234 PREVALENCE OF MEMBRANE-DAMAGED CELLS IN BOVINE EMBRYOS AFTER CULTURE TO THE BLASTOCYST STAGE IN DEFINED MEDIUM

2013 ◽  
Vol 25 (1) ◽  
pp. 264
Author(s):  
E. Wydooghe ◽  
S. Heras ◽  
A. Van Soom
Keyword(s):  
Conditioned Medium ◽  
Propidium Iodide ◽  
Poor Quality ◽  
Bovine Embryos ◽  
Arrested Development ◽  
Group Culture ◽  
Autocrine Factors ◽  
Propidium Iodide Staining ◽  
Single Culture ◽  
Negative Effect

Autocrine factors are believed to be the reason why embryos cultured in a group show superior development compared with a single culture in many species. A widespread concern with group culture is that degenerating embryos may have a negative effect on the other embryos. Previously, we were able to identify some proteins in embryo-conditioned medium by using tandem mass spectrometry, predominantly stress-related proteins. Our hypothesis was that poor-quality embryos with membrane-damaged cells passively released these identified proteins into the medium. Therefore, we wanted to investigate what percentage of membrane-damaged cells occurred in an average population of Day 8 bovine embryos. Bovine presumed zygotes (n = 606) were cultured individually in 20-µL drops of modified SOF supplemented with insulin, transferrin, and selenium and 0.1 mg mL–1 of polyvinylpyrrolidone. At 8 days post-insemination, blastocyst development was evaluated, and subsequently, all embryos were stained with Hoechst/propidium iodide. In this way, we were able to detect the total cell number (TCN) and the percentage of membrane-damaged cells (MDC ratio) because propidium iodide will stain only the nuclei of membrane-damaged cells pink. Embryo-conditioned medium was collected individually and kept at –80°C until further analysis. At 8 days post-insemination, a blastocyst rate of 27.5% was observed. After determination of the TCN per embryo, we divided the embryos in two groups: 207 embryos had a TCN of more than 64 cells (normal development; range: 64 to 313 cells) and 399 embryos had a TCN of fewer than 64 cells (arrested development; range: 1 to 61 cells). In the first group, 76.3% (138 embryos) had an MDC ratio of less than 10%; however, only 11.1% (23 embryos) showed no membrane-damaged cells at all. In the group with arrested development, only 17.8% (71 embryos) of the embryos had an MDC ratio of less than 10%, and only 13.5% (54 embryos) had no membrane-damaged cells. Of the delayed embryos, 48.6% (194 embryos) showed an MDC ratio of more than 50%, and 34.5% (138 embryos) displayed even 100% of membrane-damaged cells. In conclusion, by use of Hoechst/propidium iodide staining, we were able to detect the TCN and MDC ratio of the embryos. We observed that only a small percentage of the embryos had an MDC ratio of 0%, even if the embryos had more than 64 cells. Almost half of the arrested embryos with fewer than 64 cells had a MDC ratio of more than 50%, indicating that poor-quality embryos consist predominantly of membrane-damaged cells that can passively release proteins into the medium, with a possible negative effect on the surrounding embryos in group culture. It must be noted, however, that for logistical reasons, these results were obtained in a single culture. Further research will be done to identify the candidate autocrine factors and proteins released into the medium by poor-quality embryos by analysing embryo-conditioned medium pooled according to the results of the Hoechst/propidium iodide staining.

scholarly journals Effect of Group Culture and Embryo-culture Conditioned Medium on Development of Bovine Embryos

2006 ◽  
Vol 52 (1) ◽  
pp. 137-142 ◽  
Author(s):  
Tatsuo FUJITA ◽  
Hidenobu UMEKI ◽  
Hideaki SHIMURA ◽  
Kouji KUGUMIYA ◽  
Kazuho SHIGA
Keyword(s):  
Conditioned Medium ◽  
Embryo Culture ◽  
Bovine Embryos ◽  
Group Culture

141 BOVINE EMBRYO CULTURE IN THE MODIFIED WELL OF THE WELL SYSTEM INCREASES BLASTOCYST RATE BUT LOWERS BLASTOCYST QUALITY

2013 ◽  
Vol 25 (1) ◽  
pp. 218
Author(s):  
S. Heras ◽  
E. Wydooghe ◽  
A. Van Soom
Keyword(s):  
Embryo Culture ◽  
Hatching Rate ◽  
Bovine Embryo ◽  
Blastocyst Development ◽  
Group Culture ◽  
Autocrine Factors ◽  
Individual Culture ◽  
Blastocyst Quality ◽  
Negative Effect ◽  
Blastocyst Rate

Individual culture of cattle embryos in serum-containing medium has a detrimental effect on blastocyst development. As we previously demonstrated, this effect is not caused by the individual culture itself but by some component present in serum (Heras et al. AETE Proc. 2012). The high blastocyst rates achieved with group culture in serum-containing medium can be explained by the neutralizing effect of the produced autocrine factors. Autocrine factors produced by individually cultured embryos cannot neutralize the detrimental component due to its low amount and their diffusion in the high culture volume. Using a modified well of the well (mWOW; Vajta et al. 2000 Mol. Rep. Dev.), where embryos are singly cultured in a small well, the effect of the autocrine factors produced by individual embryos can be maximized. We hypothesized that the use of mWOW for individual embryo culture in serum-containing medium, can maximize the neutralizing effect of the autocrine factors, increasing both blastocyst rate and quality. Bovine oocytes (n = 1143, 3 replicates) were matured in TCM-199 with 20% fetal bovine serum (FBS). Presumptive zygotes were cultured in groups (Gr; 25 embryos/50-µL drops), individually (Ind; 20-µL drops), or in mWOW (20-µL drops) in SOFaa with 5% FBS. Blastocyst evaluation occurred 7 and 8 days post-insemination (dpi). Hatching rate is the proportion of hatching/hatched blastocysts out of total 8-dpi blastocysts. Blastocysts were collected at 8 dpi for differential apoptotic staining, evaluating total cell number (TCN), inner cell mass (ICM) ratio, and apoptosis (ACR; Wydooghe et al. 2011 Anal. Biol.). Developmental data were analyzed using binary logistic regression; data concerning blastocyst quality were analyzed using linear mixed model analysis. Since ACR was not normally distributed, a logarithmic transformation was performed. Differences at P < 0.05 were considered significant. Regarding blastocyst development, all groups were significantly different both at 7 dpi (Gr 33.9%, Ind 10.7%, mWOW 19.6%) and at 8 dpi (Gr 37.3%, Ind 14.0%, mWOW 29.1%). Hatching rates were significantly higher in Gr (27.9%) compared to Ind (4.0%) and mWOW (4.1%). Concerning blastocyst quality, TCN was significantly higher (P < 0.001) in Gr (233.23 ± 8.39), no differences between Ind (143.77 ± 6.32) and mWOW (125.11 ± 4.30) were observed. No differences in ICM ratio were observed (Gr 0.41 ± 0.01, Ind 0.30 ± 0.02, mWOW 0.37 ± 0.02). ACR was significantly different between all groups (Gr 0.03 ± 0.002, Ind 0.04 ± 0.003, mWOW 0.08 ± 0.005). In conclusion, the mWOW had a positive effect on blastocyst quantity, increasing blastocyst rates compared to individual culture, but still lower than in group culture. Surprisingly, mWOW had a negative effect on blastocyst quality, producing the highest ACR and the lowest TCN. This negative effect can be caused by toxic products arising from the manufacture (although the wells were rigorously flushed), or by the longer manipulation time needed. This work was funded by FWO G.0210.09N and IWT g111438.

Bovine embryos cultured in serum-poor oviduct-conditioned medium need cooperation to reach the blastocyst stage

1994 ◽  
Vol 42 (3) ◽  
pp. 445-453 ◽  
Author(s):  
L. Ferry ◽  
P. Mermillod ◽  
A. Massip ◽  
F. Dessy
Keyword(s):  
Conditioned Medium ◽  
Blastocyst Stage ◽  
Bovine Embryos

High‐Throughput Cytotoxicity Screening by Propidium Iodide Staining

2007 ◽  
Vol 41 (1) ◽  
Author(s):  
Bruce S. Edwards ◽  
Irena Ivnitski‐Steele ◽  
Susan M. Young ◽  
Virginia M. Salas ◽  
Larry A. Sklar
Keyword(s):  
High Throughput ◽  
Propidium Iodide ◽  
Propidium Iodide Staining

scholarly journals A survey of the causes of cattle organs and/or carcass condemnation, financial losses and magnitude of foetal wastage at an abattoir in Dodoma, Tanzania

2015 ◽  
Vol 82 (1) ◽  
Author(s):  
Wilfred Tembo ◽  
Hezron E. Nonga
Keyword(s):  
Public Health ◽  
Public Health Problem ◽  
Poor Quality ◽  
Significant Loss ◽  
Financial Loss ◽  
The Public ◽  
Control Programmes ◽  
Negative Effect ◽  
Strict Enforcement ◽  
Financial Losses

Slaughterhouses provide a safeguard that prevents the public from consuming meat of poor quality or meat which may be infected with zoonotic diseases. This work reviews a 3-year database of cattle that were slaughtered and inspected between 2010 and 2012 at Dodoma abattoir, Tanzania. In addition, meat inspection was undertaken for 1 month (December 2013). The aim of this study was to establish causes of organ and carcass condemnations and their financial implications as well as the magnitude of slaughter of pregnant cows at Dodoma abattoir. During retrospective study, it was found that a total of 9015 (10.5%) lungs, 6276 (7.3%) intestines, 5402 (6.3%) livers, 3291 (3.8%) kidneys and 41 (0.05%) carcasses were condemned. Pulmonary emphysema (3.4%), fasciolosis (4.5%), pimply gut (5.7%), kidney congenital cysts (1.9%) and hydatidosis (3.1%) were major causes of organ condemnations. This large number of condemned edible organs and/or carcasses implies that public health considerations result in deprivation of valuable protein. Occurrence of hydatidosis, cysticercosis, fasciolosis and tuberculosis illustrates the possible public health problem and presence of environmental infections. Of the 794 cows slaughtered in December 2013, 46% were pregnant. Financial loss as a result of organ and/or carcass condemnations was estimated at $9892. Condemnation of organs and/or carcasses and indiscriminate slaughter of pregnant cows represent a significant loss of meat and revenue and a reduction in growth of future herds, which has a negative effect on the livestock industry. This justifies appropriate surveillance and disease control programmes coupled with strict enforcement of legislation governing animal welfare to curb the slaughter of pregnant animals.

205 INCREASED PREGNANCY RATES OF POOR QUALITY BOVINE EMBRYOS BY ASSISTED HATCHING

2007 ◽  
Vol 19 (1) ◽  
pp. 219
Author(s):  
A. Taniyama ◽  
Y. Watanabe ◽  
Y. Nisino ◽  
T. Inoue
Keyword(s):  
Embryo Transfer ◽  
Zona Pellucida ◽  
Poor Quality ◽  
Pregnancy Rates ◽  
Assisted Hatching ◽  
Bovine Embryos ◽  
Production Rates ◽  
Chi Square ◽  
Chi Square Test ◽  
Rectal Palpation

Embryo transfer after superovulation is commonly used for efficient embryo and animal production and for genetic improvement in cattle. However, the quality of collected embryos varies greatly, which affects pregnancy rate. Usually, poor quality embryos are related to low pregnancy rates after embryo transfer and low viability after cryopreservation. Therefore, it is important to improve chances for survival of poor quality embryos after embryo transfer. The objective of this experiment was to improve pregnancy rates by applying the assisted hatching technique to poor quality embryos. Embryos were collected from Japanese Black cows after superovulation on Day 7 post-insemination. After being washed, embryos were morphologically classified. Embryos having more than 30% degenerated cells were assigned as poor quality embryos. The assisted hatching of embryos (cutting the zona pellucida) was performed under a stereoscope or an inverted microscope by making a cutting slit on the zona pellucida for about 20% of its circumference using a micromanipulator equipped with a cutting needle and holding pipette. After cutting, single or two embryos were transferred fresh to one uterine horn of recipient cows on Day 7 of the estrous cycle. Pregnancy and calf production rates were compared between 2 embryo transfer groups composed of fresh zona-cut embryos (ZC group) or fresh embryos with non-cut zonae pellucidae (NZC group). Pregnancy rates were determined by rectal palpation on Day 45, and calf production rates were calculated by the following formula: number of calves born/number of pregnancies. Statistical analysis was carried out using the chi-square test. Pregnancy rates of poor quality embryos in the double ET ZC group (60.3%; 44 pregnancies/73 transfers) were significantly higher (P &lt; 0.05) than those in the single ET NZC group (25.0%; 6 pregnancies/24 transfers) and in the single ET ZC group (44.0%; 37 pregnancies/84 transfers). Calf production rates were 67.3%, 45.5%, and 35.6% for the double ET ZC group, the double ET NZC group, and the single ET ZC group, respectively. Pregnancy rates of poor quality bovine embryos after double ET were remarkably improved by assisted hatching compared with those of single ET with non-assisted hatching. These results suggest that the combined methods of assisted hatching and double ET may be beneficial to produce calves from poor quality embryos.

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