Short- and long-term outcomes of the absence of protein during bovine blastocyst formation in vitro

2017 ◽  
Vol 29 (6) ◽  
pp. 1064 ◽  
Author(s):  
A. Murillo-Ríos ◽  
V. Maillo ◽  
M. Muñoz ◽  
A. Gutiérrez-Adán ◽  
S. Carrocera ◽  
...  
Keyword(s):  
Dna Methyltransferase ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Gestation Length ◽  
Individual Culture ◽  
Cell Counts ◽  
Activating Transcription Factor 4 ◽  
Short And Long Term

In cattle, individual in vitro embryo culture after Day 6 benefits development, allowing non-invasive analysis of culture medium. However, undefined supplements in culture reduce analytical reliability. In this study we assayed the short- and long-term performance of embryos after bovine serum albumin removal over a 24-h period in individual culture. The absence of protein decreased embryo development and cell counts in the inner cell mass without affecting blastocyst sex ratio. However, the absence of protein produced embryos with an improved tendency to survive vitrification after 24 h in culture (P = 0.07). After transfer to recipients, birth rates of embryos that had been cultured with protein tended to decrease (P < 0.06) mostly as a result of a higher number of miscarriages (P < 0.013), reflecting lower viability. Birthweight, gestation length, height and thorax circumference did not differ between embryos cultured with or without protein. In fresh blastocysts cultured without protein, gene expression analysis showed higher abundance (P < 0.05) of insulin-like growth factor 2 receptor (IGF2R; imprinting) and activating transcription factor 4 (ATF4) and DNA-damage-inducible transcript 3 (DDIT3; endoplasmic reticulum stress) transcripts, with DNA methyltransferase 3A (DNMT3A; imprinting) tending to increase (P = 0.062). However, in hatched blastocysts that survived cryopreservation, glucose-6-phosphate dehydrogenase (G6PD) was overexpressed in embryos cultured without protein (P < 0.01). The absence of protein results in fewer blastocysts but improved long-term viability after cryopreservation.

scholarly journals A shorter equilibration period in the VitTrans in-straw bovine embryo vitrification method improves post-warming outcomes

2020 ◽  
Author(s):  
Iris Martínez-Rodero ◽  
Tania García-Martínez ◽  
Erika Alina Ordóñez-León ◽  
Meritxell Vendrell-Flotats ◽  
Carlos Olegario-Hidalgo ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Survival Rates ◽  
Field Conditions ◽  
Bovine Embryo ◽  
Direct Transfer ◽  
Treatment Groups ◽  
Cell Counts ◽  
Hatching Rates

Abstract Background VitTrans is a device that enables the vitrification and warming/dilution of in vitro produced bovine embryos followed by their direct transfer to recipient females in field conditions. This study sought to improve the VitTrans method by comparing two equilibration times: short (SE: 3 min) and long (LE: 12 min). Outcome measures recorded in vitrified D7 and D8 expanded blastocysts were survival and hatching rates, differential cell counts, apoptosis rate and gene expression. Results While survival rates at 3 h and 24 h post-warming were reduced (P < 0.05) after vitrification, hatching rates of D7 embryos vitrified after SE were similar to those obtained in fresh non-vitrified blastocysts. Hatching rates of vitrified D8 blastocysts were lower (P < 0.05) than of fresh controls, regardless of treatment. Total cell counts, and inner cell mass and trophectoderm cell numbers were similar in hatched blastocysts derived from D7 blastocysts vitrified after SE and fresh blastocysts, while vitrified D8 blastocysts yielded lower values, regardless of treatment. The rate of apoptotic cells was significantly higher in both treatment groups when compared to fresh controls, although apoptosis rates were lower using the SE than LE protocol. No differences emerged in expression of the genes BAX, AQP3, CX43 and IFNτ between blastocysts vitrified after SE or LE, whereas a significantly higher abundance of BCL2L1 and SOD1 transcripts was observed in blastocysts vitrified after SE compared to LE. Conclusions The VitTrans device combined with a shorter exposure to the equilibration medium improves vitrification/warming outcomes facilitating the direct transfer of vitrified embryos under field conditions.

2 BOVINE EMBRYONIC STEM-LIKE CELLS DERIVED FROM IN VITRO-PRODUCED BLASTOCYSTS

2017 ◽  
Vol 29 (1) ◽  
pp. 108
Author(s):  
Y. S. Bogliotti ◽  
J. Wu ◽  
M. Vilariño ◽  
K. Suzuki ◽  
J. C. Belmonte ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Basal Medium ◽  
Primitive Endoderm ◽  
Culture Dish ◽  
Rock Inhibitor ◽  
Immunodeficient Mice

Embryonic stem cells (ESC) are derived from the inner cell mass (ICM) of preimplantation blastocysts. To date, it has been challenging to establish pluripotent ESC lines for domestic animals, which could be important for biotechnological applications, such as genetic engineering and SCNT, and biomedical research. The aim of this work was to derive and characterise bovine embryonic stem-like cells (bESC) from in vitro-produced bovine blastocysts. Embryos were produced by in vitro fertilization of in vitro-matured oocytes aspirated from abattoir ovaries and cultured in groups of 25 in 50-μL drops of KSOM (Evolve, Zenith Biotech) with 4 mg mL−1 BSA for 7 days until they reached the blastocyst stage (Ross et al., 2009 Reproduction 137, 427–437). At that point, the zona pellucida (ZP) was removed using 1 mg mL−1 Pronase (Sigma, St. Louis, MO), and ZP-free blastocysts were washed 6 times in SOF-HEPES. Three derivation approaches were tested: ZP-free whole blastocysts, mechanically isolated ICM, and immunosurgery-derived ICM. In each case, individual blastocysts/ICM were placed in 1 well of a 12-well dish seeded with a monolayer of mouse embryo fibroblasts (MEF) and cultured in mTeSR1 basal medium (without growth factors) supplemented with 20 ng mL−1 FGF2 and 2.5 μM IWR1 (CTFR) (Wu et al. 2015 Nature 521, 316–321). After 48 h, blastocysts/ICM that failed to adhere were physically pressed against the bottom of the culture dish with a 22-gauge needle under a stereoscope to aid attachment. Thereafter, the media was changed daily. Outgrowths (after 6–7 days in culture) were dissociated and passaged using TrypLE and re-seeded in the presence of ROCK inhibitor (Y-27632, 10 μM) onto newly prepared wells containing MEF. Established bESC lines were cultured on MEF and passaged every 4 to 5 days at a 1:10 split ratio. The bESC lines were characterised by immunofluorescence (IF), RNA-seq, and teratoma formation. The efficiency of cell line derivation (evaluated at passage 3) was similar for the 3 approaches: whole blastocysts (9/16, 56.3%), mechanical ICM isolation (7/12, 58.3%), and immunosurgical ICM isolation (7/16, 43.8%). The bESC were passaged and cultured long-term (more than 15 passages) and were subjected to several rounds of freezing and thawing while retaining their morphology and characteristics. IF analysis showed that long-term cultured bESC expressed the markers SOX2 and OCT4 (pluripotency), but did not express CDX2 (trophectoderm) or GATA6 (primitive endoderm). RNAseq analysis of 2 bESC lines showed that ICM markers (POU5F1, NANOG, SOX2, LIN28B, DNAMT3B, UTF1, SALL4) were expressed (RPKM > 0.4), while trophectoderm markers (CDX2, GATA2, GATA3, FGF4, TFAP2A) and primitive endoderm markers (GATA6, HNF4A) were not expressed (RPKM < 0.4). Finally, bESC lines (n = 2) were able to form teratomas in immunodeficient mice. The teratomas contained tissues representative of the 3 germ lineages and expressed lineage-specific markers (ectoderm: TUJ1, endoderm: FOXA2, and mesoderm: ASM). In conclusion, the culture condition used in this work (CTFR) enables robust derivation and long-term in vitro propagation of pluripotent bESC.

Bovine blastocyst diameter as a morphological tool to predict embryo cell counts, embryo sex, hatching ability and developmental characteristics after transfer to recipients

2006 ◽  
Vol 18 (5) ◽  
pp. 551 ◽  
Author(s):  
Michael Hoelker ◽  
Friedrich Schmoll ◽  
Hendrik Schneider ◽  
Franca Rings ◽  
Markus Gilles ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryo Cell ◽  
Cell Counts ◽  
Bull Calves ◽  
Embryo Sex ◽  
Developmental Characteristics ◽  
Number Of Cells ◽  
Bovine Blastocyst

The aim of the present study was to explore whether the blastocyst diameter and the zona thickness at 168 h after fertilisation are useful parameters to predict quality and viability of bovine in-vitro-produced (IVP)-embryos. Although significant (P < 0.05), the blastocyst diameter at 168 h correlated only poorly with the total number of cells (R2 = 0.13) and with the number of trophectoderm (TE) cells (R2 = 0.17). Hatched blastocysts (n = 66) at 216 h had a significantly greater mean diameter at 168 h (194.8 ± 16.8 µm) compared with either blastocysts that had started but not finished hatching at 216 h (n = 26, 178.4 ± 16.7 µm) or failed to commence hatching (n = 136, 162.7 ± 12.9 µm). Transfer of 101 IVP blastocysts to synchronised recipients resulted in the birth of 38 calves (38%). There were significantly more bull calves born than cow calves (P < 0.05), but this was not correlated with blastocyst diameter or zona thickness at 168 h. There was also no correlation between the diameter of blastocysts or the zona thickness at 168 h and parameters of subsequent developmental characteristics, including rates of pregnancy, resorptions and abortions, pregnancy duration, delivery to term and birthweight. Overall, the present results indicate that the blastocyst diameter and the zona thickness at 168 h are good predictors for subsequent hatching ability in vitro, but not for the number of TE cells, inner cell mass cells or total cells and neither for subsequent developmental characteristics after transfer to recipients.

63 QUALITY OF BOVINE EMBRYOS PRODUCED IN VITRO FROM IMMATURE OOCYTES TREATED WITH A SUBLETHAL HYDROSTATIC PRESSURE

2013 ◽  
Vol 25 (1) ◽  
pp. 179
Author(s):  
C. Díez ◽  
B. Trigal ◽  
J. N. Caamaño ◽  
M. Muñoz ◽  
E. Correia ◽  
...  
Keyword(s):  
Hydrostatic Pressure ◽  
Embryo Development ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Survival Rates ◽  
Pressure Treatment ◽  
Cell Counts ◽  
Development Rates ◽  
Bovine Oocytes

High hydrostatic pressure (HHP) treatment of immature porcine oocytes improves embryo development rates and cell numbers (Pribenszky et al. 2008 Anim. Reprod. Sci. 106, 200–207). However, it is unknown if similar effects can be obtained with bovine oocytes and how HHP affects cryopreservation of the developed blastocysts. In this work, we analyzed the effect of an HHP treatment (Cryo-Innovation Ltd., Budapest, Hungary) on bovine cumulus–oocyte complex (COC) as determined by their developmental ability and embryo quality. Immature COC were submitted to a pressure treatment (200 bar, 1 h at 37°C; HHP group; n = 643) in HEPES-buffered TCM199. Simultaneously, a group of COC was held at 37°C for 1 h (T group; n = 304) in HEPES-buffered TCM199, while other COC were untreated (n = 1182). After in vitro maturation, COC were fertilized in vitro (IVF) and cultured in modified SOF + 6 g L–1 BSA (Holm et al. 1999 Theriogenology 52, 683–700), and embryo development was recorded (5 replicates). Day 7 and 8 excellent- and good-quality embryos were selected for vitrification (cryologic vitrification method; Trigal et al. 2012 Theriogenology 10.1016/j.theriogenology.2012.06.018). After warming, vitrified blastocysts were cultured in modified SOF + 6 g L–1 BSA + 10% FCS for 48 h (3 replicates). Those blastocysts hatching after warming (at 24 and 48 h) were fixed and stained for differential cell counts. Data were analyzed by ANOVA and REGWQ test and are presented as least squares means ± standard error. The HHP-treated oocytes showed increased development rates on Day 3 (Day 3 ≥5-cell embryos: 64.5 ± 2.9a, 53.4 ± 3.9b, 56.7 ± 2.2b for HHP, T, and untreated groups, respectively; a v. b: P < 0.05); however, D8 blastocyst rates were not affected by the pressure treatment (28.5 ± 1.6, 26.4 ± 2.2, and 27.8 ± 1.3 for HHP, T, and untreated groups, respectively). Treatment did not affect survival rates to vitrification (2-h re-expansion rates: 100 ± 6.7, 100 ± 6.7, and 95.4 ± 6.7; 48-h hatching rates: 58.1 ± 9.4, 71.2 ± 9.4, and 62.3 ± 9.4, for HHP, T, and untreated, respectively). Embryos that hatched after warming did not differ in inner cell mass and trophectoderm cell counts (inner cell mass: 15.0 ± 1.9, 12.7 ± 3.0, and 13.0 ± 2.0; trophectoderm: 133.6 ± 8.4, 137.3 ± 12.8, and 138.4 ± 8.6 for HHP, T, and untreated groups, respectively; P > 0.05). Complementary studies are needed to analyze the effects of a sublethal stress in bovine oocytes on the subsequent embryo production and quality. Species-specific mechanisms could underlie the differences in results obtained in bovine and porcine. RTA2011-00090 (FEDER-INIA). Muñoz, Trigal, and Correia are sponsored by RYC08-03454, Cajastur, and FPU2009-5265, respectively.

scholarly journals A Shorter Equilibration Period Improves Post-Warming Outcomes after Vitrification and in Straw Dilution of In Vitro-Produced Bovine Embryos

Biology ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 142
Author(s):  
Iris Martínez-Rodero ◽  
Tania García-Martínez ◽  
Erika Alina Ordóñez-León ◽  
Meritxell Vendrell-Flotats ◽  
Carlos Olegario Hidalgo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Survival Rates ◽  
Bovine Embryos ◽  
Treatment Groups ◽  
Apoptosis Rate ◽  
Cell Counts ◽  
Hatching Rates

This study was designed to the optimize vitrification and in-straw warming protocol of in vitro-produced bovine embryos by comparing two different equilibration periods, short equilibrium (SE: 3 min) and long equilibrium (LE: 12 min). Outcomes recorded in vitrified day seven (D7) and day eight (D8) expanded blastocysts were survival and hatching rates, cell counts, apoptosis rate, and gene expression. While survival rates at 3 and 24 h post-warming were reduced (p < 0.05) after vitrification, the hatching rates of D7 embryos vitrified after SE were similar to the rates recorded in fresh non-vitrified blastocysts. The hatching rates of vitrified D8 blastocysts were lower (p < 0.05) than of fresh controls regardless of treatment. Total cell count, and inner cell mass and trophectoderm cell counts were similar in hatched D7 blastocysts vitrified after SE and fresh blastocysts, while vitrified D8 blastocysts yielded lower values regardless of treatment. The apoptosis rate was significantly higher in both treatment groups compared to fresh controls, although rates were lower for SE than LE. No differences emerged in BAX, AQP3, CX43, and IFNτ gene expression between the treatments, whereas a significantly greater abundance of BCL2L1 and SOD1 transcripts was observed in blastocysts vitrified after SE. A shorter equilibration vitrification protocol was found to improve post-warming outcomes and time efficiency after in-straw warming/dilution.

113 RETINOIC ACID DOES NOT PROTECT AGAINST THE SELECTIVE DAMAGE TO THE BOVINE INNER CELL MASS INFLICTED BY VITRIFICATION

2007 ◽  
Vol 19 (1) ◽  
pp. 174
Author(s):  
C. Díez ◽  
A. Rodríguez ◽  
C. De Frutos ◽  
J. N. Caamaño ◽  
N. Facal ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Counting ◽  
Bovine Embryo ◽  
Embryo Survival ◽  
Binding Effect ◽  
Cell Counts ◽  
Before And After

Successful cryopreservation of in vitro-produced embryos is a major objective in reproductive biotechnology. It was reported that in vitro culture with high BSA concentrations improved bovine embryo survival after vitrification (D�ez et al. 2005 Reprod. Dom. Anim. 40, 384). All-trans retinoic acid (ATRA) increases cell numbers in the inner cell mass (ICM) and the trophectoderm (TE) (Rodr�guez et al. 2006 Hum. Reprod. 21, 2149–2157). This work analyzed the effect of ATRA on bovine embryo development, survival to vitrification, and cell allocation before and after cryopreservation. Bovine cumulus–oocyte complexes were matured and fertilized in vitro, and presumptive zygotes cultured in SOF + 20 g L-1 BSA. At 139 h post-insemination (Day 6), a total of 917 morulae + early blastocysts were cultured for 24 h with: (1) 1.4 �M ATRA, (2) 0.7 �M ATRA, and (3) no ATRA (control). Embryos were subsequently cultured up to Day 9 in SOF + 20 g L-1 BSA. Development was recorded and differential cell counting was performed on Day 8 and 9 hatched blastocysts. Simultaneously, Day 7 and 8 expanded blastocysts were vitrified (OPS; Vajta 2000 Anim. Reprod. Sci. 60–61, 357–364). After warming, blastocysts were cultured for 72 h in B2 + 5% FCS with Vero cells, and cell counts were performed in fully expanded or hatched blastocysts. Data (7 replicates for cell counts before and 4 after vitrification) were processed by GLM and Duncan&apos;s test, and were expressed as LSM � SE (x,y: P = 0.01; a,b: P &lt; 0.05; α,β: P &lt; 0.002). Developmental rates did not differ among groups. Blastocysts cultured in 0.7 �M ATRA survived vitrification at rates similar to those of controls, and only hatching rates 24 h post-warming were significantly lower than those of controls (4.0 � 8.2a vs. 31.2 � 8.2b). ATRA at 1.4 �M was detrimental to survival of Day 7 embryos, whereas differences were not detected in Day 8 blastocysts. In all groups, the vitrification procedure significantly reduced the cells of the ICM (1.4 �M ATRA: 28.3 � 3.1α vs. 8.6 � 4.1β; 0.7 �M ATRA: 27.7 � 3.5α vs. 2.2 � 4.1β; Control: 31.3 � 3.1α vs. 7.0 � 5.1β). Total cell counts were: 1.4 �M ATRA: 160.0 � 9.8a vs. 130.0 � 12.2b; 0.7 �M ATRA: 165.3 � 8.8a vs. 123.2 � 11.7b; Control: 161.2 � 9.2a vs. 131.0 � 15.1b. The ratios of ICM/TE cells were: 1.4 �M ATRA: 16.9 � 2.7x vs. 6.1 � 3.2y; 0.7 �M ATRA: 17.2 � 2.3x vs. 2.0 � 3.0y; Control: 20.6 � 2.4x vs. 4.3 � 3.9y. All values are before and after vitrification, respectively. When considered together, the differences in the cell counts before and after vitrification were highly significant (*P &lt; 0.0001): 1.4 �M ATRA: 29.2 � 1.9* vs. 5.9 � 2.6; 0.7 �M ATRA: 162.5 � 5.5* vs. 127.2 � 7.6; Control: 18.3 � 1.5* vs. 4.2 � 2.0. Our results show that ATRA did not improve the embryo survival to vitrification. Although 1.4 �M ATRA was used to avoid a 'binding effect' related to an elevated protein level (Klaassen et al. 1999 Biochim. Biophys. Acta 1427, 265–275), the BSA concentrations used in culture could mask any ATRA effect. The vitrification procedure used in this study produced a selective damage within the ICM cells, which can explain the reduced survival rates obtained after warming. This work was supported by Grant AGL2005-04479.

127 BINDING RETINOID RECEPTORS BY SPECIFIC AGONISTS AFFECTS THE BOVINE BLASTOCYST DEVELOPMENT IN VITRO

2006 ◽  
Vol 18 (2) ◽  
pp. 172 ◽  
Author(s):  
E. Gómez ◽  
A. Rodríguez ◽  
C. Alonso-Montes ◽  
N. Caamaño ◽  
L. J. Royo ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Inner Cell Mass ◽  
Retinoic Acid Receptor ◽  
Cell Mass ◽  
Cell Counting ◽  
Retinoid Receptor ◽  
Blastocyst Development ◽  
Cell Counts ◽  
Development And Differentiation

Production of embryos in vitro with improved inner cell mass (ICM) and high ICM per total cell rate is a major objective in reproductive biotechnology. Exogenous all-trans retinoic acid (ATRA), a vitamin A metabolite, and endogenous retinoid regulate development and differentiation during bovine morula to blastocyst transition in vitro. ATRA binds to retinoic acid-receptor (RAR), and the ATRA isomere 9-cis-retinoic acid (9-cis-RA) binds to both RAR and the retinoid X receptor (RXR). The unspecific binding of 9-cis-RA to receptors makes it difficult to study RXR transactivation. Therefore, in this work we studied blastocyst development and cell counts by using a specific synthetic RXR agonist [LG100268 LG; a gift of Ligand Laboratories] as opossed to the effect exerted by ATRA upon RAR binding. Cumulus-oocyte complexes from slaughterhouse ovaries were matured and fertilized in vitro. Presumptive zygotes were cultured in B2 medium with Vero cells until 139 h post-insemination (Day 6), the time at which embryos [morulae (e90%) + early blastocysts] underwent treatments for 48 h in 400 �L of SOFaaci + 5% FCS. Data (5 replicates per experiment) were analyzed by CATMOD for effects, processed by GLM and Duncan's test, and expressed as LSM � SE (a,b,c P d 0.05). After a LG dose-response experiment (n = 480 morulae), blastocysts rates from LG 1 �M on Day 7 were higher than LG 10 �M, LG 0.1 �M, and LG 0 �M (Day 7: 42.8 � 4.1 vs. 34.4 � 3.7, 36.8 � 3.7, and 32.4 � 3.7, respectively). On Day 8, LG 1 �M also yielded more blastocysts than LG 0.1 �M (50 � 4.2 vs. 44.4 � 3.7, respectively). By differential cell counting (n = 113 blastocysts), hatched blastocysts with LG 10 �M showed proliferation in the ICM, while trophectoderm (TE) cells decreased conversely to LG concentration. These effects were not obvious in expanded blastocysts. In a subsequent experiment (n = 340 morulae), ATRA led to blastocysts rates on Day 8 that were higher than negative, untreated controls, but not different from LG 1 �M (42.4 � 2.4 vs. 33.1 � 2.0 and 36.0 � 2.4, respectively). ATRA and LG 1 increased TE in expanded blastocysts (n = 42) (102 � 13.2 and 96.23 � 13.2, respectively vs. 72.8 � 10.9 in the untreated group) but not in their hatched counterparts (n = 44). There were no differences in the ICM; but percentages of ICM per total cells were higher in hatched blastocysts cultured with ATRA than in expanded LG 1 �M blastocysts and expanded controls (39.5 � 5.5 vs. 24.2 � 5.7, and 20.9 � 4.7, respectively). Manipulation of retinoid receptor-specific pathways make it possible to control blastocyst development and differentiation, leading to embryos of improved quality and viability. Work is in progress to analyze gene expression in these blastocysts. This work was supported by grant MCYT, project AGL-2005-04479.

176 EFFECT OF LEUKEMIA INHIBITORY FACTOR FROM HUMAN AND MOUSE ORIGIN ON THE DEVELOPMENT OF BOVINE EMBRYOS PRODUCED IN VITRO

2007 ◽  
Vol 19 (1) ◽  
pp. 204
Author(s):  
C. De Frutos ◽  
A. Rodríguez ◽  
C. Díez ◽  
J. N. Caamaño ◽  
N. Facal ◽  
...  
Keyword(s):  
Leukemia Inhibitory Factor ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Developmental Rate ◽  
Total Cell ◽  
Inhibitory Factor ◽  
Bovine Embryos ◽  
Cell Counts

Leukemia Inhibitory Factor (LIF) is a cytokine with potential to influence embryonic quality and proliferation within the inner cell mass (ICM). However, conflicting effects of LIF have been reported with in vitro-produced (IVP) bovine embryos, in spite of LIF receptor (LIFr) and gp130 transcripts being expressed at all stages during pre-implantation development (Niemann and Wrenzycki 2000 Theriogenology 53, 21–34). As there is no commercially available bovine LIF (bLIF), researchers have used human LIF (hLIF) because of its greater sequence homology compared to murine LIF (mLIF). However, mLIF has been not compared with hLIF in culture with bovine embryos; thus this was the aim of this study. Cumulus–oocyte complexes from slaughterhouse ovaries were matured and fertilized in vitro and presumptive zygotes cultured in modified synthetic oviduct fluid with 6 g L-1 BSA. At 139 h post-insemination (Day 6), a total of 423 morulae (&gt;90%) and early blastocysts were cultured for 48 h with: (1) 100 ng mL-1 recombinant mLIF (Sigma-Aldrich Quimica SA, Madrid, Spain); (2) 100 ng mL-1 recombinant hLIF (Sigma); and (3) no LIF. Data (6 replicates) were processed by GLM and Duncan&apos;s test, and expressed as LSM � SE (ab: P &lt; 0.05; xy: P &lt; 0.01). Development was recorded up to the hatched blastocyst stage and cells were differentially counted in the ICM and trophectoderm (TE) following the method described by Thouas et al. (2001 Reprod. Biomed. Online 3, 25–29). There were no differences within developmental rate on Day 7, but reduced blastocyst rates were observed on Day 8 between hLIF (42.0 � 3.9a and 27.2 � 3.3a) and controls (57.7 � 3.9b and 38.9 � 3.3b) at the medium and expanded stages, respectively, whereas mLIF had no effect (47.4 � 3.9 and 32.3 � 3.3). Contrary to development, Day 8 blastocysts showed decreased cell counts in both the ICM and the ICM/total cell proportions in the presence of mLIF (19.1 � 3.1x and 13.8 � 2.4x vs. 32.6 � 3.0y and 24.8 � 2.3y for controls, respectively), whereas hLIF had no effect (29.7 � 3.1y and 20.9 � 2.4y). No changes were seen in TE and total cell counts. The disparate effects exhibited by hLIF and mLIF during blastocyst formation may reflect the fact that these compounds are inappropriate to replace bLIF, and/or endogenous LIF probably suffices during bovine development. In fact, mouse embryonic development and blastocyst cell numbers decrease in murine embryos injected with LIF antisense nucleotides (Cheng et al. 2004 Biol. Reprod. 70, 1270–1276). Furthermore, embryonic stem (ES)-like cell derivation in bovine is possible with (Saito et al. 2003 Biochem. Biophys. Res. Com. 309, 104–113) and without (Mitalipova et al. 2001 Cloning 3, 59–67) exogenous LIF. Therefore, strategies to investigate LIF signalling in bovine embryos and stem cells should be reconsidered. This work was supported by Grant AGL2005-04479.

124 Cell Count of Bovine In Vitro-Produced Blastocysts After In Vitro Maturation in Glass versus Plastic Vials

2018 ◽  
Vol 30 (1) ◽  
pp. 202
Author(s):  
J. O. Secher ◽  
N. Hashem ◽  
J. H. Pryor ◽  
C. R. Long ◽  
J. Docherty ◽  
...  
Keyword(s):  
Cell Count ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Total Cell ◽  
Differential Staining ◽  
Atmospheric Air ◽  
Cell Counts ◽  
Blastocyst Rate

Optimal bovine in vitro oocyte maturation (IVM) is a prerequisite for subsequent optimal blastocyst rates. Ovum pick-up (OPU), by which cumulus–oocyte complexes (COC) are collected in vivo, is performed outside a laboratory and often requires IVM to take place during transportation from the farm to the IVF laboratory. Hashem et al. (2017 Reprod. Fertil. Dev. 29, 179) demonstrated that blastocyst rates are affected by type of vial (glass v. plastic), number of COC per vial, and volume of medium per vial. This was achieved by maturing more than 2500 COC from slaughterhouse material under contrasting conditions, followed by standardised IVF and in vitro culture (IVC) and observation of blastocyst rates, morphology (1: poor; 2: good; 3: excellent), and kinetics (1: blastocyst; 2: expanded blastocyst ; 3: hatching/hatched blastocyst). Here we examined differential staining of a subset of expanded blastocysts (XB) from the previous study to assess the influence of vial material, medium volume, and number of COC per vial on total cell count, number and ratio of inner cell mass (ICM), and trophectoderm (TE) cells. In experiment 1 (4 groups), oocytes were matured in different vials without lids in an incubator at 5.5% CO2 in humidified atmospheric air at 38.5°C to assess plastic toxicity. In experiment 2 (6 groups) and experiment 3 (6 groups), the 2 best performing vials-polypropylene cryovials (Sigma-Aldrich, St. Louis, MO, USA) and glass vials (VWR International, Radnor, PA, USA)-containing 50% (Exp. 2) or 95% (Exp. 3) medium volume per vial and 5, 20, or 45 COC per vial were tested. In experiments 2 and 3, the vials were closed and incubated in atmospheric air at 38.5°C. All groups were evaluated for blastocyst rates, kinetics, and morphology. Because kinetics (range 2.01–2.25) and morphology (range 2.15–2.50) were similar in all groups, only XB were collected from each group. These were fixed and stained with CDX2 antibody and Hoechst (Wydooghe et al. 2011 Anal. Biochem. 416, 228-230) and their ICM and TE cells were counted. The cells were counted manually in blinded groups using an inverted fluorescence microscope and 16× magnification. Counts of total, ICM, and TE cells were compared between treatments by a two-way ANOVA analysis. A total of 240 XB from the 16 different vial groups were counted in the 3 experiments, with average total cell counts of 139 (110–211) and ICM cell counts of 44 (28–75). Even though the blastocyst rates differed between some of the groups, the cell counts within the XB did not differ statistically significantly between groups. In fact, the highest cell count was found in the glass vial group with the lowest blastocyst rate (45 COC per vial in 50% medium volume; blastocyst rate 28%, total cells 211, ICM cells 75). We have previously demonstrated that the type of vial, number of COC per vial, and the volume of medium per vial influence the subsequent blastocyst rates. It is concluded, however, that the embryos able to proceed to the blastocyst stages seem to be of the same quality in all groups, assessed by kinetics, morphology, and cell counts within XB.

scholarly journals 144ARE BLASTOCYST RATES A RELIABLE INDICATOR OF THE QUALITY OF AN IVP SYSTEM?

2004 ◽  
Vol 16 (2) ◽  
pp. 194 ◽  
Author(s):  
B. Avery ◽  
T. Greve
Keyword(s):  
Developmental Stage ◽  
Developmental Stages ◽  
Inner Cell Mass ◽  
Relative Proportion ◽  
Cell Mass ◽  
Tween 20 ◽  
Hatching Rate ◽  
Trophoblast Cells ◽  
Cell Counts

Normally blastocyst rates are used to document the efficiency of an IVP system, because routine transfer of all embryos is not a realistic approach. Even though pregnancies are established, there will only be a weak correlation to a given IVP system because the embryos for transfer have been highly selected. The aim of this study was to analyze the in vitro development of bovine IVM/IVF oocytes after culture in SOFaa medium with or without the presence of bovine oviduct cells (BOEC) under 5% or 20% O2 in 5% CO2 and 38.5°C in order to select the optimal IVC system under the given circumstances. The study was based on six replicates and 2373 inseminated oocytes retrieved from abattoir ovaries, and the quality markers were Day 8 blastocyst rates (BL per inseminated oocytes), morphology, kinetics, and cell count. From the relative proportion of BL, XB, and H, an average developmental stage (kinetics) could be assigned. Ranking was based on BL rate, rates of A and B graded BL, and the average developmental stage. Established standard procedures were used for IVM (23h in DMEM with 5% serum and eCG/hCG), and IVF (23h in TALP with heparin), and the inseminated oocytes were randomly allocated into four IVC groups (5% O2, 5% O2/BOEC; 20% O2, and 20% O2/BOEC) to be cultured in groups of 25 in 0.1mL oil-covered droplets of SOFaa with 5% serum (Holm P et al. 1999 Theriogenology 52, 683–700). The morphology was graded as A: compact and distinct inner cell mass, regular morphology of trophoblast cells, development corresponding to the expected; B: smaller or less distinct inner cell mass, a few degenerated trophoblast cells or slight fragmentation, development corresponding to the expected; C: dispersed or no inner cell mass, degenerated trophoblast cells or much fragmentation, developmental arrest. For cell counts the zona and cytoplasm from the individual blastocysts were lysed in 0.01M HCL and 0.1% Tween 20, leaving the isolated nuclei to be fixed in 3:1 methanol:acetic acid on a slide (Viuff D et al. 2002 Biol. Reprod. 63, 1143–1148). The kinetics were assessed as hatched per total BL at Day 8 (Fisher’s exact test, P&lt;0.01). The BL rates were significantly lower in the 20% O2 group (23% v. 31%, 32%, 33% in the other groups, respectively), while the hatching rate was significantly higher in the 5% O2 group (35% v. 12%, 10%, 18%). The frequency of A-quality blastocysts was significantly higher in the 5% O2 and 20% O2/BOEC groups (46%, 41%) than in the 20% O2 and 5% O2/BOEC groups (27%, 22%). The B-quality frequency did not differ between the four groups (41%, 40% v. 48%, 45%), whereas the C-quality inversely reflected the A-quality (13%, 19% v. 25%, 33%). There were no differences in the cell counts between the same quality grades in the four systems. An A-grade expanded BL had 134±50 cells (mean±SD), a B-grade 94±45; a hatched A-grade BL had 168±48 cells, a B-grade 143±54. This study shows that regardless of differences in average developmental stages (kinetics) and morphology, similar blastocyst rates can be obtained. Using these criteria our four IVC groups would be ranked as (1) 5% O2, (2) 20% O2/BOEC, (3) 5% O2/BOEC (4) 20% O2. In conclusion, when evaluating the suitability of an IVP system, morphology and kinetics should be considered as well as blastocyst rates.

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