73 Fatty Acid Supplementation in Culture Medium with Reduced Nutrient Concentrations Improves Bovine Blastocyst Development Compared with Standard Culture Medium

2018 ◽  
Vol 30 (1) ◽  
pp. 175
Author(s):  
R. Pasquariello ◽  
J. R. Herrick ◽  
Y. Yuan ◽  
A. F. Ermisch ◽  
J. Becker ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Culture Medium ◽  
Cell Number ◽  
Nutrient Concentrations ◽  
Blastocyst Development ◽  
Fatty Acid Supplementation ◽  
Cell Numbers ◽  
Standard Culture ◽  
Standard Culture Medium

Lipids are a potent source of cellular energy and are metabolized within mitochondria via fatty acid β-oxidation, a process that also requires carnitine. Embryos metabolize lipids during pre-implantation development, but relatively little is known about the effect of fatty acid supplementation for early bovine embryogenesis in culture. The objective of this study was to evaluate the effect of lipid supplementation (via albumin) and l-carnitine (C; 5 mM) during embryo culture in a novel medium with reduced concentrations of nutrients, compared with our standard culture medium (control). Following in vitro maturation and IVF, zygotes were cultured using a serum-free sequential media system (0-72 h step 1; 72-168 h step 2). Concentrations of salts, bicarbonate, and protein [2.5 mg mL−1 fatty acid-free (FAF) or fraction V (FrV) BSA] were the same in all treatments to maintain consistent osmolarity and pH. Nutrients (glucose/fructose, citrate, lactate, pyruvate, amino acids, vitamins, and EDTA) were diluted to 6.25% of control. In addition to the control medium (100%+FAF; n = 587), experimental treatments included 6.25%+FAF+C (essentially lipid free; n = 573) and 6.25%+FrV+C (lipid rich; n = 585). Following in vitro culture (7 reps), hatching blastocysts were stained to determine inner cell mass (ICM; SOX2+) and trophectoderm (TE; CDX2+) cell numbers. Lipid content of single expanded blastocysts was determined using gas chromatography coupled to an ISQ-LT MS/MS (GC-MS). Data (mean ± SEM) were analysed by ANOVA. Embryo cleavage did not differ between treatments. Blastocyst development (per cleaved embryo) was higher (P < 0.05) after culture in lipid rich (38.3 ± 1.5%) compared with control (29.6 ± 2.2%) and lipid free (28.1 ± 3.6%). Blastocyst hatching was reduced (P < 0.05) in lipid free (1.4 ± 0.7%) but not in lipid rich (5.2 ± 1.7) compared with control (9.8 ± 2.1). However, blastocysts developed in lipid rich and lipid free had reduced cell numbers compared with control: TE, 98.7 ± 5.9 and 98.8 ± 9.1 v. 160.3 ± 9.0; ICM, 19.2 ± 2.9 and 25.2 ± 6.1 v. 43.3 ± 4.0; and total cell number, 117.9 ± 7.3 and 124.0 ± 8.7 v. 203.6 ± 10.2, respectively. Analysis by GC-MS identified 40 annotated lipids (i.e. triacylglycerols and phosphatidyl cholines) that were significantly reduced in blastocysts cultured in lipid rich compared with control. In summary, blastocyst development was significantly improved after supplementation of fatty acids and l-carnitine to a medium with reduced nutrient concentrations. The mechanism underlying this phenomenon may be related to increased lipid metabolism in the low nutrient environment. Although more embryos developed in this novel medium, these blastocysts had reduced cell numbers even though blastocyst expansion and hatching were not affected. This reduced nutrient medium may provide an experimental model in which to independently study pathways controlling cell proliferation and blastocyst development. Future studies will investigate whether embryo cell number can be rescued while maintaining improved blastocyst development.

286 INFLUENCE OF REVERSIBLE MEIOSIS INHIBITION ON SWINE EMBRYOS PRODUCED BY IN VITRO FERTILIZATION AND PATHENOGENETIC ACTIVATION

2006 ◽  
Vol 18 (2) ◽  
pp. 250
Author(s):  
M. G. Marques ◽  
A. B. Nascimento ◽  
V. P. Oliveira ◽  
A. R. S. Coutinho ◽  
M. E. O. A. Assumpção ◽  
...  
Keyword(s):  
Culture Medium ◽  
Cell Number ◽  
Control Group ◽  
Cleavage Rate ◽  
Vitro Fertilization ◽  
Electric Pulses ◽  
Cell Numbers ◽  
Blastocyst Rate ◽  
And Control

The present work evaluated the reversible meiosis inhibition effect on the development of swine embryos produced by in vitro fertilization (IVF) or parthenogenetic activation (PA). The efficiency of PZM3 and NCSU23 embryo culture media was also evaluated. Oocytes from ovaries collected at a slaughterhouse were subjected to IVM in two different groups: CHX (cycloheximide 5 µM for 10 h) and control, both with TCM-199 + 3.05 mM glucose + 0.91 mM sodium pyruvate + 10% porcine follicular fluid (pFF) + 0.57 mM cystein + 10 ng epidermal growth factor (EGF)/mL + 10 IU eCG/mL + 10 IU hCG/mL for the initial 22 h. In the remaining period (20 h for CHX and 22 h for control), medium without hormones was utilized. After IVM, oocytes were denuded and fertilized for 6 h (IFV) or the matured oocytes were submitted to activation by electric pulses (PA) (2 DC of 1.5 kV/cm for 30 µs), incubated for 1 h in culture medium with 10 μM of CHX, and again submitted to the same electric pulses for 60 µs. Embryo development was evaluated by cleavage rate on Day 3 and blastocyst rate and blastocyst cell number on Day 7 of culture. Cleavage and blastocyst rates were analyzed by the equality-of-two-ratios test and cell number by the Kruskal-Wallis and Mann-Whitney tests (P < 0.05). In relation to IVF, the PZM3 medium was more efficient than NCSU23 for cleavage rate in the CHX group (PZM3: 68.4%, NCSU23: 44.4%) and had a better blastocyst rate in the control group (PZM3: 13.4%, NCSU23: 5.6%). With reference to PA, NCSU23 presented better cleavage and blastocyst rates than PZM3 in the CHX group (NCSU23: 89.5%, PZM3: 78.5% and NCSU23: 20.4%, PZM3: 13.0%, respectively). In the control group, only the NCSU23 blastocyst rate was higher than that for PZM3 (NCSU23: 22.5%, PZM3: 10.8%). No culture medium effect on cell number mean of IVF and PA blastocysts was observed. Maturation block improved cleavage rates in IVF groups cultured with PZM3 (68.4% and 50.6%, respectively, for CHX and control) and in PA groups cultured with NCSU23 (89.5% and 80.3%, respectively, for CHX and control), but no improvement of blastocyst rates in both groups (IVF and PA) was verified. Table 1 below shows that maturation block decreased the IVF and increased the PA blastocyst cell numbers. As older oocytes are more effectively activated, oocytes blocked with CHX achieved the maturation stage faster than the control group, therefore resulting in high-quality PA blastocysts. In conclusion, PZM3 was more efficient for IVF embryo production in contrast to NCSU23, whereas NCSU23 can be indicated for PA embryo production. Moreover, maturation blockage with CHX influenced blastocyst cell number, decreasing in IVF embryos and increasing in PA embryos. Table 1. Mean (±SD) of blastocyst cell numbers for IVF or PA groups after in vitro maturation without (control) or with cycloheximide (CHX) and cultured in NCSU23 or PZM3 medium This work was supported by FAPESP 02/10747–1.

125 EXAMINING CRITERIA FOR EXTENDING BOVINE BLASTOCYST SURVIVAL IN VITRO

2008 ◽  
Vol 20 (1) ◽  
pp. 143
Author(s):  
F. N. T. Cooke ◽  
T. M. Rodina ◽  
P. J. Hansen ◽  
A. D. Ealy
Keyword(s):  
Conditioned Medium ◽  
Culture System ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Blastocyst Development ◽  
Interferon Tau ◽  
Cell Numbers ◽  
Bovine Blastocyst

Most of the current culture procedures used for bovine in vitro embryo production terminates at the blastocyst stage. Developing procedures for extending embryo lifespan beyond this phase will provide a valuable tool for understanding events that occur during the second week of pregnancy in cattle. The overall objective of the present studies was to identify culture conditions required to support bovine blastocyst development beyond its initial formation. In the first study, individual day 8 blastocysts (day 0 = day of IVF) were cultured until day 11 in 30 µL microdrops of Potassium Simplex Optimized Medium-Bovine Embryo 2 containing 0.1 mm non-essential amino acids or Tissue Culture Medium 199 (M199). Both media were supplemented with 5% [v/v] fetal bovine serum (FBS) and incubations were in an atmosphere of either 5 or 21% (v/v) oxygen. A medium by oxygen interaction (P = 0.007) occurred when assessing cell number on day 11. Blastocysts cultured in M199 and in a 5% O2 environment had greater (P < 0.002) cell numbers (536 � 49) than blastocysts incubated in other conditions (339 � 28). Conditioned medium from blastocysts incubated in 21% O2 contained greater (P < 0.05) concentrations of bioactive interferon-tau (IFNT) than blastocysts incubated in 5% O2 regardless of medium type (70.5 � 28 v. 17.2 � 2.6 ng mL–1). In a follow-up study, blastocysts could remain morphologically viable through day 11 in M199 containing at least 2.5% FBS. To examine whether oxidative stress was responsible for the increase in IFNT production under 21% O2, blastocysts were incubated under a 5% O2 atmosphere in M199 containing 2.5% FBS and increasing concentrations of tert-butylhydroperoxide (tBH), a membrane-permeable oxidative agent. Addition of e3 nm tBH decreased cell numbers but did not increase IFNT concentrations in conditioned medium. To examine whether blastocysts could survive beyond day 11 in culture, day 11 blastocysts were transferred to 400 �L of M199 with 20% FBS under 5% oxygen and cultured from day 11 to 20–21 post-IVF. Half of the medium was replaced every 2–3 days. On day 13–14, 16.6 � 6.1% of blastocysts showed initial signs of degeneration. A portion of blastocysts (32.9 � 9.6%) began attaching to plates on days 13–15 and produced outgrowths that appeared viable on days 20–21. All of the non-attached blastocysts degenerated by day 17–18. No blastocyst elongation was detected. In conclusion, a culture system was developed that sustains blastocyst viability and IFNT production in vitro to day 11. Although this culture system allowed blastocyst survival until day 14, normal conceptus development (i.e. elongation/filamentation) was not achieved. Nonetheless, the culture system provides a useful tool for examining the initial stages of blastocyst development and IFNT production from individual bovine embryos.

scholarly journals 276INFLUENCE OF SPERM TREATMENTS ON BLASTOCYST DEVELOPMENT IN VITRO AND CELL NUMBER IN CATTLE

2004 ◽  
Vol 16 (2) ◽  
pp. 258
Author(s):  
B.G. Jeon ◽  
J.S. Moon ◽  
K.C. Kim ◽  
G.J. Rho
Keyword(s):  
Silicone Oil ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Glass Wool ◽  
Hatching Rate ◽  
Blastocyst Development ◽  
Serum Free ◽  
Cell Numbers ◽  
Bull Sperm

Experiments were designed to examine the effects of developmental rate and cell numbers in embryos produced by in vitro fertilization (IVF) using sperm from 2 bulls (sperm A and B purchased from commercial sale) isolated by three methods. Cumulus-oocyte-complexes collected from ovaries harvested from a local slaughter house were matured in 50μL droplets of serum-free M199 medium supplemented with 1μgmL−1 estradiol-17β, 10μgmL−1 LH and FSH under silicone oil at 39°C in a humidified atmosphere of 5% CO2 in air. After 24h of culture, oocytes were fertilized with the sperm treated by three different methods of isolation;; percoll gradient, swim-up and glass wool filtration;; a final concentration of 2×106 cells mL−1 was used. At 16h after fertilization, presumptive zygotes were co-cultured in serum-free M199 with BOEC for up to 192h post-insemination. At 48h and 120h post-insemination, the cultures were fed with 25μL of serum-free M199. The embryos were compared for their rates of cleavage at 48h post-insemination, development to the blastocyst stage, and hatching, and also the cell number at 192h post-insemination. Differences between treatments were analyzed using one-way ANOVA after arc-sine transformation of the proportional data of cleavage, development into blastocyst stage and hatching. Comparisons of means among treatments were performed using Tukey-Kramer multiple comparisons test. The results are summarized below. The rates of cleavage in embryos produced by IVF using sperm from 2 bulls isolated by percoll, swim-up and glass wool were not significantly different. The blastocyst development and hatching rates between sperm treatment were not significant within bull sperm A and within sperm B. However, although the hatching rate in percoll treatment of bull sperm A was higher than in bull sperm B, the difference was not statistically significant. The mean cell numbers in percoll treatment of bull sperm A (176.5±7.1) were significantly higher (P&lt;0.001) than the others. In bull sperm B the cell numbers in percoll treatment were higher than the other two treatments, but the differences were not statistically significant. In conclusion, these results support the concept that sperm preparation using percoll has beneficial effects on blastocyst cell number. Table 1 Developmental rates of in vitro embryos using sperm from 2 bulls isolated by three methods with 4 replicates

140 EFFECT OF A NOVEL BOVINE EMBRYO CULTURE MEDIUM TO IMPROVE BLASTOCYST DEVELOPMENT

2013 ◽  
Vol 25 (1) ◽  
pp. 217
Author(s):  
R. F. Gonçalves ◽  
C. Figueiredo ◽  
M. A. Achilles
Keyword(s):  
Culture Medium ◽  
Apoptotic Cell ◽  
Cell Number ◽  
Total Cell ◽  
Cell Numbers ◽  
Group 2 ◽  
Group 1 ◽  
Hatching Rates

There are still immense differences in the quality of in vitro-produced embryos compared to their in vivo-generated counterparts. These differences include a higher sensitivity of in vitro-produced embryos towards cryopreservation. The quality of such embryos has been evaluated using various parameters like morphological examination, assessment of total cell numbers, or pregnancy rates after transfer. In the present study, the effects of glycine, alanine, taurine, and glutamine addition to SOF (Achilles Genetics culture medium, Achilles Genetics®, Garça, SP, Brazil) on the in vitro development (cleavage and blastocyst rates) and quality (total cell and apoptotic cell numbers) of bovine embryos were determined. Ovaries of Nelore cows were obtained from a slaughterhouse. Cumulus–oocyte complexes (COC) were collected from follicles ≥4 mm in diameter, matured in TCM-199, and fertilized with frozen–thawed Nelore bull semen (IVF = Day 0). On Day 1, presumptive zygotes were cultured in SOF supplemented with fetal bovine serum (FBS, group 1, n = 550) or in Achilles Genetics culture medium (SOF supplemented with Achilles Mixture and FBS, group 2, n = 557) at 38.5°C and 5% CO2 in air until Day 9. Embryos were evaluated during culture: at Day 3 cleavage rates, at Day 7 blastocyst rates, and on Day 9 hatching rates. Experiments were replicated 5 times, analysed using ANOVA, followed by a comparison of means by Tukey test (P ≤ 0.05). Blastocysts at Day 8 from Group 1 (n = 75) and Group 2 (n = 75) were fixed and permeabilized for TUNEL assay (DeadEndTM Florimetric TUNEL System, Promega, Madison, WI, USA), according to the manufacturer instructions. Total cell number, apoptotic cell number, and apoptotic cell index (calculated by dividing the apoptotic cell number by total cell number) were analyzed by analysis of variance and means were compared by Student Newman Keuls test. The threshold of significance was set at P ≤ 0.05. Cleavage rates were 79.2 ± 2.5 for group 1 and 91.0 ± 2.5 for group 2. Blastocyst and hatching rates (calculated on the total of zygotes) for group 2 (47.4 ± 2.8; 82.1 ± 1.5) were significantly greater than for group 1 (39.8 ± 2.8; 74.3 ± 1.5). The total cell numbers were not different (P > 0.05) between group 1 (112.7 ± 2.9) and group 2 (111.1 ± 2.7). Blastocysts from group 2 showed lower (P < 0.05) number of apoptotic cells (10.7 ± 1.2) than those from group 1 (20.9 ± 1.2). These results indicate that the addition of glycine, alanine, taurine, and glutamine to SOF (Achilles Mixture) may be an important energy source for the bovine blastocyst and could act synergistically to enhance embryo development to the hatching stage and embryo quality. Financial support from CNPq and FAPESP.

188 THE IMPACT OF OIL SOURCE AND TYPE OF GLUTAMINE ON MEDIA EQUILIBRATION TIMES AND EMBRYO DEVELOPMENT

2007 ◽  
Vol 19 (1) ◽  
pp. 210
Author(s):  
D. W. Linck ◽  
D. K. Gardner
Keyword(s):  
Embryo Development ◽  
Embryo Culture ◽  
Culture Medium ◽  
Cell Number ◽  
Total Cell ◽  
Blastocyst Development ◽  
Total Cell Number ◽  
Cell Numbers ◽  
Oil Source ◽  
The Impact

Recently, there has been much debate involving the time necessary to effectively equilibrate an embryo culture system, i.e. dishes, media, and oil. Glutamine present in the culture medium spontaneously deaminates at 37�C to release embryo-toxic ammonium. Additionally, the source of the oil overlay can impact the culture environment. A sub-optimal oil overlay, combined with free glutamine (Gln), could effectively become embryo-toxic over a short time period (&lt;48 h). Therefore, the aim of this study was to determine how times of media equilibration, using various combinations of oil source and type of Gln, affect embryo development. Zygotes were collected from 4-week-old CF1 outbred female mice following superovulation and mating. Embryos were cultured in groups of 10 in 20-�L drops of medium G1.2. Initially, embryos were cultured in one of 4 treatment media. In this 2 � 2 factorial design, the culture medium was pre-equilibrated 18 h prior to embryo retrieval and contained free Gln or the heat-stable dipeptide alanyl-glutamine (AlaGln), combined with an oil source of either Sigma mineral oil (Sigma-Aldrich Corp., St Louis, MO, USA) or Ovoil paraffin oil (Vitrolife, Inc., Englewood, CO, USA). The initial study was then repeated using only the best and worst case groups to determine the effect of incubation time as a variable (either 2 h or 18 h). Blastocyst development and total cell numbers were analyzed after 72 h of culture, and differences between treatments were assessed using Fisher&apos;s exact test and Student&apos;s t-test. After 18 h of pre-equilibration (n e 300 embryos/treatment), blastocyst development in Ovoil + AlaGln (38.6%) was significantly greater when compared to: Ovoil + Gln: 25.5% (P &lt; 0.01), Sigma + AlaGln: 12.8% (P &lt; 0.01), and Sigma + Gln: 11.9% (P &lt; 0.01). Additionally, the total cell numbers in comparison to Ovoil + AlaGln (44.6 � 10) were significantly decreased: 35.5 � 7 (P &lt; 0.001), 34.9 � 9 (P &lt; 0.001), and 29.9 � 9 (P &lt; 0.001), respectively. In the second experiment, blastocyst development and total cell number between Ovoil + AlaGln (n = 224) and Sigma + Gln (n = 264) after 18 h of pre-equilibration were: 40.4% vs. 9.9% (P &lt; 0.01) and 46.6 � 9 vs. 29.4 � 9 P &lt; 0.001), respectively. However, after 2 h of pre-equilibration, the results between Ovoil + AlaGln (n = 260) and Sigma + Gln (n = 284) were: 42.3% vs. 18.3% (P &lt; 0.01) and 46.9 � 10 vs. 33.6 � 6 (P &lt; 0.001), respectively. Therefore, when comparing blastocyst development and total cell number between pre-equilibration times (2 h vs. 18 h), the Ovoil + AlaGln group, 42.3% vs. 40.4% and 46.9 � 10 vs. 46.6 � 9, showed no significant differences, respectively. In contrast, the Sigma + Gln group produced significant differences for both blastocyst development, 18.3% vs. 9.9% (P &lt; 0.01), and total cell number, 33.6 � 6 vs. 29.4 � 9 (P &lt; 0.05), between pre-equilibration times (2 h vs. 18 h), respectively. Data presented confirm the need for an alternative source of glutamine in embryo culture media. The data also indicate that the source of oil has a profound effect on the experimental outcome. Using the appropriate oil and form of Gln means that media can be safely equilibrated for 18 h.

scholarly journals Zinc supplementation during in vitro embryo culture increases inner cell mass and total cell numbers in bovine blastocysts1

2019 ◽  
Vol 97 (12) ◽  
pp. 4946-4950 ◽  
Author(s):  
Lydia K Wooldridge ◽  
Madison E Nardi ◽  
Alan D Ealy
Keyword(s):  
Embryo Culture ◽  
Zinc Supplementation ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Retention Rates ◽  
Bovine Embryo ◽  
Blastocyst Development ◽  
Cell Numbers

Abstract Deficiencies in current embryo culture media likely contribute to the poor blastocyst development rates and pregnancy retention rates for in vitro produced (IVP) bovine embryos. Of special concern is the lack of micronutrients in these media formulations. One micronutrient of interest is zinc, an essential trace element involved with various enzyme and transcription factor activities. The objective of this work was to describe whether zinc sulfate supplementation during in vitro embryo culture affects bovine embryo development and blastomere numbers. Either 0, 2, 20, or 40 µM zinc sulfate was supplemented to presumptive zygotes cultured in synthetic oviductal fluid containing AAs and bovine serum albumin for 8 d. None of the treatments affected cleavage rates. Percentage of blastocysts on days 7 and 8 postfertilization was not affected by supplementing 2 or 20 µM zinc but were reduced (P &lt; 0.05) with 40 µM zinc. In blastocysts harvested on day 8, inner cell mass (ICM) and total cell number were increased (P &lt; 0.05) with 2 µM zinc supplementation but not with the other zinc concentrations. Numbers of trophectoderm cells were not affected by zinc treatment. In conclusion, supplementing zinc during bovine embryo culture did not impact blastocyst development but improved ICM cell numbers. This improvement in ICM cell number may have implications for improved pregnancy retention rates after IVP embryo transfer as smaller ICM sizes are associated with poor pregnancy success in cattle.

scholarly journals The superiority of conditioned medium derived from rapidly expanded mesenchymal stem cells for neural repair

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Ya-Tzu Chen ◽  
May-Jywan Tsai ◽  
Nini Hsieh ◽  
Ming-Jei Lo ◽  
Meng-Jen Lee ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Conditioned Medium ◽  
Culture Medium ◽  
Standard Medium ◽  
Neural Repair ◽  
Cord Injury ◽  
Standard Culture ◽  
Standard Culture Medium

Abstracts Background Spinal cord injury (SCI) is a complex and severe neurological condition. Mesenchymal stem cells (MSCs) and their secreted factors show promising potential for regenerative medicine. Many studies have investigated MSC expansion efficacy of all kinds of culture medium formulations, such as growth factor-supplemented or xeno-free medium. However, very few studies have focused on the potential of human MSC (hMSC) culture medium formulations for injured spinal cord repair. In this study, we investigated the effect of hMSC-conditioned medium supplemented with bFGF, EGF, and patient plasma, namely, neural regeneration laboratory medium (NRLM), on SCI in vitro and in vivo. Methods Commercial and patient bone marrow hMSCs were obtained for cultivation in standard medium and NRLM separately. Several characteristics, including CD marker expression, differentiation, and growth curves, were compared between MSCs cultured in standard medium and NRLM. Additionally, we investigated the effect of the conditioned medium (referred to as NRLM-CM) on neural repair, including inflammation inhibition, neurite regeneration, and spinal cord injury (SCI), and used a coculture system to detect the neural repair function of NRLM-MSCs. Results Compared to standard culture medium, NRLM-CM had superior in inflammation reduction and neurite regeneration effects in vitro and improved functional restoration in SCI rats in vivo. In comparison with standard culture medium MSCs, NRLM-MSCs proliferated faster regardless of the age of the donor. NRLM-MSCs also showed increased adipose differentiative potential and reduced CD90 expression. Both types of hMSC CM effectively enhanced injured neurite outgrowth and protected against H2O2 toxicity in spinal cord neuron cultures. Cytokine arrays performed in hMSC-CM further revealed the presence of at least 120 proteins. Among these proteins, 6 demonstrated significantly increased expression in NRLM-CM: adiponectin (Acrp30), angiogenin (ANG), HGF, NAP-2, uPAR, and IGFBP2. Conclusions The NRLM culture system provides rapid expansion effects and functional hMSCs. The superiority of the derived conditioned medium on neural repair shows potential for future clinical applications.

scholarly journals 147ION COMPOSITION OF CULTURE MEDIUM INFLUENCES MITOCHONDRIAL DISTRIBUTION AND BLASTOCYST DEVELOPMENT OF PREIMPLANTATION PORCINE EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 195
Author(s):  
M.L. Conover-Sparman ◽  
R.L. Krisher
Keyword(s):  
Culture Medium ◽  
Krebs Cycle ◽  
Cell Number ◽  
Developmental Competence ◽  
Homogeneous Distribution ◽  
Distribution Data ◽  
Blastocyst Development ◽  
Developmental Potential ◽  
Mitochondrial Distribution

Elevated intracellular calcium (Ca2+) concentrations impair hamster embryo metabolism and viability (Lane M and Bavister B 1998 Biol. Reprod. 59, 1000–1007). Extracellular magnesium (Mg2+) regulates intracellular Ca2+ by controlling its uptake and release. In the present study, we examined the effects of altering Ca2+ and Mg2+ ion concentrations in Purdue Porcine Medium (PPM1) on porcine embryo mitochondrial distribution, metabolic (glycolytic and Krebs cycle) activity, and in vitro developmental potential. Cumulus-oocyte complexes collected from abattoir ovaries were matured for 40–42h, inseminated with 5×105 sperm mL−1 for 5h, and initially cultured in 1:0.4 or 2:1 ratio of Ca2+ to Mg2+ (concentrations in mM) at 38.7°C, in 6% CO2, 10% O2, balance N2. At 22–26, 46–50, and 70–74h post-insemination, 2-, 4-, and 8-cell embryos, respectively, were removed from culture to evaluate mitochondrial distribution (confocal microscopy after tetramethylrhodamine methyl ester staining) and glycolytic and Krebs cycle activity (5-[3H]-glucose and 2-[C14]-pyruvate, respectively). Remaining embryos were further cultured to determine developmental competence (2:1, n=548; 1:0.4, n=560). Cleavage was assessed on Day 3 (2:1, n=552; 1:0.4, n=560) of culture. All data were analyzed using GLM ANOVA, except mitochondrial distribution data which were analyzed using GLIMMIX. A majority (P&lt;0.05) of 2-cell (65%, 13/20) and 4-cell (67%, 22/33) embryos cultured in 2:1 displayed a homogeneous mitochondrial distribution. More (70%, 21/30; P&lt;0.05) 8-cell embryos cultured in 2:1 had a perinuclear mitochondrial distribution. When cultured in 1:0.4, a majority (61%, 14/23; P&lt;0.05) of 2-cell embryos displayed a cortical mitochondrial distribution, whereas most (P&lt;0.05) 4-cell (66%, 19/29) and 8-cell embryos (69%, 18/26) displayed a homogeneous distribution. Glycolytic and Krebs cycle activities were similar (P&gt;0.05) between treatments and across all cell stages examined. Treatment had no effect (P&gt;0.05) on cleavage or blastocyst total cell number. Unlike hamster embryos, culturing pig embryos in a higher Ca2+ concentration resulted in more embryos developing to the blastocyst stage. Culture medium containing 2mM Ca2+ and 1mMMg2+ best supports in vitro blastocyst development, possibly by supporting a more correct mitochondrial distribution. These results are not mediated via changes in glycolytic or Krebs cycle activity, thus suggesting that another cellular mechanism plays a key role in developmental competence in early pig embryos. Table 1 Effects of Ca2+:Mg2+ on porcine embryonic development and metabolic activity (mean±SEM).

62 Sequential nutrient restriction and provision during bovine in vitro embryo culture differentially affect blastocyst development and quality with oocytes from varied sources

2019 ◽  
Vol 31 (1) ◽  
pp. 156
Author(s):  
R. Pasquariello ◽  
Y. Yuan ◽  
D. Logsdon ◽  
J. Becker ◽  
L. Yao ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Embryo Culture ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Total Cell ◽  
Nutrient Concentrations ◽  
Blastocyst Development ◽  
Total Cell Number

We have demonstrated that bovine blastocyst development was improved after culture in medium with only 6.25% of standard carbohydrate and amino acid concentrations, supplemented with fatty acids. However, these blastocysts had lower cell numbers. We hypothesised that this was due to deficiencies in embryo metabolism at the time of blastocyst formation. Thus, our objectives were to (1) determine whether using a sequential combination of nutrient concentrations could rescue blastocyst cell number; and (2) investigate the efficacy of reduced nutrient medium in 2 sources of oocytes. Oocytes were in vitro matured in identical medium either in our laboratory or during shipment from a commercial supplier. Oocytes in our laboratory were derived from feedlot heifers while purchased oocytes were obtained from culled cows. Zygotes were cultured using sequential medium with fraction V BSA. In step 1/step 2, embryos were cultured using 100% (glucose 0.5 mM/fructose 3.0mM, pyruvate 0.3/0.1mM, lactate 10.0/6.0mM, NEEA 1×/1× MEM, EAA 0.25×/0.5× MEM), 25% or 6.25% of standard nutrient concentrations. On Day 3, embryos were moved to step 2 as follows: 100% to 100%, 25% to 25%, 25% to 100%, 6.25% to 25%, or 6.25% to 100%. Lipid content of single mature oocytes from both sources was determined using gas chromatography coupled to an ISQ-LT MS/MS (GC-MS; Thermo Scientific, Waltham, MA, USA). Data (mean±s.e.m.) were analysed using ANOVA (P&lt;0.05). When oocytes from feedlot heifers were used, blastocyst development and cell number did not differ between treatments. When oocytes from culled cows were used, blastocyst development was improved after embryo culture in 25-25% (45.1±3.3%) and 6.25-25% (46.6±3.2%) compared with 100-100% (34.2±3.2%). However, inner cell mass number of blastocysts cultured in 25-25% (25.6±2.5) and 6.25-25% (26.0±2.6) was reduced compared with 100-100% (41.4±4.5); TE and total cell number did not differ. Embryos cultured in 100-100%, 25-100%, and 6.25-100% were equivalent. Metabolomics revealed that 10 lipid compounds (polyunsaturated fatty acids, glycosyldiacylglycerols, and glycerophospholipids) differed in abundance between the two sources of oocytes. These results show that oocytes from different sources lead to different experimental outcomes, likely due to a combination of age, body condition, diet, and hormone treatment of the female. Oocytes from culled cows result in embryos that develop to blastocysts better in a reduced nutrient environment, although these embryos have fewer inner cell masses, suggesting that quality may be reduced. Embryos from feedlot heifer oocytes are relatively immune to nutrient fluctuations. Different endogenous fatty acid reserves in the oocyte may lead to differing metabolic strategies in the subsequent embryo, altering their response to substrate availability during in vitro culture. These results also demonstrate that reduction of nutrients during culture has no detrimental effect on blastocyst development or total cell number in either oocyte source, but that inner cell mass formation requires increased nutrient provision.

scholarly journals Quality of porcine blastocysts produced in vitro in the presence or absence of GH

Reproduction ◽  
2004 ◽  
Vol 127 (2) ◽  
pp. 165-177 ◽  
Author(s):  
A Kidson ◽  
F J Rubio-Pomar ◽  
A Van Knegsel ◽  
H T A Van Tol ◽  
W Hazeleger ◽  
...  
Keyword(s):  
Embryo Development ◽  
Reference Value ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Blastocyst Development ◽  
Cell Numbers

GH receptor (GHR) mRNA is expressed in bovine in vitro produced embryos up to the blastocyst stage and GH improves the quality of bovine embryos by increasing blastocyst cell numbers and reducing the incidence of apoptosis as evaluated by DNA strand-break labelling. Porcine in vitro produced blastocysts have lower cell numbers than in vivo blastocysts and exhibit higher incidences of apoptosis. Therefore we investigated the effects of 100 ng GH/ml NCSU23 medium during in vitro culture of presumptive in vitro fertilized sow zygotes on embryo development and blastocyst quality (defined by diameter, cell number, apoptosis and survival after non-surgical transfer). In vivo produced blastocysts were analysed concurrently as a reference value. GHR was expressed in embryos from the 2-cell to blastocyst stages. GH had no effect on blastocyst development or cell numbers, but increased the mean blastocyst diameter. The incidence of apoptosis, detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL), was decreased by GH, but when non-TUNEL-labelled apoptotic fragmented nuclei were included, no difference was seen. GH appeared to slow down the progression of apoptosis though. In vivo produced blastocysts presented no apoptotic nuclei, and contained higher cell numbers and larger diameters. Pregnancy rates on day 11 were similar for all groups, but survival was poorer for in vitro than in vivo produced blastocysts. In this study GH appeared to be beneficial only from the blastocyst stage, but the presence of GHR from early cleavage stages nevertheless indicates a role for GH throughout porcine embryo development and deserves further investigation.

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