scholarly journals 144 ROLE OF GnRH ON MOUSE PRE-IMPLANTATION EMBRYONIC DEVELOPMENT IN VITRO

2005 ◽  
Vol 17 (2) ◽  
pp. 222
Author(s):  
M. Montagner ◽  
A. Cropp ◽  
J. Swanson ◽  
R. Cederberg ◽  
P. Goncalves ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Gnrh Agonist ◽  
Critical Role ◽  
Blastocyst Stage ◽  
Dependent Manner ◽  
Blastocyst Formation ◽  
Morula Stage ◽  
Development In Vitro ◽  
Human Uterine Endometrium

The interaction between GnRH and its receptor on gonadotropes within the anterior pituitary gland represents a key point for regulation of the reproduction. In addition, GnRH can act in multiple extrapituitary tissues via autocrine/paracrine mechanisms. Protein for GnRH and mRNA for both GnRH and its receptor have been detected in human uterine endometrium and oviduct as well as in embryos at the morula/blastocyst stage in the mouse and human. Therefore, we hypothesized that GnRH may have a critical role in the development of pre-implantation embryos. To address this question, we examined the effect of a GnRH agonist and antagonist on the development of mouse embryos in vitro. For these studies, 1-cell embryos were randomly allocated to culture in KSOM containing the appropriate treatment for 144 h at 37°C in a 5% CO2 in air environment. The medium was changed every 12 h and embryos were scored daily for development. The data were compared using a χ2 test. First, we wanted to determine if a GnRH agonist, histrelin, could enhance embryonic development. Embryos were cultured with (n = 35) or without (n = 36) 10 μM histrelin. The addition of histrelin did not increase morula or blastocyst formation v. control. Second, we cultured embryos in the presence of different concentrations (0, 0.001, 0.01, 0.1, 1, and 10 μM) of the GnRH antagonist, SB-75 (cetrorelix; n = 22/treatment) in order to determine its effect on embryonic development. The 10 μM SB-75 treatment blocked embryo development beyond the compact morula stage (P < 0.001). To determine if this was a receptor mediated effect, we attempted to rescue development of SB-75 treated embryos with a histrelin challenge. Our treatments consisted of control (n = 30), 10 μM histrelin (n = 27), 10 μM SB-75 (n = 29), and 10 μM SB-75 in combination with either 1 μM (n = 27) or 10 μM (n = 25) histrelin. Both levels of histrelin partially rescued the inhibition of blastocyst formation by SB-75 (P < 0.01). Next, we were interested in examining the signaling cascade activated following binding of GnRH to its receptor in pre-implantation embryos. Toward this end, we treated embryos with inhibitors of either PKC or PKA. First, embryos were cultured in the presence of 0 (n = 33), 0.1 (n = 35), 1 (n = 35), or 10 (n = 35) μM GF109203X (GFX), a PKC inhibitor. Similar to the results obtained with SB-75, treatment with 10 μM GFX significantly reduced development to the compact morula stage and completely blocked blastocyst formation. Second, we treated embryos (n = 15 to 17/treatment) with different concentrations (0, 0.01, 0.1, 0.5, or 1 mM) of the PKA inhibitor, SQ22536. In contrast to treatment with GFX, rates of blastocyst formation were decreased only by 35% (P < 0.05) at the highest concentration of SQ22536. The percentage of embryos developing to the hatched blastocyst stage was decreased in a dose-dependent manner following SQ22536 treatment (P < 0.05); however, this effect was not consistent with SB-75 inhibition of blastocyst formation. We suggest that GnRH has an important autocrine effect on early embryonic development, potentially signaling via PKC. Funding for M Montagner was provided by CAPES, Brazil.

scholarly journals 131 MODIFICATION OF AMINO ACID CONCENTRATIONS IN MEDIUM BY PIG EMBRYOS FROM THE ZYGOTE TO THE BLASTOCYST STAGE

2005 ◽  
Vol 17 (2) ◽  
pp. 216
Author(s):  
P. Booth ◽  
T. Watson ◽  
H. Leese
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Embryonic Development ◽  
Amino Acid Profile ◽  
Blastocyst Stage ◽  
Morula Stage ◽  
Amino Acid Profiles ◽  
The Difference ◽  
Amino Acid Concentrations

Pre-implantation embryos can produce and consume amino acids in a manner dependent upon stage of embryonic development (Partridge and Leese 1996 Reprod. Fert. Dev. 8, 945) that may also be predictive of subsequent viability (Houghton et al. 2002 Hum. Reprod. 17, 999). To examine these relationships in the pig, the appearance or depletion of 18 amino acids from a presumptive near-physiological mixture was determined by HPLC in porcine in vitro-produced embryos from the zygote to the blastocyst stage. Cumulus oocyte complexes derived from slaughterhouse prepubertal pig ovaries were matured for 40 h in modified TCM-199 before being fertilized (Day 0) with frozen thawed semen in tris-based medium. After 6 h, presumptive zygotes were denuded and cultured in groups of 20 in NCSU medium modified to contain a physiological mixture of 18 amino acids including 0.1 mM glutamine (NCSUaa). Groups of 2–10 embryos (dependent on stage) were removed on Day 0 (1 cell), Day 1 (2- and 4-cell), Day 4 (compact morula), and Day 6 (blastocyst) and placed in 4 μL NCSUaa for 24 h. After incubation, the embryos were removed and the medium analyzed by HPLC. Each stage was replicated 3–9 times. Since amino acid profiles of 2- and 4-cell embryos were not different, data were combined. Overall, arginine (1.19 ± 0.33), glutamine (0.78 ± 0.34) and threonine (0.05 ± 0.04) were significantly (P < 0.01) depleted from the medium whereas alanine (0.21 ± 0.1), glycine (0.20 ± 0.06), asparagine (0.13 ± 0.5), lysine (0.1 ± 0.03), isoleucine (0.08 ± 0.01), valine (0.05 ± 0.01), leucine (0.04 ± 0.02), phenylalanine (0.03 ± 0.01), and histidine (0.02 ± 0.04) significantly (P < 0.05) accumulated (mean of the 4 sampling timepoints; all values pmol/embryo/h ± SEM). The difference between amino acid accumulation and depletion (balance) was approximately equivalent between Day 0 and the morula stage although turnover (sum of depletion and accumulation) steadily decreased during this period from 3.1 on Day 0 to 1.35 pmol/embryo/h at the morula stage. However, at the blastocyst stage, turnover and balance increased to 6.32 and 2.42 pmol/embryo/h, respectively, i.e. net appearance occurred. Notable changes in amino acid profile during development included decreases in accumulation of asparagine, glutamate, and glycine in the medium and the depletion of glutamine over Days 0, 1, and 4, followed by reversal of these trends by Day 6. These data suggest that pig embryos can alter the accumulation and depletion rates of amino acids in a manner that is dependent on the specific amino acid and the stage of embryonic development. This work was supported by BBSRC.

scholarly journals K+ efflux through two-pore domain K+ channels is required for mouse embryonic development

Reproduction ◽  
2012 ◽  
Vol 143 (5) ◽  
pp. 625-636 ◽  
Author(s):  
Chang-Gi Hur ◽  
Eun-Jin Kim ◽  
Seong-Keun Cho ◽  
Young-Woo Cho ◽  
Sook-Young Yoon ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Mammalian Cells ◽  
Selective Serotonin Reuptake Inhibitors ◽  
Blastocyst Stage ◽  
Ruthenium Red ◽  
Channel Blockers ◽  
Blastocyst Formation ◽  
Cell Stage ◽  
Wide Range ◽  
Morula Stage

Numerous studies have suggested that K+ channels regulate a wide range of physiological processes in mammalian cells. However, little is known about the specific function of K+ channels in germ cells. In this study, mouse zygotes were cultured in a medium containing K+ channel blockers to identify the functional role of K+ channels in mouse embryonic development. Voltage-dependent K+ channel blockers, such as tetraethylammonium and BaCl2, had no effect on embryonic development to the blastocyst stage, whereas K2P channel blockers, such as quinine, selective serotonin reuptake inhibitors (fluoxetine, paroxetine, and citalopram), gadolinium trichloride, anandamide, ruthenium red, and zinc chloride, significantly decreased blastocyst formation (P<0.05). RT-PCR data showed that members of the K2P channel family, specifically KCNK2, KCNK10, KCNK4, KCNK3, and KCNK9, were expressed in mouse oocytes and embryos. In addition, their mRNA expression levels, except Kcnk3, were up-regulated by above ninefold in morula-stage embryos compared with 2-cell stage embryos (2-cells). Immunocytochemical data showed that KCNK2, KCNK10, KCNK4, KCNK3, and KCNK9 channel proteins were expressed in the membrane of oocytes, 2-cells, and blastocysts. Each siRNA injection targeted at Kcnk2, Kcnk10, Kcnk4, Kcnk3, and Kcnk9 significantly decreased blastocyst formation by ∼38% compared with scrambled siRNA injection (P<0.05). The blockade of K2P channels acidified the intracellular pH and depolarized the membrane potential. These results suggest that K2P channels could improve mouse embryonic development through the modulation of gating by activators.

scholarly journals 205.The effect of FSH concentration during IVM and gamete co-incubation length during IVF on the development of unstimulated prepubertal ewe oocytes

2004 ◽  
Vol 16 (9) ◽  
pp. 205 ◽  
Author(s):  
K. M. Morton ◽  
W. M. C. Maxwell ◽  
G. Evans
Keyword(s):  
Blastocyst Stage ◽  
Developmental Competence ◽  
Blastocyst Formation ◽  
In Vitro Development ◽  
Oocyte Collection ◽  
Chi Squared ◽  
Development In Vitro ◽  
Stage 1 ◽  
Vitro Development

The developmental competence of prepubertal oocytes can be increased by the administration of gonadotrophins prior to oocyte collection (1); but this is not possible with abattoir-sourced oocytes, and modifications to the IVP system may increase in vitro development. Experiments were conducted to determine the effects of FSH concentration (10, 20 or 60 μg mL-1) during IVM (5 replicates) and gamete co-incubation length (short: 2-3 h, long: 18-20 h) during IVF (6 replicates) on subsequent embryonic development. For both experiments ovaries were collected from prepubertal lambs (16-24 weeks) slaughtered at an abattoir and embryos produced in vitro (1). Data were analysed by chi-squared test. Oocyte cleavage at 48 hours post-insemination (hpi) was higher for oocytes matured in medium containing 20 (60/77; 77.9%) and 60 (56/73; 76.7%) than 10 μg mL-1 (40/67; 59.7%) FSH. Blastocyst formation (% cultured oocytes) on Day 7 (Day 0 = IVF) was higher for oocytes matured with 20 (31/77; 40.3%) than 10 (16/67; 23.9%) or 60 μg mL-1 (20/73; 27.4%). Oocyte cleavage at 48 hpi was reduced for short (36/57; 63.2%) compared with long (49/55; 89.1%) co-incubation, although blastocyst formation (% cultured oocytes; Day 7) did not differ between groups (22/57; 38.6% and 23/55; 41.8%, respectively). These results demonstrate that increasing the FSH concentration above normal levels during IVM of prepubertal lamb oocytes improves development in vitro. Gamete co-incubation length did not influence the proportion of oocytes progressing to the blastocyst stage. (1) Morton et al. (2003) Proc. Soc. Reprod. Fert. P18.

Knockdown of regulator of G-protein signalling 2 (Rgs2) leads to abnormal early mouse embryo development in vitro

2015 ◽  
Vol 27 (3) ◽  
pp. 557 ◽  
Author(s):  
Yan Zhu ◽  
Ya-Hong Jiang ◽  
Ya-Ping He ◽  
Xuan Zhang ◽  
Zhao-Gui Sun ◽  
...  
Keyword(s):  
Embryonic Development ◽  
G Protein ◽  
Embryo Development ◽  
Expression Patterns ◽  
Critical Role ◽  
Α Subunit ◽  
G Protein Signalling ◽  
Early Embryos ◽  
Development In Vitro

Regulator of G-protein signalling 2 (Rgs2) is involved in G-protein-mediated signalling by negatively regulating the activity of the G-protein α-subunit. In the present study, the expression patterns of Rgs2 in mouse ovarian tissues and early embryos were determined by semiquantitative reverse transcription–polymerase chain reaction, immunohistochemistry and immunofluorescent analyses. Rgs2 expression was observed in the ovarian tissues of adult female mice, with an almost equal expression levels during different stages of the oestrous cycle. Rgs2 was abundant in the cytoplasm, membrane, nuclei and spindles of intact polar bodies in mouse early embryos at different developmental stages from the zygote to blastocyst. The effect of Rgs2 knockdown on early embryonic development in vitro was examined by microinjecting Rgs2-specific short interfering (si) RNAs into mouse zygotes. Knockdown of endogenous Rgs2 expression led to abnormal embryonic development in vitro, with a considerable number of early embryos arrested at the 2- or 4-cell stage. Moreover, mRNA expression of three zygotic gene activation-related genes (i.e. Zscan4, Tcstv1 and MuERV-L) was decreased significantly in 2-cell arrested embryos. These results suggest that Rgs2 plays a critical role in early embryo development.

279 IMPROVEMENT OF RAT IN VITRO FERTILIZATION PROTOCOL USING CRYOPRESERVED SPERM AND ITS APPLICATION TO OTHER STRAINS: A PRELIMINARY STUDY

2010 ◽  
Vol 22 (1) ◽  
pp. 296
Author(s):  
J. Ito ◽  
Y. Seita ◽  
S. Sugio ◽  
K. Fujiwara ◽  
N. Kashiwazaki
Keyword(s):  
In Vitro Fertilization ◽  
Embryonic Development ◽  
Tyrosine Phosphorylation ◽  
Blastocyst Formation ◽  
Vitro Fertilization ◽  
Culture Time ◽  
Initial Culture ◽  
Preliminary Study ◽  
Development In Vitro

In rats, the success of in vitro fertilization (IVF) was reported almost 40 years ago. Although it had been demonstrated in papers that these IVF oocytes using sperm freshly collected from cauda epididymides can be developed to term via embryo transfer, successful IVF with cryopreserved rat sperm has never been reported. Very recently, we reported establishment of a successful IVF system using frozen-thawed spermatozoa treated with a phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthin (IBMX), in Wistar rats (Seita et al. 2009 Biol. Reprod.). The objectives in this study were (1) improvement of the IVF system to a more convenient and simple protocol and (2) a preliminary study for applying our IVF system to inbred rat strains Fischer 344 (F344) and Brown-Norway (BN). In experiment 1, we examined the effect of preincubation time for frozen-thawed sperm on fertilization (2 pronuclei formation). Frozen-thawed sperm were preincubated up to 6 h and then used for IVF according to our previous report. Data showed that sperm preincubated for 5 h contributed to higher fertility than those for other preincubation times. In experiment 2, we examined the effect of co-culture time up to 10 h for IVF on fertility and embryonic development in vitro. The oocytes co-cultured with sperm beyond 6 h showed higher fertilization and blastocyst formation rates than those in 2 and 4 h. In experiment 3, we examined the effect of initial culture period in fertilization medium (310 mOsm modified R1ECM; O h et al. 1998 Biol. Reprod.) on embryonic development in vitro. After IVF, oocytes were cultured in fertilization medium for 6, 12, or 24 h and further cultured in R1ECM up to 120 h. Cleavage rates were not affected by initial culture time in fertilization medium. However, oocytes cultured in fertilization medium for 12 h showed higher blastocyst formation rate than those for 0, 6 and 24 h. In experiment 4, we examined whether the IVF protocol can be applied to F344 and BN rats. Fresh and frozen-thawed sperm collected from cauda epididymides in F344 and BN were used for detection of capacitation-associated tyrosine phosphorylation. In fresh F344 sperm, tyrosine phosphorylation was induced in a time-dependent manner. Although tyrosine phosphorylation was inhibited in frozen-thawed F344 sperm, it was dramatically accelerated by IBMX treatment as well as frozen-thawed Wistar sperm. However, tyrosine phosphorylation in fresh and frozen-thawed BN sperm was suppressed and the phosphorylation in frozen-thawed sperm was not improved by IBMX. Our data demonstrate that (1) a more efficient IVF system using frozen-thawed rat sperm was developed and (2) the IVF system can be applied to at least F344 strain. The work was supported in part by Grant-in-Aid for Scientific Research from JSPS (KAKENHI) (21789253) to J.I. This work was also supported in part by the Promotion and Mutual Aid Corporation for Private Schools of Japan through a Grant-in-Aid for Matching Fund Subsidy for Private Universities to J.I. and N.K.

Rat embryo development in vitro

1981 ◽  
Vol 96 (4) ◽  
pp. 546-551 ◽  
Author(s):  
S. K. Roy ◽  
Jayasree Sengupta ◽  
S. K. Manchanda
Keyword(s):  
Bovine Serum Albumin ◽  
Bovine Serum ◽  
Culture Medium ◽  
Synthetic Medium ◽  
Blastocyst Formation ◽  
Rat Embryo ◽  
Rat Embryos ◽  
Morula Stage ◽  
Development In Vitro

Abstract. We report the first successful culture of 8-cell/morula stage rat embryos in a fully synthetic medium supplemented with 3% crystalline bovine serum albumin. Eighty-four per cent of morulae developed to blastocysts, showing that this is a highly efficient culture medium for in vitro studies on rat pre-implantation embryos. Blastocyst formation was severely inhibited by antioestrogen (nafoxidine 3 μg/ml) but no further reversal was obtained by giving oestrogen to culture medium containing this antagonist.

scholarly journals Development of cultured bovine embryos after exposure to high temperatures in the physiological range

Reproduction ◽  
2001 ◽  
pp. 107-115 ◽  
Author(s):  
RM Rivera ◽  
PJ Hansen
Keyword(s):  
Heat Shock ◽  
Embryonic Development ◽  
High Temperatures ◽  
Deleterious Effect ◽  
Blastocyst Stage ◽  
The Body ◽  
Body Temperatures ◽  
Cleavage Rate ◽  
Development In Vitro

Embryonic development is inhibited by exposure of cultured embryos to high temperatures. However, culture temperatures used to demonstrate the effects of heat on development have been higher than the body temperatures experienced typically by heat-stressed cows. The aim of this study was to determine whether exposing bovine oocytes and embryos to temperatures characteristic of body temperatures of heat-stressed cows would affect embryonic development in vitro. The CO2 percentage of the gas phase was adjusted in all experiments to prevent pH changes in the medium caused by decreased solubility of CO2 at high temperatures. Fertilization of oocytes at 41.0 degrees C reduced cleavage rate and the percentage of oocytes that became blastocysts compared with at 38.5 degrees C. There was no deleterious effect of fertilization at 40.0 degrees C. When putative zygotes and two-cell embryos were exposed to a range of temperatures from 38.5 to 41.0 degrees C for 3, 6, 9 or 12 h, heat shock reduced the number that developed to the blastocyst stage but only after exposure to 41.0 degrees C for 9 or 12 h. In addition, it was tested whether low O2 tension would reduce the detrimental effects of heat shock. The deleterious effect of 41.0 degrees C was not dependent upon oxygen content or the gas mixture used for culture (5% versus 20.95% O2), indicating that the deleterious effects of heat shock did not depend upon a high O2 environment. In the final experiment, embryos were exposed to 24 h fluctuations in temperature designed to mimic the rectal temperatures of cows exposed to heat stress. Exposure of embryos to this pattern of temperatures starting after fertilization reduced development when embryos were exposed to this environment for 8 days but not when embryos were exposed for 1 day only. These findings indicate that embryonic development can be disrupted by a short-term severe or a prolonged mild heat shock and that the effects of heat shock are not artefacts of changes in pH or high oxygen tension.

3 SEX INFLUENCES REGULATION OF GENE EXPRESSION BY DICKKOPF 1 IN THE BOVINE MORULA

2015 ◽  
Vol 27 (1) ◽  
pp. 94
Author(s):  
A. C. Denicol ◽  
K. B. Dobbs ◽  
P. J. Hansen
Keyword(s):  
Gene Expression ◽  
Embryonic Development ◽  
Cell Junctions ◽  
Dependent Manner ◽  
Stage Of Development ◽  
Expression Of Genes ◽  
Regulated Expression ◽  
Morula Stage ◽  
Dickkopf 1

Successful embryonic development depends upon molecules secreted by the reproductive tract. Among such molecules is the protein dickkopf 1 (DKK1), an antagonist of canonical WNT signalling that can also activate the noncanonical, planar cell polarity (PCP) pathway. DKK1 increases the proportion of cells that are trophectoderm and hypoblast in the blastocyst and increases competence of embryos to establish pregnancy after transfer to recipients. The objective was to determine whether DKK1 affects cell fate by regulating expression of genes that promote differentiation at the morula stage, possibly by activating the PCP pathway. A second objective was to determine if actions of DKK1 on the embryonic transcriptome were dependent on embryo sex. Bovine oocytes were fertilized in vitro with pools of 3 bulls for which X- and Y-sorted sperm was available. Embryos were treated with 100 ng mL–1 DKK1 or vehicle at Day 5 of development and harvested 24 h later. Embryos were pooled in 5 replicates of 20 embryos each. Following RNA reverse-transcription and amplification, cDNA was used for microarray analysis of global gene expression using the Affymetrix® Bovine Gene 1.0 ST array (Affymetrix, Santa, Clara, CA, USA). Statistical analysis was performed by ANOVA using JMP® Genomics (SAS Institute, Cary, NC, USA). A total of 9931 transcripts were identified as being expressed. Differentially expressed genes (DEG) were considered as those associated with P < 0.05 and fold change ≥1.5 or <0.66. There were 124 DEG between females and males (91 up-regulated in females and 33 up-regulated in males). A total of 68% of the genes up-regulated in females were located in the X chromosome. Treatment with DKK1 resulted in 132 DEG in females (68 up-regulated and 64 down-regulated) and 136 DEG in males (90 up-regulated and 46 down-regulated). Of these, 34 genes were regulated by DKK1 in both sexes: 14 in the same direction and 20 in opposite directions. Analysis by Ingenuity® Pathway software indicated that changes in gene expression caused by DKK1 would increase formation of actin fibers in females and inhibit formation in males. DKK1 inhibited expression of AMOT in male embryos, indicating that DKK1 may inhibit Hippo signalling at this stage of development. Evidence for regulation of the PCP pathway by DKK1 was the finding that DKK1 regulated expression of genes involved in cell polarization and differentiation in both females and males. In both sexes, DKK1 regulated expression of many genes associated with HNF4A, a marker of hypoblast cells that promotes formation of cell junctions. In conclusion, female and male embryos developing in vitro have different transcriptomic profiles at the morula stage. DKK1 regulates cell differentiation and embryonic development in a sex-dependent manner and effects may be mediated, at least in part, by activation of the PCP pathway.Supported by USDA AFRI 2011-67015-30688 and NIH R03 HD080855.

scholarly journals 148 AN EFFECT OF MELATONIN ON DEVELOPMENT OF BOVINE EMBRYOS CULTURED IN VITRO UNDER OPTIMAL OR ENHANCED OXYGEN TENSIONS

2005 ◽  
Vol 17 (2) ◽  
pp. 224 ◽  
Author(s):  
O. Poleszczuk ◽  
K. Papis ◽  
E. Wenta-Muchalska
Keyword(s):  
In Vitro Culture ◽  
Oxygen Concentration ◽  
Embryo Development ◽  
Blastocyst Stage ◽  
Free Radical Scavengers ◽  
Control Group ◽  
Bovine Embryo ◽  
Blastocyst Formation ◽  
Development In Vitro

Many different systems of free radical scavengers have been investigated during the last few years for in vitro culture of mammalian embryos. Melatonin is a potent reactive oxygen species scavenger and has been tested in the promotion of mouse embryo development in vitro (Ishizuka et al. 2000 J. Pin. Res. 28, 48–51). An effect of melatonin on bovine embryo development in vitro is described here. Slaughterhouse-derived oocytes were subjected to standard in vitro maturation and fertilization procedures. Presumptive zygotes randomly allocated to experimental groups were cultured for 3 days (Day 1–Day 3) in CR1aaLA medium (Papis et al. 2000 Theriogenology 54, 651–658) supplemented with two different concentrations of melatonin (10−6 M or 10−4 M; Sigma, St. Louis, MO, USA) or without melatonin (control). Culture was performed under two different gas atmospheres containing 4% CO2 and either normal (7%) or enhanced (20%) oxygen concentration (2 × 3 factorial analysis). At the end of Day 3, embryos from each treatment group, developed to at least the 4-cell stage, were collected and cultured without melatonin until Day 10 at optimum 4% CO2 and 7% O2 atmosphere. The numbers of blastocysts at Day 8 and hatching/hatched blastocysts at Day 10 were recorded. Five replicates of each treatment were performed. Blastocyst formation rates of presumptive zygotes and of Day 3, 4-cell embryos were calculated for each group. Differences between groups were analyzed using chi-square and/or Fisher's exact tests where appropriate. P < 0.05 was considered statistically significant. Out of 100, 100, and 101 presumptive zygotes cultured for the first 3 days in 7% oxygen with 10−4 M, 10−6 M, or no melatonin, 31 (31%), 40 (40%), and 44 (43.5%) developed to blastocyst stage and 25 (25%), 33 (33%), and 36 (36%) to hatching/hatched blastocyst stage, respectively. On the other hand, out of 102, 102, and 100 zygotes cultured in the same concentrations of melatonin, but under 20% of oxygen, an opposite tendency was observed, as 42 (41%), 25 (24.5%), and 32 (32%) blastocysts and 26 (25.5%), 21 (20.6%), and 25 (25%) hatching/hatched blastocysts developed, respectively. No statistical significance was reached here. However, out of 4-cell embryos put into in vitro culture after initial treatments in different melatonin concentrations, a decreased ratio of blastocyst formation was observed in the 10−4 M melatonin group (31/65, 47.7%) compared to that of the control (44/65, 67.7%; P = 0.0327) when the lower oxygen concentration was applied. However, a beneficial effect of melatonin was observed in the presence of 20% oxygen. Out of 61 embryos, 42 (68.9%) developed to the blastocyst stage after treatment in 10−4 M melatonin concentrations, vs. 32/63 (50.8%; P = 0.0458) blastocysts developed in control group. In conclusion, a beneficial or a harmful effect of melatonin on bovine embryo in vitro development was observed depending on the oxygen concentration during the treatment. Results presented seem to confirm a potent free radicals scavenging activity of melatonin in a bovine embryo culture system.

scholarly journals 277ENERGY REQUIREMENT DURING DEVELOPMENT TO THE BLASTOCYST STAGE OF PORCINE EMBRYOS PRODUCED IN VITRO

2004 ◽  
Vol 16 (2) ◽  
pp. 259
Author(s):  
K. Kikuchi ◽  
M. Ozawa ◽  
D.-I. Fuchimoto ◽  
J. Noguchi ◽  
H. Kaneko ◽  
...  
Keyword(s):  
Energy Source ◽  
Embryonic Development ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Energy Sources ◽  
In Vitro Production ◽  
Vitro Fertilization ◽  
Development In Vitro ◽  
Vitro Production

A successful in vitro production (IVP) of porcine blastocysts, which enables piglet production after transfer to recipients, was reported (Kikuchi et al., 2002 Biol. Reprod. 66, 1033–1041). Generally, in the IVP system, both glucose and glutamine as energy sources were included in vitro culture (IVC) medium from Day 2 (Day 0=the day of in vitro fertilization) until Day 6. However, the exact requirement of these substances for the development to the blastocyst stage of IVP embryos has not yet been clarified. The objective of the present study was to evaluate whether these two substances are necessary for embryonic development to the blastocyst stage in culture during the period. Porcine cumulus-oocyte complexes were matured for 46h and fertilized in vitro as reported by Kikuchi et al. (see above). After removal of cumulus cells and spermatozoa, the oocytes were cultured subsequently in NCSU-37 supplemented with pyruvate and lactate (IVC-PyrLac) for 2 days. Then they were cultured until Day 6 in other IVC medium prepared as follows (1–6); Basic IVC medium (BM) was a modified NCSU-37 consisting of 108.7mM NaCl, 4.8mM KCl, 1.7mM CaCl2, 1.2mMKH2PO4, 1.2mM MgSO4, 25.1mM NaHCO3 and 4mgmL−1 fatty acid-free BSA. Then one or more of the following energy sources were supplemented to BM;; (1) 12mM sorbitol (SigmaUltra), 5.55mM glucose (Wako special grade) and 1.0mM glutamine (Sigma) (NCSU-37/Gln+), (2) 19.2mM sorbitol and 1.0mM glutamine (IVC-Sorbitol/Gln+); (3) 19.2mM mannitol (SigmaUltra) and 1.0mM glutamine (IVC-Mannitol/Gln+), (4) 12mM sorbitol and 5.55mM glucose (NCSU-37/Gln−); 5) 19.2mM sorbitol (IVC-Sorbitol/Gln−); and 6) 19.2mM mannitol (IVC-Mannitol/Gln−). The osmolarity of these media was adjusted to 283–285 osmolg−1. All embryos were fixed as whole mounts, stained and evaluated. The rate of blastocysts in NCSU-37/Gln+ (26.8%) was significantly higher (P&lt;0.05; by analysis of variance and Duncan’s multiple range test) than those in IVC-Sorbitol/Gln+, IVC-Mannitol/Gln+ and NCSU-37/Gln− (19.0%, 17.0% and 15.5%, respectively). A remarkable decrease in the rates in IVC-Sorbitol/Gln− and IVC-Mannitol/Gln− (P&lt;0.05; 1.4% and 2.0%, respectively) was observed. The cell numbers of NCSU-37/Gln+, IVC-Sorbitol/Gln+, IVC-Mannitol/Gln+ and NCSU-37/Gln− (55.5, 52.0, 49.6 and 58.7, respectively) had a tendency to be higher than those of IVC-Sorbitol/Gln− and IVC-Mannitol/Gln− (38.0 and 35.2, respectively). These results confirm that the supplementation of maturation medium with at least one energy source (glucose or glutamine) promotes embryonic development in vitro to the blastocyst stage, that the combination of both sources improves the chance of the embryonic survival, and that porcine embryos do not utilize sorbitol or mannitol as an energy source. The importance of glucose and glutamine is suggested for the development to the blastocyst stage of porcine IVP embryos.

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