP hardening, nor nuclear maturation nor fertilization were altered by roscovitine culture for 48 h. A higher proportion of oocytes had a type III cortical granule pattern (premature translocation to the oolemma) after roscovitine culture for 48 h. However, embryo development was not compromised as cleavage, development to 8–16 cell and blastocyst stages were at least comparable in control and roscovitine-treated oocytes. In conclusion, the studies have shown that S-roscovitine reversibly maintained bovine oocytes at the GV stage for 48 h. However, maintenance of oocytes in static culture for 48 h was not sufficient to improve development above non-treated controls.

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Effects of resazurin on fertilization of bovine oocytes and preimplantation embryo development in vitro

Theriogenology ◽

10.1016/s0093-691x(98)90575-1 ◽

1998 ◽

Vol 49 (1) ◽

pp. 222

Author(s):

S. Wang ◽

R.G. Holyoak ◽

G. Liu ◽

R.C. Evans ◽

T.D. Bunch

Keyword(s):

Embryo Development ◽

Preimplantation Embryo ◽

Preimplantation Embryo Development ◽

Development In Vitro ◽

Bovine Oocytes

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Analysis of Morphokinetic Parameters of Feline Embryos Using a Time-Lapse System

Animals ◽

10.3390/ani11030748 ◽

2021 ◽

Vol 11 (3) ◽

pp. 748

Author(s):

Joanna Kochan ◽

Agnieszka Nowak ◽

Barbara Kij ◽

Sylwia Prochowska ◽

Wojciech Niżański

Keyword(s):

Embryo Development ◽

Embryo Quality ◽

Time Lapse ◽

Blastocyst Stage ◽

Cavity Formation ◽

Non Invasive ◽

Morphokinetic Parameters ◽

Morphological Defects ◽

Development In Vitro

The aim of this study was to analyze the morphokinetic parameters of feline embryos using a time lapse system. Oocytes matured in vitro were fertilized (IVF) and in vitro cultured in a time lapse-system (Primo Vision®, Gothenburg, Sweden). The first cell division of embryos occurred between 17 h post insemination (hpi) and 38 hpi, with the highest proportion of embryos (46%) cleaving between 21 and 24 hpi. The timing of the first cleavage significantly affected further embryo development, with the highest development occurring in embryos that cleaved at 21–22 hpi. Embryos that cleaved very early (17–18 hpi) developed poorly to the blastocyst stage (2%) and none of the embryos that cleaved later than 27 hpi were able to reach the blastocyst stage. Morphological defects were observed in 48% of the embryos. There were no statistically significant differences between the timing intervals of the first cleavage division and the frequency of morphological defects in embryos. Multiple (MUL) morphological defects were detected in more than half (56%) of the abnormal embryos. The most frequent single morphological defects were cytoplasmic fragmentation (FR) (8%) and blastomere asymmetry (AS) (6%). Direct cleavage (DC) from 1–3 or 3–5 blastomeres, reverse cleavage (RC) and vacuoles were rarely observed (2–3%). The timing of blastocyst cavity formation is a very good indicator of embryo quality. In our study, blastocyst cavity formation occurred between 127–167 hpi, with the highest frequency of hatching observed in blastocysts that cavitated between 142–150 hpi. Blastocysts in which cavitation began after 161 h did not hatch. In conclusion, the timing of the first and second cleavage divisions, the timing of blastocyst cavity formation and morphological anomalies can all be used as early and non-invasive indicators of cat embryo development in vitro.

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