29 Co-Incubation of Equine Cloned Embryos with Sialic Acid: Effect on Pregnancy Rate

2018 ◽  
Vol 30 (1) ◽  
pp. 154
Author(s):  
D. Vichera ◽  
R. Olivera ◽  
V. Arnold ◽  
J. Vergara ◽  
R. Jordan ◽  
...  

In vitro-produced equine embryos have certain morphological characteristics that differ from embryos produced in vivo. One of them is the absence or inadequate formation of the embryo capsule. This capsule is composed of mucin-like glycoproteins produced by the trophectoderm with high proportion of sialic acid, which confers anti-adhesive properties. This characteristic is necessary for the intrauterine embryo migration process to occur, which is vital and fundamental for pregnancy recognition. For this reason, inadequate formation of the glycoprotein capsule could result in a lower pregnancy rate. In this study, we aimed to evaluate the effect of cloned equine embryo co-incubation with sialic acid on blastocyst and pregnancy rates. To achieve this, equine oocytes obtained from slaughterhouse ovaries were matured in TCM-199 HEPES medium with 2% fetal bovine serum, 2% antibiotics, and 1 μg mL−1 FSH, incubated at 39°C for 24 h. Matured oocytes were denuded with pronase, enucleated, and fused to donor bone marrow mesenchymal cells according to Olivera et al. (2016 PLoS One 11, e0164049, 10.1371/journal.pone.0164049). Chemical activation was induced using 8.7 μM ionomycin for 4 min and embryos were incubated with 1 mM 6-DMAP and 5 mg mL−1 cycloheximide for 4 h. Afterwards embryos were cultured in microwells for 8 days in DMEM-F12 medium. On Day 6, cloned equine embryos were exposed to 5 μM sialic acid for 48 h (SA group). On Day 8, blastocysts were transferred to recipient mares 5 days post-ovulation and pregnancy was confirmed 15 days post-transfer by transrectal ultrasound. Embryo clones generated without sialic acid exposure were used as a control (C) group. Fisher test was used to analyse both blastocyst and pregnancy rates. Blastocyst rates were 14% (46/328) and 15% (62/413) and pregnancy rates were 30.4% (7/23) and 19.4% (6/31) for SA and C groups, respectively. No statistical differences were observed between groups for the analysed parameters, even though pregnancy rates tended to be higher in the SA group. This effect could be a consequence of higher concentrations of the glycoprotein involved in the formation of the embryo capsule.

1995 ◽  
Vol 7 (2) ◽  
pp. 281 ◽  
Author(s):  
SJ Silber ◽  
P Devroey ◽  
H Tournaye ◽  
Steirteghem AC Van

For men with uncorrectable obstructive azoospermia, their only hope of fathering a child is microsurgical epididymal sperm aspiration (MESA) combined with in vitro fertilization (IVF). In 1988, proximal epididymal sperm were demonstrated to have better motility than senescent sperm in the distal epididymis, and it was thought that retrieval of motile sperm from the proximal epididymis would yield reliable fertilization and pregnancy rates after conventional IVF. However, the results to date have been poor, and although a minority of patients achieved good fertilization rates with IVF, the vast majority (81%) had consistently poor or no fertilization and the pregnancy rate averaged only 9%. Recently, intracytoplasmic sperm injection (ICSI) has been successfully used to achieve fertilization and pregnancies for patients with extreme oligoasthenozoospermia. ICSI has therefore been applied to cases of obstructive azoospermia and, in this report, 67 MESA-IVF cases are compared with 72 MESA-ICSI cases. The principle that motile sperm from the proximal segments of the epididymis should be used for ICSI was followed, although in the most severe cases in which there was an absence of the epididymis (or absence of sperm in the epididymis), testicular sperm were obtained from macerated testicular biopsies. These sperm only exhibited a weak, twitching motion. In 72 consecutive MESA cases, ICSI resulted in fertilization and normal embryos for transfer in 90% of the cases, with an overall fertilization rate of 46%, a cleavage rate of 68%, and ongoing or delivered pregnancy rates of 46% per transfer and 42% per cycle. The pregnancy and take-home baby rates increased from 9% and 4.5% with IVF to 53% and 42% with ICSI. There were no differences between the results for fresh epididymal, frozen epididymal or testicular sperm, and the number of eggs collected did not affect the outcome. The results were also unaffected by the aetiology of the obstruction such as congenital absence of the vas deferens or failed vasoepididymostomy. The only significant factor which affected the pregnancy rate was female age. It is concluded that although complex mechanisms involving epididymal transport may be beneficial for conventional fertilization of human oocytes (in vivo or in vitro), none of these mechanisms are required for fertilization after ICSI. Given the excellent results with epididymal and testicular sperm, ICSI is obligatory for all future MESA patients. Finally, the use of ICSI with testicular sperm from men with non-obstructive azoospermia is also discussed.


2007 ◽  
Vol 19 (1) ◽  
pp. 297
Author(s):  
S. Li ◽  
W. Yu ◽  
J. Fu ◽  
Y. Bai ◽  
F. Jin ◽  
...  

Data collected from commercial embryo transfer programs in 63 farms in China during June 2002 to December 2005 was analyzed to examine the effects of various factors (biopsy, freezing, sample size, embryo development and quality, in vitro culture, and recipient quality) on pregnancy rates of in vivo-biopsied embryos. Embryos were flushed from superovulated dairy cattle and subjected to a biopsy for sexing determination using protocols and sexing kits supplied by AB Technology Ltd. Fresh embryos were implanted on the same day or frozen with AG freeze medium (AB Technology Ltd., Pullman, WA, USA) for later transfer. Recipients were synchronized with CIDA + PG protocols. Embryos were cultured in 6-well dishes containing 1.3 mL of holding medium (AB Technology Ltd.) in each well at room temperature (20–25�C) for examination of embryo survival in vitro. The chi-square test was used in statistic analysis. The implantation of fresh embryos after biopsy did not affect pregnancy rates (49.6%, 257/518) compared to that of non-biopsied fresh and frozen–thawed embryo groups (52.9%, 47/140 and 46.6%, 177/380, respectively). However, for biopsied embryos subjected to frozen and thawed procedures before implantation, particularly for those subjected to the removal of a larger biopsy, a reduced pregnancy rate was observed (41.8%, 297/710; P < 0.01). Pregnancy rates among biopsied embryos at 3 different development stages (morula-early blastocyst, blastocyst, and expanded blastocyst) were not different. Similar results were found between embryo groups of grade 1 and 2. A significant decrease in pregnancy rate (0/10) was observed with embryos held in vitro for a longer period of time (>5 h), suggesting detrimental effects of in vitro conditions on embryo survival. The highest pregnancy rate (68.0%) was observed in recipients synchronized for the first time before being implanted with biopsied embryos. Significant decreases in such rates were found in recipients synchronized for the second or third times or those with an abortion history at the first or second synchronization-implantation treatment (P < 0.01). Better pregnancy rates (45.6%, 41/90; 46.1%, 76/165; and 45.5%, 5/11) were obtained for recipients implanted with biopsied embryos at Days 7.5, 8.0, and 8.5 post-heat detection, respectively, compared to 16% at Day 7 (3/18, P < 0.05). It is concluded that mechanical treatment (cutting) does not reduce the survival of biopsied embryos; however, cryopreservation reduces their ability to survive in vivo. The analyses also suggest that holding embryos in vitro should not be longer than 5 h unless more favorable in vitro conditions can be provided. To achieve better results of implantation of biopsied embryos, embryo transfer should be performed during 7.5–8.5 days post-estrus, and the healthy recipients synchronized for the first time should be used.


2017 ◽  
Vol 29 (1) ◽  
pp. 157
Author(s):  
T. Nishisouzu ◽  
A. Abe ◽  
S. Matoba ◽  
O. Dochi ◽  
K. Okamura

Despite the rapid expansion of in vitro embryo production (IVP) technology for genetic improvement in the cattle industry in the last decades, pregnancy rates by the transfer of IVP embryos are still lower than those derived from in vivo-produced embryos. The objective of this study was to analyse factors affecting pregnancy rates after the transfer of IVP Japanese Black cattle embryos under farm conditions. Holstein heifers (n = 4,475) and cows (n = 8,541) were selected as recipients. Most cows (80%) were managed in tie-stall barns and most heifers (80%) were managed in pens. Embryo transfers were performed for 9 years, from 2004 to 2012. The embryos were produced from oocytes derived from a local abattoir and semen from 14 proven bulls by the Livestock Improvement Association of Japan (Hamano and Kuwayama 1993 Theriogelogy 39, 703–712). The fresh IVP embryos (quality; IETS code 1) that reached the blastocyst stage after 7 to 8 days (insemination = Day 0) were transported by an airplane (2 h) and subsequently by a car (1.5 h). Embryos were non-surgically transferred to each recipient on Day 7 to 9 of their natural oestrous cycle on farms. Pregnancy was diagnosed on Day 40 to 60 after oestrus. Pregnancy results were statistically analysed using the GLM procedures of SAS. The following variables were included in the model: recipient parity (0, 1, 2, or 3), day (7, 8, or 9) of the oestrous cycle at the time of embryo transfer, oestrus behaviour (increased activity observed by farmers), presence of mucus at oestrus, presence of blood after oestrus, and year (1, 2, 3, 4, 5, 6, 7, 8, or 9) and season (April–June as spring, July–September as summer, October–December as fall, or January–March as winter) of embryo transfer. The Bonferroni correction was used to counteract the problem of multiple comparisons. Heifers had significantly higher pregnancy rates than cows (51.0% v. 37.9%, respectively; P < 0.01), and first parity cows had higher pregnancy rates than third parity cows (42.9% v. 35.7%, respectively; P < 0.01). Pregnancy rates were significantly higher in recipients that received an embryo transfer on Day 8 of their oestrous cycle, than on Day 7 (46.6% v. 42.4%, respectively; P < 0.01). Recipients without oestrus behaviour had higher pregnancy rates than those with oestrus behaviour (46.3% v. 43.4%, respectively; P < 0.01). The presence of mucus and/or blood after oestrus and the season of transfer were not found to significantly affect pregnancy rates. The results of this study indicated that performing IVP embryo transfers on Day 8 of a recipient’s oestrous cycle will improve the pregnancy rate, season does not have an effect on pregnancy rate, and the detection of oestrus by monitoring increased activity is not always reliable and instead should be determined by multiple symptoms on farm conditions.


2008 ◽  
Vol 20 (1) ◽  
pp. 150 ◽  
Author(s):  
S. Menges ◽  
H. Wei ◽  
D. Faber ◽  
D. Kraemer ◽  
C. Long

In vitro-produced (IVP) bovine embryos are known to produce a lower pregnancy rate when compared to conventional in vivo-produced embryos. The inability of the IVP embryo to hatch from the zona pellucida (ZP) after embryo transfer is thought to be one contributing factor. This study was designed to evaluate the utilization of a microscope objective-mounted laser to cut the ZP to assist hatching prior to transfer into the recipient. Preliminary data were acquired to evaluate the effect of laser treatment on in vitro development and blastomere survival following treatment. In six replicates, bovine oocytes were in vitro-matured, fertilized, and cultured as per standard laboratory procedures (TransOva Genetics, Sioux Center, IA, USA). On Days 5, 6, and 7 of in vitro culture, embryos were randomly divided into 3 treatment groups: no treatment (Control; n = 63), sham ZP cut (Sham; n = 68), or ZP cut (Cut; n = 70). Control embryos were immediately returned to the incubator following selection. Sham embryos were exposed to all conditions as Cut except laser-assisted hatching. The XYClone� system is a 300-mW, class 1 laser that emits a 3.5-µm beam at a wavelength of 1480 nm (Hamilton Thorne Biosciences, Beverly, MA, USA). This laser was used to produce the Cut group, using a pulse strength of 90% and pulse length of 600 µs. Embryos were returned to culture until Day 8 when rates of embryonic development and the percentage of live cells were determined. Chi-square was used to analyze all data. No significant effect of treatment or day of exposure was noted in either the total number of developing embryos or the ratio of live cells in each embryo. Mean live cells ranged from 89 to 96% across all treatments regardless of day of treatment. To investigate IVP embryo viability after laser-assisted hatching, commercially produced embryos (TransOva Genetics, Sioux Center, IA, USA) were randomly divided into two groups on the day of transfer, Control or Cut. The ZP of treated embryos were cut with slightly reduced laser exposure of 80% pulse strength and pulse length of 500 µs on Day 7, immediately prior to transfer into estrus-synchronized recipients. Pregnancy rates were determined via ultrasonagraphy at Day 30 (n = 337) and, due to the commercial nature of this project, only a subset of the Day 30 pregnant cows was checked at Day 60 (n = 289). The 30-day pregnancy rates were 49.2% and 54.1% for Control (n = 189) and Cut (n = 148) embryos, respectively, and were not statistically different (P > 0.05). However, at Day 60, the pregnancy rates for the Control (45.7%; n = 166) and Cut groups (57.7%; n = 123) were statistically different (P < 0.05). These results demonstrate that laser-assisted hatching using the XYClone system can improve 60-day pregnancy rates for in vitro-produced embryos.


1993 ◽  
Vol 70 (04) ◽  
pp. 676-680 ◽  
Author(s):  
H F Kotzé ◽  
V van Wyk ◽  
P N Badenhorst ◽  
A du P Heyns ◽  
J P Roodt ◽  
...  

SummaryPlatelets were isolated from blood of baboons and treated with neuraminidase to remove platelet membrane sialic acid, a process which artificially ages the platelets. The platelets were then labelled with 111In and their mean life span, in vivo distribution and sites of Sequestration were measured. The effect of removal of sialic acid on the attachment of immunoglobulin to platelets were investigated and related to the Sequestration of the platelets by the spleen, liver, and bone marrow. Removal of sialic acid by neuraminidase did not affect the aggregation of platelets by agonists in vitro, nor their sites of Sequestration. The removal of 0.51 (median, range 0.01 to 2.10) nmol sialic acid/108 platelets shortened their life span by 75 h (median, range 0 to 132) h (n = 19, p <0.001), and there was an exponential correlation between the shortening of the mean platelet life span and the amount of sialic acid removed. The increase in platelet-associated IgG was 0.112 (median, range 0.007 to 0.309) fg/platelet (n = 25, p <0.001) after 0.79 (median, range 0.00 to 6.70) nmol sialic acid/108 platelets was removed (p <0.001). There was an exponential correlation between the shortening of mean platelet life span after the removal of sialic acid and the increase in platelet-associated IgG. The results suggest that platelet membrane sialic acid influences ageing of circulating platelets, and that the loss of sialic acid may have exposed a senescent cell antigen that binds IgG on the platelet membrane. The antibody-antigen complex may then provide a signal to the macrophages that the platelet is old, and can be phagocytosed and destroyed.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Maria-Argyro Karageorgou ◽  
Dimosthenis Stamopoulos

AbstractRadiolabeled magnetic nanoparticles are promising candidates as dual-modality-contrast-agents (DMCA) for diagnostic applications. The immunocompatibility of a new DMCA is a prerequisite for subsequent in vivo applications. Here, a new DMCA, namely Fe3O4 nanoparticles radiolabeled with 68Ga, is subjected to immunocompatibility tests both in vitro and in vivo. The in vitro immunocompatibility of the DMCA relied on incubation with donated human WBCs and PLTs (five healthy individuals). Optical microscopy (OM) and atomic force microscopy (AFM) were employed for the investigation of the morphological characteristics of WBCs and PLTs. A standard hematology analyzer (HA) provided information on complete blood count. The in vivo immunocompatibility of the DMCA was assessed through its biodistribution among the basic organs of the mononuclear phagocyte system in normal and immunodeficient mice (nine in each group). In addition, Magnetic Resonance Imaging (MRI) data were acquired in normal mice (three). The combined OM, AFM and HA in vitro data showed that although the DMCA promoted noticeable activation of WBCs and PLTs, neither degradation nor clustering were observed. The in vivo data showed no difference of the DMCA biodistribution between the normal and immunodeficient mice, while the MRI data prove the efficacy of the particular DMCA when compared to the non-radiolabeled, parent CA. The combined in vitro and in vivo data prove that the particular DMCA is a promising candidate for future in vivo applications.


2016 ◽  
Vol 41 ◽  
pp. 70 ◽  
Author(s):  
P.M. McCue ◽  
R.A. Ferris ◽  
J. Stokes ◽  
J. Hatzel ◽  
D. Trundell ◽  
...  

2004 ◽  
Vol 16 (2) ◽  
pp. 213 ◽  
Author(s):  
J. Small ◽  
M. Colazo ◽  
D. Ambrose ◽  
R. Mapletoft ◽  
J. Reeb ◽  
...  

The objective was to evaluate the effect of pLH treatment on pregnancy rates in recipients receiving in vivo- or in vitro-produced bovine embryos. Heifers (n=37) and lactating (n=28) and non-lactating (n=150) beef cows were treated at random stages of the cycle with 100μg GnRH i.m. (Cystorelin, Merial Canada Inc., Victoriaville, Quebec, Canada) on Day −9, 500μg cloprostenol i.m. (PGF; Estrumate, Schering Plough Animal Health, Pointe-Claire, Quebec, Canada) on Day —2 and GnRH on Day 0 (66h post-PGF; without estrus detection). Cattle were placed at random, by class, into three groups: no further treatment (Control; n=71), or 12.5mg pLH (Lutropin-V, Bioniche Animal Health, Belleville, Ontario, Canada) on Day 5 (n=72) or on Day 7 (n=72) after the second GnRH. On Day 7, cattle with a CL &gt;10mm in diameter (determined ultrasonically) received in vivo-produced, fresh (Simmental) or frozen (Holstein), or in vitro-produced frozen (Holstein) embryos (embryo type balanced among groups). Embryos were cryopreserved in 10% ethylene glycol; in vivo-produced frozen embryos were thawed 5 to 10s in air, 15s in a water-bath at 30°C and then “direct-transferred” nonsurgically. In vitro-produced frozen embryos (donated by IND Lifetech Inc., Delta, British Columbia, Canada) were thawed in a water-bath at 27°C for 10s and placed in ViGro Holding Plus medium (AB Technology, Pullman, WA, USA) at room temperature, evaluated and then transferred nonsurgically. Pregnancy was determined by ultrasonography on Day 35. Data were analyzed with CATMOD, chi-square and GLM procedures (SAS Institute, Cary, NC, USA.). Twenty cattle (9.3%) did not receive embryos; five heifers had cervical problems, and five heifers and 10 cows did not have a CL &gt;10mm. Overall, 7.1% of the recipients had two CL on the day of embryo transfer. There was no effect (P&gt;0.05) of treatment, embryo type (or interaction) or class of recipient on pregnancy rate (overall, 44.1%, 86/195; Table 1). Similarly, mean (±SD) CL diameter and luteal area did not differ (P&gt;0.05) among groups or between pregnant and open recipients (overall, 22.0±3.4mm and 352.0±108.7mm, respectively). However, recipients with a CL diameter ≥18mm tended (P&lt;0.1) to have a higher pregnancy rate (45.8 vs 25.0%). In a subset of 40 recipients examined ultrasonically on Day 12, 50% of those treated on Day 5 and 70% of those treated with pLH on Day 7 had two CL. In summary, overall pregnancy rate in GnRH-synchronized recipients receiving in vitro- or in vivo-produced embryos by nonsurgical transfer was 44.1%. Embryo survival to Day 35 was not affected by type of embryo or treatment with pLH 5 or 7 days after ovulation. Table 1 Pregnancy rate in recipients on Day 35 based on pLH treatment and embryo-type


2019 ◽  
Vol 5 (2) ◽  
pp. 89-99
Author(s):  
Polina A. Golubinskaya ◽  
Marina V. Sarycheva ◽  
Svetlana Y. Burda ◽  
Maksim V. Puzanov ◽  
Natalya A. Nadezhdina ◽  
...  

Introduction: Valproic acid (VA) is carboxylic acid with a branched chain, which is used as an antiepileptic drug. Valproic acid influence on cells in vivo: VA, which is an antiepileptic drug, is also a teratogen, which causes defects of a neural tube and an axial skeleton, although the mechanisms are not yet fully clear. Valproic acid influence on mesenchymal stem cells (MSC) in vitro: It is shown that valproic acid reduces the intracellular level of oxygen active forms. Valproic acid effect on tumor cells: VA inhibits tumor growth through several mechanisms, including the cell cycle stop, differentiation induction and inhibition of growth of tumor vessels. Valproic acid influence on enzymes: It affects mainly GSK-3. Valproic acid influence on animals’ cells: It is shown that VA can significantly improve an ability to develop in vitro and improve nuclear reprogramming of embryos. Erythropoietin (EPO): Is an hypoxia-induced hormone and a cytokine, which is necessary for normal erythropoiesis. EPO is widely used in in vitro experiments. Conclusion: Thus, the influence of VA and EPO on cells can be used in cell technologies.


Animals ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 1359
Author(s):  
Jordana S. Lopes ◽  
Estefanía Alcázar-Triviño ◽  
Cristina Soriano-Úbeda ◽  
Meriem Hamdi ◽  
Sebastian Cánovas ◽  
...  

The increasing use of in vitro embryo production (IVP) followed by embryo transfer (ET), alongside with cryopreservation of embryos, has risen concerns regarding the possible altered pregnancy rates, calving or even neonatal mortality. One of the hypotheses for these alterations is the current culture conditions of the IVP. In an attempt to better mimic the physiological milieu, embryos were produced with female reproductive fluids (RF) as supplements to culture medium, and another group of embryos were supplemented with bovine serum albumin (BSA) as in vitro control. Embryos were cryopreserved and transferred while, in parallel, an in vivo control (artificial insemination, AI) with the same bull used for IVP was included. An overview on pregnancy rates, recipients’ hormonal levels, parturition, and resulting calves were recorded. Results show much similarity between groups in terms of pregnancy rates, gestation length and calves’ weight. Nonetheless, several differences on hormonal levels were noted between recipients carrying AI embryos especially when compared to BSA. Some calving issues and neonatal mortality were observed in both IVP groups. In conclusion, most of the parameters studied were similar between both types of IVP derived embryos and the in vivo-derived embryos, suggesting that the IVP technology used was efficient enough for the safe production of calves.


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