scholarly journals Genetic influence on the reduction in bovine embryo lipid content by L-carnitine

2016 ◽  
Vol 28 (8) ◽  
pp. 1172 ◽  
Author(s):  
Luis Baldoceda ◽  
Dominic Gagné ◽  
Christina Ramires Ferreira ◽  
Claude Robert
Keyword(s):  
Lipid Content ◽  
Culture Medium ◽  
Cell Damage ◽  
Mitochondrial Activity ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Intracellular Lipid ◽  
Embryo Culture Medium

The decreased rate of pregnancy obtained in cattle using frozen in vitro embryos compared with in vivo embryos has been associated with over-accumulation of intracellular lipid, which causes cell damage during cryopreservation. It is believed that the higher lipid content of blastomeres of bovine embryos produced in vitro results in darker-coloured cytoplasm, which could be a consequence of impaired mitochondrial function. In this study, l-carnitine was used as a treatment to reduce embryonic lipid content by increasing metabolism in cultured bovine embryos. We have observed previously that in vivo embryos of different dairy breeds collected from cows housed and fed under the same conditions differed in lipid content and metabolism. As such, breed effects between Holstein and Jersey were also examined in terms of general appearance, lipid composition, mitochondrial activity and gene expression. Adding l-carnitine to the embryo culture medium reduced the lipid content in both breeds due to increased mitochondrial activity. The response to l-carnitine was weaker in Jersey than in Holstein embryos. Our results thus show that genetics influence the response of bovine embryos to stimulation of mitochondrial metabolism.

scholarly journals Isolation and Characterization of Functionally Active Extracellular Vesicles from Culture Medium Conditioned by Bovine Embryos In Vitro

2018 ◽  
Vol 20 (1) ◽  
pp. 38 ◽  
Author(s):  
Krishna Pavani ◽  
An Hendrix ◽  
Wim Van Den Broeck ◽  
Liesbeth Couck ◽  
Katarzyna Szymanska ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Embryo Culture ◽  
Culture Medium ◽  
Culture Media ◽  
Apoptotic Cell ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Isolation And Characterization ◽  
Embryo Culture Medium

Extracellular vesicles (EVs) play a possible role in cell–cell communication and are found in various body fluids and cell conditioned culture media. The aim of this study was to isolate and characterize EVs in culture medium conditioned by bovine embryos in group and to verify if these EVs are functionally active. Initially, ultracentrifuged bovine serum albumin (BSA) containing medium was selected as suitable EV-free embryo culture medium. Next, EVs were isolated from embryo conditioned culture medium by OptiPrepTM density gradient ultracentrifugation. Isolated EVs were characterized by nanoparticle tracking analysis, western blotting, transmission, and immunoelectron microscopy. Bovine embryo-derived EVs were sizing between 25–230 nm with an average concentration of 236.5 ± 1.27 × 108 particles/mL. Moreover, PKH67 EV pre-labeling showed that embryo-secreted EVs were uptaken by zona-intact bovine embryos. Since BSA did not appear to be a contaminating EV source in culture medium, EV functionality was tested in BSA containing medium. Individual embryo culture in BSA medium enriched with EVs derived from conditioned embryo culture medium showed significantly higher blastocyst rates at day 7 and 8 together with a significantly lower apoptotic cell ratio. In conclusion, our study shows that EVs play an important role in inter embryo communication during bovine embryo culture in group.

161 LIPID CONTENT, RESISTANCE TO CRYOPRESERVATION, AND SEX RATIO OF IN VITRO BOVINE BLASTOCYSTS PRODUCED IN A SERUM-FREE SYSTEM

2006 ◽  
Vol 18 (2) ◽  
pp. 188
Author(s):  
F. George ◽  
C. Daniaux ◽  
G. Genicot ◽  
F. Focant ◽  
B. Verhaeghe ◽  
...  
Keyword(s):  
Sex Ratio ◽  
Lipid Content ◽  
Culture Medium ◽  
Nile Red ◽  
Embryo Cryopreservation ◽  
Hatching Rate ◽  
Free System ◽  
Serum Free

In vitro-produced (IVP) bovine blastocysts are known to be more sensitive to cryopreservation than their in vivo counterparts. Removing serum from the culture medium decreases sanitary risk and could improve embryo resistance to cryopreservation by preventing the accumulation of intracellular lipids. Our objectives were to evaluate the lipid content, resistance to cryopreservation, and sex ratio of IVP embryos cultured in a serum-free system. Oocytes from slaughterhouse ovaries were matured in a serum-free enriched medium (Donnay et al. 2004 Reprod. Fertil. Dev. 16, 274) and cultured in 5% O2 in modified SOF supplemented with 5% FCS (FCS) or with insulin-transferrin-selenium (ITS) and 0.1 mg/mL polyvinylpyrrolidone (PVP) (ITS-PVP) or 4 mg/mL BSA (ITS-BSA) (Daniaux et al. 2005 Reprod. Fertil. Dev. 17, 217). Day 5 morulae were stained with the fluorescent dye Nile Red in order to evaluate their lipid content (Genicot et al. 2005 Theriogenology 63, 1181). Day 7 blastocysts (diameter ≥160 µm) were selected, classified according to their size, and frozen in HEPES-SOF containing 1.5 M ethylene glycol, 0.1 M sucrose, and 1.8 mg/mL wheat peptones (George et al. 2002 Reproduction 29, 51). The lipid content was significantly lower in morulae cultured in ITS-BSA compared with the two other media (320 ± 10 arbitrary fluorescence units vs. 383 ± 12 in FCS and 406 ± 10 in ITS-PVP; n = 271; ANOVA2: P < 0.01). After cryopreservation, a higher total hatching rate was found 24 h post-thawing in blastocysts cultured in ITS-BSA and for both serum-free conditions at 48 h (Table 1). In particular, embryos ≤180 µm cultured in FCS were less resistant to cryopreservation than embryos of the same size produced without serum. Expanded blastocysts cultured in ITS-BSA were sexed by PCR (Grisart et al. 1995 Theriogenology 43, 1097) and a higher proportion of male embryos was found (62.7%; n = 51). In conclusion, a complete serum-free system was set up from oocyte maturation to embryo cryopreservation that gave high quality embryos resistant to cryo-preservation. Embryos produced in ITS-BSA presented a lower lipid content, but a shift of the expanded blastocyst sex ratio toward males was observed. Table 1. Hatching rates post-thawing as a function of the blastocyst size and the culture medium

83 VITAMIN K2 SUPPLEMENTATION IMPROVES BLASTOCYST RATE BY RECOVERY OF MITOCHONDRIA IN IN VITRO-CULTURED BOVINE EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 155
Author(s):  
L. Baldoceda ◽  
C. Vigneault ◽  
P. Blondin ◽  
C. Robert
Keyword(s):  
In Vitro Culture ◽  
Fluorescent Microscopy ◽  
Vitamin K2 ◽  
Control Group ◽  
Mitochondrial Activity ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Blastocyst Rate ◽  
Mitotracker Red

Mitochondria play an important role during early mammalian embryo development through their diverse cellular functions, in particular creating balance between production of ATP by electron transport chain and oxidative stress. Embryonic mitochondria are inherited maternally and independently of the nuclear genome. They show limited activity during the early developmental stages before embryonic genome activation. It has been shown that in vitro culture (IVC) has an adverse effect on mitochondrial function in embryos. So far several attempts have been performed to improve and rescue the impaired mitochondria. It has been shown that vitamin K2 (a membrane-bound electron carrier, similar to ubiquinone) was used to rescue mitochondrial dysfunction and resulted in more efficient ATP production in eukaryotic cells (Vos et al. 2012 Science 336, 1306–1310). Therefore, the aim of the present study was to investigate the effects of supplementation of vitamin K2 on mitochondrial activity and blastocyst rate. Cumulus–oocytes complexes (n = 687) recovered from slaughtered animals, were matured and fertilized in vitro according to our standard procedures. After fertilization, zygotes were cultured in SOF media supplemented with 10 mg mL–1 BSA. At 96 h post-fertilization, vitamin K2 was added to the culture media (n = 448 oocytes). On Day 7, treatment embryos were compared with untreated controls (n = 239 oocytes). In vitro culture was carried out at 38.5°C under 5% CO2, 7% O2, and 88% N2. Differences among groups in blastocyst yield were analysed by ANOVA. Mitochondrial activity data was analysed by unpaired 2-tailed t-tests. Results show that the vitamin K2-treated group had a significantly (P < 0.05) higher blastocyst rate (+8.6%), expanded blastocyst rate (+7.8%), as well as better morphological quality compared with the control group. Furthermore, to evaluate mitochondria activity, pools of embryos of each treatment were labelled with a specific dye for active mitochondria (Mitotracker Red). A significantly higher intensity of Mitotracker Red (P < 0.05) was observed in the vitamin K2 treatment versus control group, as measured by fluorescent microscopy. In conclusion, for the first time, our data prove that supplementation of vitamin K2 during IVC of bovine embryos increases blastocyst rates and embryo quality. Future studies will focus on gene expression to identify targets implicated in impaired mitochondrial activity in in vitro bovine embryo production.

76 Effects of serum type in maturation medium on in vitro development of bovine embryos

2019 ◽  
Vol 31 (1) ◽  
pp. 163
Author(s):  
A. Mesalam ◽  
S. Zhang ◽  
K.-L. Lee ◽  
S.-H. Song ◽  
L. Xu ◽  
...  
Keyword(s):  
Lipid Content ◽  
Cumulus Cell ◽  
Cell Number ◽  
Mitochondrial Activity ◽  
Bovine Embryos ◽  
Gap Junctional Communication ◽  
Junctional Communication ◽  
Maturation Medium ◽  
Gap Junctional

This study investigated the effect of bovine serum albumin (BSA), charcoal:dextran stripped fetal bovine serum (CDS FBS), and heat-inactivated FBS (HI FBS) in maturation medium on their ability to support in vitro oocyte maturation, cumulus cell-oocyte gap junctional communication, and development of bovine embryos. Charcoal:dextran treatment of FBS removes lipophilic chemicals, certain steroid hormones, and certain growth factors; however, HI FBS have a lot-to-lot variation in steroid hormones level that can affect the reproducibility of experimental findings. Oocytes were cultured in TCM-199 supplemented with either 8% (w/v) BSA, 10% (v/v) CDS FBS, or 10% (v/v) HI FBS and 1µg mL−1 oestradiol-17β, 10µg mL−1 FSH, 10ng mL−1 epidermal growth factor, 0.6mM cysteine, 0.2mM sodium pyruvate, and followed by IVF, and the zygotes were cultured in SOF-BE1 medium. The developmental ability and quality of bovine embryos were determined by assessing their cell number, lipid content, mitochondrial activity, gene expression, immunocytochemistry, and cryo-tolerance. The differences in embryo development between experimental groups were analysed by 1-way ANOVA. The Duncan’s multiple range tests were used to test the differences between the treatments. The level of statistical significance was set at P&lt;0.05. We have shown that CDS FBS significantly improved (P&lt;0.05) the percentage of MII oocytes compared with that in the BSA supplemented group (77.2±1.0% v. 69.3%±2.3%, respectively). Moreover, CDS FBS had a higher significant (P&lt;0.05) effect on the rate of blastocyst formation compared with HI FBS and BSA (45.2±0.7% v. 37.4±1.5% and 31.1±3.9%, respectively; 6 replicates were performed). Culture of oocytes with CDS FBS increased (P&lt;0.05) the expression of gap junction proteins, CX37 and CX43, at both transcriptional and translation levels as determined by quantitative RT-PCR and immunofluorescence analysis, respectively. We also found that CDS FBS significantly increased total cell number and decreased the apoptotic index in Day-8 blastocysts compared with the BSA group. Furthermore, the beneficial effects of CDS FBS on embryos were associated with significantly reduced intracellular lipid content and increased mitochondrial activity in both oocytes and blastocysts as identified by Nile red and MitoTracker Green staining, respectively. Taken together, these data suggest that supplementation of maturation medium with CDS FBS, as an alternative to HI FBS, affected cumulus cell-oocyte gap junctional communication, and subsequently improved in vitro developmental competence of bovine oocytes and embryos. Research was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through Agri-Bio industry Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (grant numbers: 117029-3 and 315017-5).

Forskolin effect on the cryosurvival of in vitro-produced bovine embryos in the presence or absence of fetal calf serum

Zygote ◽  
2012 ◽  
Vol 22 (2) ◽  
pp. 146-157 ◽  
Author(s):  
Daniela Martins Paschoal ◽  
Mateus José Sudano ◽  
Midyan Daroz Guastali ◽  
Rosiára Rosária Dias Maziero ◽  
Letícia Ferrari Crocomo ◽  
...  
Keyword(s):  
Culture Medium ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Similar Rate ◽  
Bovine Embryos ◽  
Embryo Survival ◽  
Vitrification Solution ◽  
Oviductal Fluid

SummaryThe objective of this study was to assess the viability and cryotolerance of zebu embryos produced in vitro with or without the addition of fetal calf serum (FCS) and forskolin (F). Embryos produced in vivo were used as a control. Presumptive zygotes were cultured in modified synthetic oviductal fluid supplemented with amino acids (SOFaa), bovine serum albumin (BSA) and with (2.5%) or without (0%) FCS. On day 6 of growth, the embryos from each group were divided into treatments with or without 10 μM F to induce embryonic lipolysis, comprising a total of four experimental groups: 2.5% FCS, 0% FCS, 2.5% + F and 0% + F. For vitrification, embryos were exposed to vitrification solution 1 (5 M EG (ethylene glycol)) for 3 min and then transferred to vitrification solution 2 (7 M EG, 0.5 M galactose solution and 18% (w/v) Ficoll 70) before being introduced to liquid nitrogen. The presence of FCS in the culture medium resulted in the production of embryos with a similar rate of damaged cells compared with in vivo-produced embryos. After vitrification, the 2.5% FCS group had a significantly higher rate of damaged cells when compared with the other groups (P < 0.05). The results of this experiment indicated that the omission of FCS and the addition of forskolin do not have deleterious effect on embryo production rates. In addition, embryos produced in the presence of FCS had greater sensitivity to cryopreservation, but this effect was reversed when forskolin was added to the medium, which improved embryo survival without affecting embryo development and quality after vitrification.

Effects of amino acids on development in vitro of cleavage-stage bovine embryos into blastocysts

1996 ◽  
Vol 8 (5) ◽  
pp. 835 ◽  
Author(s):  
T Pinyopummintr ◽  
BD Bavister
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Embryo Development ◽  
Culture Medium ◽  
Essential Amino Acids ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Development In Vitro ◽  
Defined Culture

Effects of amino acids on early bovine embryo development in vitro were examined using a chemically-defined, protein-free culture medium. Bovine embryos produced in vitro were cultured from 18 h to 72 h post insemination in a simple medium containing lactate as the only energy source except for the amino acid treatments. Subsequently, embryos were transferred to TCM-199 supplemented with serum for blastocyst development to substantiate their developmental competence. Treatments were: (1) non-essential amino acids from TCM-199 (NEA); (2) essential amino acids from TCM-199 (EA); (3) NEA+EA; (4) Eagle's minimum essential medium amino acids (MEM AA); (5) 11 amino acids present in HECM-6 (11 AA); and (6) 0.2 mM glutamine (GLN). A higher proportion of embryos (percentage of inseminated ova) cleaved to the > or = 8-cell stage by 72 h post insemination in NEA (56.7%), EA (41.2%), 11 AA (40.3%) and GLN (51.1%) than in either NEA+EA (30.0%) or MEM AA (33.1%). However, after transfer to complex medium, embryos that had developed in EA, as well as those in MEM AA or NEA+EA, produced significantly fewer blastocysts (37.1%, 34.4% and 25.6% respectively) than those in NEA (56.7%), GLN (48.9%) or 11 AA (37.7%). The ability of blastocysts to hatch from their zonae pellucidae was also affected by amino acid treatment during cleavage stages. The present study indicated that the addition of NEA or GLN or 11 AA to a chemically-defined culture medium during the cleavage phase of bovine embryo development increases their subsequent ability to reach the blastocyst stage. These data have implications for understanding the nutritional needs of bovine embryos produced in vitro and for optimizing the composition of culture media to support their development.

scholarly journals Cryopreservation of in vitro-produced bovine embryos by vitrification: In pursuit of a simplified, standardized procedure that improves pregnancy rates to promote cattle industry use

2020 ◽  
Vol 36 (3) ◽  
pp. 251-270
Author(s):  
Van Do ◽  
Andrew Taylor-Robinson
Keyword(s):  
Slow Rate ◽  
Survival Rates ◽  
Slow Freezing ◽  
Embryo Cryopreservation ◽  
Human Embryos ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cattle Industry

The goal of cryopreservation is to retain the original stage of gametes and embryos after they have endured cooling and warming. Slow freezing is a standard method for in vivo-derived bovine embryo cryopreservation, threefifths of such embryos being frozen by this method globally. However, it is evident that slow freezing is not efficient for cryopreserving in vitro-produced bovine embryos. Hence, only one-third of in vitro-produced bovine embryos are cryopreserved. Vitrification is a preferred method for storage of human embryos; consequently, it has been explored as a novel means to store in vitro-produced bovine embryos, for which it shows considerable promise as an alternative to slow freezing. This is due to several reasons: vitrification is often less time-consuming than slow freezing; it does not need expensive slow rate freezing machines; and it has been proven to have comparatively higher survival rates. Yet, in the cattle industry vitrification continues to present shortcomings, such as possible toxicity of vitrification solutions and failure to standardize methods, which pose a challenge for its application to in vitro-produced bovine embryos. Therefore, determining the most suitable procedure is crucial to make vitrification more practical in commercial settings.

51 ARTIFICIAL DORMANCY OF BOVINE EMBRYOS FOR A MAXIMUM OF 7 DAYS USING A SIMPLE MEDIUM

2014 ◽  
Vol 26 (1) ◽  
pp. 139 ◽  
Author(s):  
K. Tsuchiya ◽  
A. Ideta ◽  
Y. Nishimiya ◽  
S. Tsuda ◽  
Y. Aoyagi
Keyword(s):  
Pregnancy Rate ◽  
Storage Period ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Hypothermic Preservation ◽  
Mammalian Embryos ◽  
Simple Medium ◽  
Hypothermic Storage

The worldwide pregnancy rate using cryopreserved mammalian embryos has not improved over the past 2 decades, probably because the freeze-thawing processes cause significant damage. Therefore, it is now relevant to examine the feasibility of short-term non-freezing preservation, and whether this could be applied to embryos that have high vitality and are to be transferred into recipients within several days. We introduce here an artificial dormancy fluid that can extend the hypothermic storage period of bovine embryos for a maximum of 7 days. First, to examine the effect of different basal media and the optimal concentration of fetal bovine serum (FBS) for hypothermic preservation, bovine blastocysts produced in vitro were stored at 4°C in a plastic ministraw in 1 of the following 3 media: PBS, medium 199, or Leibovitz L15 with various amount of FBS (0, 5, 20, 50, or 100%) for 3 days. Second, to examine the effect of Good's buffers, bovine embryos produced in vivo (morula to blastocyst stages) were stored at 4°C in a plastic ministraw in medium 199 plus 50% FBS supplemented with various Good's buffers [HEPES, TES, piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), MOPS, and 4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS)] for 7 days. Following hypothermic preservation, the chilled embryos were squeezed out of the straw into PBS and washed 3 times in the same medium. Subsequently, the embryos were cultured in CR1aa medium supplemented with 5% FBS for 48 h at 38.5°C under 5% CO2 in air with high humidity. The viability rate of the embryos was assessed at the end of the culture period. Finally, to observe the pregnancy rate of chilled embryos, 32 embryos produced in vivo were stored at 4°C for 7 days in medium 199 plus 50% FBS supplemented with HEPES. Following hypothermic preservation, the chilled embryos were transferred into recipient heifers (1 embryo per recipient). Pregnancy was determined by real-time B-mode ultrasonography (Convex scanner HS-1500, Honda electronics Co. Ltd, Toyohashi, Japan) on Day 60 of gestation. Data were analysed using the chi-squared test. The viability rate of the embryos after hypothermic storage for 3 days was significantly increased for medium 199 plus 50% FBS [27/30 (90%)] compared with PBS [18/30 (60%)] or Leibovitz L15 [15/30 (50%)] plus 50% FBS (P < 0.05). Chilled embryos stored for 7 days in medium 199 plus 50% FBS supplemented with HEPES had much higher survival than embryos stored in the same medium with other Good's buffers. The pregnancy rate of the chilled embryos stored for 7 days was extremely high [24/32 (75%)] and normal live calves were delivered at term. In conclusion, maintaining artificial dormancy of bovine embryos for 7 days using a simple medium appears to be feasible. This is the first documented success of storing chilled mammalian embryos in a viable state for 7 days. To be of practical value, bovine embryo preservation at hypothermic temperatures must be able to maintain viability for periods longer than 7 days. This work was supported by the Program for Promotion of Basic and Applied Research for Innovations in Bio-Oriented Industry.

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