84 Actions of DKK1 on the bovine embryo during the morula-to-blastocyst stage of development on pregnancy outcomes and placental hormone secretion after embryo transfer

2022 ◽  
Vol 34 (2) ◽  
pp. 279
T. F. Amaral ◽  
J. G. V. Grázia ◽  
A. M. Gonella-Diaza ◽  
L. A. G. Martinhão ◽  
D. Heredia ◽  
K.H. Lu ◽  
I. Gordon ◽  
M.P. Boland ◽  
T.F. Crosby

The development of an efficient laboratory procedure which would enable cattle ovarian oocytes to be matured in vitro, fertilized and cultured in vitro to the blastocyst stage of development could have important practical and scientific implications. The commercial exploitation of certain embryo transfer techniques applicable in cattle (eg., twinning by embryo transfer) might be facilitated by the development of such a procedure and there would be many advantages to having a cheap source of embryos available for research purposes. The present report deals with some of the studies recently carried out in this laboratory aimed at utilising follicular oocytes recovered from the ovaries of cattle slaughtered for beef at the abattoir. Such studies have been undertaken over a period of almost twenty years, starting with the work of Sreenan (1968)* but it now realised that the oocytes of farm mammals are incapable of normal development until after the completion of complex changes during maturation.

2020 ◽  
Vol 98 (11) ◽  
Peter J Hansen

Abstract Typically, bovine embryos are transferred into recipient females about day 7 after estrus or anticipated ovulation, when the embryo has reached the blastocyst stage of development. All the biological and technical causes for failure of a female to produce a blastocyst 7 d after natural or artificial insemination (AI) are avoided when a blastocyst-stage embryo is transferred into the female. It is reasonable to expect, therefore, that pregnancy success would be higher for embryo transfer (ET) recipients than for inseminated females. This expectation is not usually met unless the recipient is exposed to heat stress or is classified as a repeat-breeder female. Rather, pregnancy success is generally similar for ET and AI. The implication is that either one or more of the technical aspects of ET have not yet been optimized or that underlying female fertility that causes an embryo to die before day 7 also causes it to die later in pregnancy. Improvements in pregnancy success after ET will depend upon making a better embryo, improving uterine receptivity, and forging new tools for production and transfer of embryos. Key to accelerating progress in improving pregnancy rates will be the identification of phenotypes or phenomes that allow the prediction of embryo competence for survival and maternal capacity to support embryonic development.

2014 ◽  
Vol 26 (1) ◽  
pp. 175
M. S. Ortega ◽  
J. B. Cole ◽  
T. S. Sonstegard ◽  
P. J. Hansen

The objective was to identify patterns of expression during the pre-implantation period of several genes associated with genetic variation in fertility (CWC15) or development to the blastocyst stage (C1QB, MON1B, PARM1, PCCB, PMM2, TBC1D24, and WBP1). These genes are involved in cellular processes such as mRNA splicing, immune protection, fatty acid oxidation, resistance to apoptosis, glycoprotein synthesis, and intracellular transport. Embryos were produced in vitro from slaughterhouse oocytes and semen using a mix of Bos taurus and Bos indicus cows and bulls. Pools of 40 matured oocytes or embryos at the 2-cell [27–31 h post-insemination (hpi)], 3- to 4-cell (46–52 hpi), 5- to 8-cell (49–59 hpi), 9- to 16-cell (72–75 hpi), morula (120–123 hpi), and blastocyst (168–171 hpi) stages were collected. The RNA was purified and synthesised into cDNA for real-time qPCR analysis. The YWHAZ, GAPDH, and SDHA were used as steady-state controls of expression. A total of 5 pools were analysed for each of the 6 stages. The C1QB was not detected at any stage; however, transcript amounts for the other genes were affected by stage of development (P < 0.05). The WBP1 remained low from the oocyte to the 5- to 8-cell stage (fold-change relative to matured oocytes: 1.0 ± 0.2 v. 1.4 ± 0.2), increased at the 9- to 16-cell stage (14.8 ± 0.2), and decreased to the blastocyst stage (7.1 ± 0.2). The expression pattern of PARM1 was similar, with greatest expression at the 9- to 16-cell stage. In contrast, expression of PMM2 and TBC1D24 was highest at the 2-cell stage and decreased at the morula and blastocyst stages. Expression of CWC15, MON1B, and PCCB decreased steadily from the oocyte to the blastocyst stage. Given that the major round of embryonic genome activation occurs at the 8- to 16-cell stage, it is possible that PARM1 and WBP1 play important roles around this time. The PMM2 and TBC1D24 may represent genes activated before the 8- to 16-cell stage. The CWC15 has been identified as a lethal gene; results suggest lethality occurs after the blastocyst stage. Further research will clarify the role and importance of these genes in the early development of the bovine embryo. The authors acknowledge support from AFRI Grant No. 2013–68004–20365 from USDA NIFA.

2013 ◽  
Vol 25 (1) ◽  
pp. 182
R. Morató ◽  
T. Mogas

Although slow freezing continues to be the most widely used technique of cryopreservation for bovine in vivo- and in vitro-produced embryos, vitrification has been tested in different species with good results, especially when dealing with in vitro-produced embryos. Vitrification represents a minor expense in time and equipment associated with cryopreservation compared with conventional slow freezing. However, vitrification, which is the most common method for human embryo cryopreservation, has not been widely adopted by embryo-transfer practitioners for commercial use in cattle. In general, vitrification requires gradual cryoprotectant dilution in a laboratory setting, and it is difficult to perform in the field. The objective of this study was to develop a one-step dilution method suitable for one-step bovine embryo transfer using the cryotop vitrification method. Embryos produced in vitro by standard procedures were vitrified at the blastocyst stage at Day 7 post-insemination in a mixture of 15% ethylene glycol + 15% dimethyl sulfoxide + 0.5 M sucrose using cryotop devices. Embryos were randomly assigned to 1 of 3 warming methods: (1) W3: warming was carried out following the cryotop method (1 M sucrose for 1 min, 0.5 M sucrose for 3 min, and 0 M sucrose for 6 min); (2) W1/0.5: embryos were warmed directly in 0.5 M sucrose for 3 min; and (3) W1/0: embryos were warmed directly in 0 M sucrose for 5 min. Survival rates were assessed in terms of blastocyst re-expansion, hatching, and hatched status at 3 and 24 h after warming. Data were analyzed using the statistical analysis systems package (SAS, v9.1). Data from at least 3 replicates were collected. Comparisons of vitrified–warmed blastocyst survival rates between groups were performed using the chi-squared test. The level of statistical significance was set at P < 0.05. When embryo survival was evaluated at 3 h postwarming, embryos warmed using the 3-step dilution protocol and those warmed directly in 0.5 M sucrose showed higher percentages of survival (W3: 89.8%, n = 98; W1/0.5: 87.5%, n = 64; P < 0.05) than those blastocysts that were warmed directly in 0 M sucrose (W1/0: 66.4%, n = 146). However, similar rates irrespective of the warming procedure were observed at 24 h postwarming (W3: 85.7%, W1/0.5: 88.2%, W1/0: 70.5%). Warmed in vitro-produced embryos exposed to W3 (47.6%) and W1/0.5 (35.6%) achieved higher percentages of embryos developing to the hatched blastocyst stage after 24 h of culture than those embryos warmed in W1/0 (20.4%; P < 0.05). Our results indicate that direct warming and dilution of cyotop-vitrified embryos in 0.5 M sucrose for 3 min may enable one-step bovine embryo transfer without requirement of a microscope or other laboratory equipment, simplifying the embryo-transfer procedure of vitrified embryos on farm at the same level of complexity as carrying out AI. Support came from Spanish MEC (RZ2010-00015-0-00; AGL2010-19069) and Generalitat de Catalunya (2009 SGR 621).

2004 ◽  
Vol 16 (2) ◽  
pp. 254 ◽  
R.C. Fry ◽  
C.R. Earl ◽  
F.K. Hollinshead ◽  
D. Wild ◽  
W. Lindemans

Previously we demonstrated that sex-sorted sperm could produce IVF embryos from juvenile and adult cattle at rates similar to those for unsorted sperm (Fry et al., 2003 Theriogenology 52, 198). In this study we investigated the pregnancy rates of recipient cattle following the transfer of frozen/thawed IVF embryos generated from young heifers using sex-sorted and unsorted sperm. COCs collected from FSH-stimulated Senepol or Beefex heifers by TVR were matured, fertilized with either sex-sorted or unsorted Senepol sperm and cultured for 6 days under our standard laboratory conditions (Fry et al., 2003 Theriogenology 59, 446, Earl et al., 1997 Theriogenology 47, 255). Embryos reaching the blastocyst or expanded blastocyst stage of development were frozen by the CL-V method of vitrification. Briefly, embryos were equilibrated for 5–10min in HEPES-199 media containing 20% FCS (HM), placed in HM containing 10% EG, 10% DMSO for approximately 2 minutes and then in HM containing 20% EG, 20% DMSO for between 20–60sec (Vatja et al., 1997 Cryoletters 18, 191). Vitrification was achieved by collecting between 5–10 IVF embryos in a 3-μL droplet and securing this droplet to a coded CL-V holder. The droplet was vitrified using the CL-V kit (Lindemans et al., 2004 Theriogenology in press) and then sealed in a precooled ‘‘straw’’ for storage in liquid nitrogen. To thaw, the ‘‘straw’’ with specimen was removed from storage;; the specimen droplet was withdrawn from the ‘‘straw’’ and placed directly into HM containg 0.2M sucrose (SM). After approximately 5–10min each embryo was assessed, loaded into a tomcat catheter in SM and transferred surgically into a recipeint cow within 10–15min of thaw. Of 129 Brahman and Brahman cross cows receiving 2 injections of 125μg cloprostenol 11 days apart, 60 exhibited oestrus 2–4 days after the second injection and 53 were deemed suitable for embryo transfer. Pregnancy was determined by ultrasound on Day 40. No difference in pregnancy rate was found between treatment groups (P&gt;0.05; Table 1). The low submission rate (60/129) and pregnancy rate for the in vivo control group indicate that the fertility of the recipient cows may have been compromised by the drought conditions predominating in Central Queensland. Notwithstanding, the CL-V method for the vitrification of IVF embryos produced by either sex-sorted or unsorted sperm gave similar and very promising pregnancy results of around 40%. This provides new opportunities for the rapid banking of large numbers of sexed IVF embryos generated from elite cattle by TVR for user friendly embryo transfer programs. Table 1 Pregnancies from IVF embryos derived from sex-sorted and unsorted sperm and frozen by the CL-V method of vitrification, or from in vivo embryos frozen in glycerol

2021 ◽  
Aimé Jazmín Garza Arredondo ◽  
Diana Elisa Zamora Ávila ◽  
Uziel Castillo Velásquez ◽  
Gustavo Moreno Degollado ◽  
José Fernando De La Torre Sánchez ◽  

Abstract Endogenous heat shock cognate 71 kDa protein (HSC70) has a vital role in early embryonic development. This study assessed the effects of exogenous HSC70 on bovine embryo development and expression of genes associated with apoptosis. Expression analyses of HSPA1A, HSPA8, Bcl-2, and Bax genes were performed in bovine embryos in vivo on day 7 of development. Subsequently, expression of HSPA1A and HSPA8 were associated with apoptotic genes (Bcl-2 and Bax) in cultured bovine embryos in vitro that were supplemented with various concentrations (0 or control group, 50, and 100 ng) of HSC70. The results indicated that the control group (0 ng) in vitro embryos had higher expression of HSPA8, Bax, and Bcl-2 genes, compared with the vivo embryos (P < 0.01). In vitro-produced embryos supplemented with 50 ng or 100 ng HSC70 had higher expression of HSPA1A, HSC70, Bcl-2, and Bax genes, compared with the control group (P < 0.01). Embryos supplemented with 100 ng had greater expression of the HSPA8 gene compared with the control group and the group supplemented with 50 ng. However, embryos supplemented with 50 ng had better characteristics (i.e., stage of development and quality) than the control and 100-ng groups. In conclusion, supplementation of in vitro culture medium with HSC70 promoted development to the blastocyst stage and improved blastocyst quality.

2020 ◽  
Qing Li ◽  
Liming Ruan ◽  
Lingling Zhu ◽  
Zengyu Yang ◽  
Maoling Zhu ◽  

Abstract Objective: The aim of this study was to evaluate the association between serum estradiol (E2) and pregnancy outcomes of cleavage- or blastocyst-stage frozen embryo transfer (FET) cycles using hormone replacement therapy.Methods: A total of 776 FET cycles (669 couples) performed from January 2016 to December 2019 were included in the present retrospective cohort study. The impact of progesterone-initiation-day serum E2 levels on the ongoing pregnancy/live birth (OP/LB) rates was determined, and cleavage-stage embryo transfers and blastocyst-stage embryo transfers were analyzed separately. Results: Regarding cleavage-stage embryo transfer cycles, serum E2 levels on progesterone initiation day were significantly lower in the OP/LB group than in the non-OP/LB group (214.75 ± 173.47 vs. 253.20 ± 203.30 pg/ml; P = 0.023). In addition, there were downward trends in implantation, clinical pregnancy and OP/LB rates with increasing E2 levels. However, in blastocyst-stage embryo transfer cycles, such trends were not observed, and there was no significant difference between the OP/LB group and the non-OP/LB group. Logistic regression analysis revealed that E2 levels on progesterone initiation day in cleavage-stage embryo transfer cycles were independently associated with OP/LB (odds ratio = 1.000, 95% confidence interval: 1.000-1.001, P = 0.008). The areas under the receiver operating characteristic curve were 0.55 in cleavage-stage embryo transfer cycles and 0.53 in blastocyst-stage embryo transfer cycles.Conclusions: The association of low OP/LB rates with elevated E2 levels on the progesterone initiation day in cleavage-stage embryo transfer cycles suggests that E2 levels should be monitored during artificial cleavage-stage embryo transfer cycles. However, it is not necessary to monitor serum E2 levels when transferring blastocysts in artificial FET cycles.

2020 ◽  
Vol 46 (4) ◽  
pp. 595-605 ◽  
Yi‐xin Li ◽  
Jin Wang ◽  
Tian‐ze Sun ◽  
Mo‐qi Lv ◽  
Pan Ge ◽  

Reproduction ◽  
2017 ◽  
Vol 153 (4) ◽  
pp. 405-419 ◽  
Paula Tribulo ◽  
James I Moss ◽  
Manabu Ozawa ◽  
Zongliang Jiang ◽  
Xiuchun (Cindy) Tian ◽  

The bovine was used to examine the potential for WNT signaling to affect the preimplantation embryo. Expression of seven key genes involved in canonical WNT signaling declined to a nadir at the morula or blastocyst stage. Expression of 80 genes associated with WNT signaling in the morula and inner cell mass (ICM) and trophectoderm (TE) of the blastocyst was also evaluated. Many genes associated with WNT signaling were characterized by low transcript abundance. Seven genes were different between ICM and TE, and all of them were overexpressed in TE as compared to ICM, including WNT6, FZD1, FZD7, LRP6, PORCN, APC and SFRP1. Immunoreactive CTNNB1 was localized primarily to the plasma membrane at all stages examined from the 2-cell to blastocyst stages of development. Strikingly, neither CTNNB1 nor non-phospho (i.e., active) CTNNB1 was observed in the nucleus of blastomeres at any stage of development even after the addition of WNT activators to culture. In contrast, CTNNB1 associated with the plasma membrane was increased by activators of WNT signaling. The planar cell polarity pathway (PCP) could be activated in the embryo as indicated by an experiment demonstrating an increase in phospho-JNK in the nucleus of blastocysts treated with the non-canonical WNT11. Furthermore, WNT11 improved development to the blastocyst stage. In conclusion, canonical WNT signaling is attenuated in the preimplantation bovine embryo but WNT can activate the PCP component JNK. Thus, regulation of embryonic development by WNT is likely to involve activation of pathways independent of nuclear actions of CTNNB1.

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