Influence of glucose on the sex ratio of bovine IVM/IVF embryos cultured in vitro

2001 ◽  
Vol 13 (6) ◽  
pp. 361 ◽  
Author(s):  
A. Gutiérrez-Adán ◽  
J. Granados ◽  
B. Pintado ◽  
J. De La Fuente
Keyword(s):  
Sex Ratio ◽  
In Vitro Culture ◽  
Culture Media ◽  
Calf Serum ◽  
Blastocyst Stage ◽  
Fluid Medium ◽  
Morula Stage ◽  
Effect Of Glucose ◽  
Glucose Supplementation

The effect of glucose in the medium used during in vitro culture on the sex ratio of bovine blastocysts derived from in-vitro-matured andin-vitro-fertilized oocytes was evaluated. Oocytes were matured and inseminated with mixed sperm from three bulls and were cultured in vitro in modified synthetic oviducal fluid medium with 10% fetal calf serum, with or without glucose supplementation. The overall rate of cleaved embryos that developed to expanded blastocyst in the medium without glucose (27.0%) was significantly greater (P<0.05) than the percentage observed when embryos were cultured in medium with glucose (17.5%). Analysis of variance was performed to analyse the effect of glucose on the proportion of male embryos reaching the blastocyst stage (or arrested at the morula stage) during Days 7 to 10. Regardless of the presence or absence of glucose in the medium, significantly (P<0.05) more male than female embryos were harvested as expanded blastocysts on Day 7 and on Day 8 of culture. On Days 9 plus 10 of culture, a sex ratio imbalance only occurred in the absence of glucose in the culture medium (P<0.05). Glucose did not produce any significant effect on the sex ratio of the overall number of expanded blastocysts harvested by Day 10 of in vitro culture. However a significantly greater proportion of females (P<0.01) were found among those embryos that developed only to the morulae stage after 10 days in vitro. These results show that glucose supplementation of culture media produces a preferential loss of female embryos during culture to the blastocyst stage.

scholarly journals Parthenogenetic development of rabbit oocytes after electrical stimulation

2011 ◽  
Vol 51 (No. 9) ◽  
pp. 400-405
Author(s):  
A. Wierzchos
Keyword(s):  
Pulse Generator ◽  
Electric Pulse ◽  
Calf Serum ◽  
Functional Condition ◽  
Blastocyst Stage ◽  
White Female ◽  
Foetal Calf Serum ◽  
Electric Pulses ◽  
Morula Stage

The aim of this study was to determine the effect of electric pulses on the structural and functional condition of rabbit oocytes. The New Zealand White female rabbits at 3&ndash;5 months of age and at 3&ndash;4 kg body weight served as oocyte donors. Oocytes after flushing from the oviducts were placed between two electrodes in an electroporation chamber which was filled with a dielectric solution. Following a short incubation in B2 medium, oocytes were subjected to an electric pulse released by an electrical pulse generator. Oocytes were then incubated in 500 &micro;l of B2 medium supplemented with 20% foetal calf serum (FCS) at 38&deg;C in an atmosphere of 5% CO<sub>2 </sub>in air. Oocytes were cultured until the morula/blastocyst stage (approx. 72 h). The experiment was conducted using 430 oocytes obtained post mortem. In vitro cultured oocytes not subjected to an electric pulse were the control. Each group was subdivided into replications according to electric current intensity. The analysis of experimental variants shows that in the first variant all embryos developed to the morula stage but only 10% of them continued to develop to the blastocyst stage. In the second variant we observed that 5&ndash;10% of oocytes developed to the blastocyst stage after treatment with 2.0 and 2.5 kV/cm pulse but in the group of 1.0 kV/cm pulse 35% of oocytes developed only to the 2&ndash;12 b stage. In the third variant only 1 oocyte (5%) continued to develop to the blastocyst stage, but in the fourth variant oocyte development stopped at the morula stage. In the fifth variant, called an &ldquo;extreme&rdquo; one, oocytes stopped to develop at the stage of 2&ndash;12 b (about 25%) and the percentage of degenerated oocytes dramatically increased (about 60%). &nbsp;

132 POST-HATCHING BOVINE EMBRYO DEVELOPMENT IN VITRO: RELATIONSHIP BETWEEN SEX AND SIZE

2008 ◽  
Vol 20 (1) ◽  
pp. 146
Author(s):  
G. M. Machado ◽  
C. R. Laender ◽  
M. M. Franco ◽  
L. O. Leme ◽  
R. Rumpf ◽  
...  
Keyword(s):  
Sex Ratio ◽  
Well Being ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Fluid Medium ◽  
Developmental Potential ◽  
Vitro Fertilization ◽  
The Difference ◽  
And Gender

In vitro post-hatching embryos culture is a procedure that allows the establishment of more accurate tools for evaluating embryo developmental potential without the need of transferring them to recipient animals (Vejlsted et al. 2006 Theriogenology, 65, 153–165). It is well established that in the in vitro embryo production (IVP) technique, the sex ratio is imbalanced in favor of male embryos. The difference in sex ratio observed in the blastocyst stage at day 7 may be attributed to a variety of factors including developmental speed. However, whether or not this difference in sex ratio and speed of development continues after hatching is not known. The objective of this study was to evaluate post-hatching embryonic development until day 11 after in vitro fertilization (day 0) associating embryo size and gender. A total of 468 oocytes, obtained from abattoir-derived ovaries, were used. They were matured, fertilized, and cultured in vitro for 8 days in synthetic oviduct fluid medium (SOF Nutricell�) and incubated at 39�C in 5% CO2 in air. Degenerated embryos on day 8 and non-hatching embryos on day 9 were removed from culture droplets, and only hatched blastocysts were kept. Then, embryos were measured using a graduated ocular and post-hatching development (PHD) medium (Brand�o et al. 2005 Biol. Reprod. 71, 2048–2055) was added in each well, being the final medium 1:1 of SOF:PHD. On day 11, the embryos were evaluated under stereomicroscope and only morphologically normal blastocysts were measured and frozen at –80�C, for gender diagnosis. The DNA from frozen samples was extracted with trizol reagent, sodium citrate solution (0.1 m), and ethanol. Sex embryos determination was performed by PCR and visualized in 2% agarose gel. Data were analyzed using the Mann-Whitney test. The results show that the majority (69%) of the embryos that reached blastocyst stage at day 7 developed in the PHD system until day 11. From the initial oocytes, 144 embryos (30.1%) and 146 (31.1%) embryos had reached the blastocyst stage at days 7 and 8, respectively. At day 9, 89 (19%) embryos were hatched and 65 embryos (13.9%) developed until day 11, of which only 48 embryos (73.8%) had a clear trophoblast. No difference (P > 0.05) in the percentage of male and female embryos was observed when embryos were evaluated at day 11 of culture. In addition the mean size was similar (P > 0.05) for female (467.24 � µm, n = 19) and male (478.84 � 190.21 µm, n = 29) embryos. The results suggest that after post-hatching culture the differences in sex ration and in gender development in IVP bovine embryos are not evident. The development until day 11 showed that post-hatching in vitro culture of bovine blastocyst can be used for embryo evaluation in later phase of development However, several questions still remain to be investigated regarding post-hatching culture of bovine blastocysts before it can be used as a tool to evaluate in vitro embryos. Supported by Embrapa and UnB, Brazil.

71 The Effect of Two Different In Vitro Culture Media and Mice Embryo Groupings on Hatchability After 24 Hours of Culture

2018 ◽  
Vol 30 (1) ◽  
pp. 174
Author(s):  
N. C. Negota ◽  
M. L. Mphaphathi ◽  
L. P. Nethenzheni ◽  
T. L. Rammutla ◽  
N. R. Serota ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Assisted Reproductive Technologies ◽  
Culture Media ◽  
Reproductive Technologies ◽  
Blastocyst Stage ◽  
Hatching Rate ◽  
Significant Difference ◽  
Autocrine Signalling ◽  
Blastocyst Hatching

Mammalian blastocysts must hatch out from the zona pellucida before implantation. In vitro embryo culture and grouping of mice blastocysts are conducive options of assisted reproductive technologies (ART) to speed up the hatching rate of mice embryos. The number of embryos per unit volume has the greatest impact on hatching rates due to autocrine signalling. The study aimed to determine the effect of two in vitro culture (IVC) media (TCM-199 and Ham’s F10) and embryo groupings (1, 2, 3, and 4 embryos per 50-µL droplet) after 24 h of culture on hatching rate. Breeds of C57BL/6 (n = 10) and BALB/c (n = 10) were raised until they reached maturity and bred naturally to produce the first filial generation. The photoperiod was 14 h of light followed by 10 h of darkness in the breeding house, and feed and water were provided ad libitum. Female mice were superovulated using eCG and hCG. The first filial generations from 2 breeds were used for the collection of 160 blastocysts and randomly allocated into 2 IVC media (80 embryos for TCM-199 and 80 embryos for Ham’s F10) and again subjected to 4 embryo groupings (1, 2, 3, and 4 embryos per droplet) treatments. Four replicates were done per treatment group. The general linear model of Minitab version 17 (Minitab Inc., State College, PA, USA) was used to analyse the data. The hatching rate of blastocyst stage was significantly higher for TCM-199 (56.9 ± 27.2) compared with Ham’s F10 (50.0 ± 35.1%). The comparison of all embryo groupings, 1 (20.0 ± 40.5), 2 (28.8 ± 29.7), 3 (59.1 ± 38.8), and 4 (43.8 ± 32.4%) per 50-µL droplet showed significant differences, irrespective of IVC medium and breed. In TCM-199, groupings of 1 (20.0 ± 41.0), 2 (30.0 ± 29.9), 3 (63.3 ± 40.3), and 4 (42.5 ± 33.5%) had a significant difference on blastocyst hatching percent. In Ham’s F10, groupings of 1 (20.0 ± 41.0), 2 (27.5 ± 30.2), 3 (55.0 ± 37.9), and 4 (45.0 ± 32.0%) were significantly different on blastocyst hatching rate. However, an increase in hatching rate was observed for the interaction of media and embryo groupings and especially when embryos were increased per droplet in all breeds. In conclusion, the use of TCM-199 and grouping of 3 embryos per 50-µL droplet during culture had the highest hatching rate compared with the use of Ham’s F10.

scholarly journals Comparative Study of Different Assisted Hatching Techniques and In Vitro Culture Media on Mice Embryo Hatching Rate

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Negota NC ◽  
◽  
Mphaphathi ML ◽  
Nethenzheni LP ◽  
Rammutla TL ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Culture Media ◽  
Abdominal Cavity ◽  
Blastocyst Stage ◽  
Assisted Hatching ◽  
Hatching Rate ◽  
Female Mice ◽  
Significant Difference ◽  
F1 Generation

The study investigated the influence of Assisted Hatching (AH) techniques (mechanical, chemical, enzymatic thinning and laser) and two in vitro culture media (Ham’s F10 and TCM-199) on hatching rate of mice embryos (blastocyststage) following 24 hours of culture. The C57BL/6-black (n=30) and BALB/cwhite (n=30) mouse breeds were raised until maturity and naturally bred to produce a F1 generation. The light in the breeding house was controlled and the mice were fed ad libitum. Female mice (n=30) were injected (peritoneal) with 0.1 ml (5 IU) of eCG into the abdominal cavity with 1 ml syringe and 0.5x16 mm needle to stimulate follicular growth and 46-49 h later was injected with 0.1 ml (5 IU) of hCG to cause ovulation, maintain the corpus luteum and stimulate it to secrete progesterone for maintenance of pregnancy. After the injection, the male and female (1:1) could mate overnight. Female mice with vaginal plugs were observed and kept separately for blastocyst-stage embryos collection on day three following successful mating. They were euthanized, and abdomen sterilized with 70% ethanol. Using a sterile surgical scissor, a fine cut was made, holding the skin firmly above and below the incision, the skin was pulled apart using forceps. The embryos were flushed from the uterus using a 30-gauge needle with culture media. Following the AH techniques, embryos were cultured in TCM-199 or Ham’s F10 for 24 hours and zonal thickness of all hatched embryos were measured. Immediately after assisted hatching, the embryos were cultured into two different in vitro culture media. All embryos hatched were stained and the zonal thickness of embryos were measured. The number of blastomeres were counted and recorded 24 h later. Data collected were subjected to analysis of variance using PROC General Linear Model. The Tukey’s test was used to separate the means. A significant difference was observed between the thickness of Zona Pellucida (ZP) pre and post treatment after 24 hours of culture. However, there was no significance difference among blastocyst hatching rate and the blastomeres nuclei counted after staining. The thickness of the ZP decreased with individual AH techniques. The interaction between AH techniques and in vitro culture was found to be significantly different on blastocyst hatchability. However, laser AH technique had highest hatchability (56.3%) when embryos were cultured in TCM-199 followed by mechanical AH techniques (52.6%). The hatchability rate (33.3%) was recorded in the chemical AH technique group. The blastomeres nuclei counted under interaction of AH techniques and culture media was not differently affected, with the values ranging from 69 to 76%. In conclusion, the use of different AH techniques resulted in varying effect and increase outcomes towards the hatching rate.

223 RELATIONSHIP BETWEEN THE LENGTH OF CELL CYCLES, CLEAVAGE PATTERN, AND DEVELOPMENTAL COMPETENCE DURING IN VITRO CULTURE OF IN VITRO-MATURED/IN VITRO-FERTILIZED BOVINE OOCYTES

2009 ◽  
Vol 21 (1) ◽  
pp. 209 ◽  
Author(s):  
T. Somfai ◽  
Y. Inaba ◽  
Y. Aikawa ◽  
M. Ohtake ◽  
S. Kobayashi ◽  
...  
Keyword(s):  
Cell Division ◽  
In Vitro Culture ◽  
Cell Mass ◽  
Calf Serum ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Cleavage Pattern ◽  
Cell Cycles ◽  
Bovine Oocytes

In in vitro embryo production systems, there is a need to select embryos with good developmental competence at the early stages. This study was conducted to determine whether there was any relationship between the duration of the first 3 cell cycles, the cleavage pattern of the first cell division, and the developmental competence of embryos during in vitro culture. A total of 320 in vitro-matured and in vitro-fertilized bovine oocytes were cultured in microdrops of CR1aa medium supplemented with 5% calf serum covered by mineral oil in 5% CO2 and 20% O2 at 38.5°C. The kinetics of embryo development were measured by time-lapse cinematography. Embryos were classified according to their cleavage pattern at the first cell division. Of 285 cleaved embryos, 119 had 2 blastomeres of the same size (normal cleavage: NC), 49 had 2 blastomeres with multiple small fragments (multiple fragments: MF), 34 had 2 blastomeres and a protrusion (protrusion: PT), 45 showed direct cleavage from 1 cell to 3 or 4 blastomeres (3–4BL), and 60 oocytes cleaved to 2 blastomeres of different sizes (unequal blastomeres: UB). (Twenty-two embryos belonged to 2 classes.) After 175 h of culture, blastocysts were either subjected to differential inner cell mass/trophectoderm (ICM/TE) staining or karyotyped. The first and second cell cycles (mean ± SEM) of viable embryos (that could develop to the blastocyst stage) were significantly shorter than those of nonviable embryos (24.9 ± 0.3 h and 8.7 ± 0.1 h v. 26.6 ± 0.7 h and 10.0 ± 0.1 h, respectively); however, the length of the third cell cycle did not differ (P < 0.05, paired t-test). The duration of 1 cell stage in the NC group was significantly shorter than that of MF, PT, 3–4BL, and UB groups (24.7 ± 0.4 h, 26.6 ± 0.5 h, 26.3 ± 0.6 h, 26.0 ± 0.2 h, and 27.7 ± 0.9 h, respectively). The length of the second and third cell cycles did not differ among the groups. The percentage of NC embryos that developed to the blastocyst stage was similar to that of the 3–4BL group (66.9 and 56.7%, respectively) but was significantly higher than those of the MF, PT, and UB groups (40.5, 26.5, and 35.6%, respectively; P < 0.05, ANOVA). The mean cell numbers of NC blastocysts did not differ from those of the MF, 3–4BL, and UB groups but were higher than those of PT embryos (147.1, 155.6, 121.6, 146.4, and 115.1, respectively). There was no difference in ICM/TE rates between the groups. Unlike NC, MF, PT, and UB embryos, most (6 of 8 karyotyped) 3–4BL blastocysts had abnormal ploidy, such as haploid, triploid, mixoploid, or chaotic chromosome numbers, in blastomeres. Our results revealed that not only the length of the first cell cycles, but also the cleavage pattern during first cell division can be a marker of developmental competence and should be considered for the selection of good-quality embryos for embryo transfer. This work was supported by the Research and Development Program for New Bio-industry Initiatives.

144 Sex ratio of in vitro-produced goat embryos

2019 ◽  
Vol 31 (1) ◽  
pp. 197 ◽  
Author(s):  
M. Stoltzfus ◽  
J. Wayman ◽  
R. Stilz ◽  
D. Bresnahan
Keyword(s):  
Sex Ratio ◽  
Dna Extraction ◽  
Culture Media ◽  
The United States ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Pcr Analysis ◽  
Significant Difference ◽  
The Impact

Goats are important livestock species because they produce meat, milk, and fibre and are also easily maintainable on small farms. Although goats provide many products and consumption of goat meat is increasing in the United States, the industry lags compared with many species with regard to IVF techniques to enhance goat production. It has been demonstrated in other species that male IVF embryos tend to develop faster than those of females. This may be due to increased tolerance of male embryos to inadequate conditions, particularly, glucose concentrations in culture media. However, the sex ratio of goat embryos produced utilising IVF remains unknown. The aim of this study was to determine the sex ratio of goat embryos utilising a commercially available media suite (IVF Biosciences, Falmouth, UK). Oocytes were harvested from ovaries obtained from 2 local abattoirs and matured in vitro. Frozen sperm from 1 of 2 billy goats were randomly assigned for each round of IVF. Embryos were evaluated daily from Days (D) 6 through 9 of in vitro culture. On the day an embryo reached the expanded blastocyst stage, it was removed from culture and placed into DNA extraction buffer (PicoPureTM DNA Extraction Kit, Applied Biosystems, Waltham, MA, USA) and stored at −20 for PCR analysis, typically within one month of collection. In all unknown samples, positive male (sperm) and female (uterus) controls, the amelogenin gene was amplified and products were evaluated on a 1.5% agarose gel with ethidium bromide. Embryos with 2 bands (202 and 262bp) were classified as male, and those with 1 band (262bp) were classified as female. Embryos with no bands were not included in analysis. Embryos reached the expanded blastocyst stage on D6 (n=29), D7 (n=39), and D8/9 (n=35, combined for evaluation). A chi-squared analysis comparing the percentage of male and female embryos to the expected 50% was completed for each time point (D6, D7, D8/9), as well as overall ratios (D6-9). In total, 350 oocytes were utilised in 6 rounds of IVF resulting in a mean blastocyst rate of 32% (range 17-47%). There was no significant difference in the number of embryos that were male on D6 (55%) and D7 (46%). However, on D8/9 significantly fewer embryos were male (29% male; P=0.01). Overall, there was no significant difference (P=0.14) in the sex ratio, with 41% male and 59% female embryos. Our findings are somewhat consistent with other species, in that male goat embryos produced via IVF develop more quickly in culture conditions; however, female embryos were still able to tolerate culture conditions. Delayed blastocyst development may not necessarily be an indication of a reduced quality embryo but one that is slower to develop based on its sex. This could be due to expression of X-linked genes being unbalanced during pre-implantation embryo development stages and warrants further study. One influencer of sex ratio we are currently investigating is the impact of glucose during culture, to further understand metabolism in IVF embryos.

Effects of gonadotrophins, growth hormone and prolactin on developmental competence of domestic cat oocytes matured in vitro

1995 ◽  
Vol 7 (5) ◽  
pp. 1061 ◽  
Author(s):  
RD Schramm ◽  
BD Bavister
Keyword(s):  
Growth Hormone ◽  
Calf Serum ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Domestic Cat ◽  
Developmental Competence ◽  
Developmental Potential ◽  
Nuclear Maturation ◽  
Morula Stage

Specific aims were to (1) examine the developmental capacity of felid oocytes matured in vitro and (2) determine the effects of gonadotrophins, growth hormone and prolactin on nuclear and cytoplasmic maturation oocytes in vitro. Oocytes were obtained from excised ovaries of 21 cats, and were matured for 45-46 h in modified CMRL-1066 culture medium (1 mM glutamine, 1 mM pyruvate and 20% bovine calf serum), with one of the following: (1) gonadotrophins (1.0 micrograms mL-1 hFSH+10 micrograms mL-1 hLH), (2) gonadotrophins+10 micrograms mL-1 growth hormone, (3) gonadotrophins+10 micrograms mL-1 prolactin, or (4) no hormones. Oocytes were inseminated with ejaculated cat sperm capacitated in TALP medium. Embryos were cultured in modified CMRL-1066 medium until developmental arrest, then stained with Hoechst 33342 to assess nuclear status or cell number. Gonadotrophins enhanced (P < or = 0.05) the incidence of nuclear maturation, but neither gonadotrophins, growth hormone nor prolactin improved fertilization or developmental potential of oocytes matured in vitro. Mean percentages of mature oocytes that were fertilized and cleaved to or beyond the 2, 4, 8 and 16-cell stages were 80, 77, 66, 42 and 24%, respectively. Three embryos progressed to 40-60 cells, but none developed a blastocoel. Thus, although gonadotrophins enhance nuclear maturation of oocytes in vitro, and mature oocytes are capable of fertilization and development to the morula stage, culture with growth hormone, prolactin or gonadotrophins during maturation in vitro does not enhance developmental competence or overcome the morula-to-blastocyst-stage block in development of domestic-cat oocytes matured in vitro.

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