In vitro culture and non-invasive metabolic profiling of single bovine embryos

2019 ◽  
Vol 31 (2) ◽  
pp. 306
Author(s):  
Monika Nõmm ◽  
Rando Porosk ◽  
Pille Pärn ◽  
Kalle Kilk ◽  
Ursel Soomets ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Metabolic Profiling ◽  
Culture Medium ◽  
Culture Media ◽  
Blastocyst Stage ◽  
Growth Media ◽  
Low Molecular Weight ◽  
Bovine Embryos ◽  
Non Invasive

Selecting high-quality embryos for transfer has been a difficult task when producing bovine embryos invitro. The most used non-invasive method is based on visual observation. Molecular characterisation of embryo growth media has been proposed as a complementary method. In this study we demonstrate a culture medium sampling method for identifying potential embryonic viability markers to predict normal or abnormal embryonic development. During single embryo culture, 20µL culture media was removed at Days 2, 5 and 8 after fertilisation from the same droplet (60µL). In all, 58 samples were analysed using liquid chromatography–mass spectrometry. We demonstrate that it is possible to remove samples from the same culture medium droplets and not significantly affect blastocyst rate (25.2%). Changes in any single low molecular weight compound were not predictive enough. Combining multiple low molecular weight signals made it possible to predict Day 2 and 5 embryo development to the blastocyst stage with an accuracy of 64%. Elevated concentrations of lysophosphatidylethanolamines (m/z=453, 566, 588) in the culture media of Day 8 well-developing embryos were observed. Choline (104m/z) and citrate (215m/z) concentrations were increased in embryos in which development was retarded. Metabolic profiling provides possibilities to identify well-developing embryos before transfer, thus improving pregnancy rates and the number of calves born.

85 LOW-MOLECULAR-WEIGHT METABOLITES IN BOVINE IN VITRO PRODUCTION CULTURE MEDIA AS EMBRYO QUALITY MARKERS

2015 ◽  
Vol 27 (1) ◽  
pp. 135
Author(s):  
M. Nõmm ◽  
E. Mark ◽  
K. Kilk ◽  
S. Kõks ◽  
Ü. Jaakma
Keyword(s):  
Molecular Weight ◽  
Embryo Quality ◽  
Culture Media ◽  
Principal Component ◽  
Blastocyst Stage ◽  
Low Molecular Weight ◽  
In Vitro Production ◽  
Quality Markers ◽  
Vitro Production

The need for noninvasive embryo quality assessment techniques has increased as the in vitro production of cattle embryos has become more popular and necessary in the beef and milk production industries. In this study, we assessed the metabolomic profile of embryo culture media to determine whether it is possible to evaluate differences in low-molecular-weight metabolites in the culture media composition of morula stage embryos compared with embryos that develop to the blastocyst stage. Single bovine embryos were cultured in 60-µL SOF+0.4% BSA droplets under mineral oil. Twenty microliters of culture media was removed at Day 2, 5, and 8 post-fertilization. Cultured droplets without a zygote served as the control samples. A total of 42 samples were analysed using liquid chromatography-mass spectrometry (Q-Trap 3200, Ab Sciex, Framingham, MA, USA), followed by principal component analysis. Our preliminary results indicated significant differences (P < 0.00001) in 10 low-molecular-weight compounds between the groups. Three of those compounds (588, 589, and 702 Da) were represented in higher concentrations only in embryos that advanced into the blastocyst stage. These first results could allow the identification of embryos with improved viability and give better understanding of the development of pre-implantation embryo.This study was supported by CCRMB, Project ANIREP (3.2.0701.12–0036) and institutional grant IUT8–1.

Biochemical markers for pregnancy in the spent culture medium of in vitro produced bovine embryos

2021 ◽  
Author(s):  
Gabriela de Oliveira Fernandes ◽  
Marcella Pecora Milazzotto ◽  
Andrei Antonioni Guedes Fidelis ◽  
Taynan Stonoga Kawamoto ◽  
Ligiane de Oliveira Leme ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Biochemical Markers ◽  
Culture Medium ◽  
Culture Media ◽  
Ionization Mass ◽  
Metabolic Profiles ◽  
Bovine Embryos ◽  
The Media

Abstract The present study aimed to identify biomarkers to assess the quality of in vitro produced (IVP) bovine embryos in the culture media. IVP embryos on Day (D) 5 of development were transferred to individual drops, where they were maintained for the last 48 h of culture. Thereafter, the medium was collected and the embryos were transferred to the recipients. After pregnancy diagnosis, the media were grouped into the pregnant and nonpregnant groups. The metabolic profiles of the media were analyzed via electrospray ionization mass spectrometry, and the concentrations of pyruvate, lactate, and glutamate were assessed using fluorimetry. The spectrometric profile revealed that the media from embryos from the pregnant group presented a higher signal intensity compared to that of the nonpregnant group; the ions 156.13 Da [M + H]+, 444.33 Da [M + H]+, and 305.97 Da [M + H]+ were identified as biomarkers. Spent culture medium from expanded blastocysts (Bx) that established pregnancy had a greater concentration of pyruvate (p = 0.0174) and lesser concentration of lactate (p = 0.042) than spent culture medium from Bx that did not establish pregnancy. Moreover, pyruvate in the culture media of Bx can predict pregnancy with 90.9% sensitivity and 75% specificity. In conclusion, we identified markers in the culture media that helped in assessing the most viable IVP embryos with a greater potential to establish pregnancy.

scholarly journals Effective medium for in vitro sprouting of the buds and multiplication of the plantlets, and identification of the fungal contaminant associated with the explants of Gnetum

2021 ◽  
Vol 16 (2) ◽  
pp. 001-013
Author(s):  
Abwe Mercy Ngone ◽  
Lawrence Monah Ndam ◽  
Rita Mungfu Njilar ◽  
Doungous Oumar ◽  
Thomas Eku Njock
Keyword(s):  
Culture Medium ◽  
Culture Media ◽  
Fungal Growth ◽  
Growth Media ◽  
Fungal Contamination ◽  
Surface Sterilization ◽  
Pure Cultures ◽  
Nodal Cuttings ◽  
Baseline Information

Plant tissue culture requires the optimization of growth media. Gnetum, known locally in Cameroon as “Eru” is an indigenous gymnospermous vegetable with diverse medicinal, nutritional, cultural and socio-economic values. This resource is over-exploited and expected to neighboring countries, resulting to increased scarcity in the forest. Preliminary work on the in vitro culture of nodal cuttings was faced by the problem of fungal contamination. It was therefore necessary to isolate and identify the fungal contaminant, optimize the surface sterilization of field material and compose an appropriate medium for sprouting. Pure cultures of the fungus were obtained and grown on Potato Dextrose Agar (PDA) and Sabouraud Dextrose Agar (SDA). The identification was based on the appearance of the fungal growth on plates and also on the microscopic view. This was affected by the use of keys. Gnetum explants were disinfected with the various concentrations of disinfectants, preceded in some instances by pre-treatments, as well as incorporating fungicides in the culture medium. Two different culture media were employed: the Woody Plant Medium (WPM) and the Murashige and Skoog (MS) based establishment medium (Y-1). Gnetum was found to live in association with a complex of Microsporum species. The level of contamination of cultures was reduced from 100% to 40% when pre-treated before disinfection and even lower to 10% by incorporating fungicides in the medium. Sprouting was observed in WPM. This study provides baseline information on the in vitro propagation of Gnetum and thus opens up avenues for more research to be carried out in this field.

scholarly journals MicroRNAs from Extracellular Vesicles Secreted by Bovine Embryos as Early Biomarkers of Developmental Competence

2020 ◽  
Vol 21 (23) ◽  
pp. 8888
Author(s):  
Bárbara Melo-Baez ◽  
Yat S. Wong ◽  
Constanza J. Aguilera ◽  
Joel Cabezas ◽  
Ana C. F. Mançanares ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Fatty Acid Biosynthesis ◽  
Culture Media ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Target Cells ◽  
Bovine Embryos ◽  
Developmental Potential ◽  
Maternal Communication

During early development, embryos secrete extracellular vesicles (EVs) that participate in embryo–maternal communication. Among other molecules, EVs carry microRNAs (miRNAs) that interfere with gene expression in target cells; miRNAs participate in embryo–maternal communication. Embryo selection based on secreted miRNAs may have an impact on bovine breeding programs. This research aimed to evaluate the size, concentration, and miRNA content of EVs secreted by bovine embryos with different developmental potential, during the compaction period (days 3.5–5). Individual culture media from in vitro–produced embryos were collected at day 5, while embryos were further cultured and classified at day 7, as G1 (conditioned-culture media by embryos arrested in the 8–16-cells stage) and G2 (conditioned-culture media by embryos that reached blastocyst stages at day 7). Collected nanoparticles from embryo conditioned culture media were cataloged as EVs by their morphology and the presence of classical molecular markers. Size and concentration of EVs from G1 were higher than EVs secreted by G2. We identified 95 miRNAs; bta-miR-103, bta-miR-502a, bta-miR-100, and bta-miR-1 were upregulated in G1, whereas bta-miR-92a, bta-miR-140, bta-miR-2285a, and bta-miR-222 were downregulated. The most significant upregulated pathways were fatty acid biosynthesis and metabolism, lysine degradation, gap junction, and signaling pathways regulating pluripotency of stem cells. The characteristics of EVs secreted by bovine embryos during the compaction period vary according to embryo competence. Embryos that reach the blastocyst stage secrete fewer and smaller vesicles. Furthermore, the loading of specific miRNAs into the EVs depends on embryo developmental competence.

284 COMPARATIVE FERTILITY OF FRESHLY-COLLECTED VERSUS FROZEN - THAWED SPERMATOZOA FOR IN VITRO FERTILIZATION IN THE FISHING CAT (PRIONAILURUS VIVERRINUS)

2006 ◽  
Vol 18 (2) ◽  
pp. 249
Author(s):  
G. Magarey ◽  
J. Herrick ◽  
K. Thiangtum ◽  
W. Tunwattana ◽  
W. Swanson
Keyword(s):  
Sperm Motility ◽  
Culture Medium ◽  
Culture Media ◽  
Egg Yolk ◽  
Ovarian Follicles ◽  
Blastocyst Stage ◽  
Motile Sperm ◽  
Vitro Fertilization ◽  
International Transport

Wild populations of fishing cats (Prionailurus viverrinus) in Southeast Asia are in decline, primarily due to habitat loss. Because the fishing cat population in North American zoos is small (n = 69) and inbred (F = 0.17) with relatively low genetic variation (86%), infusion of new founder genes from Asia is a conservation priority. Importation of cryopreserved semen for use with IVF and ET may offer one alternative to the international transport of living animals. In this study, our objectives were to (1) compare motility longevity of fresh vs. frozen-thawed fishing cat spermatozoa in two culture media, (2) evaluate ovarian responses to exogenous gonadotropins, and (3) assess development of IVF embryos produced with fresh vs. frozen-thawed spermatozoa. Raw semen was collected via electroejaculation from male fishing cats (n = 4), divided into groups, and washed. Two sperm pellets were resuspended in either Ham's F10 medium (HF10; with 5% FBS) or our feline optimized culture medium (FOCM; with 0.4% BSA); another pellet was diluted in TEST egg yolk, cooled to 5�C over 3 h, glycerated (4%), and cryopreserved in straws over LN2 vapor. Frozen sperm samples were thawed, washed, and diluted in either HF10 or FOCM. Fresh and frozen-thawed sperm motility (percent motile, rate of forward progress) in each medium (10 � 106 motile sperm/mL) was assessed (at 0, 1, 3, and 6 h) in microdrops under oil during culture (38�C; 6% CO2 in air). Female fishing cats (n = 10) were treated with exogenous gonadotropins (150 IU eCG, 100 IU hCG, 85-h interval) and ovarian follicles were aspirated laparoscopically. Recovered oocytes were inseminated with fresh (2 � 105 motile sperm/mL) or frozen-thawed (5 � 105 motile sperm/mL) spermatozoa in FOCM microdrops; resulting embryos were either cryopreserved or cultured in FOCM (with 5% FBS added at 72 h post-insemination) for 7 days. Sperm motility over time did not differ (P > 0.05) between media for either fresh or frozen-thawed samples; however, across media, frozen-thawed sperm motility was lower (P < 0.05) and declined faster (P < 0.05) compared to fresh spermatozoa. Females produced an average (�SEM) of 9.8 � 2.9 mature ovarian follicles, allowing recovery of 7.3 � 2.6 high-quality oocytes per female. Oocyte cleavage percentage at 42 h p.i. was lower (P < 0.05) with frozen-thawed spermatozoa (38%, 11/29) compared to freshly collected spermatozoa (68%, 17/25). Overall, 35% (6/17) of cultured embryos developed to blastocysts with no difference (P > 0.05) between embryos produced with frozen-thawed (4/11) vs. fresh (2/6) spermatozoa. Although fishing cat sperm motility and fertility appear compromised after cryopreservation, our results demonstrate the ability of frozen-thawed spermatozoa to produce IVF embryos that are capable of developing to blastocyst stage in vitro. This work was supported by (NIH RR015388).

98 EXPERIMENTAL TRANSFER OF BOVINE IVF-DERIVED 32-CELL STAGE EMBRYOS INTO THE UTERUS: ENVIRONMENTAL EFFECTS ON DEVELOPMENTAL CHARACTERISTICS

2016 ◽  
Vol 28 (2) ◽  
pp. 179
Author(s):  
M. Hoelker ◽  
D. Salilew-Wondim ◽  
F. Rings ◽  
D. Tesfaye ◽  
K. Schellander
Keyword(s):  
Culture Media ◽  
Uterine Cavity ◽  
Blastocyst Stage ◽  
Considerable Proportion ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Developmental Rates ◽  
Uterine Environment

Usually, in vitro-produced bovine embryos are cultured in vitro in static culture systems for 7 to 9 days in media composed according the oviducal fluid although it is well accepted that around Day 4.5–5 the bovine embryo enters the uterine cavity, providing environmental conditions different from the oviduct. Therefore, one has to raise the question whether changing culture media properties after Day 5 of culture could have beneficial effects on early development of bovine embryos. To answer that question, we transferred bovine IVF derived 32-cell stage embryos into the uterine cavity of synchronized recipients. All embryos had been matured and fertilized under routine standard conditions and were cultured in synthetic oviducal fluid supplemented with essential and nonessential amino acids (SOFaa) supplemented with either 0.3% fatty acid free bovine serum albumin (BSAfaf/Uterus) or 10% serum (serum/uterus) at 38.5°C, 5% O2, and 5% CO2 in humidified air prior transfer into the uterine environment, allowing further development to the blastocyst stage within the physiological environment prior recollection at Day 7 by routine uterine flushing followed by comparison with statically in vitro-developed embryos cultured in media supplemented with serum (serum/serum group) or BSAfaf (BSAfaf/BSAfaf group). All in all, a total of 1031 in vitro-derived 32-cell stage embryos were transferred to 21 synchronized Simmental recipient heifers. Of these, a total of 680 embryos (66%) could be recollected at Day 7. Embryos of the serum/serum group reached a higher blastocyst rate compared with embryos of the BSAfaf/BSAfaf group (68% v. 41%; P < 0.05, ANOVA, Tukey test), whereas the developmental rate to the blastocyst stage did not differ after 9 days of in vitro culture, indicating higher developmental kinetics of bovine 32-cell stage embryos when culture media is supplemented with serum. Moreover, embryos of the serum/uterus group reached significantly lower developmental rates to the blastocyst stage until Day 7 compared with embryos of the serum/serum group (12.9% v. 68.4%). Likewise, embryos in the BSAfaf/uterus group reached significantly lower developmental rates to the blastocyst stage until Day 7 compared with embryos in the BSAfaf/BSAfaf group (16.0% v. 40.1%). When allowed to develop for additional 48h in vitro, developmental rates to the blastocyst stage at Day 9 were still higher in BSAfaf/BSAfaf treatment compared with the BSAfaf/uterus treatment (91.4% v. 74.4%) and the serum/serum treatment compared with the serum/uterus treatment (92.5% v. 56.0%). Taken together, the results of our study demonstrate that uterine transfer of bovine 32-cell stage embryos results in reduction of developmental kinetics as well as lower developmental rates compared with embryos statically cultured in vitro. That might indicate, that a considerable proportion of bovine 32-cell stage embryos might not be able to adapt to the uterine environment.

scholarly journals Lysophosphatidic Acid Signaling in Late Cleavage and Blastocyst Stage Bovine Embryos

2014 ◽  
Vol 2014 ◽  
pp. 1-11 ◽  
Author(s):  
Ana Catarina Torres ◽  
Dorota Boruszewska ◽  
Mariana Batista ◽  
Ilona Kowalczyk-Zieba ◽  
Patricia Diniz ◽  
...  
Keyword(s):  
Lysophosphatidic Acid ◽  
Culture Medium ◽  
Marker Gene ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Human Embryos ◽  
Lipid Mediator ◽  
Bovine Embryos ◽  
Pluripotency Marker

Lysophosphatidic acid (LPA) is a known cell signaling lipid mediator in reproductive tissues. In the cow, LPA is involved in luteal and early pregnancy maintenance. Here, we evaluated the presence and role of LPA in bovine early embryonic development. In relevant aspects, bovine embryos reflect more closely the scenario occurring in human embryos than the mouse model. Transcription of mRNA and protein expression of enzymes involved in LPA synthesis (ATX andcPLA2) and of LPA receptors (LPAR1–4) were detected in Days 5 and 8in vitroproduced embryos. Embryonic LPA production into culture medium was also detected at both stages of development. Supplementation of culture medium with LPA (10−5 M) between Days 2 and 8 had no effect on embryo yield and quality and on blastocyst relative mRNA abundance of genes involved in prostaglandin synthesis (PTGS2,PGES, andPGFS) and steroidogenesis (3βHSD). However, LPA treatment affected transcription levels of embryo quality markers, decreasingBAX(apoptotic) and increasingBCL2(antiapoptotic) andIGF2R(growth marker) gene transcription levels. Blastocyst transcription ofOCT4(pluripotency marker) was not affected by LPA stimulation. In conclusion, LPA is an early bovine embryonic autocrine/paracrine signaling mediator, and LPA action may be relevant in early embryo-maternal interactions leading to embryonic survival.

Supplementation of culture medium with L-carnitine improves development and cryotolerance of bovine embryos produced in vitro

2013 ◽  
Vol 25 (4) ◽  
pp. 589 ◽  
Author(s):  
Toshikiyo Takahashi ◽  
Yasushi Inaba ◽  
Tamas Somfai ◽  
Masahiro Kaneda ◽  
Masaya Geshi ◽  
...  
Keyword(s):  
Lipid Metabolism ◽  
Culture Medium ◽  
Apoptotic Cell ◽  
Survival Rates ◽  
Blastocyst Stage ◽  
Bovine Embryos ◽  
High Lipid ◽  
Zygote Development ◽  
Vitro Development

High lipid content in embryos is associated with low freezing tolerance. This study assessed the effects of exogenous l-carnitine, an enhancer of lipid metabolism, on the in vitro development and freezing survival of bovine embryos. Also, effects on metabolic activity, reactive oxygen species (ROS) and apoptosis were investigated. Supplementation of embryo culture medium with 1.518 mM or 3.030 mM l-carnitine significantly increased the rates of zygote development to the blastocyst stage and blastocyst cell numbers whereas 6.072 mM of this compound did not improve embryo development. Survival rates after slow freezing of blastocysts were significantly higher when embryos were cultured in the presence of 1.518 mM or 3.030 mM l-carnitine compared with the control. A lower density of lipid droplets was detected in l-carnitine-treated blastocysts compared with the control. l-carnitine significantly reduced ROS levels in 2-cell embryos but did not reduce ROS levels at later stages. The apoptotic cell rate was not different between control and l-carnitine-treated blastocysts. l-carnitine significantly increased ATP levels in 2-cell embryos but not at the 8-cell or blastocyst stages. l-carnitine increased the expression of metabolism-related ATP6 and COX1 genes in blastocysts. In conclusion, l-carnitine supplementation enhanced lipid metabolism in embryos resulting in improved development and cryotolerance of bovine blastocysts produced in vitro.

168 CORRELATION OF DEVELOPMENT KINETICS AND SEX OF IN VITRO-PRODUCED BOVINE EMBRYOS

2012 ◽  
Vol 24 (1) ◽  
pp. 196
Author(s):  
A. R. Buzzo ◽  
A. R. Pupulim ◽  
J. Mazucheli ◽  
F. V. Meirelles ◽  
I. P. Emanuelli
Keyword(s):  
In Vitro Culture ◽  
Culture Medium ◽  
Culture Media ◽  
Proteinase K ◽  
Bovine Embryos ◽  
Male And Female ◽  
Stage Of Development ◽  
Males And Females ◽  
Kinetics Of

Approaches to improve the culture medium for in vitro production (IVP) of bovine embryos have been continuous because of the high commercial demand and a portion of this attempts the production of female cattle (dairy cows and stud cattle). However, in some embryonic in vitro culture systems, the development kinetics is faster in male than in female embryos (Avery 1992 Mol. Reprod. Dev. 32, 265–70; Xu 1992 Mol. Reprod. Dev. 31, 249–50). The aim of this work was to relate the kinetics of blastocyst expansion with the production rates of male and female embryos. Cumulus–oocyte complexes (n = 917; classes I and II) of cows from a slaughterhouse were matured with TCM-199 bicarbonate and 10% FCS (38.5°C, 5% CO2) for 24 h and fertilized with frozen-thawed semen in TALP-IVF medium for 18 h. Presumptive zygotes were culture in SOF medium supplemented with 10% FSB (5% O2, 38.5°C). Seven days after IVF, embryos were divided in 2 groups according to their kinetic stage of development: nonexpanded blastocysts (n = 175), or hatched and expanded blastocysts (n = 146). Hence, embryos were individually frozen in LN and stored in cryotubes. After thawing, Proteinase K (16 mg mL–1) was added to each tube and the tubes were incubated for 60 min at 37°C. Proteinase was denatured at 98°C for 10 min and the contents of each tube were divided into 2 samples (A and B) and subjected to the PCR technique. Two pairs of primers for the specific sequence of the Y chromosome were used to amplify the sequence of 210 and 250 bp for the male bovine and 1 pair of primers was used for the autosomal bovine sequence with a 280-bp fragment. Female embryos with a 280-bp product were observed in sample A and none were observed in sample B. The presence of 2 amplicons (280 and 210 bp) in sample A and 1 amplicon of 250 bp in sample B indicated that the embryo was male. A chi-square test was used to evaluate homogeneity. An analysis of the percentage of males and females between the experimental groups was performed by logistic regression and significance was considered when P < 0.05. There was no difference in the proportions of males and females in the nonexpanded blastocyst group (49.71 and 50.29%; P > 0.05). In the hatched and expanded blastocyst group, the proportion of males (65.75%) was statistically different from the proportion of females (34.25%); that is, the chance of the embryo being male was twice as high (P < 0.0038). These results suggest that there is a difference in the kinetics of embryo development between male and female embryos and that blastocyst expansion can point that out. In vitro culture media with FCS support the development of expanded male blastocysts. Further research in culture medium modifications (FCS, the energy source, amino acids and others) are needed to respond to the trend in the production of sex-defined embryos.

114 DEVELOPMENT OF AN OPTIMIZED WELL IN WELL CULTURE SYSTEM: EFFECT OF MINIWELL CALIBER AND CULTURE DROP VOLUME ON DEVELOPMENTAL COMPETENCE OF BOVINE EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 157
Author(s):  
M. Hoelker ◽  
N. Gahnem ◽  
C. Phatsara ◽  
K. Schellander ◽  
D. Tesfaye
Keyword(s):  
Treatment Group ◽  
Culture System ◽  
Culture Medium ◽  
Culture Media ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Bovine Embryos ◽  
System Effect ◽  
Developmental Problems ◽  
Developmental Rates

To overcome developmental problems due to the scientific need to track single embryos or due to embryo culture in small groups, the Well in Well culture system has been developed previously. Here we aimed to examine the effects of different MiniWell caliber and different embryo densities on developmental rates. Cumulus oocyte complexes (COC) aspirated from small follicles (2–8 mm) were cultured in modified TCM (TCM199, Sigma, Taufkirchen, Germany) supplemented with 12% heat inactivated oestrus cow serum and 10 μg mL–1 FSH (FSH-p, Sheering, Kenilworth, NJ, USA) for 24 h at 39°C in a humidified atmosphere with 5% CO2 in air. Fertilization was performed in Fert-TALP medium. After 20 h coincubation with sperm cells, presumptive zygotes were allocated into MiniWells of different diameter (0.3, 0.4, 0.5, and 0.7 mm) and depth (0.3, 0.5, and 0.7 mm) to investigate the effect of MiniWell diameter and depth on further development of the embryos. To investigate the effect of embryo density we compared the development of zygotes placed in MiniWells with different total volumes of culture medium per well. For all experiments bovine embryos cultured in groups of 16 and groups of 50 served as controls. Developmental rates of in vitro-produced embryos were analysed by chi-square test. Differences of P < 0.05 were considered to be significant. When embryos were cultured in a 4 × 3 factorial design (n = 240 per treatment group in 16 replicates), MiniWell diameter (0.3 mm v. 0.4 mm v. 0.5 mm v. 0.7 mm) significantly affected developmental rates to the blastocyst stage (21.5% v. 26.9% v. 32.5% v. 31.3%, respectively) and MiniWell depth (0.3 mm v. 0.5 mm v. 0.7 mm) influenced development (24.4% v. 27.4% v. 29.3%, respectively). When embryos (n = 160 per treatment group in 10 replicates) were cultured in different total volumes of culture media per Well (0 μL, v. 150 μL v. 500 μL) development of embryos to the blastocyst stage (3.1% v. 13.1% v. 33.1%, respectively) differed significantly. When a total of 240 embryos cultured in 15 replicates in group of 16 were compared with a total of 750 embryos cultured in 15 replicates in group of 50, embryos cultured in group of 16 reached the blastocyst stage at a significantly lower level than zygotes cultured in the group of 50 (22.2% v. 30.3%), whereas zygotes cultured in MiniWells were able to compensate against low embryo densities reaching a blastocyst rate as high as embryos cultured in group of 50 (31.3 v. 30.3%). The best developmental rate of bovine zygotes to the blastocyst stage was observed in MiniWells with a diameter of 0.7 mm and 0.7 mm depth in 500 μL culture medium per well. In conclusion, we successfully optimized the Well in Well culture system by exploring the most suitable MiniWell properties supporting improved developmental rates to the blastoyst stage by compensating against negative effects of low embryo densities allowing to track each individual embryo over the complete culture period.

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