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.

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.

scholarly journals Leukemia Inhibitory Factor Stimulates Primitive Endoderm Expansion in the Bovine Inner Cell Mass

2021 ◽  
Vol 2 ◽  
Author(s):  
Lydia K. Wooldridge ◽  
Alan D. Ealy
Keyword(s):  
Leukemia Inhibitory Factor ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Inhibitory Factor ◽  
Primitive Endoderm ◽  
Total Cell Number ◽  
Cell Numbers ◽  
Bovine Blastocyst

Previous work determined that bovine interleukin-6 (IL6) increases inner cell mass (ICM), primitive endoderm (PE), and total cell number in in vitro produced (IVP) bovine blastocysts. Another IL6 family member, leukemia inhibitory factor (LIF), has the potential to produce the same effects of IL6 due to the presence of its receptor in bovine blastocysts. We compared the abilities of LIF and IL6 to increase ICM cell numbers in day 7, 8, and 9 IVP bovine blastocysts. Supplementation with 100 ng/ml LIF from day 5 onward improved blastocyst formation rates on days 7 and 8 similar to what was observed when supplementing 100 ng/ml IL6. However, LIF supplementation did not cause an increase in ICM numbers like was observed after supplementing IL6. On day 9, increases in PE cell numbers were detected after LIF supplementation, but 300 ng/ml LIF was required to achieve the same effect on PE numbers that was observed by providing 100 ng/ml IL6. Collectively, these results show that LIF can mimic at least some of the effects of IL6 in bovine blastocyst.

scholarly journals 127APOPTOSIS IN BOVINE BLASTOCYSTS FROM FIVE DIFFERENT PRODUCTION SYSTEMS

2004 ◽  
Vol 16 (2) ◽  
pp. 186
Author(s):  
J.O. Gjørret ◽  
P. Maddox-Hyttel
Keyword(s):  
Production Systems ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Embryo Cell ◽  
Developmental Potential ◽  
Cell Numbers ◽  
Tunel Reaction

Regulation of apoptosis may be affected by factors during preimplantation development, and this is possibly related to embryo developmental potential. Here we investigate differences in the incidence of apoptotic nuclei in Day 7 bovine blastocysts produced by two different in vivo and three different in vitro methods. In vivo embryos were produced either by a regular superovulation procedure (reg group; n=29; Laurincik et al., 2003, Mol. Reprod. Dev. 65, 73–85), or by postponement of the LH surge (pp group; n=35; van de Leemput et al., 2001, Therio. 55, 573–592). In vitro embryos were derived from systems using either co-culture (cc group; n=30, Avery and Greve 2000, Mol. Reprod. Dev. 55, 438–445), or culture in synthetic oviduct fluid (SOF) with (S+group; n=35) or without serum (S− group; n=38; Holm et al., 1999, Theriogenology, 52, 683–700). Embryos were collected at approx. 168h post ovulation/insemination and subjected to chromatin staining and detection of DNA degradation by TUNEL reaction. The total number of nuclei, number of nuclei displaying apoptotic morphology (+M), number of nuclei displaying TUNEL reaction (+T), and number of nuclei displaying both markers simultaneously (M&amp;T) were scored according to J.O. Gjørret et al. (2003 Biol. Reprod. 69. in press). Only M&amp;T nuclei were regarded as apoptotic, and +M, +T, and apoptotic (M&amp;T) indices (%) were calculated for the trophoblast (tb), inner cell mass (i) and the total blastocysts (t) in each group. Significant differences were observed for all parameters when all groups were compared (ANOVA, P ranging from 0.024 to&lt;0.0001). Highest number of total nuclei were observed in the S+ group, whereas the lowest indices were observed in the pp group, which had significant lower indices in the i and t than in the reg., S+ and S− groups P&lt;0.05; Tukey’s post test for ANOVA). Highest indices were generally observed in the S− group. The results demonstrate that not only embryo cell numbers but also incidences of apoptotic markers are affected by the mode of production. However, in Day 7 bovine blastocysts high cell number is not consistent with a low incidence of apoptosis. Even though cell numbers appeared comparable in the two in vivo groups, their incidences of apoptosis were different, and the reg group displayed indices comparable to the in vitro groups, highlighting the importance of ovulation protocols when in vivo embryos are used as reference material in general. Table 1

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.

scholarly journals Leukemia inhibitory factor stimulates primitive endoderm expansion in the bovine inner cell mass

2021 ◽  
Author(s):  
Lydia K. Wooldridge ◽  
Alan D. Ealy
Keyword(s):  
Leukemia Inhibitory Factor ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Inhibitory Factor ◽  
Primitive Endoderm ◽  
Total Cell Number ◽  
Cell Numbers ◽  
Bovine Blastocyst

Abstract Previous work determined that bovine interleukin-6 (IL6) increases inner cell mass (ICM), primitive endoderm (PE) and total cell number in in vitro produced (IVP) bovine blastocysts. Another IL6 family member, leukemia inhibitory factor (LIF), has the potential to produce the same effects of IL6 due to the presence of its receptor in bovine blastocysts. We compared the abilities of LIF and IL6 to increase ICM cell numbers in day 7, 8 and 9 IVP bovine blastocysts. Supplementation with 100 ng/ml LIF from day 5 onward improved blastocyst formation rates on days 7 and 8 similar to what was observed when supplementing 100 ng/ml IL6. However, LIF supplementation did not cause an increase in ICM numbers like was observed after supplementing IL6. On day 9, increases in PE cell numbers were detected after LIF supplementation, but 300 ng/ml LIF was required to achieve the same effect on PE numbers that was observed by providing 100 ng/ml IL6. Collectively, these results show that LIF can mimic at least some of the effects of IL6 in bovine blastocyst.

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.

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 Analysis of DNA fragmentation in bovine somatic nuclear transfer embryos using TUNEL

Reproduction ◽  
2002 ◽  
pp. 813-819 ◽  
Author(s):  
M Fahrudin ◽  
T Otoi ◽  
NW Karja ◽  
M Mori ◽  
M Murakami ◽  
...  
Keyword(s):  
Dna Fragmentation ◽  
Nuclear Transfer ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Cell Stage ◽  
Vitro Fertilization ◽  
Fragmented Nucleus ◽  
Number Of Cells

The production of cloned animals is an inefficient process because of early or late embryonic losses. This study focused on the DNA fragmentation that occurs during embryonic development. The occurrence of DNA fragmentation was examined in bovine embryos produced by in vitro fertilization (IVF) and somatic cell nuclear transfer (NT) using the terminal deoxynucleotidyl transferase (TdT) nick-end labelling (TUNEL). IVF and NT embryos at the two-cell to blastocyst stage were stained by TUNEL for the analysis of DNA-fragmented nuclei and with propidium iodide for determination of the total number of cells. DNA fragmentation was first detected in NT embryos at the four-cell stage, but in IVF embryos at the six- to eight-cell stage. The percentage of embryos with at least one DNA-fragmented nucleus increased with the advance of the developmental stage of embryos in both IVF and NT groups. The DNA-fragmented nucleus index in NT embryos that developed beyond the four-cell stage was significantly higher (P<0.01) than that of IVF embryos at the same stage. In the both IVF and NT groups, TUNEL-labelled cells were detected in almost all blastocysts and were mainly observed in presumptive inner cell mass (ICM) cells of embryos. The DNA-fragmented nucleus index was negatively correlated with the total number of cells in NT blastocysts, but this relationship was not observed in IVF blastocysts. These results suggest that the high occurrence of DNA fragmentation observed in NT embryos may be related to early embryonic loss after transfer.

Sign in / Sign up

Export Citation Format

Share Document